JP4335272B2 - Novel transferases and amylases, their production and use, and genes for the novel enzymes - Google Patents

Description

Background of the Invention

TECHNICAL FIELD The present invention relates to an I-new transferase, a method for producing the same, a method for producing an oligosaccharide using the enzyme, and a gene for the enzyme and use thereof, II-a novel amylase, a method for producing the same, and an α using the enzyme. , Α-trehalose, and the enzyme gene and use thereof.

More details
I-The present invention uses a saccharide of 3 or more sugars having at least 3 or more glucose residues as α-1,4 bonds from the reducing end as a substrate, and α-1,4 bonds at the reducing end as α-1,4. The present invention relates to a novel transferase having an action of transferring to α-1 bond and a method for producing the same, and more particularly to the above-mentioned enzyme produced by Archaebacteria of the order of Sulfolobales, for example, bacteria such as Sulfolobus genus and Acidianus genus.

  The present invention also relates to a novel process for producing trehalose oligosaccharide and the like using the above-described novel enzyme, and more specifically, efficient and high yield using malto-oligosaccharide or the like as a raw material, for example, glucosyl trehalose. And a method for producing trehalose oligosaccharides such as maltooligosyl trehalose.

  The present invention also relates to a DNA fragment encoding the above novel transferase and the genetic engineering use of this DNA fragment.

  II-The present invention uses a saccharide of three or more saccharides in which at least three or more saccharides are composed of glucose units from the reducing end as a substrate, and hydrolyzes from the reducing terminus to mainly release monosaccharides and / or disaccharides. The present invention relates to a novel amylase having activity and a method for producing the same. Specifically, in the present invention, at least the reducing end trisaccharide is composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond, The α-1,4 bond between the 2nd and 3rd glucose is formed using a saccharide of 3 or more sugars whose bond between the 2nd and 3rd glucose is α-1,4 bond as a substrate. The present invention relates to a novel amylase having a main activity of hydrolyzing and releasing α, α-trehalose and a method for producing the same. The enzyme is a novel amylase having the activity of hydrolyzing α-1,4 bonds in the molecular chain of the substrate in an endo form, and can be produced by bacteria belonging to the genus Sulfolobus. This enzyme is useful in industries such as starch sugar industry, textile industry and food industry.

The present invention also relates to a method for producing α, α-trehalose, characterized in that the novel amylase and the novel transferase are allowed to act in combination. Specifically, α, α-trehalose is produced in high yield using starch, starch degradation products, malto-oligosaccharides alone or a mixture of maltooligosaccharides as raw materials, and using the novel transferase and amylase of the present invention as enzymes. Regarding the method.
The present invention also relates to a DNA fragment encoding the above novel amylase and the genetic engineering utilization of this DNA fragment.

Background Art Background Art of Enzyme Transferase Various glycosyltransferases, cyclodextrin glucanotransferases (CGTase), etc. have been found so far for glycosyltransferases acting on starch and starch degradation products such as maltooligosaccharides (biological organisms). Chemistry Experimental Method 25, Starch / Related Glycoenzyme Experimental Method, published by Academic Publishing Center, and Bioindustry, Vol. 9, No. 1 (1992) 39-44). These enzymes are enzymes that transfer the glucosyl group to the α-1,2, α-1,3, α-1,4, α-1,6 positions, but transfer to the α-1, α-1 positions. Such an enzyme is not yet known. As an enzyme that acts on α-1, α-1 bonds, there is trehalase, which is a trehalose-degrading enzyme. However, it is a degrading enzyme that uses trehalose as the only substrate, and its equilibrium and reaction rate are biased toward the degradation reaction side. Yes.

  In recent years, oligosaccharides have been found to have physicochemical properties such as moisture retention, shape retention, viscosity, browning prevention, and physiological activities such as low calorie, anti-cariogenic, and bifido activity. Oligosaccharides such as maltooligosaccharides, branched oligosaccharides, fructooligosaccharides, galactooligosaccharides, and xylooligosaccharides have been developed (sweetener (1989 version) published by Medical Research, monthly food chemical (February 1993) 21-29, etc. reference).

  Among oligosaccharides, oligosaccharides having no reducing end include fructo-oligosaccharides produced by fructosyl transferase using sucrose having no reducing ability as a skeleton. In addition, starch degradation products such as malto-oligosaccharides that do not have a reducing end include cyclodextrins produced by CGTase, α, β-trehalose (neotrehalose), and oligos that have been hydrogenated at the reducing end by chemical synthesis. There are sugar reduction products (oligosaccharide alcohol) and the like. These oligosaccharides having no reducing terminal have various physicochemical properties and physiological activities that are not found in conventional chickenpox and maltooligosaccharides. Therefore, since the oligosaccharide does not have a reducing end with respect to an oligosaccharide having a α-1, α-1 bond on the reducing end side in malto-oligosaccharide, the above oligosaccharide having no reducing end is It can be expected to have such physicochemical properties and physiological activity.

  By the way, the malto-oligosaccharide having the α-1, α-1 bond on the reducing end side as described above can be considered as a trehalose oligosaccharide in which glucose or malto-oligosaccharide is bound to α, α-trehalose. Therefore, in addition to the physicochemical properties and physiological activities of oligosaccharides having no reducing end, such trehalose oligosaccharides have a characteristic activity (eg, special table) as seen in α, α-trehalose. It can also be expected to have (see Sho 63-500562).

  Regarding trehalose oligosaccharide, there is a report (Biosci. Biotech. Biochem., 57 (7), 1220-1221 (1993)) that there is a trace amount in yeast in nature. Although it has been reported (1994 Agricultural Chemistry Conference, Abstracts of Lectures, 247), the method uses expensive trehalose as a raw material, and it is not possible to expect an inexpensive supply.

  By the way, Lama et al. Have found that there is a heat-resistant starch-degrading activity in a cell extract of Sulfobus solfataricus strain MT-4 strain (DSM 5833 strain), which is a kind of archaea (Biotech. Forum. Eur. 8, 4, 2-1 (1991)). They further report that this activity is the activity of producing trehalose and glucose from starch. However, the above report does not mention at all about the presence of trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose. Furthermore, no investigation has been made on archaea other than the above strains.

  In addition, it can be said that it is necessary to establish an efficient method for obtaining this novel transferase for efficient production of trehalose oligosaccharide.

  Therefore, for mass production of trehalose oligosaccharide, further mass acquisition of this novel transferase is desired. For this purpose, it is preferable to obtain genes of these enzymes and produce them by genetic engineering. Furthermore, if a gene can be obtained, an enzyme with improved heat resistance, pH resistance, and increased reaction rate can be expected using protein engineering techniques. However, no report on gene cloning for such an enzyme has yet been made.

  The present invention relates to a novel transferase having a catalytic action for the reaction of producing trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose from sugars such as maltooligosaccharide, a method for producing the same, and the use of the enzyme. An object of the present invention is to provide a novel method for producing trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose in an efficient and high yield from raw materials such as maltooligosaccharides.

  As a result of intensive studies on trehalose-producing activity of archaea, the present inventors have found that a wide range of archaeal cell extracts belonging to the order of Sulfolobales, specifically belonging to the genus Sulfolobus, Acidianus, etc., have maltotriose as a substrate. As found to produce glucosyl trehalose. At this time, although the production of trehalose and glucose was confirmed by the activity measurement method by Lama et al. Using starch as a substrate, it was found that confirmation of the production of trehalose oligosaccharides such as glucosyl trehalose was very difficult. In addition, it has been found that the production activity of trehalose found by Lama et al. Disappears during the purification process of archaeal cell extracts, and the purification and specification of the enzyme itself having such activity as a whole is virtually impossible. I found it possible.

Under such circumstances, the present inventors have further researched, and a new activity measurement method using a malto-oligosaccharide (eg, maltotriose) as a substrate and an activity of producing a trehalose oligosaccharide (eg, glucosyl trehalose) as an index. As a result of this measurement method, it was found that trehalose oligosaccharide (for example, glucosyl trehalose) can be easily detected, and further, purification of an enzyme having this activity with respect to various strains was attempted. Surprisingly, the enzyme thus obtained acts on a sugar of maltotriose or more in which three or more glucose residues are α-1,4 bonds from the reducing end to convert the reducing end glucose residue to α-1 , Α-1, a completely new transferase having the ability to produce trehalose oligosaccharides such as glucosyl trehalose Was knowledge that it is a hydrolase. The presence of trehalose oligosaccharide in which the reducing end glucose residue such as maltooligosaccharide was transferred to α-1, α-1 was confirmed by ¹ H-NMR and ¹³ C-NMR (Examples I-1, 7 and 8).

  The present inventors have further found that trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose can be produced in large quantities from sugars such as maltooligosaccharide by using such a novel enzyme. The invention has been completed.

  The present inventors have further established a method for isolating a gene for such a novel enzyme and producing a recombinant novel transferase using this gene by genetic engineering.

II. Background Art of Enzyme Amylase Amylase is a general term for enzymes that hydrolyze starch, in which α-amylase is an enzyme that has an activity to hydrolyze α-1,4 glucoside bonds in an endo manner. α-Amylase exists widely in the living world, and is found in saliva and pancreatic juice as a digestive enzyme in mammals. In plants, it is often observed in malt and the like. It is also widely distributed in the microbial world, and in particular, fungi belonging to the genus Aspergillus and α-amylase produced by bacteria belonging to the genus Bacillus are used in industry (Amylase, supervised by Michinori Nakamura, published by the Society Publishing Center, 1986 Year).

  These α-amylases are used in various industrial applications. For example, they are widely used in the starch liquefaction process in the starch sugar industry and in the desizing process in the textile industry. It ’s an enzyme. According to “Knowledge of Enzyme Application” (Toshiaki Komaki, published by Kosho Shobo, 1986), as important in the starch liquefaction process, 1) dissolve starch molecules as completely as possible, 2) liquefaction products It is convenient for the purpose of the next saccharification step, 3) the conditions are such that the liquefied product does not age, and 4) it is carried out at as high a concentration as possible (30-35%) from an economic standpoint. ing. The starch liquefaction process is carried out by, for example, a continuous isothermal liquefaction method, a jet cooker method, etc., and usually a high-concentration starch emulsion containing α-amylase is instantaneously raised to a high temperature (around 85 to 110 ° C.). At the same time it begins to swell and swell, it is liquefied by the action of α-amylase. That is, the starch liquefaction process requires a temperature sufficient to swell the starch so that the enzyme can act. Examples of enzymes that can be used in such a field include thermostable α-amylases produced by the aforementioned Aspergillus genus Bacillus and Bacillus genus bacteria. In order to further improve the heat resistance of these enzymes, it may be necessary to add calcium. If starch micelles that have been swollen and cleaved in the starch liquefaction process are not affected by α-amylase, once the temperature falls, the starch will re-aggregate and new micelles that are less liquified by α-amylase (slightly soluble starch) Will make. As a result, the problem is that the resulting sugar solution becomes turbid and difficult to filter. In order to prevent such a situation, a method of increasing the degree of liquefaction (DE = Dextrose Equivalent) to some extent is employed. However, there are cases where it is necessary to keep this DE as low as possible (that is, the degree of polymerization of the sugar chain is high) in order to maintain a high yield, as in the production process of enzymatic maltose. Therefore, after the starch liquefaction step, when the enzyme is further acted in the next step, if a thermostable enzyme that can be acted on while maintaining a high temperature is used, even if the starch concentration is high, it is used to produce a hardly soluble starch. It can be said that the reaction can be carried out without causing the contamination, and at the same time, the risk of microbial contamination can be reduced, which is advantageous in terms of process management and hygiene management. In addition, in order to use the enzyme repeatedly, it is said that it is important that the enzyme has high stability, particularly heat resistance, when it is immobilized and used as a bioreactor. That is, it may be exposed to a relatively high temperature for immobilization, but if it has low heat resistance, it may be deactivated during the immobilization operation. From the above, it can be said that an enzyme having high heat resistance can be used very advantageously in various industries including, for example, a starch liquefaction process.

  In recent years, thermophilic and highly thermophilic bacteria have been widely screened for obtaining thermostable enzymes including amylase. Among them, archaea belonging to the genus Thermococcales and Pyrococcus are also targeted, and it has been reported that α-amylase is produced (Applied and Environmental Microbiology, 1985-1991 (1990); JP-A-6-62869, etc.) . In addition, archaea belonging to the genus Sulfolobus are also targeted, and isolation of thermostable enzymes has been reported. Here, archaea belonging to the genus Sulfolobus are taxonomically highly hyperthermic (temperature: grows in the range of 55 ° C. to 88 ° C.), acidophilic (grows in the range of pH: 1-6), aerobic, It refers to archaea defined as sulfur bacteria (cocci (irregular): diameter 0.6-2 μm). Therefore, if an archaea belonging to the genus Sulfolobus can produce amylase, this amylase is also expected to have heat resistance. Lama et al. Have found that the cell extract of Sulfolobus solfacialus strain MT-4 strain (DSM 5833 strain), a kind of archaea, has heat-resistant amylolytic activity (Biotech. Forum. Eur. 8, 4). 2-1 (1991)). This document reports that this activity is the activity to produce α, α-trehalose and glucose from starch. However, this active substance is only partially purified, and the active substance is not specified. Also, the enzymological properties of the activity are not elucidated at all. According to the study by the present inventors (details will be described later), the active substance derived from the above strain which Lama et al. Acted on starch is a mixture of a plurality of enzymes, and the final product obtained by using this is a mixture. Was α, α-trehalose and glucose.

  By the way, α-amylase has an activity of reducing iodine starch reaction in the initial stage, that is, an activity of hydrolyzing α-1,4 glucan into an endo type (liquefaction activity). This liquefied amylase also has various modes in reaction mechanism. That is, the activity of hydrolyzing in the endo form is common, but it is known that each has its own characteristics when the degradation pattern of maltooligosaccharide is examined. For example, there are those that recognize and hydrolyze a specific position from the non-reducing end side, and those that recognize and hydrolyze a specific position from the reducing end side, and the decomposed main product is glucose Or those that are maltose or maltooligosaccharides. Specifically, the pancreatic α-amylase hydrolyzes the second or third α-1,4 bond from the reducing end (starch and related carbohydrate enzyme test method, Michinori Nakamura and Junji Kakinuma, Published by Academic Publishing Center, 1989). In addition, Bacillus subtilis-derived α-amylase hydrolyzes the α-1,4 bond that is the sixth from the non-reducing end or the third from the reducing end (Knowledge of Enzyme Application, Toshiaki Komaki, published by Koshobo, (1986). It is said that the difference in the reaction pattern of α-amylase is caused by the structure of each enzyme, and the subsite theory has been proposed for the interpretation of these phenomena. In addition, it has been recognized that there are those having a transfer activity and a condensation activity, and also a special α-amylase that generates cyclodextrin.

  On the other hand, α, α-trehalose is an α-1, α-1 bond of two molecules of glucose between its reducing groups, and is present in many living organisms, plants and microorganisms in the natural world, and freezing biological membranes. It is known to have many functions, such as protecting cells from drought and drying, and an energy source for insects. In recent years, this α, α-trehalose has been studied in the fields of pharmaceuticals, cosmetics, foods, etc. as a stabilizer against freezing and drying of proteins (Japanese Patent Publication No. 5-81232, Japanese Patent Laid-Open No. Sho 63-500562, etc.). However, there are few examples of actual use because of the fact that an inexpensive mass production method has not been established so far.

  Conventional methods for producing α, α-trehalose include, for example, yeast extraction (Japanese Patent Laid-Open No. 5-91890, Japanese Patent Laid-Open No. 4-360692 and the like), and method based on intracellular production using yeast (Japanese Patent Laid-Open No. 5-292986, European Patent). No. 0451896, etc.), a production method using microorganisms belonging to the genus Sclerothium or the genus Rhizoctonia (Japanese Patent Laid-Open No. 3-130084) has been attempted. However, since these methods are microbial production, a multi-step purification process is required for disrupting microbial cells and removing contaminants. In addition, microorganisms belonging to the genus Arthrobacter (Agrib. Biol. Chem., 33, No. 2, 190, 1969, Suzuki T, et al), microorganisms belonging to the genus Nocardia (Japanese Patent Application Laid-Open No. Sho) 50-154485), and production of bacterial cells by fermentation using glutamic acid-producing bacteria (French Patent 2671099, Japanese Patent Laid-Open No. 5-21882, etc.) has also been studied. Furthermore, examination of the production of α, α-trehalose by a biosynthetic enzyme gene has also been attempted (PCT Patent 93-17093). Each of these methods uses a sugar source such as glucose and utilizes a metabolic system that requires ATP and UTP as energy. Therefore, a complicated purification process of α, α-trehalose from the culture solution is required. In addition, attempts have been made to investigate production by enzymatic methods such as a method using trehalose phosphorylase (Japanese Patent Publication No. 63-60998) and a method using trehalase (Japanese Patent Laid-Open No. 7-51063). There are problems such as stability. All of the conventional methods as described above have problems such as low yield, complicated purification process, low production amount, complicated enzyme preparation, and the like. At present, it is strongly desired to establish a method for producing a good α, α-trehalose.

  α, α-trehalose is widely found in nature as described above, and its presence has been confirmed in archaea (System. Appl. Microbiol. 10, 215. 1988). Specifically, as described above, Lama et al. Have found that the cell extract of Sulfobus sofaraticus strain MT-4 strain (DSM 5833 strain), which is a kind of archaea, has heat-resistant amylolytic activity. The presence of α, α-trehalose is confirmed in the product (Biotech. Forum. Eur. 8, 4, 2-1 (1991)). Although this document reports that this activity is an activity to produce α, α-trehalose and glucose from starch, it actually only gives an example of using 0.33% soluble starch as a substrate. The amount of α, α-trehalose produced at that time was very small, and the production ratio of α, α-trehalose: glucose was 1: 2. Therefore, it was necessary to separate a large amount of glucose as a by-product, and the purpose of mass production of α, α-trehalose could not be achieved.

  As described above, the present inventors have used archaea belonging to the order of Sulfolobales as a substrate of at least three sugars having three or more glucose residues α-1,4 bonds from the reducing end as a substrate. Found to produce a transferase having the action of transferring the terminal α-1,4 bond to α-1, α-1 bond, and using this enzyme, such as glucosyl trehalose from maltooligosaccharide, maltooligosyl trehalose, etc. Invented a method for producing trehalose oligosaccharides. Trehalose oligosaccharide refers to a maltooligosaccharide having an α-1, α-1 bond on the reducing end side.

  On the other hand, among various conventionally known enzymes, trehalose oligosaccharide in which the reducing end of maltooligosaccharide is α-1, α-1 bond is converted to α-α next to the α-1, α-1 bond. As far as the present inventors know, there is no report about an enzyme having an action of specifically hydrolyzing at the position of 1,4 bond and releasing α, α-trehalose with high yield. That is, it is not possible with conventional amylases to specifically hydrolyze the α-1,4 bond between the second and third glucoses on the reducing end side of trehalose oligosaccharide to release α, α-trehalose. It was possible. Therefore, if an amylase having an activity of hydrolyzing α-1,4 bonds in starch or a starch degradation product molecular chain and capable of catalyzing the α, α-trehalose production reaction as described above can be developed, There will be significant benefits in mass production of α, α-trehalose.

  In addition, for mass production of α, α-trehalose, it is desired to obtain a large amount of this new amylase. For this purpose, it is preferable to obtain genes of these enzymes and produce them by genetic engineering. Furthermore, if a gene can be obtained, an enzyme with improved heat resistance, pH resistance, and increased reaction rate can be expected using protein engineering techniques.

  The present invention has trehalose oligo having an activity of hydrolyzing α-1,4 bonds in starch or starch degradation product molecular chains in an endo-type, and the reducing end of maltooligosaccharide becomes α-1, α-1 bond. Provided is a novel amylase capable of catalyzing a reaction of specifically hydrolyzing an α-1,4 bond between the second and third glucoses on the reducing end side of a sugar to release α, α-trehalose. And providing a method for producing the enzyme, and using the enzyme, α, α-trehalose can be effectively and in high yield from inexpensive raw materials such as starch, starch degradation products, or maltooligosaccharides. An object is to provide a novel method of manufacturing.

  As a result of intensive studies on the amylolytic activity of archaea, the present inventors have found that a wide range of archaeal cell extracts belonging to the order of Sulfolobales, in particular, belonging to the genus Sulfolobus, have heat-resistant amylolytic activity. I found it. At that time, it was confirmed that the sugars produced by the decomposition of starch were mainly glucose and α, α-trehalose as described in the literature of Lama et al. Therefore, when the properties of amylolytic activity were further investigated for various strain extracts, the enzymes produced by these strains were liquefied amylase, in terms of enzyme activity such as amylolytic activity and α, α-trehalose producing activity, It has been found that the enzyme mixture is composed of endo-type amylases such as glucoamylase, exo-type amylases, and transferases. Moreover, the enzyme activity was found to be due to the synergistic action of the activities of these multiple enzymes. Furthermore, when attempting to purify individual enzymes, the activity measurement method by Lama et al. Using the decrease in blue color due to the iodine starch reaction as an index is probably because the sensitivity and quantification are low, the purification of enzymes having such enzyme activity as a whole. Was found to be low in terms of yield and very difficult to operate. Further, according to the detailed examination by the present inventors, it was found that the partial purification method described in the literature of Lama et al. Was not purified or isolated at the protein level.

  Under such circumstances, the present inventors have further researched and developed a new activity measurement method using trehalose oligosaccharide (for example, maltotriosyl trehalose) as a substrate and the activity of releasing α, α-trehalose as an index. As a result of thinking, when this measurement method was carried out, it was found that amylase activity could be easily detected, and further, purification of an enzyme having this activity for various strains was attempted. It succeeded in separation and purification. In addition, when the enzymatic properties of the isolated and purified amylase were examined, surprisingly, the enzyme thus obtained has an activity of hydrolyzing starch or starch degradation products in an endo form, It has the activity of hydrolyzing starch degradation products or maltooligosaccharides from the reducing end side to produce monosaccharides and / or disaccharides. In particular, the bond between the first and second glucoses on the reducing end side is α Reactivity with three or more sugars (for example, trehalose oligosaccharide) in which the bond between the second and third glucoses at the terminal side is an α-1,4 bond in the α-1,1 bond These are higher than the corresponding malto-oligosaccharides, using such saccharides of 3 or more sugars as substrates, hydrolyzing the α-1,4 bonds between the second and third glucoses from the reducing end side. Activity to release α, α-trehalose That they have to meet, and found that an enzyme having a novel mechanism of action at all.

  The present inventors further established a method for isolating a gene for such a novel enzyme and producing a recombinant novel amylase by genetic engineering using this gene.

I. Novel transferase The present invention uses, as a substrate, a saccharide of three or more sugars in which at least three or more glucose residues are α-1,4 bonds from the reducing end, and α-1,4 bond at the reducing end is α-1,4. The present invention provides a novel transferase having an action of transferring to α-1 bond (hereinafter also referred to as the novel transferase of the present invention, or simply the enzyme of the present invention or the present enzyme).

  In another aspect of the present invention, a maltooligosaccharide in which all glucose residues are α-1,4 bonds is used as a substrate, and α-1,4 bonds at the reducing end thereof are changed to α-1, α-1 bonds. The present invention provides a novel transferase having an action of transferring.

  The present invention is further based on an activity measurement method in which a bacterium capable of producing such a transferase having the ability to produce transferase is cultured in a medium, and from the culture, malto-oligosaccharide is used as a substrate and trehalose oligosaccharide is generated as an index. The present invention provides a method for producing the novel transferase of the present invention, which comprises isolating and purifying the transferase.

  Furthermore, the present invention uses the enzyme of the present invention to allow at least three sugar residues from the reducing end to act as a substrate with three or more sugars having α-1,4 bonds, and at least at the reducing end side. A trisaccharide is composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond between the second and third glucoses on the terminal side. The present invention provides a method for producing a saccharide in which the terminal disaccharide is an α-1, α-1 bond, wherein the saccharide is an α-1,4 bond.

  The present invention still further provides a method for producing trehalose oligosaccharide, wherein the enzyme of the present invention is used and each maltooligosaccharide alone or a mixture thereof is allowed to act as a substrate.

Another object of the present invention is to provide a novel transferase gene.
Another object of the present invention is to provide a novel recombinant transferase using the gene and a method for producing the same.

  A further object of the present invention is to provide an efficient method for producing trehalose oligosaccharides such as glucosyl trehalose and maltoglucosyl trehalose using a novel recombinant transferase.

  Therefore, the DNA fragment according to the present invention uses, as a substrate, a saccharide of three or more sugars in which at least three glucose residues from the reducing end are α-1,4 bonds, and the α-1,4 bond at the reducing end is defined as α. -1, comprising a gene encoding a novel transferase having an action to transfer to α-1 bond.

  Moreover, the recombinant novel transferase according to the present invention is an expression product of the above DNA fragment.

Furthermore, the method for producing a recombinant novel transferase according to the present invention comprises:
Culturing a host cell transformed with the gene, producing the recombinant novel transferase in the culture, and collecting it.

II. Novel amylase The present invention uses a saccharide of three or more saccharides in which at least three or more saccharides are composed of glucose units from the reducing end as a substrate, and hydrolyzes mainly from the reducing end to release monosaccharides and / or disaccharides. It is intended to provide a novel amylase having the activity of

  In another aspect of the present invention, at least three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond. , Using a saccharide of 3 or more sugars in which the bond between the second and third glucoses on the terminal side is an α-1,4 bond as a substrate, α-1, between the second and third glucoses, The present invention provides a novel amylase having a main activity of hydrolyzing four bonds and releasing α, α-trehalose.

  Furthermore, in another aspect, the present invention provides a novel amylase having the above-described activity and also having an activity of hydrolyzing α-1,4 bonds in the molecular chain of the substrate in an endo form. Is.

  The present invention further comprises culturing a bacterium having the ability to produce the amylase of the present invention as described above in a medium, and using the activity to produce α, α-trehalose from the culture using trehalose oligosaccharide as a substrate. The present invention provides a method for producing the amylase characterized in that the amylase is isolated and purified based on an activity measurement method.

  The inventors of the present invention, when the amylase of the present invention as described above and the above-described transferase of the present invention are combined with a saccharide raw material such as starch, starch degradation product, and maltooligosaccharide, efficiently, In addition, it was found that α, α-trehalose was produced in a high yield.

  Therefore, the present invention further provides a method for producing α, α-trehalose characterized in that the amylase and transferase of the present invention as described above are used in combination.

Another object of the present invention is to provide a novel amylase and its gene.
Another object of the present invention is to provide a novel recombinant amylase using the gene and a method for producing the same.
A further object of the present invention is to provide a method for producing α, α-trehalose using a recombinant novel amylase.

Therefore, the amylase gene according to the present invention is
(1) Activity to hydrolyze α-1,4 glucoside bond in sugar chain in endo form,
(2) At least three or more saccharides from the reducing end are composed of glucose units, and the bonds are α-1,4 linkages of three or more saccharides as substrates, mainly hydrolyzing from the reducing end side. Activity to release monosaccharides and / or disaccharides, and (3) at least three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is α-1 , Α-1 bond, and the second and third sugars are made from three or more sugars whose substrates are α-1,4 bonds between the second and third glucoses on the terminal side. Which comprises a DNA sequence encoding a novel amylase having the main activity of hydrolyzing the α-1,4 bond between the two glucose groups and releasing α, α-trehalose.

  Moreover, the recombinant novel amylase according to the present invention is an expression product of the above gene.

Furthermore, the method for producing α, α-trehalose according to the present invention includes:
The above recombinant novel amylase and at least three or more sugar residues having α-1,4 bonds at least three glucose residues from the reducing end as a substrate, and the α-1,4 bond at the reducing end as α-1 , A novel transferase having an action of transferring to α-1 bond,
It comprises a step of contacting at least 3 or more glucose residues from the reducing end with 3 or more sugars which are α-1,4 bonds.

Deposit of microorganisms The new strain KM1 described later, which was isolated from the natural world by the present inventors , was deposited under the accession number FERM BP-4626 on April 1, 1994. Deposited at the Institute of Biotechnology, AIST.
E. coli strain E. coli transformed with plasmid pKT22 (see Example I-14 described below) containing the novel transferase gene according to the present invention. E. coli JM109 / pKT22 was transformed into Escherichia coli transformed with plasmid p09T1 (see Example I-16 below) under the accession number FERM BP-4843 on October 21, 1994. E. E. coli JM109 / p09T1 has been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on May 9, 1995 under the accession number FERM BP-5093.

  In addition, E. coli strain E. coli transformed with plasmid pKA2 (see Example II-19 described later) containing the novel amylase gene according to the present invention. E. coli JM109 / pKA2 was transformed into Escherichia coli transformed with plasmid p09A1 (see Example II-22 described later) under the accession number FERM BP-4857 on October 31, 1994. E. E. coli JM109 / p09A1 was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-5092 on May 9, 1995.

I. New transferase
Microorganisms that produce the novel transferase of the present invention Archaebacteria that can be used in the present invention include the Sulfobus sofaraticus ATCC 35091 (DSM 1616) strain, Sulfobus solfataricus DSM 5833 strain, the Sulfobus solfataricus KM1 natural strains from the nature of the present inventors And the following new strains isolated in pure form), Sulfolobus acidocaldarius ATCC 33909 (DSM 639) strain, Acidianus brierleyi DSM 1651 strain, and the like.

  Microorganisms producing the novel transferase of the present invention are thus considered to cover a fairly wide range of archaea belonging to the order of Sulfolobus and Acidianus belonging to the taxonomics. Here, the Sulfolobales is an archaebacteria defined taxonically as highly acidophilic thermophilic, aerobic, sulfur bacteria (cocci). The above-mentioned Acidianus brierleyi DSM 1651 strain belonging to the genus Acidianus is a bacterium previously classified as Sulfolobus brierleyi DSM 1651 strain, and the above-mentioned Sulfolobus solfatricus DSM 5833 strain is called cell gal. . Therefore, it is needless to say that all bacteria that are genetically or taxonomically related to the archaebacteria and can produce the same kind of enzymes can be used in the present invention.

In the illustrated microorganisms in Sulfolobus solfataricus KM1 strain above, Sulfolobus solfataricus KM1 strain a strain which the present inventors have isolated from a hot spring in Gunma Prefecture, shows a such following manner nature.
(1) Morphological properties Shape and size of bacteria: Cocci (irregular) Diameter 0.6-2 μm
(2) Growth optimum condition pH: Grows in the range of 3 to 5.5, and the optimum growth pH is 3.5 to 4.5.
Temperature: grows in the range of 55 ° C to 85 ° C, optimal growth temperature is 75 ° C to 80 ° C
Can metabolize sulfur (3) Distinguish between aerobic and anaerobic: aerobic

  Based on the above properties, the strain was identified according to Bergey's Manual of Systematic Bacteriology Volume 3 (1989), and as a result, this strain belongs to the Sulfobus sulfaraticus, and thus this strain was named Sulfobusus sulFatK.

In culturing the above strain, the medium may be liquid or solid, and usually, shaking culture or aeration and agitation culture using a liquid medium is performed. Thus, the medium is not particularly limited as long as it is suitable for growth. For example, the Catalog of Bacteria and Pages 18th Edition (1992) published by American Type Culture Collection (ATCC) and Deutsche Samling von
Sulfolobus solfaricus Medium described in Catalog of Strains 5th edition (1993) issued by Mikroorganismen and Zellkulturen GmbH (DSM) can be preferably used. Furthermore, starch, maltooligosaccharide, etc. may be added as a sugar source. In addition, the culture conditions are not particularly limited as long as the culture temperature and pH are as described above.

Cultivation of microorganisms producing the novel transferase of the present invention The culture conditions for producing the novel transferase of the present invention may be appropriately selected within a range where the transferase can be produced. In the case of liquid shaking culture or aeration and agitation culture, culture for 2 to 7 days is appropriate at the pH and culture temperature at which each microorganism grows. As the medium, for example, Sulfolobus solfataricus Medium according to American Type Culture Collection (ATCC) issued Catalogue of Bacteria and Phages 18 Ed. (1992) and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) issued Catalog of Strains 5 edition (1993) Etc. can be preferably used. Furthermore, starch, maltooligosaccharide, etc. may be added as a sugar source.

Purification of novel transferase of the present invention Extraction of the novel transferase of the present invention produced by the above-mentioned microorganisms is carried out by first obtaining cells from a culture obtained by the above-described culture method by a known method, for example, centrifugation. Is suspended in an appropriate buffer, and the cells are disrupted by freeze-thawing, sonication, grinding, etc., and the cell extract containing the transferase is obtained by centrifugation or filtration.

  Purification of the novel transferase of the present invention present in this bacterial cell extract can be performed by appropriately combining known separation and purification methods. For example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing molecular weight differences such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, and ion exchange chromatography. Methods using specific charge differences, methods using specific affinity such as affinity chromatography, methods using hydrophobic differences such as hydrophobic chromatography and reverse phase chromatography, and isoelectric focusing And a method using the difference in isoelectric point. Specific examples thereof are as shown in Examples I-2 to I-5 described later. Finally, an enzyme showing a single band is obtained as a purified enzyme by native polyacrylamide gel, SDS polyacrylamide gel or isoelectric focusing.

  Regarding the activity measurement of the enzyme or enzyme-containing material separated in the various purification processes as described above, the activity measurement method in the method of Lama et al. Uses starch as a substrate, and according to this, the production of trehalose and glucose is Although it can be confirmed, the production of trehalose oligosaccharide cannot be detected at all, and this method has a major problem that even the trehalose producing activity disappears during the purification and cannot be confirmed. It was impossible. However, when the present inventors adopted a new activity measurement method using malto-oligosaccharide (for example, maltotriose) as a substrate and using as an index the activity of producing trehalose oligosaccharide (for example, glucosyl trehalose), the target enzyme is the first time. Finally, the purification and specification of the novel transferase activity main body of the present invention could be realized.

註１：経時変化
マルトトリオースを基質として用いた場合、主反応であるグルコシルトレハロースの生成とともに、副反応としてマルトース及びグルコースが等モル生成された。
マルトテトラオース以上の重合度ｎを持つ糖では、主反応として、還元末端のグルコース単位がα−１、α−１結合した糖が生成され、同様に副反応として等モルずつの重合度（ｎ−１）糖及びグルコースが生成された。
註２：酵素作用／酵素反応様式
還元末端がα−１，４結合でグルコースが３つ結合したマルトトリオース以上の糖の還元末端の糖を、転移によりα−１，α−１で結合させる活性を有する酵素と考えられる。また基質濃度が低い場合、及び長時間反応させた場合等においては、副反応、即ち、グルコースポリマーからグルコースを遊離する活性も有する。この詳細は、実施例Ｉ−７の具体例において示す通りである。 Various properties of the novel transferase of the present invention Examples of the enzyme of the present invention include Sulfobus solfataricus KM1 strain, Sulfobus solfataricus DSM 5833 strain, Sulfobus acidocaldarius ATCC 33909 strain, and Acidianus bridase strain M Various properties are summarized in Table 1 below. The data in the table is based on the specific examples shown in Examples I-6 and 7.

Note 1: Change with time When maltotriose was used as a substrate, equimolar amounts of maltose and glucose were produced as side reactions along with the production of glucosyltrehalose, which was the main reaction.
For sugars having a degree of polymerization n equal to or higher than maltotetraose, a sugar having α-1 and α-1 linked glucose units at the reducing end is produced as the main reaction, and similarly, the degree of polymerization (n -1) Sugar and glucose were produced.
註 2: Enzyme action / enzyme reaction mode The sugar at the reducing end of maltotriose or higher sugar in which the reducing end is α-1,4 bond and three glucoses are bound by α-1, α-1 by transfer It is considered an enzyme having activity. In addition, when the substrate concentration is low and when the reaction is performed for a long time, it also has a side reaction, that is, an activity of releasing glucose from the glucose polymer. The details are as shown in the specific example of Example I-7.

  The properties of the enzyme have been described above. As described in the section of enzyme action / enzyme reaction, the activity of the enzyme is maltotriose with α-1,4 bonds at the reducing end and three glucose bonds. This is the activity of binding the sugar at the reducing end of the above sugar at α-1, α-1 by transfer, which is a completely new enzyme activity. However, as will be apparent from the following examples, there are some differences in the properties of the present enzyme other than the enzyme activity due to the genus or species of the strain.

Production of trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose The present invention uses the present enzyme and at least three or more sugars having α-1,4 bonds at least three glucose residues from the reducing end as a substrate. And at least the three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond, and the two sugars on the terminal side are Provided is a method for producing a sugar in which the terminal disaccharide is an α-1, α-1 bond, characterized in that a sugar having an α-1,4 bond between the eye and the third glucose is produced. However, the production method of the present invention will be described below with the most typical specific examples, that is, production methods of trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose.

  The method for producing trehalose oligosaccharides such as glucosyl trehalose and malto-oligosyl trehalose in the present invention is based on the present enzyme produced by archaea, from sugars such as maltooligosaccharides, typically from each of maltooligosaccharides alone or a mixture thereof. Trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose are produced. Therefore, as long as the present enzyme produced by archaea is capable of acting on a sugar such as maltooligosaccharide, the manner of contact between the present enzyme transferase and a sugar such as maltooligosaccharide is not particularly limited. Specifically, in general, crude enzymes are obtained from archaeal cells or disrupted cells, and then purified enzymes obtained in various purification steps or directly isolated and purified through various purification means are directly used. What is necessary is just to make it act on sugars, such as maltooligosaccharide. Alternatively, the enzyme may be immobilized on a carrier according to a conventional method, and contacted with a sugar such as maltooligosaccharide as the immobilized enzyme. In addition, two or more present enzymes obtained from a plurality of archaebacteria may coexist and contact with a sugar such as maltooligosaccharide.

  A mixture of malto-oligosaccharides, which is a typical substrate raw material in the production method of the present invention described above, is obtained by, for example, subjecting starch to degradation or acid degradation by endo-amylase, debranching enzyme, etc., and at least three glucose residues from the reducing end. It can be produced by appropriately decomposing so that the group is an α-1,4 bond. In this case, for example, bacteria such as Bacillus genus, fungi such as Aspergillus genus, plant-derived enzymes such as malt, and the like can be used as the endo-amylase. As the debranching enzyme, for example, pullulanase derived from bacteria such as Bacillus genus and Klebsiella genus, isoamylase derived from Pseudomonas genus, and the like can be used. Further, these enzymes can be used in combination.

  The use concentration of a sugar such as maltooligosaccharide may be appropriately selected in consideration of the specific activity of this enzyme, the reaction temperature, etc., as long as the sugar used can be dissolved. It is common to set it as 0.5 to 70% of range, Preferably it is 5 to 40% of range. The reaction temperature and pH conditions in the reaction between the sugar and the enzyme are preferably performed under the optimum conditions for the present enzyme transferase. Therefore, it is common to carry out under conditions of about 50 to 85 ° C. and pH of about 3.5 to 6.5, preferably 60 to 80 ° C. and pH of 4.5 to 6.0.

  The produced reaction solution containing trehalose oligosaccharide such as glucosyl trehalose or maltooligosyl trehalose can be purified according to a known method. For example, the obtained reaction solution is desalted with an ion exchange resin, and the target sugar fraction is separated by chromatography using activated carbon, ion exchange resin (HSO3 type) or cation exchange resin (Ca type) as a separating agent. Alternatively, it is possible to obtain trehalose oligosaccharide of high purity by concentrating and crystallizing.

Gene encoding novel transferase According to the present invention, a gene encoding the above-described novel transferase is further provided.

  Examples of the DNA fragment comprising the gene encoding the novel transferase according to the present invention include the DNA fragment represented by the restriction enzyme map shown in FIG. 26 or FIG.

  This DNA fragment can be obtained from archaea belonging to the order of Sulfolobales, preferably from archaea belonging to the genus Sulfolobus, more preferably from Sulfolobus solfaricus KM1 or Sulfolobus acidocaldarius ATCC 33909, which will be described later. The preferred isolation method from Sulfolobus solfaricus KM1 and Sulfolobus acidocaldarius ATCC 33909 is described in detail in the examples which follow.

  Specific examples of the origin from which this DNA fragment may be obtained include Sulfolobus solfataricus DSM 5354 strain, DSM 5833 strain, ATCC 35091 strain, ATCC 35092 strain, Sulfolobus acidoidarius ATCC49426 strain, Sulfolobus shiMidba strain 89 it can. The fact that these archaea can be the origin of the DNA fragments according to the present invention is that, in the hybridization test of Example I-17 described later, a novel transferase gene derived from Sulfolobus solfatricus KM1 is hybridized with the chromosomal DNA of these archaea. It is also clear from the fact that the properties of the enzyme itself are very similar as described above. Furthermore, it can be said that the result of this Example shows that the novel transferase gene according to the present invention is highly conserved specifically in archaea belonging to the order of Sulfolobales.

  According to a preferred aspect of the present invention, there is provided a DNA fragment comprising a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or 4 as a preferred specific example of a gene encoding the novel transferase according to the present invention. The Furthermore, preferred specific examples of the DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2 include the nucleotide sequences from the 335th to 2518th of the base sequence shown in SEQ ID NO: 1. Preferable specific examples of the DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 4 include the nucleotide sequences from 816 to 2855 of the base sequence shown in SEQ ID NO: 3.

  In general, if an amino acid sequence of a protein is given, the base sequence encoding it can be easily determined with reference to a so-called codon table. Accordingly, various base sequences encoding the amino acid sequence shown in SEQ ID NO: 2 or 4 can be appropriately selected. Therefore, in the present invention, the “DNA sequence encoding the amino acid shown in SEQ ID NO: 2” means that having the sequence from the 335th to 2518th of the base sequence shown in SEQ ID NO: 1, and codons that are degenerate A base sequence that has the same base sequence and encodes the amino acid shown in SEQ ID NO: 2 except that is used. In addition, the “DNA sequence encoding the amino acid shown in SEQ ID NO: 4” means that the nucleotide sequence shown in SEQ ID NO: 3 has the sequence from 816 to 2855 and codons that are degenerate. It also means a base sequence that has the same base sequence except that it encodes the amino acid shown in SEQ ID NO: 4.

  Further, as described later, the novel transferase according to the present invention includes an equivalent sequence of the amino acid sequence shown in SEQ ID NO: 2 or 4. Therefore, the DNA fragment according to the present invention further includes a base sequence encoding this equivalent sequence.

  As a result of investigating the presence of a sequence having homology with the base sequence shown in SEQ ID NO: 1 or 3 using sequence analysis software genetics (software development) through the base sequence data bank (EMBL), We have confirmed that there is no such sequence.

  The DNA fragments having the sequences from 335 to 2518 of the base sequence shown in SEQ ID NO: 1 and the DNA fragments having the sequences from 816 to 2518 of the base sequence shown in SEQ ID NO: 3 according to the present invention have a fixed base sequence. Therefore, one means for obtaining the DNA fragment is to produce it according to a nucleic acid synthesis technique.

  This sequence can be obtained from the archaebacteria belonging to the above-mentioned Sulfolobales, preferably Sulfobus solfaricus KM1 strain, or Sulfobus acidocaldarius ATCC 33909 strain using genetic engineering techniques. For example, it can be preferably carried out by the method described in Molecular Cloning: A Laboratory Manual (Sambrook, Maniatis et al., Cold Spring Harbor Laboratory Press (1989)). Specific methods are described in detail in the examples described below.

Recombinant novel transferase As described above, since a novel transferase gene is provided, the present invention provides a recombinant novel transferase that is the expression product of this gene.

  Preferable specific examples of the recombinant novel transferase according to the present invention include the expression product of the DNA fragment represented by the restriction enzyme map shown in FIG. 26 or FIG.

  Furthermore, preferred specific examples include a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing or an equivalent sequence thereof. Here, the “equivalent sequence” is an amino acid sequence represented by SEQ ID NO: 2 or 4, in which some amino acids are inserted, substituted or deleted, or added to both ends, and It shall mean what still retains the novel transferase action. The retention of the novel transferase action in the equivalent sequence means that, in an actual usage mode using the action, the polypeptide having all the sequences shown in SEQ ID NO: 2 or 4 can be used in substantially the same manner under the same conditions. It shall be said that a certain degree of activity is maintained. Such an “equivalent sequence” is specifically calculated in Example I-18 when the homology of the amino acid sequence of the novel transferase was calculated in consideration of the gap between the Sulfolobus solfacialus KM1 strain and the Sulfobus acidocaldarius ATCC33909 strain Even if it is 49%, the same activity is retained, so that by referring to the sequences shown in SEQ ID NOs: 2 and 4, those skilled in the art can select and manufacture without particular difficulty. It is clear.

  As clarified in Example I-17 to be described later, DNA fragments having the sequences shown in SEQ ID NOs: 1 and 3 are other than the Sulfolobus solfacialus KM1 strain and the Sulfolobus acidocaldarius ATCC33909 strain that are the origins of the DNA fragments. It forms a hybrid with a DNA fragment derived from another strain. On the other hand, as described above, the existence of a novel transferase having very similar properties was confirmed from these strains. Further, as will be clarified in Example I-18, which will be described later, the homology of the amino acid sequence of the novel transferase was calculated in consideration of the gap between the two strains, Sulfobus solfataricus KM1 and Sulfobus acidocaldarius ATCC 33909. 49 %. Therefore, it can be said to those skilled in the art that a novel transferase activity can be retained in a sequence having a certain degree of homology with the amino acid sequence shown in SEQ ID NO: 2 or 4.

  Regarding the presence of sequences having homology with the amino acid sequences shown in SEQ ID NOs: 2 and 4, using amino acid sequence data banks (Swiss proto and NBRF-PFB) using sequence analysis software genetics (software development) As a result of examination, the present inventors have confirmed that such a sequence does not exist.

Expression of a gene encoding a novel transferase A DNA molecule comprising a DNA fragment encoding a novel transferase according to the present invention in a state capable of being replicated in the host cell and capable of expressing the gene, particularly an expression vector, in the form of a host cell Once transformed, the recombinant novel transferase according to the present invention can be produced in the host cell.

  Accordingly, the present invention further provides a DNA molecule, particularly an expression vector, containing a gene encoding the novel transferase according to the present invention. This DNA molecule can be obtained by incorporating a DNA fragment encoding the novel transferase according to the present invention into a vector molecule. According to a preferred embodiment of the invention, this vector is a plasmid.

  The preparation of the DNA molecule according to the present invention can be carried out in accordance with the method described in the above-mentioned Molecular Cloning: A Laboratory Manual.

  The vector utilized in the present invention can be appropriately selected from viruses, plasmids, cosmid vectors, etc., taking into account the type of host cell to be used. For example, when the host cell is Escherichia coli, λ phage bacteriophage, pBR and pUC plasmids, Bacillus subtilis pUB plasmids, and YEp and YCp vectors when yeast is used.

  This plasmid preferably contains a selectable marker for the transformant, and a drug resistance marker or an auxotrophic marker gene can be used as the selectable marker.

  Furthermore, the DNA molecule as an expression vector according to the present invention comprises a DNA sequence necessary for the expression of a novel transferase gene, such as a transcription regulatory signal such as a promoter, a transcription initiation signal, a ribosome binding site, a translation termination signal, and a transcription termination signal, translation regulation. It is preferable to have a signal or the like.

  As a promoter, not only a promoter that can function in a host contained in an inserted fragment, but also a promoter such as lactose operon (lac) and tryptophan operon (trp) in E. coli, alcohol dehydrogenase gene (ADH) in yeast, acid Promoters such as phosphatase gene (PHO), galactose gene (GAL), glyceraldehyde 3-phosphate dehydrogenase gene (GPD) can be preferably used.

  Here, the base sequence from 1 to 2578 of the base sequence shown in SEQ ID NO: 1 and the base sequence from 1 to 3467 of the base sequence shown in SEQ ID NO: 3 are the sequences necessary for the above expression. It is also preferable to use this sequence as it is because it is believed to contain it.

  In addition, when the host cell is Bacillus subtilis or yeast, it is also advantageous to secrete a recombinant novel transferase enzyme outside the cell body using a secretory vector.

  As a host cell, in addition to Escherichia coli, Bacillus subtilis, yeast, and higher eukaryotes can be used. As the Bacillus subtilis, for example, a microorganism belonging to the genus Bacilus is preferably used. In this genus, it is known that there are strains that secrete a large amount of protein outside the cell. Therefore, by using a secretory vector, a large amount of recombinant novel amylase can be secreted into the culture solution. Furthermore, purification from the culture supernatant is facilitated, which is preferable. In addition, strains that hardly secrete protease outside the cells are also known in the genus, and using such strains is preferable because the recombinant novel amylase according to the present invention can be efficiently produced. When an organism that does not produce glucoamylase is selected as a host cell, the recombinant novel transferase according to the present invention is obtained in the state of a bacterial cell extract or a simple purified crude enzyme, and this is directly used as a trehalose oligo described later. Since it can be used for manufacture of sugar, it is very advantageous.

  A recombinant novel transferase produced by the transformant described above can be obtained as follows. First, the above host cells are cultured under appropriate conditions, and bacterial cells are obtained from the obtained culture by a known method, for example, by centrifugation, suspended in an appropriate buffer, frozen, thawed, and sonicated. The bacterial cells are crushed by treatment, grinding, etc., and a bacterial cell extract containing a recombinant novel transferase is obtained by centrifugation or filtration.

  Purification of the recombinant new transferase present in the bacterial cell extract can be performed by appropriately combining known separation and purification methods. For example, methods that utilize differences in heat resistance such as heat treatment, methods that utilize differences in solubility such as salt precipitation and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that use different molecular weights, methods that use charge differences such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, hydrophobic chromatography, and reverse phase chromatography Examples include a method using a difference in hydrophobicity, and a method using a difference in isoelectric point such as isoelectric focusing. Since this recombinant novel transferase has heat resistance, it can be removed as a precipitate by denaturing the host protein by heat treatment, so that purification can be performed very easily.

Production of Trehalose Oligosaccharides Using Recombinant Novel Transferases Furthermore, according to the present invention, there is provided a method for producing so-called trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose using the above-mentioned recombinant novel transferases.

  That is, in the method according to the present invention, at least the trisaccharide on the reducing terminal side is composed of glucose units, and the bond between the first and second glucoses on the terminal side is α-1, α-1 bond, A method for producing a trehalose oligosaccharide in which the linkage between the second and third glucoses on the terminal side is an α-1,4 linkage, wherein the recombinant novel transferase is treated with at least three or more glucose from the reducing end. Contacting the residue with a trisaccharide or higher sugar wherein the residue is an α-1,4 bond.

  There are no particular limitations on the sugars of three or more sugars in which at least three or more glucose residues are α-1,4 bonds from the reducing end, and starch, starch degradation products, maltooligosaccharides and the like can be mentioned. Here, as a starch degradation product, starch is subjected to degradation or acid degradation by endo-amylase, debranching enzyme, etc., and at least three or more glucose residues from the reducing end are appropriately α-1,4 bonds. What was manufactured by decomposing | disassembling is mentioned. Examples of endo-amylase used herein include bacteria such as Bacillus genus, fungi such as Aspergillus genus, plant-derived enzymes such as malt, and the like. As the debranching enzyme, for example, pullulanase derived from bacteria such as Bacillus genus, Klebsiella genus, isoamylase derived from Pseudomonas genus and the like can be used. Further, these enzymes can be used in combination.

  The contact mode and the conditions of the recombinant novel transferase according to the present invention and at least three or more sugars in which at least three glucose residues are α-1,4 bonds from the reducing end are as follows. It is not particularly limited as long as it is a mode capable of acting on the liquid crystal. A preferred embodiment in the case of contacting in a solution is as follows. That is, the concentration of sugars such as maltooligosaccharides may be appropriately selected in consideration of the specific activity of the present enzyme, reaction temperature, etc., as long as the sugar to be used can be dissolved. It is common to set it as a range, Preferably it is 5 to 40% of range. The reaction temperature and pH conditions in the reaction between the sugar and the enzyme are preferably carried out under the optimum conditions for the recombinant novel transferase. Therefore, it is common to carry out under conditions of about 50 to 85 ° C. and pH of about 3.5 to 6.5, preferably 60 to 80 ° C. and pH of 4.5 to 6.0.

  In addition, the degree of purification of the recombinant novel transferase can also be selected as appropriate, it can be used as a crude enzyme from the disrupted cells of the transformant, and can be used as a purified enzyme obtained in various purification steps. May be. Further, it may be used as an enzyme isolated and purified through various purification means.

  Furthermore, the enzyme may be immobilized on a carrier according to a conventional method and contacted with sugar in the immobilized state.

  The produced trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose can be obtained by purifying the reaction solution according to a known method. For example, the obtained reaction solution is desalted with an ion exchange resin, and the target sugar fraction is separated by chromatography using activated carbon, ion exchange resin (HSO3 type) or cation exchange resin (Ca type) as a separating agent. Alternatively, it is possible to obtain trehalose oligosaccharide of high purity by concentrating and crystallizing.

II. New amylase
Microorganisms that produce the novel amylase of the present invention Archaebacteria that can be used in the present invention include Sulfolobus solfaricus KM1 strain (the above-mentioned new strain isolated by the present inventors in a substantially pure form), Sulfolobus solifericus DSM 5833 strain, Sulfolobus acidocaldarius ATCC 33909 (DSM 639) strain, and the like.

  Microorganisms producing the novel amylase of the present invention are thus considered to cover a fairly wide range of archaea belonging to the order of Sulfolobales. Here, Sulfolobales are taxonomically highly thermophilic (temperature: grows in the range of 55 ° C. to 88 ° C.), acidophilic (grows in the range of pH: 1-6), aerobic, sulfur bacteria (cocci) (Random): Archaea defined as 0.6-2 μm in diameter). The above-mentioned Sulfolobus solfaricus DSM 5833 strain is a bacterium previously designated as Caldariella acidophila. Therefore, from these facts, bacteria that are genetically or taxonomically related to the above archaea and that can produce the same kind of enzymes, and strains of these bacteria are treated with various mutagens. It goes without saying that all the mutant strains obtained can be used in the present invention.

  Among the microorganisms exemplified above, Sulfolobus solfaricus KM1 strain is a strain isolated by the present inventors from a hot spring in Gunma Prefecture, and its properties, culture method and deposit are as described in detail above. is there.

Cultivation of microorganisms producing the novel amylase of the present invention The culture conditions for producing the novel amylase of the present invention may be appropriately selected within a range where the amylase can be produced. In the case of liquid shaking culture or aeration and agitation culture, culture for 2 to 7 days is appropriate at the pH and culture temperature at which each microorganism grows. Examples of media include American Type Culture Collection (ATCC) published by Catalog of Bacteria and Pages 18th edition (1992) and Deutsche Sammlung von Mikorgangen Zellkul Can be used. Furthermore, starch, maltooligosaccharide, etc. may be added as a sugar source.

Purification of the novel amylase of the present invention Extraction of the novel amylase of the present invention produced by the above-mentioned microorganisms is carried out by first obtaining a microbial cell from a culture obtained by the culture method as described above, for example, by centrifugation, This is suspended in an appropriate buffer, and the cells are disrupted by freeze-thawing, sonication, grinding, etc., and a cell extract containing the amylase is obtained by centrifugation or filtration.

  Purification of the novel amylase of the present invention present in this bacterial cell extract can be performed by appropriately combining known separation and purification methods. For example, methods using solubility such as salt precipitation and solvent precipitation, methods using molecular weight differences such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, ion exchange chromatography A method using a difference in charge such as a method using a specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as hydrophobic chromatography or reverse phase chromatography, and isoelectric focusing For example, a method using the difference in isoelectric point such as electrophoresis. Specific examples thereof are as shown in Examples II-2 to II-4 described later. Finally, an enzyme showing a single band is obtained as a purified enzyme by native polyacrylamide gel, SDS polyacrylamide gel or isoelectric focusing.

  Regarding the activity measurement of the enzyme or enzyme-containing material separated in the various purification processes as described above, the activity measurement method in the method of Lama et al. Uses starch as a substrate, and according to this, there are a plurality of various amylases. Although the degradation of starch can be detected in the state where the amylase is separated, both detection sensitivity and quantitativeness are low in the state where each amylase is separated, and in this method, the starch degradation activity is almost lost during purification and cannot be confirmed. As a result, the enzyme activity body could not be purified and specified. However, the present inventors adopted a new activity measurement method using trehalose oligosaccharide (for example, maltotriosyl trehalose) as a substrate and using as an index the activity of degrading into α, α-trehalose and malto-oligosaccharide (for example, maltotriose). As a result, the specificity, detection sensitivity, and quantification are very high, and the target enzyme can be isolated and purified for the first time. Finally, the purification and specification of the novel amylase activity body of the present invention can be realized. It was.

註１：経時変化
可溶性デンプンを基質として用いた場合、反応初期にヨウ素デンプン反応が速やかに消失し、引き続き、マルトース、グルコースを主成分として若干量のマルトトリオース、マルトテトラオースを生成するように分解した。
註２：酵素作用／酵素反応様式
本酵素はデンプン、デンプン分解物、及びマルトオリゴ糖を基質とした場合、加水分解反応により主にグルコース、マルトースを生成し、少量のマルトトリオース、マルトテトラオースを生成する。本活性の作用機作についてはこれらの基質に対してエンド型に作用するアミラーゼ活性と共にその還元末端側から主に単糖、２糖を生成する活性を有する。 Properties of the novel amylase of the present invention As examples of the enzyme of the present invention, the archaebacteria of each of the enzymes produced by Sulfolobus solfataricus KM1 strain, Sulfolobus solfataricus DSM 5833, and Sulfolobus acidocaldarius ATCC 33909 (DSM 639) The various properties are summarized in Table 2 below. The data in the table is based on the specific example shown in Example II-5.

Note 1: Change over time When soluble starch is used as a substrate, the iodine starch reaction disappears rapidly at the beginning of the reaction, and subsequently, a certain amount of maltotriose and maltotetraose are mainly produced mainly from maltose and glucose. Disassembled.
註 2: Enzyme action / Enzyme reaction mode When starch, starch degradation products, and maltooligosaccharides are used as substrates, this enzyme mainly produces glucose and maltose by hydrolysis, and produces a small amount of maltotriose and maltotetraose. Generate. The mechanism of action of this activity has the activity of producing monosaccharides and disaccharides mainly from the reducing end side, together with amylase activity acting in an endo-type on these substrates.

In particular, the bond between the first and second glucoses at the reducing end is an α-1, α-1 bond, and the bond between the second and third glucoses at the reducing end is α-1, High reactivity with 3 or more sugars (for example, trehalose oligosaccharide) that are 4 bonds, and when this is used as a substrate, α-1,4 bonds between the 2nd and 3rd glucose from the reducing end side Has the activity of specifically releasing α, α-trehalose at the beginning of the reaction.
Therefore, this enzyme is considered to be a novel amylase. The details are as shown in the specific example of Example II-5.

  The properties of the enzyme have been described above. As is apparent from Table 2 and the examples described later, the properties of the enzyme other than the enzyme activity are slightly different depending on the genus or species of the strain. It is recognized that there is.

The transferase used in the production of α, α-trehalose The transferase used in the production method of α, α-trehalose of the present invention includes the aforementioned I.I. The transferase of the present invention described in detail in the section of novel transferase can be used. Specifically, for example, Sulfobus solfataricus ATCC 35091 (DSM 1616) strain, Sulfobus solfataricus DSM 5833 strain, Sulfolobus solfataricus KM1 strain, Sulbobus acidocal33 Can do.

  The transferase can be produced, for example, according to the method shown in Examples I-2 to I-5 described later. The transferase thus obtained has various properties as shown in Example I-6 described later.

Production of α, α-trehalose The present invention provides a method for producing α, α-trehalose using the novel amylase and transferase of the present invention. The production method of the present invention is most typical. A specific example, that is, a method for producing α, α-trehalose from sugar raw materials such as starch, starch degradation products, and maltooligosaccharide will be described below. The mechanism by which the above two enzymes act on starch or the like is probably as follows. That is, the novel amylase of the present invention acts on starch, starch degradation product, or maltooligosaccharide first by its endo-type activity, which is decomposed into amylose or maltooligosaccharide, and then the amylose or maltooligosaccharide by the action of transferase. The α-1,4 bond at the reducing end of the sugar is rearranged to the α-1, α-1 bond, and the novel amylase acts again to react with α, α-trehalose and amylose or malto-oligo reduced in degree of polymerization by two. It is considered that sugar is produced and the above reaction is repeated on the amylose or maltooligosaccharide thus produced, so that α, α-trehalose is produced in a high yield.

  Such an action mechanism is that the binding between the first and second glucoses on the reducing end side is an α-1, α-1 bond, and the binding between the second and third glucoses on the terminal end side. Reactivity with 3 or more sugars (for example, trehalose oligosaccharide) in which is α-1,4 bond is higher than the reactivity with the corresponding malto-oligosaccharide, respectively, and the reducing terminal of the above 3 or more sugars Due to the specific reaction mode of the novel amylase of the present invention, which specifically hydrolyzes the α-1,4 bond between the second and third glucoses on the side to release α, α-trehalose It is thought to do.

  As far as the present inventors know, malto-oligosyl trehalose in which the reducing end of maltooligosaccharide is α-1, α-1 bond in a conventionally known amylase, becomes the α-1, α-1 bond. There is no specific hydrolyzing at the α-1,4 bond position of the α-1, α-trehalose in a high yield, so that α, α-trehalose is produced in a high yield. It was almost impossible.

  The production method of α, α-trehalose in the present invention is as long as the amylase of the present invention (the present enzyme) produced by archaea is in a mode capable of acting on sugars such as starch, starch degradation products, and maltooligosaccharides. The mode of contact between the transferase and sugars such as starch, starch degradation products, and maltooligosaccharides is not particularly limited. Specifically, in general, crude enzymes are obtained from archaeal cells or disrupted cells, and then purified enzymes obtained in various purification steps or directly isolated and purified through various purification means are directly used. What is necessary is just to make it act on sugars, such as starch, a starch degradation product, or maltooligosaccharide. Alternatively, the enzyme thus obtained may be immobilized on a carrier and contacted with a sugar such as starch, starch degradation product, or maltooligosaccharide as the immobilized enzyme. Two or more of the present enzymes obtained from a plurality of archaebacteria may coexist and contact with sugars such as starch, starch degradation products, or maltooligosaccharides.

  In the method for producing α, α-trehalose of the present invention, the amylase and transferase should be used within the optimum range. In other words, excess amylase acts on starch, starch degradation products, or maltooligosaccharides whose reducing ends are not affected by transferase to produce glucose and maltose, while excess transferase is produced by the enzyme. This is because the produced trehalose oligosaccharide such as malto-oligosyl trehalose is decomposed by side reaction to produce glucose.

  Specifically, the concentration of amylase and transferase relative to the substrate is 1.5 U / ml and 0.1 U / ml or more, preferably 1.5 U / ml and 1.0 U / ml or more, respectively, more preferably 15 U / ml, respectively. ml and 1.0 U / ml or more, and the concentration ratio of amylase to transferase is 100 to 0.075, preferably 40 to 3.

  The concentration of sugars such as starch, starch degradation products, and maltooligosaccharides may be appropriately selected in consideration of the specific activity of the enzyme used, reaction temperature, etc., as long as the sugar used can be dissolved. It is common to set it as 0.5 to 70% of range, Preferably it is 5 to 40% of range. The reaction temperature and pH conditions in the reaction between the sugar and the enzyme are preferably performed under the optimum conditions for amylase and transferase. Therefore, it is common to carry out under conditions of about 50 to 85 ° C. and pH of about 3.5 to 8, preferably 60 to 75 ° C. and pH of 4.5 to 6.0.

  Moreover, when the sugar raw material to be used is starch, starch decomposition products, etc. with a high degree of polymerization, the production of α, α-trehalose can be promoted by additionally using another endo-type liquefied amylase in combination. Furthermore, debranching enzymes such as pullulanase and isoamylase can also be used. In this case, endo-amylase, pullulanase, isoamylase and the like do not need to be enzymes produced by archaea. Therefore, although not particularly limited, for example, bacteria such as Bacillus genus, fungi such as Aspergillus genus, and plant-derived amylases such as malt can be used. As the debranching enzyme, for example, pullulanase derived from bacteria such as Bacillus genus and Klebsiella genus (including thermostable pullulanase), isoamylase derived from Pseudomonas genus and the like can be used. Further, these enzymes can be used in combination.

  However, addition of an excessive amount of amylase may produce glucose and maltose that are not utilized by transferase. Similarly, in the debranching enzyme, addition of an excessive amount causes a decrease in the solubility of the substrate due to cleavage of 1,6-bonds, resulting in a high-viscosity insoluble substance (amylose) that is not used. Therefore, the amount of amylase and debranching enzyme used must be carefully adjusted so as not to produce excess glucose, maltose, or insoluble substances. For example, with respect to the debranching enzyme, the concentration used should be appropriately selected in consideration of the specific activity of the amylase of the present invention, the reaction temperature, etc., and in the range that does not produce insoluble substances. Specifically, in the case of treatment at 40 ° C. for 1 hr, the range is generally 0.01 to 100 U / ml, preferably 0.1 to 25 U / ml with respect to the substrate. . (Refer to Examples II-6, 13, and 14 for the definition of the activity of the debranching enzyme.) When the debranching enzyme is used, the substrate is previously introduced with the debranching enzyme before the α, α-trehalose production reaction. Any method may be used, such as a treatment method or a method of coexisting with amylase and transferase at any stage during the production reaction of α, α-trehalose. In this case, the debranching enzyme is preferably used in combination at least once in any stage, and more preferably used in combination at least once in the initial stage. When a thermostable debranching enzyme is used, the same effect can be obtained by adding it once at any stage or initial stage in the α, α-trehalose production reaction.

  The produced reaction solution containing α, α-trehalose can be purified according to a known method. For example, the obtained reaction solution is desalted with an ion exchange resin and subjected to chromatography using an activated carbon, an ion exchange resin (HSO 3 type), or a cation exchange resin (Ca type) as a separating agent. Can be separated or further subsequently concentrated and crystallized to obtain highly pure α, α-trehalose.

Gene encoding novel amylase According to the present invention, a gene encoding the novel amylase is further provided.
Specific examples of the DNA fragment containing the gene encoding the novel amylase according to the present invention include the DNA fragment represented by the restriction enzyme map shown in FIG. 34 or FIG.

  This DNA fragment can be obtained from archaea belonging to the order of Sulfolobales, preferably from archaea belonging to the genus Sulfolobus, more preferably from Sulfolobus solfaricus KM1 or Sulfolobus acidocaldarius ATCC 33909, which will be described later. The preferred isolation method from Sulfolobus solfaricus KM1 and Sulfolobus acidocaldarius ATCC 33909 is described in detail in the examples which follow.

  Specific examples of the origin from which this DNA fragment is considered to be obtainable include Sulfobus solfataricus DSM5354 strain, DSM5833 strain, ATCC35091 strain, ATCC35092 strain Sulfolobus acididalusDSB strain 89 The fact that these archaea can be the origin of the DNA fragment according to the present invention is that, in the hybridization test of Example II-24 described later, novel amylase genes derived from Sulfolobus solfataricus KM1 and Sulfolobus acidocaldarius ATCC33909 were used. It is also clear from the fact that it forms a hybrid with the chromosomal DNA, and also shows that the properties of the enzyme itself are very similar as described above. Furthermore, it can be said that the result of this Example shows that the novel amylase gene according to the present invention is highly conserved specifically in archaea belonging to the order of Sulfolobales.

  According to a preferred aspect of the present invention, there is provided a DNA fragment comprising a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 6 or 8 as a preferred specific example of a gene encoding the novel amylase according to the present invention. The Furthermore, preferred specific examples of the DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 6 include the base sequence from 642 to 2315 of the base sequence shown in SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 8. Preferred specific examples of the DNA fragment to be encoded include the base sequence Nos. 1176 to 2843 shown in SEQ ID No. 7.

  In general, if an amino acid sequence of a protein is given, the base sequence encoding it can be easily determined with reference to a so-called codon table. Therefore, various base sequences encoding the amino acid sequence shown in SEQ ID NO: 6 or 8 can be appropriately selected. Therefore, in the present invention, the “DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 6” has the base sequence shown in SEQ ID NO: 5 and the degenerate relationship thereof. A base sequence having the same base sequence except that a codon is used and encoding the amino acid shown in SEQ ID NO: 6 is also meant. The “DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 8” includes a nucleotide sequence shown in SEQ ID NO: 7 having a sequence of Nos. 1176 to 2843, and a codon in its degenerate relationship. In addition, the nucleotide sequence having the same nucleotide sequence and encoding the amino acid shown in SEQ ID NO: 8 is also meant.

  Furthermore, as described later, the novel amylase according to the present invention includes an equivalent sequence of the amino acid sequence shown in SEQ ID NO: 6 or 8. Therefore, the DNA fragment according to the present invention further includes a base sequence encoding this equivalent sequence.

  Further, the novel amylase according to the present invention includes a sequence in which Met is further added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 6. Therefore, the DNA fragment containing the novel amylase according to the present invention includes those having the nucleotide sequence from 639 to 2315 of the nucleotide sequence shown in SEQ ID NO: 5.

As a result of investigating the presence of sequences having homology with the nucleotide sequences shown in SEQ ID NOs: 5 and 7, using sequence analysis software genetics (software development) through the nucleotide sequence data bank (EMBL), We have confirmed that there is no such sequence.
The DNA fragment having the nucleotide sequence 639 or 642 to 2315 of the nucleotide sequence shown in SEQ ID NO: 5 or the DNA fragment having the nucleotide sequence of 1176 to 2843 of the nucleotide sequence shown in SEQ ID NO: 7 according to the present invention is a base. Since the sequence is fixed, one means for obtaining the DNA fragment is to produce it in accordance with the method of nucleic acid synthesis.

  This sequence can be obtained from the archaebacteria belonging to the above-mentioned Sulfolobales, preferably Sulfobus solfaricus KM1 strain, or Sulfobus acidocaldarius ATCC 33909 strain using genetic engineering techniques. For example, it can be preferably carried out by the method described in Molecular Cloning: A Laboratory Manual (Sambrook, Maniatis et al., Cold Spring Harbor Laboratory Press (1989)). Specific methods are described in detail in the examples described below.

Recombinant Novel Amylase As described above, a novel amylase gene is provided. Therefore, according to the present invention, a recombinant novel amylase that is an expression product of this gene is provided.

  Preferable specific examples of the recombinant novel amylase according to the present invention include an expression product of a DNA fragment represented by the restriction enzyme map shown in FIG. 34 or FIG.

  Furthermore, preferable specific examples include a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or 8 in the sequence listing or an equivalent sequence thereof. Here, the “equivalent sequence” is the amino acid sequence shown in SEQ ID NO: 6 or 8, in which some amino acids are inserted, substituted, or deleted, or added to both ends, And what still holds the above-mentioned novel amylase activity. The retention of the novel amylase activity in the equivalent sequence means that in the actual usage mode using the activity, the polypeptide having all the sequences shown in SEQ ID NO: 6 or 8 can be used in substantially the same manner under the same conditions. It shall be said that a certain degree of activity is maintained. Such an “equivalent sequence” was specifically calculated in Example II-23 in which the homology of the novel amylase was taken into account at the amino acid sequence level between the Sulfolobus solfacialus KM1 strain and the Sulfobus acidocaldarius ATCC33909 strain. Even in the case of 59%, the same activity is retained, so that by referring to the sequence shown in SEQ ID NO: 6 or 8, those skilled in the art can select and manufacture without particular difficulty Obviously.

Furthermore, according to another aspect of the present invention, there is further provided an amino acid sequence in which Met is further added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 6. The novel amylase according to the present invention had the sequence shown in SEQ ID NO: 6 in its natural form. However, when a new amylase is obtained from the gene information isolated as described later by the gene recombination technique using the sequence, Met is further added to the N-terminus of the amino acid sequence of SEQ ID NO: 6. It can be seen that Furthermore, it is clear that this sequence has a novel amylase activity, and therefore the amino acid sequence to which this Met is added is also included in the present invention.
As clarified later in Example II-24, the DNA fragment having the sequence from No. 1393 to No. 2116 shown in SEQ ID NO: 7 is the Sulfolobus acidocaldarius ATCC 33909 strain or Sulfolobus solfatalicus which is the origin of this DNA fragment. It forms a hybrid with a DNA fragment derived from another strain other than the KM1 strain. On the other hand, as described above, the presence of a novel amylase having very similar properties was confirmed from these strains. Further, as will be clarified in Example II-23 to be described later, the homology of the amino acid sequence of the novel amylase was calculated in consideration of the gap between the two strains of Sulfolobus solfataricus KM1 and Sulfolobus acidocaldarius ATCC33909. %. Therefore, it can be said to those skilled in the art that a novel amylase activity can be retained in a sequence having a certain degree of homology with the amino acid sequence shown in SEQ ID NO: 6 or 8.

  Regarding the presence of sequences having homology with the amino acid sequences shown in SEQ ID NOs: 6 and 8, using amino acid sequence data banks (Swiss proto and NBRF-PFB), using sequence analysis software genetics (software development) As a result of examination, the present inventors have confirmed that such a sequence does not exist.

Expression of a gene encoding a novel amylase A DNA molecule comprising a DNA fragment encoding a novel amylase according to the present invention in a state capable of being replicated in the host cell and capable of expressing the gene, particularly an expression vector, in the form of a host cell Once transformed, the novel amylase according to the present invention can be produced in the host cell.

  Accordingly, the present invention further provides a DNA molecule, particularly an expression vector, containing a gene encoding the novel amylase according to the present invention. This DNA molecule can be obtained by incorporating a DNA fragment encoding the novel amylase according to the present invention into a vector molecule. According to a preferred embodiment of the invention, this vector is a plasmid.

  The preparation of the DNA molecule according to the present invention can be carried out in accordance with the method described in the above-mentioned Molecular Cloning: A Laboratory Manual.

  The vector utilized in the present invention can be appropriately selected from viruses, plasmids, cosmid vectors, etc., taking into account the type of host cell to be used. For example, when the host cell is Escherichia coli, λ phage-type bacteriophage, pBR, pUC-type plasmids, Bacillus subtilis, pUB-type plasmids, yeast, YEp, YCp-type vectors.

  This plasmid preferably contains a selectable marker for the transformant, and a drug resistance marker or an auxotrophic marker gene can be used as the selectable marker.

  Furthermore, the DNA molecule as an expression vector according to the present invention comprises a DNA sequence necessary for the expression of a novel amylase gene, for example, a transcription regulatory signal such as a promoter, a transcription initiation signal, a ribosome binding site, a translation termination signal, a transcription termination signal, and a translation regulation. It is preferable to have a signal or the like.

  As a promoter, not only a promoter that can function in a host contained in an inserted fragment, but also a promoter such as lactose operon (lac) and tryptophan operon (trp) in E. coli, alcohol dehydrogenase gene (ADH) in yeast, acid Promoters such as phosphatase gene (PHO), galactose gene (GAL), glyceraldehyde 3-phosphate dehydrogenase gene (GPD) can be preferably used.

  Here, the base sequence from 1 to 2691 in the base sequence shown in SEQ ID NO: 5 and the base sequence from 1 to 3600 in the base sequence shown in SEQ ID NO: 7 efficiently express a novel amylase in Escherichia coli. . Therefore, since the base sequences shown in SEQ ID NOs: 5 and 7 seem to contain at least a sequence necessary for expression in E. coli, it is preferable to use this sequence as it is.

  In addition, when the host cell is Bacillus subtilis or yeast, it is also advantageous to secrete a novel amylase outside the bacterial body using a secretory vector.

  As a host cell, in addition to Escherichia coli, Bacillus subtilis, yeast, and higher eukaryotes can be used. As the Bacillus subtilis, for example, a microorganism belonging to the genus Bacilus is preferably used. In this genus, it is known that there are strains that secrete a large amount of protein outside the cell. Therefore, by using a secretory vector, a large amount of recombinant novel amylase can be secreted into the culture solution. Furthermore, purification from the culture supernatant is facilitated, which is preferable. In addition, strains that hardly secrete protease outside the cells are also known in the genus, and using such strains is preferable because the recombinant novel amylase according to the present invention can be efficiently produced. Further, when an organism that does not produce glucoamylase is selected as a host cell, a recombinant novel amylase according to the present invention is obtained in the state of a bacterial cell extract or a crude enzyme that has been subjected to simple purification, and this is directly described later as α, Since it can be used for production of α-trehalose, it is extremely advantageous.

  The recombinant novel amylase produced by the transformant described above can be obtained as follows. First, the above host cells are cultured under appropriate conditions, and bacterial cells are obtained from the obtained culture by a known method, for example, by centrifugation, suspended in an appropriate buffer, frozen, thawed, and sonicated. The bacterial cells are crushed by treatment, grinding, etc., and a bacterial cell extract containing recombinant novel amylase is obtained by centrifugation or filtration.

  Purification of the recombinant new amylase present in this bacterial cell extract can be performed by appropriately combining known separation and purification methods. For example, methods that utilize differences in heat resistance such as heat treatment, methods that utilize differences in solubility such as salt precipitation and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that use different molecular weights, methods that use charge differences such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, hydrophobic chromatography, and reverse phase chromatography Examples include a method using a difference in hydrophobicity, and a method using a difference in isoelectric point such as isoelectric focusing. Since this recombinant novel amylase has heat resistance, it can be removed as a precipitate by denaturing the host protein by heat treatment, so that purification can be performed very easily.

Production of α, α-trehalose using recombinants According to the present invention, there is provided a method for producing α, α-trehalose using the above-mentioned recombinant novel amylase and the above-described recombinant novel transferase.
According to a preferred embodiment of the method for producing α, α-trehalose, the recombinant novel amylase according to the present invention and the recombinant novel transferase may be mixed and contacted with sugars such as starch, starch degradation products, maltooligosaccharides at the same time. . It is also preferable to replace either one of the recombinant novel transferase and the recombinant novel amylase with a naturally occurring enzyme.

  The concentration of sugars such as starch, starch degradation products, maltooligosaccharides and the like may be appropriately selected in consideration of the specific activity of the enzyme, reaction temperature, etc. as long as the sugar to be used can be dissolved. Generally it is made into the range of -70%, Preferably it is the range of 5-40%. The reaction temperature and pH conditions in the reaction between the sugar and the enzyme are preferably performed under the optimum conditions for the recombinant novel amylase and the recombinant novel transferase according to the present invention. Therefore, about 50 to 85 ° C. and about pH 3.5 to 8 are common, preferably 60 to 75 ° C. and pH 4.5 to 6.0.

  In addition, in sugars such as starch having a high degree of polymerization and starch degradation products, production of α, α-trehalose can be promoted by using an endo-type liquefying amylase or debranching enzyme as an auxiliary. Examples of such endo-type liquefying amylase include bacteria such as Bacillus genus, fungi such as Aspergillus genus, plant-derived enzymes such as malt, and the like. As the debranching enzyme, for example, pullulanase derived from bacteria such as Bacillus genus, Klebsiella genus, isoamylase derived from Pseudomonas genus and the like can be used. Further, these enzymes can be used in combination.

  However, addition of an excessive amount of endo-type liquefying amylase produces glucose and maltose that are not utilized by the novel transferase. Similarly, in pullulanase, addition of an excessive amount causes a decrease in the solubility of the substrate due to cleavage of the α-1,6 bond, resulting in a high viscosity insoluble matter that is not used. Therefore, it is preferable that the amounts of endo-type liquefying amylase and pullulanase used in this case are adjusted so as not to generate excessive glucose, maltose, or insoluble matter.

  When pullulanase is used, either a method of pretreating the substrate with pullulanase in advance or a method of using a recombinant novel amylase and a novel transferase coexisting at any stage during the production reaction of α, α-trehalose. It may be.

  The produced α, α-trehalose can be obtained by purifying the reaction solution according to a known method. For example, the obtained reaction solution is desalted with an ion exchange resin, and the target sugar fraction is obtained by chromatography using activated carbon, ion exchange resin (HSO3 type), or cation exchange resin (Ca type) as a separating agent. High purity α, α-trehalose can be obtained by separation or further subsequent concentration and crystallization.

  Hereinafter, specific examples will be shown and the present invention will be described in more detail. Needless to say, the present invention is not limited to these examples.

Example I-1 Glucosyl trehalose producing activity of archaea The glucosyl trehalose producing activity of the strains shown in Table 3 below was examined. As a method, the cultured cells of each strain are subjected to ultrasonic disruption treatment and centrifugation, and malttriose as a substrate is finally added to the supernatant so as to become 10%, and reacted at 60 ° C. for 24 hours. After stopping the reaction by heat treatment at 100 ° C. for 5 minutes, the produced glucosyl trehalose was measured by HPLC analysis under the following conditions.
Column: TOSOH TSK-gel Amide-80 (4.6 x 250mm)
Solvent: 75% acetonitrile Flow rate: 1.0 ml / min
Temperature: Room temperature Detector: Differential refractometer
As the enzyme activity, the enzyme activity for converting maltotriose into 1 μmol of glucosyl trehalose per hour was shown as 1 unit. However, in Table 3, it showed as activity per microbial cell.

The HPLC chart is as shown in FIG. As shown in the figure, the main reactant appeared as a single peak without anomer on the HPLC chart with a slight delay from the unreacted substrate. The main product was fractionated on a TSK-gel amide-80 HPLC column and analyzed by ¹ H-NMR and ¹³ C-NMR, and confirmed to be glucosyl trehalose. The chemical formula is as follows.

As a result, it was found that the cell extract of a strain belonging to the order of Sulfolobales has glucosyl trehalose producing activity, that is, the present enzyme transferase activity.

Example I-2 Purification of the Enzyme Transferase from Sulfolobus solfaticalus KM1 Sulfobus sulphataricus KM1 strain was published by American Type Culture Collection (ATgc), which contains 2 g / l of soluble starch and 2 g / l of yeast extract. The culture was performed at 75 ° C. for 3 days in the medium of medium No. 1304 described in the edition (1992). The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 3.3 g / liter.

  200 g of the microbial cells obtained as described above are suspended in 400 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by sonication for 15 minutes at 0 ° C., and then centrifuged. To obtain a supernatant solution. To this, ammonium sulfate was added so as to be 60% saturated.

  The precipitate obtained by centrifugation was dissolved in 50 mM sodium acetate buffer (pH 5.5) containing 1M ammonium sulfate and 5 mM EDTA, and equilibrated with the same buffer. Hydrophobic chromatography (column: TOSOH TSK-gel) Phenyl-TOYOPEARL 650S 800 ml). The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 10 mM sodium acetate buffer (pH 5.5) and desalted.

  Subsequently, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 300 ml) equilibrated with the same buffer. The column was washed with the same buffer and subsequently the target transferase was eluted with a linear gradient of 900 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target transferase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), washed with 50 mM sodium acetate buffer (pH 5.5), and desalted.

  Next, ammonium sulfate was dissolved in the desalted and concentrated solution to 1 M, and passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-5PW HPLC) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a 30 ml linear gradient of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 10 mM sodium acetate buffer (pH 5.0) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE 5PW HPLC) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a 30 ml linear gradient from 0M to 0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000).

Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.
The activity measurement was performed in the same manner as in Example I-1.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 4 below.

Example I-3 Purification of the Enzyme Transferase from Sulfobus solfataricus DSM5833 Sulfobus solfataricus DSM5833 Strain was obtained from the American Type Culture Collection CC Cultured at 75 ° C. for 3 days in the medium No. 1304 described in the 18th edition (1992). The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 1.7 g / liter.

  56 g of the cells obtained as described above are suspended in 100 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by ultrasonic disruption at 0 ° C. for 15 minutes, and then centrifuged. To obtain a supernatant solution.

  Then, ammonium sulfate is dissolved in the supernatant solution to 1M and equilibrated with 50 mM sodium acetate buffer (pH 5.5) containing 1M ammonium sulfate and 5 mM EDTA (column: TOSOH TSK-gel Phenyl). -Passed through TOYOPEARL 650S 200ml). The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 300 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 900 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

  Next, ammonium sulfate was dissolved to 1 M in the desalted concentrated solution, and passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-TOYOPEARL 650S 200 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target transferase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000) and subsequently dialyzed with 25 mM bis-Tris HCl buffer (pH 6.7).

Next, it was passed through chromatofocusing (column: Pharmacia Mono P HR / 5/20) equilibrated with the same buffer. Immediately after injection of the sample, the target transferase was eluted with 10% polybuffer 74HCl (pH 5.0, Pharmacia). The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000) and subsequently dialyzed with 25 mM bis-Tris HCl buffer (pH 6.7).
Further, chromatofocusing was performed under the same conditions to elute the target transferase. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.
The activity measurement was performed in the same manner as in Example I-1.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 5 below.

Example I-4 Purification of the enzyme transferase from Sulfolobus acidocaldarius ATCC 33909 and Phages 18th Edition (1992) and cultured at 75 ° C. for 3 days in the medium No. 1304 medium (pH 3.0). The cells were collected by centrifugation and stored at -80 ° C. The yield of the bacterial cells was 2.9 g / liter.

  92.5 g of the cells obtained as described above are suspended in 200 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by ultrasonic disruption for 15 minutes at 0 ° C., and then centrifuged. Separation was performed to obtain a supernatant solution.

  Then, ammonium sulfate is dissolved in the supernatant solution to 1M and equilibrated with 50 mM sodium acetate buffer (pH 5.5) containing 1M ammonium sulfate and 5 mM EDTA (column: TOSOH TSK-gel Phenyl). -TOYOPEARL 650S 400 ml). The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 300 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 900 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

  Next, ammonium sulfate was dissolved to 1 M in the desalted concentrated solution, and passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-TOYOPEARL 650S 200 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target transferase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000) and subsequently dialyzed against 25 mM Bis-Tris HCl buffer (pH 6.7).

  Next, it was passed through chromatofocusing (column: Pharmacia Mono P HR / 5/20) equilibrated with the same buffer. Immediately after injection of the sample, the target transferase was eluted with 10% polybuffer 74HCl (pH 5.0, Pharmacia). The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000) and subsequently dialyzed against 25 mM bis-Tris HCl buffer (pH 6.7).

  Further, chromatofocusing was performed under the same conditions to elute the target transferase. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.
The activity measurement was performed in the same manner as in Example I-1.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 6 below.

Example I-5 Purification of this enzyme transferase from Acidianus brierleyi DSM 1651 strain Acidianus brierleyi DSM 1651 strain (published by Deutsche Sammlung von Mikrugenismenund Zellkultrum, Germany) And then cultured at 70 ° C. for 3 days. The cells were collected by centrifugation and stored at -80 ° C. The yield of the bacterial cells was 0.6 g / liter.

  12 g of the cells obtained as described above are suspended in 120 ml of 50 mM sodium acetate buffer solution (pH 5.5) containing 5 mM EDTA, lysed by sonication for 15 minutes at 0 ° C., and then centrifuged. A supernatant solution was obtained.

  Then, ammonium sulfate is dissolved in the supernatant solution to 1M and equilibrated with 50 mM sodium acetate buffer (pH 5.5) containing 1M ammonium sulfate and 5 mM EDTA (column: TOSOH TSK-gel Phenyl). -Passed through TOYOPEARL 650S 200ml). The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut: 13000), then washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 300 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target transferase was eluted with a linear gradient of 900 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target transferase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000) and subsequently dialyzed against 25 mM Bis-Tris HCl buffer (pH 6.7).

  Next, it was passed through chromatofocusing (column: Pharmacia Mono P HR5 / 20) equilibrated with the same buffer. Immediately after injection of the sample, the target transferase was eluted with 10% polybuffer 74HCl (pH 5.0, Pharmacia). The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.
The activity measurement was performed in the same manner as in Example I-1.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 7 below.

Example I-6 Examination of various properties of the present enzyme transferase The enzymatic properties of the purified enzyme obtained in Example I-2 were measured.

(1) Molecular weight The molecular weight of the purified enzyme in its native state was measured by gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg). Molecular weights of 200,000; 97,400; 68,000; 43,000; 29,000; 18,400; 14,300 were used as marker proteins.
As a result, the molecular weight of the transferase was 54,000.
Molecular weight was measured by SDS polyacrylamide gel electrophoresis with a gel concentration of 6%. The molecular weights of 200,000; 116,300; 97,400; 66,300; 55,400; 36,500; 31,000; 21,500; 14,400 were used as marker proteins.
As a result, the molecular weight of the transferase was 76,000.
There was a difference in the molecular weight measurements between gel filtration chromatography and SDS polyacrylamide gel electrophoresis, which is probably due to some interaction between the gel filtration column packing and the protein. It is done. Therefore, the molecular weight value obtained by gel filtration does not necessarily indicate the molecular weight in the native state of the enzyme.

(2) Isoelectric point As a result of agarose gel isoelectric focusing, the isoelectric point was 6.1.

(3) Stability The stability of the obtained purified enzyme at each temperature and pH is shown in FIGS. 2 and 3, respectively. The measurement was carried out using a glycine hydrochloride buffer between pH 3 and 5, a sodium acetate buffer between pH 4 and 6, a sodium phosphate buffer between pH 5 and 8, and a tris hydrochloride between pH 8 and 9. The system buffer was sodium bicarbonate buffer between pH 9 and 10, and KCl-NaOH buffer between pH 11 and 13, respectively.
This enzyme was stable by treatment at 85 ° C. for 6 hours, and was stable by treatment at pH 4.0-10.0 for 6 hours at room temperature.

(4) Reactivity The reactivity of the obtained purified enzyme at each temperature and pH is shown in FIGS. 4 and 5, respectively. The measurement is performed using glycine hydrochloride buffer (□) during pH 3 to 5, sodium acetate buffer (●) during pH 4 to 5.5, and sodium phosphate buffer during pH 5 to 7.5. For (Δ), between pH 8 and 9, Tris-HCl buffer (緩衝) was used. This enzyme has a reaction optimum temperature in the vicinity of 60 to 80 ° C., and a reaction optimum pH in the vicinity of pH 5.0 to 6.0.

(5) Effects of various activators and inhibitors In the method for measuring the glucosyl trehalose producing activity of Example I-1, the substances shown in Table 8 below were added together with the substrate, and the activity measurement in each case was carried out in Example I. -1 was performed and the presence or absence of activation or inhibition was examined. As a result, it was found that copper ions and SDS were inhibited. Many sugar-related enzymes are activated by calcium ions, but this enzyme is not activated by calcium ions.

(6) Substrate specificity This purified enzyme was allowed to act on the substrates shown in Table 9 below to examine the presence or absence of α-1, α-1 transfectants. The activity measurement was performed in the same manner as in Example I-1.

  As a result, for this purified enzyme, the production of trehalose oligosaccharide was confirmed from maltotriose (G3) to maltoheptaose (G7). Moreover, it reacts with any of isomaltotriose, isomalttetraose, isomaltopentaose, and panose having α-1,6 bonds at the first to fourth or second from the reducing end. There wasn't.

  In addition, the enzymatic properties of the purified enzymes derived from Sulfobus solfataricus DSM 5833, Sulfobus acidocaldarius ATCC 33909, and Acidianus brierleyi DSM 1651 obtained in Examples I-3 to I-5 were investigated by the same method. Is shown in Table 1 above.

Example I-7 Production of glucosyl trehalose and malto-oligosyl trehalose from maltooligosaccharide 100 mM maltotriose (G3) to maltoheptaose (G7), and purified enzyme 13.5 Units obtained in Example I-2 / ml (enzyme activity when maltotriose was used as a substrate) was allowed to act to produce the corresponding α-1, α-1 transposants. Each product was analyzed by the method of Example I-1, and the yield and enzyme activity were examined. In addition, the enzyme activity in Table 10 indicates the enzyme activity for converting each maltooligosaccharide into 1 μmol of the corresponding α-1, α-1 transferant per hour as 1 unit. The results are as shown in Table 10 below.

  From the results in the table, the substrate was the most active when it was G5, and showed about 8 times that of G3. The yield was 44.6% for G3, but it was 63.5-73.1% for G4 and above.

  Moreover, when the composition of the reaction product obtained by using G3, G4, and G5 as a substrate was examined, the results were as shown in FIGS.

  That is, when maltotriose was used as a substrate, equimolar amounts of maltose and glucose were produced as side reactions along with the production of glucosyl trehalose as the main reaction.

  When a sugar having a degree of polymerization n equal to or higher than that of maltotetraose is used as a substrate, as a main reaction, a sugar having a degree of polymerization n in which the glucose unit at the reducing end is α-1, α-1 linked is first produced. As a reaction, an equimolar degree of polymerization (n-1) sugar and glucose were produced. When the reaction of these saccharides further progressed, the same reaction proceeded secondarily from the saccharide of degree of polymerization (n-1) (in addition, the saccharides indicated as trisaccharide or tetrasaccharide in FIGS. In addition, a similar reaction proceeds secondarily with unreacted maltotriose or maltotetraose, and sugars having α-1, α-1 linked at the ends are included). Moreover, the production | generation of the sugar more than a polymerization degree (n + 1), ie, the transition body between molecules, was not recognized. In addition, it was recognized that the hydrolysis which is a side reaction decreases when the chain length becomes G4 or more.

As examples of these main reactants, trisaccharide, tetrasaccharide, and pentasaccharide, which are main products from the substrates G3, G4, and G5, are fractionated on a TSK-gel amide-80 HPLC column, and ¹ H- Analysis was performed by NMR and ¹³ C-NMR. As a result, each showed a structure in which one glucose residue at the reducing end was bonded at α-1, α-1, and each of them showed glucosyl trehalose (α-D-maltosyl α-D-glucopyranoside), maltosyl trehalose (α- D-maltotriosyl α-D-glucopyranoside) and maltotriosyl trehalose (α-D-maltotetraosyl α-D-glucopyranoside). Each of these chemical formulas is as follows.

  From the above results, the enzyme of the present invention is an enzyme having an activity of binding the reducing end of a glucose polymer of maltotriose or higher in which glucose is bound at α-1,4 by α-1, α-1 by transfer. It is concluded that It was also found that as a side reaction, as is often observed in glycosyltransferases, a hydrolysis reaction is carried out using a water molecule as an acceptor, and one bond on the reducing end side is cleaved to release one glucose molecule.

Example I-8 Production of glucosyl trehalose and maltooligosyl trehalose from a mixture of maltooligosaccharide trehalose Using the purified enzyme 10 Units / ml obtained in Example I-2, the substrate was soluble starch (manufactured by Nacalai Tesque, special grade) α -Degradation product of amylase (decomposed to oligosaccharide without showing iodine starch reaction: α-amylase used here is derived from Sigma A-0273 Aspergillus oryzae), glucosyl trehalose and Attempts were made to produce various types of maltooligosyl trehalose. The reaction solution was analyzed by HPLC analysis under the following conditions.
Column: BIORAD AMINEX HPX-42A (7.8 x 300mm)
Solvent: Water Flow rate: 0.6ml / min
Temperature: 85 ° C
Detector: Differential refractometer
FIG. 9 shows an analysis chart by HPLC (A). As a control, an HPLC chart when the enzyme transferase was not added was shown (B). As a result, since the reducing end of the oligosaccharide of the reaction product was transferred to α-1 and α-1, an oligosaccharide having a shorter retention time than the product of the control amylase alone was generated. As examples of these reactants, trisaccharide, tetrasaccharide, and pentasaccharide were fractionated in the same manner as in Example I-7 and analyzed by ¹ H-NMR and ¹³ C-NMR. Also shows a structure in which one glucose residue at the reducing end is linked by α-1 and α-1, and each has glucosyl trehalose (α-D-maltosyl α-D-glucopyranoside) and maltosyl trehalose (α-D-maltotrio). Syl α-D-glucopyranoside) and maltotriosyltrehalose (α-D-maltotetraosyl α-D-glucopyranoside). Each of these chemical formulas is as follows.

The following reagents or raw materials used in the following Examples II-1 to II-14 (including Comparative Examples II-1 to II-2 and Reference Examples II-1 to II-4) are all from the following manufacturers. I got it.
α, α-trehalose: Sigma soluble starch: Nacalai Tesque, special grade
Klebsiella pneumoniae-derived pullulanase: Wako Pure Chemical Industries, 165-15651
Paindex # 1 and Paindex # 3: Maltose manufactured by Matsutani Chemical (G2): Maltotriose (G3), Maltotetraose (G4), Maltopentaose (G5), Maltohexaose (G6) manufactured by Wako Pure Chemical, Maltoheptaose (G7) and Amylose DP-17: Hayashibara Biochemical Amylopectin: Nacalai Tesque, Special Grade Isomaltose: Wako Pure Chemicals Isomalt Triose: Wako Pure Chemicals Isomalt Tetraose: Seikagaku Corporation Isomalt pentaose manufactured by Seikagaku Corporation Panose: manufactured by Tokyo Chemical Industry

Example II-1 Measurement of Trehalose Oligosaccharide Decomposing Activity and Starch Liquefaction Activity of Archaea The activity of the strains shown in Table 11 below was examined. As a method, cultured bacterial cells of each strain are subjected to ultrasonic disruption treatment and centrifugation, and the supernatant is used as a crude enzyme solution, to which maltotriosyl trehalose as a substrate is finally added to 10 mM. After the reaction at ℃, pH 5.5 (50 mM sodium acetate buffer), the reaction was stopped by heat treatment at 100 ℃ for 5 minutes, and then the produced α, α-trehalose was analyzed by HPLC analysis under the conditions shown below. It was measured.
Column: TOSOH TSK-gel Amide-80 (4.6 x 250mm)
Solvent: 72.5% acetonitrile Flow rate: 1.0 ml / min
Temperature: Room temperature Detector: Differential refractometer Trehalose oligosaccharide decomposition activity showed 1 unit of enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose. However, in Table 11, it showed as activity per microbial cell. In addition, maltotriosyl trehalose was prepared by adding a purified transferase derived from Sulfolobus solifericus KM1 to 10% maltopentaose containing 50 mM acetic acid (pH 5.5) at 10 Units / ml and reacting at 60 ° C. for 24 hours. Thereafter, the fractionation was carried out using a TSK-gel Amide-80 HPLC column under the above conditions. In addition, the activity of the purified transferase derived from Sulfolobus solfariticus KM1 is defined as 1 Unit as the enzyme activity that produces 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  The HPLC chart is as shown in FIG. As shown in the figure, a peak showing the same retention time as α, α-trehalose without anomer and a peak showing the same retention time as maltotriose appeared on the HPLC chart. The first product was fractionated on a TSK-gel amide-80 HPLC column, and as a result of analysis by 1H-NMR and 13C-NMR, it was confirmed to be α, α-trehalose.

  Further, using the same crude enzyme solution (supernatant) as described above, the supernatant was appropriately diluted to 0.5 ml of 100 mM sodium acetate buffer (pH 5.5) containing 2% soluble starch, and 60 ml was added. The reaction was performed at ° C. Sampling was performed over time, and the reaction was stopped by adding twice the amount of 1N hydrochloric acid to this sample. Next, a 0.1% potassium iodide solution containing 2/3 times the amount of 0.01% iodine was added, and 1.8 times the amount of water was further added. Finally, the absorbance at 620 nm was measured, and the activity was measured from the change over time.

The analysis of the saccharide | sugar produced | generated after reaction was measured by the HPLC analysis method on conditions shown below, after processing for 5 minutes at 100 degreeC and stopping reaction.
Column: BIO-RAD AMINEX HPX-42A (7.8 x 300mm)
Solvent: Water Flow rate: 0.6ml / min
Temperature: 85 ° C
Detector: Differential refractometer The amylolytic activity was defined as 1 Unit for the amount of enzyme that reduces the absorbance at 620 nm by the blue-violet color of the starch-iodine complex by 10% in 10 minutes. However, in Table 11, it showed as activity per microbial cell g.

  The analysis result by AMINEX HPX-42A HPLC of the reaction product by the crude enzyme solution derived from Sulfolobus solfatricus KM1 strain was as shown in FIG.

  As a result, the cell extract of a strain belonging to the genus Sulfolobus has the activity of decomposing trehalose oligosaccharides to release α, α-trehalose and the activity of hydrolyzing starch to mainly release monosaccharides and disaccharides. I found it.

Example II-2 Purification of the enzyme amylase from Sulfolobus solfaticalus KM1 Sulfobus sulphataricus KM1 strain was obtained from the American Type Culture Collection (ATCC), which contains 2 g / l of soluble starch and 2 g / l of yeast extract. Cultured at 75 ° C. for 3 days in the medium No. 1304 described in the 18th edition (1992). The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 3.3 g / liter.

  200 g of the cells obtained as described above are suspended in 400 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by ultrasonic crushing treatment at 0 ° C. for 15 minutes, and then centrifuged. A supernatant solution was obtained. To this, ammonium sulfate was added so as to be 60% saturated.

  The precipitate obtained by centrifugation was dissolved in 50 mM sodium acetate buffer (pH 5.5) containing 1M ammonium sulfate and 5 mM EDTA, and equilibrated with the same buffer. Hydrophobic chromatography (column: TOSOH TSK-gel) Phenyl-TOYOPEARL 650S 800 ml). The column was washed with the same buffer and then the target amylase was eluted with a linear gradient of 600 ml of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut: 13000), then washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 300 ml) equilibrated with the same buffer. The column was washed with the same buffer and subsequently the target amylase was eluted with a linear gradient of 900 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was loaded on a gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target amylase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 25 mM bis-tris-HCl buffer (pH 6.3) and desalted.

  Next, this desalted concentrated solution was loaded on chromatofocusing (column: Pharmacia Mono P HR5 / 20) equilibrated with the same buffer, and 10% Polybuffer 74 (manufactured by Pharmacia, adjusted to pH 4.0 with hydrochloric acid). ) To elute the target amylase. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), washed with 10 mM sodium acetate buffer (pH 6.8), and desalted.

  Next, a 1/4 volume of sample buffer (62.5 mM Tris-HCl buffer (pH 6.8), 10% glycerol, 2% SDS, 0.0125% bromophenol blue) is added to the desalted and concentrated solution. The target amylase was eluted by% SDS polyacrylamide gel electrophoresis (SDS-PAGE) (device: BIO-RAD prepcell model 491). The active fraction was collected, concentrated with an ultrafiltration membrane (molecular weight cut 13000), washed with 10 mM sodium acetate buffer (pH 5.5) and desalted.

  Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.

  For the activity measurement in this purification, maltotriosyl trehalose was used as a substrate, and other methods were performed in the same manner as the HPLC analysis method of TSK-gel Amide-80 shown in Example II-1.

The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 12 below.

Example II-3 Purification of the Enzyme Amylase from Sulfolobus solfataricus DSM5833 Sulfobus solfataricus DSM5833 Strain was obtained from the American Type Culture Collection (ATCC) from the American Type Culture Collection, which contains 2 g / l of soluble starch and 2 g / l of yeast extract. Cultured at 75 ° C. for 3 days in the medium No. 1304 described in the 18th edition (1992). The cells were collected by centrifugation and stored at -80 ° C. The yield of the bacterial cells was 1.2 g / liter.

  25 g of the microbial cells obtained as described above are suspended in 50 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by sonication for 15 minutes at 0 ° C., and then centrifuged. A supernatant solution was obtained.

  Hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-) was added to this supernatant solution to a concentration of 1 M and equilibrated with 50 mM sodium acetate buffer (pH 5.5) containing 1 M ammonium sulfate and 5 mM EDTA. TOYOPEARL 650S 100 ml). The column was washed with the same buffer and the target amylase was then eluted with a 300 ml linear gradient of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut: 13000), then washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 100 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target amylase was eluted with a linear gradient of 300 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was loaded on a gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target amylase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 25 mM bis-tris-iminodiacetic acid buffer (pH 7.1) and desalted.

  Next, this desalted concentrated solution was loaded on chromatofocusing (column: Pharmacia Mono P HR5 / 20) equilibrated with the same buffer solution, and 10% Polybuffer 74 (manufactured by Pharmacia, adjusted to pH 4.0 with iminodiacetic acid). ) To elute the target amylase. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 25 mM bis-tris-iminodiacetic acid buffer (pH 7.1) and desalted.

  Next, this desalted concentrated solution was loaded on chromatofocusing (column: Pharmacia Mono P HR5 / 20) equilibrated with the same buffer solution, and adjusted to pH 4.0 with 10% Polybuffer 74 (manufactured by Pharmacia, iminodiacetic acid). The target amylase was eluted. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was loaded on gel filtration chromatography (column: TSK-gel G3000SW HPLC), and the target amylase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

  Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.

  For the activity measurement in this purification, maltotriosyl trehalose was used as a substrate, and other methods were performed in the same manner as the HPLC analysis method of TSK-gel Amide-80 shown in Example II-1.

The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 13 below.

Example II-4 Purification of the enzyme amylase from Sulfolobus acidocaldarius ATCC33909 Sulfobus acidocaldarius ATCC33909 strain was published by American TypeCultureCultureCultureCultureCulture Cultured at 75 ° C. for 3 days in the medium No. 1304 described in the 18th edition (1992). The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 2.7 g / liter.

  25 g of the microbial cells obtained as described above are suspended in 50 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by sonication for 15 minutes at 0 ° C., and then centrifuged. A supernatant solution was obtained.

  Hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-) was added to this supernatant solution to a concentration of 1 M and equilibrated with 50 mM sodium acetate buffer (pH 5.5) containing 1 M ammonium sulfate and 5 mM EDTA. TOYOPEARL 650S 100 ml). The column was washed with the same buffer and the target amylase was then eluted with a 300 ml linear gradient of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut: 13000), then washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Next, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 100 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the target amylase was eluted with a linear gradient of 300 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA.

  Next, the desalted concentrated solution was loaded on a gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target amylase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), washed with 50 mM sodium acetate buffer (pH 5.5), and desalted.

  Next, ammonium sulfate was dissolved in the desalted and concentrated solution to 1 M, and passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-5PW HPLC) equilibrated with the same buffer. The column was washed with the same buffer and then the target amylase was eluted with a 30 ml linear gradient of 1M to 0M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 25 mM bis-tris-iminodiacetic acid buffer (pH 7.1) and desalted.

  Next, this desalted concentrated solution was loaded on chromatofocusing (column: Pharmacia Mono P HR5 / 20) equilibrated with the same buffer solution, and adjusted to pH 4.0 with 10% Polybuffer 74 (manufactured by Pharmacia, iminodiacetic acid). The target amylase was eluted. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), and then washed and desalted with 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA.

  Finally, a purified enzyme showing a single band was obtained by native polyacrylamide gel, SDS polyacrylamide gel, and isoelectric focusing.

  For the activity measurement in this purification, maltotriosyl trehalose was used as a substrate, and other methods were performed in the same manner as the HPLC analysis method of TSK-gel Amide-80 shown in Example II-1.

The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 14 below.

Example II-5 Examination of Properties of the Enzyme Amylase The enzymatic properties of the purified enzyme obtained in Example II-2 were measured.

(1) Molecular weight Molecular weight was measured by SDS polyacrylamide gel electrophoresis with a gel concentration of 6%. The molecular weights of 200,000; 116,300; 97,400; 66,300; 55,400; 36,500; 31,000; 21,500; 14,400 were used as marker proteins.
As a result, the molecular weight of the amylase was 61,000.

(2) Isoelectric point As a result of agarose gel isoelectric focusing, the isoelectric point was 4.8.

(3) Stability The stability of the obtained purified enzyme at each temperature and pH is shown in FIGS. 12 and 13, respectively. The enzyme activity was measured according to the measurement method using maltotriosyl trehalose in Example II-1, and a glycine hydrochloride buffer solution was used between pH 3 and 5, and a sodium acetate buffer solution was used between pH 4 and 6. Between pH 5-8, sodium phosphate buffer, between pH 8-9 tris hydrochloric acid buffer, between pH 9-10 sodium bicarbonate buffer, pH 11-11
A KCl-NaOH buffer solution was used for 13.5.
This enzyme was stable by treatment at 85 ° C. for 6 hours, and was stable by treatment at room temperature for 6 hours at pH 3.5 to 10.0.

(4) Reactivity The reactivity of the purified enzyme obtained at each temperature and pH is shown in FIGS. 14 and 15, respectively. Enzyme activity was measured according to the measurement method using maltotriosyl trehalose in Example II-1, between pH 2 and 4 with sodium citrate buffer (□) and between pH 4 and 5.5. Used a sodium acetate buffer (●), a pH 5 to 7.5 sodium phosphate buffer (Δ), and a pH 8 to 9 tris hydrochloric acid buffer (◇).
This enzyme has a reaction optimum temperature in the vicinity of 70 to 85 ° C. and a reaction optimum pH in the vicinity of pH 4.5 to 5.5.

(5) Effects of various activators and inhibitors In the method for measuring the maltotriosyl trehalose degradation activity of Example II-1, the substances shown in Table 15 below were added together with the substrate, and the activity was measured in each case. It carried out similarly to II-1 and investigated the presence or absence of activation or inhibition. As a result, it was found that copper ions and sodium dodecyl sulfate (SDS) were inhibited. However, for the inhibition by SDS, the enzyme activity was recovered by removing SDS by a method such as dialysis or ultrafiltration. Many sugar-related enzymes are activated by calcium ions, but this enzyme is not activated by calcium ions.

(6) Substrate specificity The purified enzyme 25.0 Units / ml (enzyme activity when maltotriosyltrehalose is used as a substrate) is a 10 mM substrate (amylopectin, soluble starch, 2. 8%) and analyzed for degradability and decomposition products. For various maltooligosaccharides, amylose DP-17, amylopectin, soluble starch, various isomaltooligosaccharides, and panose, the production activity of monosaccharide + disaccharide was used as an index, and various trehalose oligosaccharides, amylose DP-17α-1, α- Α, α-trehalose formation for 1-transition (oligosaccharide with α-1, α-1 bond between the first and second glucose residues on the reducing end side of amylose DP-17) Using the activity as an index, maltose and α, α-trehalose were analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1 using glucose production activity as an index.
In addition, the enzyme activity in Table 16 indicates the enzyme activity that liberates 1 μmol of monosaccharide and disaccharide per hour as 1 Unit.

註：グルコシルトレハロース、マルトシルトレハロース、マルトテトラオシルトレハロース、マルトペンタオシルトレハロース及びアミロースDP-17,α-1, α-1転移体は、いずれも実施例II−１におけるマルトトリオシルトレハロースの調製法に準じて製造したものである。 The results are as shown in Table 16 below and FIGS.

註: Glucosyl trehalose, maltosyl trehalose, maltotetraosyl trehalose, maltopentaosyl trehalose, and amylose DP-17, α-1, α-1 transposants are all of maltotriosyl trehalose in Example II-1. It is manufactured according to the preparation method.

  The results of analysis of reaction products from maltopentaose, amylose DP-17 and soluble starch by AMINEX HPX-42A HPLC are shown in FIGS. 17A, 17B and 17C, respectively, as well as maltotriosyl trehalose and maltopentaosyl trehalose. The results of analysis of the reaction product from TSK-gel Amide-80 HPLC are shown in FIGS. 18 and 19, respectively.

  As a result, for this purified enzyme, it acts extremely well on trehalose oligosaccharides such as maltotriosyl trehalose in which the glucose residue on the reducing end side is α-1, α-1 linked, and the degree of polymerization with α, α-trehalose. Was confirmed to produce a corresponding maltooligosaccharide with a decrease of two. Further, it was confirmed that glucose or maltose was mainly liberated from maltose (G2) to maltoheptaose (G7), amylose, and soluble starch. However, it does not react with α-1, α-trehalose which is α-1, α-1 bond, and isomaltose, isomaltotriose, isomalttetraose and isomalt where all bonds are α-1,6 bonds. It did not react with panose or panose having α-1,6 bond second from the reducing end.

(7) End-type amylase activity Disappearance of iodine coloration when it is made to act on soluble starch by the purified enzyme 200 Units / ml (enzyme activity when maltotriosyl trehalose is used as a substrate), and monosaccharide and disaccharide The time course change of the starch hydrolysis rate determined from the production amount of the starch was analyzed by the amylolytic activity measurement method shown in Example II-1 and the analysis method by AMINEX HPX-42A HPLC.
As a result, as shown in FIG. 20, with this purified enzyme, the hydrolysis rate at the point when the iodine reaction coloration disappeared by 50% was as low as 3.7%, and thus the properties of endo-amylase were exhibited. confirmed.

(8) Examination of mechanism of action Uridinediphosphoglucose glucose [glucose -6 - ³ H], and maltotetraose in glycogen synthase (from rabbit skeletal muscle, Sigma Co. G-2259) by the action of, the non-reducing end glucose Maltopentaose whose residue was radiolabeled with ³ H was synthesized and purified by preparative purification. Next, 10 mM maltopentaose labeled with this radioactivity was used as a substrate, and 10 Units / ml of a purified transferase derived from Sulfolobus sofaraticus KM1 (enzyme activity when maltotriose was used as a substrate) was added at 60 ° C. Maltotriosyl trehalose, which was allowed to act for 3 hours and whose non-reducing end glucose residue was radioactively labeled with ³ H, was synthesized and purified. (Note that glucoamylase (derived from Rhizopus, manufactured by Seikagaku Corporation) was allowed to act on this product to completely decompose it into glucose and α, α-trehalose. These were separated by thin layer chromatography and separated into liquids. When the radioactivity was measured with a scintillation counter, no radioactivity was observed in the α, α-trehalose fraction, and radioactivity was observed in the glucose fraction, and thus the non-reducing end glucose residue was radiolabeled. Confirmed.)
Using the maltopentaose prepared by radiolabeling the non-reducing terminal glucose residue with ³ H and the non-reducing terminal glucose residue radiolabeled with ³ H prepared as described above as substrates. These were allowed to act on the purified enzyme obtained in Example II-2 at 50 Units / ml and 5 Units / ml, respectively, and sampled before the reaction and at 60 ° C., 0.5, 1 and 3 hours. This reaction product was developed by thin layer chromatography (Kieselgel 60 Merck, solvent: butanol-ethanol-water = 5: 5: 3). A portion corresponding to each of the obtained sugars was collected, and the radioactivity was measured with a liquid scintillation counter. The results are shown in FIGS. 21 and 22, respectively.

  As is apparent from FIGS. 21 and 22, when maltopentaose is used as a substrate, no radioactivity is observed in glucose and maltose fractions which are hydrolysis products, and radioactivity is present in maltotetraose and maltotriose fractions. Was recognized. When maltotriosyl trehalose was used as a substrate, no radioactivity was observed in the α, α-trehalose fraction that was a hydrolyzate, and radioactivity was observed in the maltotriose fraction.

  Therefore, it was found that the mechanism of action of this purified enzyme has an activity of mainly producing monosaccharides and disaccharides from the reducing end side, together with amylase activity acting on the endo type.

  In addition, the enzymatic properties of each of the purified enzymes derived from Sulfolobus solfataricus DSM5833 and Sulfolobus acidocaldarius ATCC33909 obtained in Examples II-3 and 4 were examined in the same manner, and the results are shown in Table 2 above. Indicated.

Comparative Example II-1 Degradability of Various Oligosaccharides by Pancreatic α-Amylase and Analysis of Degradation Products Porcine pancreatic α-amylase is known to hydrolyze maltooligosaccharides from the reducing end in disaccharide or trisaccharide units. (Starch and related carbohydrate enzyme test method P135, Michinori Nakamura, Junji Kakinuma, Academic Publishing Center). Therefore, as a comparative example of the novel amylase of the present invention, α-amylase derived from porcine pancreas (Sigma, A-6255) 1 Units / ml (corresponding to 1 mg maltose for 3 minutes at pH 6.9 and 20 ° C. using starch as a substrate) The amount of the enzyme that produces the reducing sugar is 1 Unit) and 10 mM substrate shown in Table 17 below was allowed to act at pH 6.9 and 20 ° C. to analyze the degradability and degradation products. The enzyme activity was analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, using the production activity of disaccharide + trisaccharide as an index.
The enzyme activity in Table 17 is shown as 1 Unit for the enzyme activity that liberates 1 μmol of each oligosaccharide per hour.

The results are as shown in Table 17 below and FIGS.

註: Glucosyl trehalose, maltosyl trehalose, maltotetraosyl trehalose and maltopentaosyl trehalose are all produced according to the method for preparing maltotriosyl trehalose in Example II-1.

  The analysis result by TSK-gel Amide-80 HPLC of the reaction product from maltopentaosyl trehalose is shown in FIG.

  As a result, pancreatic amylase produces malto-oligosaccharides having a degree of polymerization reduced by two or three from maltotetraose (G4) to maltoheptaose (G7), but glucose on the reducing end side. It was confirmed that α, α-trehalose was not liberated from trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose having α-1, α-1 bonded residues, and the reactivity was low.

Example II-6 Production of α, α-trehalose from soluble starch and various starch degradation products 150 Units / ml of the purified enzyme obtained in Example II-2 and 10 Units / ml of purified transferase derived from Sulfolobus solfaricus KM1 strain As a starch degradation product, Klebsiella pneumoniae-derived pullulanase (manufactured by Wako Pure Chemical Industries, Ltd.) at 25 Units / ml under conditions of 40 ° C. and 1 hr in advance. Soluble starch that had been decomposed in 1,6 linkages; As another starch degradation product, α-amylase derived from Bacillus amylolichefaciens (Boehringer Mannheim) 12.5 Units / ml under conditions of 30 ° C. and 2.5 hours Soluble starch previously partially decomposed with potato; Paindex # 1; Paindex # 3 (both Matsutani G3-G7 malto-oligosaccharides alone; and amylose DP-17 (all manufactured by Hayashibara Biochemical), respectively, were used after being reacted for about 100 hr under conditions of a final concentration of 10%, 60 ° C., and pH 5.5. An attempt was made to produce α, α-trehalose utilizing the synergistic action of enzymes.

  The reaction solution was analyzed by the AMINEX HPX42A HPLC analysis method shown in Example II-1, taking the case where the substrate was soluble starch as an example.

  Further, after the unreacted substrate was decomposed with glucoamylase, the analysis by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1 was performed, and the yield of α, α-trehalose produced was examined.

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  With respect to the activity of pullulanase, the enzyme activity that produces 1 μmol of maltotriose per minute at pH 6.0 and 30 ° C. using pullulan as a substrate was shown as 1 Unit.

The results are shown in Table 18 below.

  The analysis result of the reaction product from soluble starch by AMINEX HPX42A HPLC is shown in FIG.

  As a result, when soluble starch was used as a substrate, α, α-trehalose was produced at a yield of 37.0%. Moreover, in various starch degradation products, when soluble starch obtained by hydrolyzing α-1,6 bonds with pullulanase is used as a substrate, 62.1%, various maltooligosaccharides in which all bonds are α-1,4 bonds, and amylose In DP-17, they were 36.4 to 67.1% and 83.5%, respectively. These results indicate that the yield of α, α-trehalose in the final product is higher as the substrate having a longer α-1,4 bond linear chain is used.

Example II-7 Production of α, α-trehalose at various enzyme concentrations from soluble starch Using the present purified enzyme obtained in Example II-2 and a purified transferase derived from Sulfolobus solfaricus KM1 and using Klebsiella pneumoniae as a substrate Pullulanase (manufactured by Wako Pure Chemical Industries, Ltd.) derived from soluble starch pretreated at 25 ° C. for 1 hour at 25 ° C., and the enzyme (final concentration 10%) was added with the enzymes shown in Table 19 respectively. The reaction was carried out for about 100 hours at 60 ° C. and pH 5.5 to try to produce α, α-trehalose using the synergistic action of these enzymes. In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, and the yield of the produced α, α-trehalose was examined. .

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  With respect to the activity of pullulanase, the enzyme activity that produces 1 μmol of maltotriose per minute at pH 6.0 and 30 ° C. using pullulan as a substrate was shown as 1 Unit.

The results are shown in Table 19 below.

  From the results of the table, it can be seen that the yield of α, α-trehalose was maximum when transferase was 20 Units / ml and amylase was 150 Units / ml, reaching 65.1%.

Comparative Example II-2 Production of α, α-trehalose using amylase of other biological origin As a comparison of the novel amylase of the present invention, amylase derived from Bacillus subtilis, Bacillus licheniformis and Aspergillus oryzae (100100, manufactured by Seikagaku Corporation, respectively) Sigma A-3403 and A-0273 (both active at 60 ° C.), and in combination with a purified transferase derived from Sulfobus sofaraticus KM1, the substrate was pulled from Klebsiella pneumoniae (manufactured by Wako Pure Chemical) The soluble starch (final concentration 10%) pretreated at 25 Units / ml under the conditions of 40 ° C. and 1 hr was added to the substrate with the enzyme at the concentration shown in Table 20 to 60 ° C. and pH 5.5. For about 100 hours, and an attempt was made to produce α, α-trehalose utilizing the synergistic action of these enzymes. In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by TSK-gel Amide-80 HPLC analysis method shown in Example II-1. The yield of α, α-trehalose produced was examined. .

  In addition, the enzyme activity of amylase is shown as 1 Unit, which is reacted under the same conditions as in Example II-1 to reduce the absorbance at 620 nm by the blue-violet color of the starch-iodine complex by 10% in 10 minutes. .

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  With respect to the activity of pullulanase, the enzyme activity that produces 1 μmol of maltotriose per minute at pH 6.0 and 30 ° C. using pullulan as a substrate was shown as 1 Unit.

The results are shown in Table 20 below.

  The results in the table show that α, α-trehalose can be produced using amylase of other biological origin, but the yield is lower than that when using the novel enzyme of the present invention. It was.

Example II-8 Production of α, α-trehalose at various enzyme concentrations from amylose DP-17 Using the purified enzyme obtained in Example II-2 and a purified transferase derived from Sulfolobus solfaricus KM1 Amylose DP-17 (manufactured by Hayashibara Biochemical) (final concentration 10%) was added to the substrate with the enzyme having the concentration shown in Table 21 and reacted at 60 ° C. and pH 5.5 for about 100 hours. An attempt was made to produce α, α-trehalose utilizing the synergistic action of enzymes. In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, and the yield of the produced α, α-trehalose was examined. .

  In addition, the novel amylase activity of this invention showed the enzyme activity which releases 1 micromol alpha, alpha-trehalose in 1 hour from maltotriosyl trehalose as 1 Unit like Example II-1.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

The results are shown in Table 21 below.

  From the results in the table, when amylose DP-17 having a linear α-1,4 bond is used as a substrate, the yield of α, α-trehalose is maximum when transferase is 10 Units / ml and amylase is 150 Units / ml. It can be seen that it reached 83.4%.

Example II-9 Production of α, α-trehalose at various soluble starch concentrations Using the present purified enzyme obtained in Example II-2 and a purified transferase derived from Sulfolobus solfaricus KM1 as a soluble starch, the substrate The final concentrations of 5%, 10%, 20%, and 30% were added with the enzymes shown in Table 22 and reacted at 60 ° C. and pH 5.5 for about 100 hours. An attempt was made to produce α, α-trehalose using synergism. During the reaction, pullulanase (a product derived from Klebsiella pneumoniae, manufactured by Wako Pure Chemical Industries, Ltd.) was added so that the total amount was 5 units / ml every 12 hr from 0 hr to 96 hr, including 0 hr, at 40 ° C. for 1 hr. Processed under conditions.

  In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, and the yield of the produced α, α-trehalose was examined. .

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  With respect to the activity of pullulanase, the enzyme activity that produces 1 μmol of maltotriose per minute at pH 6.0 and 30 ° C. using pullulan as a substrate was shown as 1 Unit.

The results are shown in Table 22 below.

  From the results of the table, even when the concentration of the soluble starch of the substrate was changed to 5 to 30%, the yield of α, α-trehalose was improved by using the amylase and transferase concentrations under the optimum conditions. It can be seen that it can be made 70% or more.

Example II-10 Production of α, α-trehalose from soluble starch containing various pullulanase treatments Using the present purified enzyme obtained in Example II-2 and a purified transferase derived from Sulfolobus solfaricus KM1 As starch (final concentration: 10%), enzymes having the concentrations shown in Table 23 were added to the substrate and reacted at 60 ° C. and pH 5.5 for about 120 hours. Α, α utilizing the synergistic action of these enzymes -Attempted to produce trehalose. At this time, once in the course of 24 hr (a), once in 48 hr (b), once in 72 hr (c), once in 96 hr (d), between 24 hr and 96 hr, a total of 4 times every 24 hr ( e) Between 0 hr and 96 hr, a total of 9 times every 12 hr including 0 hr (f), and at a reaction initial stage of 0 hr to 12 hr, a total of 5 times every 3 hr including 0 hr, and thereafter 24 hr to 96 hr During this period, pullulanase (a product derived from Klebsiella pneumoniae) was added so that the concentration shown in the table was reached under conditions of 7 times (g) every 12 hours. In each case, the conditions were 40 ° C. and 1 hour after the addition. Was treated with pullulanase.

  In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, and the yield of the produced α, α-trehalose was examined. .

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  With respect to the activity of pullulanase, the enzyme activity that produces 1 μmol of maltotriose per minute at pH 6.0 and 30 ° C. using pullulan as a substrate was shown as 1 Unit.

The results are shown in Table 23 below.

  From the results in the table, the yield can be improved by introducing pullulanase treatment in the middle of the reaction, and in the method of performing the treatment multiple times, or the method of performing the treatment multiple times in the initial reaction stage, It was found that the yield of α, α-trehalose can be further improved. Under the conditions of transferase 10 Units / ml, amylase 150 Units / ml, pullulanase treatment method (g), and pullulanase 5 Units / ml, the yield of α, α-trehalose was maximized and reached 80.9%.

Example II-11 Production of α, α-trehalose at various amylose DP-17 concentrations and various reaction temperatures The purified enzyme obtained in Example II-2 and the purified transferase derived from Sulfolobus solfaricus KM1 were each 320 Units / g-substrate and 20 Units / g-substrate were added, and the substrate was treated as amylose DP-17 at a substrate concentration and reaction temperature shown in Tables 24 and 25 for about 100 hours, respectively. An attempt was made to produce α, α-trehalose utilizing the action.

  The reaction solution decomposes the unreacted substrate with glucoamylase, and then analyzes by TSK-gel Amide-80 HPLC analysis method shown in Example II-1. The yield and reaction rate of the α, α-trehalose produced. I investigated.

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

The results are shown in Tables 24 and 25 below.
In addition, the reaction rate in Table 24 indicates the rate of liberating 1 μmol of α, α-trehalose per hour as 1 Unit.

From the results in the table, it was found that the reaction rate increased in a temperature-dependent manner when the reaction temperature was raised to 40-80 ° C. Also, at low temperatures (40-50 ° C), the substrate concentration is high (30-
40%) insolubilized, and the yield was remarkably reduced. However, when the temperature was raised, the substrate was dissolved and the yield could be maintained high. The yield reached 75.1%.

  From the results of this example, by using the amylase of the present invention having high heat resistance, it is possible to prepare a high temperature and high concentration, which is advantageous from the viewpoint of ease of handling economically and α, α-trehalose. It is understood that a manufacturing method can be provided.

Example II-12 Production of α, α-trehalose using thermostable pullulanase at various soluble starch concentrations and various reaction temperatures The purified enzyme obtained in Example II-2, a purified transferase derived from Sulfolobus solfaricus KM1, And commercially available thermostable pullulanase (Debranching Enzyme Amano (Derived from Bacillus sp., Amano Pharmaceutical Co., Ltd.); In addition, hydrophobic chromatography TOSOH TSK-gel Phenyl-TOYOPEARL 650S removes mixed glucoamylase activity and α-amylase activity And 280 Units / g-substrate, 80 Units / g-substrate, and 32 Units / ml, respectively, and the substrate as soluble starch at the substrate concentrations and reaction temperatures shown in Tables 26 and 27, respectively. About 100 hours of reaction, synergistic action of these enzymes Using α, I tried to production of α- trehalose.

  The reaction solution decomposes the unreacted substrate with glucoamylase, and then analyzes by TSK-gel Amide-80 HPLC analysis method shown in Example II-1. The yield and reaction rate of the α, α-trehalose produced. I investigated.

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  The activity of pullulanase was shown as 1 Unit for the enzyme activity that produces 1 μmol of maltotriose per minute at pH 5.5 and 60 ° C. using pullulan as a substrate.

The results are shown in Tables 26 and 27 below.
In addition, the reaction rate in Table 26 shows the rate of liberating 1 μmol of α, α-trehalose per hour as 1 Unit.

  The reaction yield was 35.0% when the reaction was carried out under the above conditions at a substrate concentration of 10% and a reaction temperature of 60 ° C., except that no thermostable pullulanase was added.

From the results shown in the table, it was found that the heat-stable pullulanase can be improved in yield only by adding it once in the reaction, and the reaction rate increases in a temperature-dependent manner when the reaction temperature is raised to 40-60 ° C. In addition, when the substrate concentration is high (20-30%) at low temperatures (40-50 ° C.), the substrate becomes insoluble and the yield decreases remarkably. However, at high temperatures (60 ° C.), the substrate dissolves and the yield is high. I was able to maintain it. The yield reached 74.1%.

Example II-13 Production of α, α-trehalose from soluble starch containing isoamylase treatment Using the present purified enzyme obtained in Example II-2 and a purified transferase derived from Sulfolobus solfaricus KM1 as a substrate, soluble starch (Final concentration 10%), the substrate was added so as to be 1280 Units / g-substrate and 80 Units / g-substrate, respectively, and reacted at 60 ° C. and pH 5.0 for about 100 hrs. The production of α, α-trehalose used was attempted. At this time, isoamylase (a product derived from Pseudomanas amyloderamosa under the conditions of 0 to 12 hours in the initial reaction stage, including 0 hours and 3 times in total for 3 hours, and then 24 to 96 hours and 3 times in total for 24 hours, Seikagaku Kogyo Co., Ltd.) was added so as to have the concentration shown in Table 28, and in each case, after addition, isoamylase treatment was performed under conditions of 40 ° C. and 1 hr.

  In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, and the yield of the produced α, α-trehalose was examined. .

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  The isoamylase activity was determined by mixing 0.1 ml of 0.5 M acetate buffer pH 3.5 and 0.1 ml of enzyme solution with 0.5 ml of 1% soluble glutinous starch and reacting at 40 ° C. The absorbance at 610 nm due to blue-violet color was measured with a cell of 1 cm width (starch, related carbohydrate enzyme experiment method, Michinori Nakamura, Junji Kakinuma, published by Academic Publishing Center, 1989). Defined as 1 Unit.

The results are shown in Table 28 below.

  From the results in the table, as in the case of pullulanase (a product derived from Klebsiella pneumoniae), the yield can be improved by introducing an isoamylase treatment during the reaction, and the yield of α, α-trehalose is 75. It reached 7%.

Example II-14 Production of α, α-trehalose from soluble starch containing a debranching enzyme treatment from Sulfolobus solfaricus KM1 The present purified enzyme obtained in Example II-2, a purified transferase from Sulfolobus solfaricus KM1, and The debranching enzyme of Sulfolobus solfaricus KM1 strain (separated from the bacterial cell extract according to the method of Reference Example II-3) is shown in 1280 Units / g-substrate, 80 Units / g-substrate, and the table below, respectively. Add to the concentration, and react the substrate as soluble starch (final concentration 10%) at 60 ° C and pH 5.5 for about 100 hr, and try to produce α, α-trehalose using the synergistic action of these enzymes It was.

  In the reaction solution, an unreacted substrate was decomposed with glucoamylase, and then analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1, and the yield of the produced α, α-trehalose was examined. .

  In addition, the activity of the novel amylase of the present invention is shown as 1 Unit in the same manner as in Example II-1, in which the enzyme activity that liberates 1 μmol of α, α-trehalose per hour from maltotriosyl trehalose.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  The activity of the purified debranching enzyme derived from Sulfolobus solfaricus KM1 was prepared by mixing 0.5 ml of 1% soluble glutinous starch with 0.1 ml of 0.5 M acetate buffer pH 5.0 and 0.1 ml of enzyme solution at 60 ° C. The absorbance at 610 nm of the amylose-iodine complex due to the blue-violet color was measured in a 1 cm-wide cell, and the amount of enzyme to be increased by 0.1 per hour was defined as 1 Unit.

The results are shown in Table 29 below.

  From the results of the table, as in the case of the thermostable pullulanase (debranching enzyme Amano, a product derived from Bacillus sp.), The debranching enzyme derived from the Sulfolobus solfatalicus KM1 strain can be improved by adding it once in the reaction. And the yield of α, α-trehalose reached 69.8%.

Reference Example II-1 Production of transfer oligosaccharides by transferase at various amylose DP-17 concentrations and various reaction temperatures Purified transferase from Sulfobus sofaraticus KM1 was added so as to be 20 Units / g-substrate, and the substrate was amylose DP- 17, the reaction was carried out for about 100 hours at the substrate concentration and reaction temperature shown in Tables 30 and 31, respectively, to produce the corresponding trehalose oligosaccharide in which the glucose residue on the reducing end side was α-1, α-1 linked. .

  The corresponding trehalose oligosaccharide in which the reducing-side glucose residue is α-1, α-1 linked is a dinitrosalicylic acid method (starch and related carbohydrate enzyme experiment method, Michinori Nakamura and Shinji Kakinuma, published by the Society Publishing Center, 1989), the amount of the reducing end was measured, and the yield and reaction rate were examined from the reduced amount.

  The activity of the purified transferase derived from Sulfolobus sofaraticus KM1 showed 1 Unit as the enzyme activity for producing 1 μmol of glucosyl trehalose per hour at pH 5.5 and 60 ° C. using maltotriose as a substrate.

  The results are shown in Tables 30 and 31 below.

In addition, the reaction rate in Table 30 shows the rate of releasing 1 μmol of α, α-trehalose per hour as 1 Unit.

  From the results in the table, it was found that the reaction rate increased in a temperature-dependent manner when the reaction temperature was raised to 40-80 ° C. Further, when the substrate concentration was high (particularly 40%) at a low temperature (40 to 50 ° C.), the substrate was insolubilized and the yield was remarkably reduced. However, when the temperature was high, the substrate was dissolved and the yield could be kept high. The molar yield reached 76.6%.

Reference Example II-2 Measurement of Solubility of Amylose DP-17 in Water Amylose DP-17 was dissolved by heating to a 5, 10, 20, 30, 40% (w / vol) solution, and then 35, 40, 50 , Put in a high-temperature bath at 60, 70, 80 ° C., sample over time, filter the insoluble matter produced, determine the amylose DP-17 concentration in the supernatant solution, and saturate the equilibrium concentration The solubility was determined as a point.

The results are shown in Table 32 below.

Reference Example II-3 Purification of a debranching enzyme derived from Sulfolobus solfatricus KM1 Sulfolobus solfatricus KM1 strain was published by American Type Culture Collection (ATCC), which contains 2 g / liter of soluble surname starch and 2 g / liter of yeast extract. Cultured at 75 ° C. for 3 days in the medium of medium number 1304 described in Pages 18 (1992). The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 3.3 g / liter.

  82 g of the microbial cells obtained as described above are suspended in 400 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by sonication for 15 minutes at 0 ° C., and then centrifuged. To obtain a supernatant solution.

  Hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-) was added to this supernatant solution to a concentration of 1 M and equilibrated with 50 mM sodium acetate buffer (pH 5.5) containing 1 M ammonium sulfate and 5 mM EDTA. TOYOPEARL 65OS 800 ml). The column was washed with the same buffer, and the target debranching enzyme was recovered in the flow-through fraction. Since the amylase, transferase, and glucoamylase contained in the supernatant solution are retained and adsorbed by the column filler Phenyl-TOYOPEARL 65OS, they were separated from the target debranching enzyme. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  Subsequently, it was passed through ion exchange chromatography (column: TOSOH TSK-gel DATE-TOYOPEARL 65OS 300 ml) equilibrated with the same buffer. The column was washed with the same buffer, and then the target debranching enzyme was eluted with a linear gradient of 900 ml of 0M to 0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M sodium chloride and 5 mM EDTA to desalinate.

  This desalted concentrated solution was loaded on a gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg), and the target debranching enzyme was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 25 mM bis-tris-iminodiacetic acid buffer (pH 7.1) for desalting.

  This desalted concentrated solution was loaded on chromatofocusing (column: Pharmacia Mono P HR5 / 20) equilibrated with the same buffer, and 10% Polybuffer 74 (manufactured by Pharmacia, adjusted to pH 4.0 with iminodiacetic acid). The target debranching enzyme was eluted. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000), then washed with 10 mM Tris-HCl buffer (pH 7.5) and desalted.

  This desalted concentrate was passed through ion exchange chromatography (column: TOSOH TSK-gel DATE 5PW HPLC) equilibrated with the same buffer. The column was washed with the same buffer, and then the target debranching enzyme was eluted with a linear gradient of 30 ml of 0M-0.3M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 13000) to obtain a partially purified product (liquid product) of the target debranching enzyme.

  In the above purification method, the target debranching enzyme was detected by using 5% soluble starch as a substrate, and adding 2 Units / ml and 32 Units / ml of the purified amylase and purified transferase derived from Sulfolobus solfaricus KM1 together with the test solution, respectively. Then, the reaction was performed at pH 5.5 and 60 ° C., and the activity of increasing the amount of α, α-trehalose produced in comparison with the case where no test solution was added was used as an index.

The activity of the partially purified debranching enzyme derived from Sulfolobus solfaricus KM1 obtained by the above purification method was as follows: 0.5 ml of 1% soluble glutinous starch, 0.1 ml of 0.5 M acetate buffer pH 5.0, enzyme solution 0. 1 ml was mixed and reacted at 60 ° C., the absorbance at 610 nm due to the blue purple color of the amylose-iodine complex was measured in a 1 cm-wide cell, and the amount of enzyme to be increased by 0.1 per hour was defined as 1 Unit.
The specific activity of the debranching enzyme partially purified by the above purification operation was 495 Units / mg.

Reference Example II-4 Examination of various properties of the debranching enzyme derived from Sulfolobus solfariticus KM1 The enzymatic properties of the partially purified debranching enzyme obtained in Reference Example II-3 were measured.

(1) Action and Substrate Specificity The reactivity and action against each substrate were examined using the substrates shown in Table 33 below and each activity measurement method.

  The dinitrosalicylic acid method (starch and related carbohydrate enzyme experiment method, Michinori Nakamura and Junji Kakinuma, published by the Society Press, 1989) is a method for quantifying the increase in the amount of reducing end due to the hydrolysis reaction of α-1,6 bond. is there.

  The iodine coloration method is performed in the same manner as in Reference Example II-3, and the increase in linear amylose due to the hydrolysis reaction of the α-1,6 bond is quantified by the increase in absorbance at 610 nm due to the blue purple of the amylose-iodine complex. Is the method.

Analysis of degradation products by liquid chromatography (HPLC method) was performed by the Bio-Rad AMINEX HPX-42A HPLC analysis method shown in Example II-1, and the resulting oligosaccharides were examined.

  As is clear from the above results, the main debranching enzyme produces 1) reducing ends from pullulan and various starches, 2) increases the iodine coloration degree of various starches, and 3) maltotri from pullulan. Aus can be generated. Furthermore, 4) As shown in Example II-14, when soluble starch was used as a substrate and reacted with a purified amylase and purified transferase derived from Sulfobus sofaraticus KM1, compared with the case where no main debranching enzyme was added. Significantly increase the amount of α, α-trehalose produced. Therefore, it is understood from these facts that this enzyme hydrolyzes α-1,6 bonds of starch and pullulan.

(2) Stability Table 34 shows the stability of the obtained partially purified enzyme in the treatment for 3 hours at each temperature.

  This enzyme had a residual activity of 73.3% after treatment at 60 ° C. for 3 hours.

(3) Reactivity The reactivity of the obtained partially purified enzyme at each pH and each temperature is shown in Tables 35 and 36, respectively. For the measurement, a glycine hydrochloride buffer solution was used during pH 3 to 5, a sodium acetate buffer solution was used during pH 4 to 5.5, and a sodium phosphate buffer solution was used during pH 5 to 7.5.

This enzyme has a reaction optimum temperature in the vicinity of 60 to 65 ° C. and a reaction optimum pH in the vicinity of pH 4.0 to 5.5.

(4) Isoelectric point As a result of measuring the pH of the debranching enzyme fraction separated by chromatofocusing, the isoelectric point was 4.4.

(5) Effects of various activators and inhibitors The substances shown in Table 37 below were added together with the substrate, and the activity in each case was measured in the same manner as in Reference Example II-3 to determine the presence or absence of activation or inhibition. Examined. As a result, it was found that the copper ions were inhibited. Many sugar-related enzymes are activated by calcium ions, but this enzyme is not activated by calcium ions.

Example I-9 Determination of partial amino acid sequence of a novel transferase derived from Sulfolobus solfaricus KM1 The determination of the partial amino acid sequence of the purified enzyme obtained in Example I-2 was performed by Iwamatsu (Biochemical 63, 139 (1991)) and others. By the method. That is, the purified novel transferase is suspended in an electrophoresis buffer (10% glycerol, 2.5% SDS, 2% 2-mercaptoethanol, 62 mM Tris-HCl buffer (pH 6.8)), and SDS polyacrylamide electrolysis is performed. It was subjected to electrophoresis. After the electrophoresis, the enzyme was subjected to electroblotting (Salto blot IIs type (Sartorius)) for 1 hour at 160 mA from a gel on a polyvinylidene difluoride (PVDF) membrane (ProBlot, (Applied, Biosystems)).

  After the transfer, the membrane of the transferred part of the enzyme is cut out and about 300 μl reducing buffer (6M guanidine hydrochloride, 0.5M Tris-HCl buffer (pH 3.5), 0.3% EDTA, 2% acetonitrile) is used. After immersion, 1 mg of dithiothreitol was added thereto, and reduction was performed at 60 ° C./about 1 hour under argon. To this was added 2.4 mg monoiodoacetic acid dissolved in 10 μl of 0.5N sodium hydroxide solution, and the mixture was stirred for 20 minutes under light shielding. The PVDF membrane was taken out, thoroughly washed with 2% acetonitrile, and then stirred in 0.1% SDS for 5 minutes. Next, the PVDF membrane was lightly washed with water, then immersed in 0.5% polyvinylpyrrolidone-40, 100 mM acetic acid, and allowed to stand for 30 minutes. Thereafter, the PVDF membrane was lightly washed with water and cut into about 1 mm square. This was immersed in a digestion buffer (8% acetonitrile, 90 mM Tris-HCl buffer (pH 9.0)), 1 pmol of Achromobacter Protease I (Wako Pure Chemical Industries, Ltd.) was added, and digested at room temperature for 15 hours. This digest was separated by reverse phase HPLC using a C8 column (Nippon Millipore Ltd., μ-Bondashere 5C8, 300A, 2.1 × 150 mm) to obtain 10 types of peptide fragments. As a peptide elution solvent, A solvent (0.05% trifluoroacetic acid) and B solvent (2-propanol / acetonitrile 7: 3 containing 0.02% trifluoroacetic acid) were used. Was eluted at a flow rate of 0.25 ml / min for 40 minutes. About the obtained peptide fragment, the amino acid sequence was determined by the automatic Edman degradation method using the gas phase peptide sequencer (Applied Biosystems model 470 type).

  Further, the peptide fragment digested with Achromobacter Protease I was further subjected to secondary digestion with Asp-N, and the obtained peptide fragment was separated in the same manner as described above to determine the amino acid sequence.

As a result, the partial amino acid sequence was as follows.
Achromobacter protease digested peptide fragment AP-1: Val Ile Arg Glu Ala Lys (SEQ ID NO: 9)
AP-2: Ile Ser Ile Arg Gln Lys (SEQ ID NO: 10)
AP-3: Ile Ile Tyr Val Glu (SEQ ID NO: 11)
AP-4: Met Leu Tyr Val Lys (SEQ ID NO: 12)
AP-5: Ile Leu Ser Ile Asn Glu Lys (SEQ ID NO: 13)
AP-6: Val Val Ile Leu Thr Glu Lys (SEQ ID NO: 14)
AP-7: Asn Leu Glu Leu Ser Asp Pro Arg Val Lys (SEQ ID NO: 15)
AP-8: Met Ile Ile Gly Thr Tyr Arg Leu Gln Leu Asn Lys (SEQ ID NO: 16)
AP-9: Val Ala Val Leu Phe Ser Pro Ile Val (SEQ ID NO: 17)
AP-10: Ile Asn Ile Asp Glu Leu Ile Ile Gln Ser Lys (SEQ ID NO: 18)
AP-11: Glu Leu Gly Val Ser His Leu Tyr Leu Ser Pro Ile (SEQ ID NO: 19)
Asp-N digested peptide fragment DN-1: Asp Glu Val Phe Arg Glu Ser (SEQ ID NO: 20)
DN-2: Asp Tyr Phe Lys (SEQ ID NO: 21)
DN-3: Asp Gly Leu Tyr Asn Pro Lys (SEQ ID NO: 22)
DN-4: Asp Ile Asn Gly Ile Arg Glu Cys (SEQ ID NO: 23)
DN-5: Asp Phe Glu Asn Phe Glu Lys (SEQ ID NO: 24)
DN-6: Asp Leu Leu Arg Pro Asn Ile (SEQ ID NO: 25)
DN-7: Asp Ile Ile Glu Asn (SEQ ID NO: 26)
DN-8: Asp Asn Ile Glu Tyr Arg Gly (SEQ ID NO: 27)

Example I-10 Preparation of Chromosomal DNA of Sulfolobus solfaticalus KM1 Sulfobus solfatricus KM1 strain cells were obtained according to the method of Example I-2.

  To 1 g of the cells, 10 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 25% sucrose, 1 mg / ml lysozyme, 1 mM EDTA, 150 mM NaCl was added and suspended, and left at room temperature for 30 minutes. To this, 0.5 ml of 10% SDS and 0.2 ml of 10 mg / ml proteinase K (manufactured by Wako Pure Chemical Industries, Ltd.) were added and left at 50 ° C. for 2 hours. The solution was then extracted with phenol / chloroform and the aqueous phase was removed and ethanol precipitated. The precipitated DNA was wound up with a sterilized glass rod, washed with 70% ethanol, and dried under reduced pressure. Finally, 1.5 mg of chromosomal DNA was obtained.

Example I-11 Preparation of DNA probe based on partial amino acid sequence and confirmation of probe by PCR method Oligonucleotide DNA based on information on partial amino acid sequence of a novel transferase derived from Sulfobus solfaricus KM1 determined in Example I-9 Primers were prepared using a DNA synthesizer (Applied Biosystems Model 381). The sequence was as follows.
DN-1
Amino acid sequence N-terminal AspGluValPheArgGluSe C-terminal DNA primer 5 ′ TTCACGAAAAACCCTCAT 3 ′ (SEQ ID NO: 28)
Base sequence C T TG T T
DN-8
Amino acid sequence N-terminal AspAsnIleGluTyrArgGly C-terminal DNA primer 5 ′ GATAACATAGAATACAGAGG 3 ′ (SEQ ID NO: 29)
Base sequence T T G T G
PCR was performed using 100 ng of each of these DNA primers and 100 ng of chromosomal DNA of Sulfolobus solfalicus KM1 prepared in Example I-10. The PCR apparatus is a GeneAmp PCR system model 9600 manufactured by PerkinElmer, Inc. One cycle is performed at 94 ° C. for 30 seconds, 50 ° C. for 1 minute and 72 ° C. for 2 minutes, 30 cycles and a total volume of 100 μl did.

  10 μl of the obtained reaction solution was analyzed by 1% agarose electrophoresis. As a result, a DNA fragment having a length of about 1.2 kb was specifically amplified.

  The ends of this PCR product were blunted and subcloned into the HincII site of pUC118. The DNA sequence of the inserted fragment of this plasmid was determined using a DNA sequencer / GENESCAN model 373A manufactured by Applied Biosystems. As a result, a DNA sequence corresponding to the amino acid sequence obtained in Example I-9 was found.

Example I-12 Cloning of a Novel Transferase Gene Derived from Sulfobus solfaricus KM1 100 μg of the chromosomal DNA of Sulfobus solfaricus KM1 prepared in Example I-10 was partially digested with the restriction enzyme Sau3Al. A 5 to 10 kb DNA fragment was separated and purified by ultracentrifugation of the reaction solution using a sucrose density gradient. On the other hand, the plasmid vector pUC118 was digested with BamHI, the end was dephosphorylated with alkaline phosphatase, and the chromosomal DNA fragment of the above-mentioned 5-10 kb Sulfolobus solfaricus KM1 strain was ligated with T4 DNA ligase. Using a mixture containing the pUC118 plasmid vector containing this insert, E. coli JM109 cells were transformed. This was seeded on an LB agar plate medium containing 50 μg / ml ampicillin to form colonies to prepare a DNA library.

  Screening of a recombinant plasmid containing a novel transferase gene in this DNA library was performed by PCR as follows.

  First, colonies were scraped off and suspended in TE buffer. This was treated at 100 ° C. for 5 minutes to disrupt the cells, and PCR was performed by the method described in Example I-11.

  Subsequently, 10 μl of the obtained PCR reaction solution was analyzed by 1% agarose electrophoresis, and a clone in which a DNA fragment having a length of about 1.2 kb was amplified was defined as a positive clone.

  As a result, one positive clone was obtained from 600 transformants. When the plasmid extracted from it was analyzed, it had an inserted fragment of about 8 kb. This plasmid was designated “pKT1”.

  Further, the insert fragment was partially digested with the restriction enzyme Sau3AI, and PCR was performed in the same manner as described above to reduce the insert fragment. As a result, transformants carrying plasmids having inserts of about 3.8 kb and about 4.5 kb were obtained. These plasmids were designated “pKT21” and “pKT11”, respectively.

Restriction enzyme maps of the inserted fragments of these plasmids were as shown in FIG.
In addition, as a restriction enzyme used by the above Example, all the commercial items (purchased from Takara Shuzo Co., Ltd.) were utilized.

Example I-13 Determination of the DNA sequence of a novel transferase gene derived from Sulfolobus solfaricus KM1 The DNA base sequence of the common portion of the inserted fragment in the plasmids pKT11 and pKT21 obtained in Example I-12 was determined.

  First, a deletion plasmid was prepared from this plasmid DNA using Takara Shuzo Co., Ltd.'s kilosequence deletion kit. Next, the DNA sequence of the inserted fragment of this plasmid was determined by using Perkin Elmer Japan, PRISM, Sequenase Dye Primer Sequencing Kit, Taq Dye Deoxy ™ Terminator Cycle Sequencing Kit and Applied Biosystems 3 using DNA Sequencer G / ESA N / S

  The base sequence between the ends of SphI-pKT21 (between A and B in FIG. 26) in the consensus sequence and the amino acid sequence deduced therefrom were as shown in SEQ ID NOs: 1 and 2.

  All sequences corresponding to the partial amino acid sequence obtained in Example I-9 were found in this amino acid sequence. This amino acid sequence was 728 in length and was thought to encode a protein with an estimated molecular weight of 82 kDa. This molecular weight almost coincided with the molecular weight value obtained by subjecting a purified enzyme of a novel transferase derived from Sulfolobus solfaricus KM1 to SDS-PAGE.

Example I-14 Expression of novel transferase in transformant pKT21 obtained in Example I-12 was cleaved with SphI and XbaI and ligated to pUC119 (Takara Shuzo) cleaved with the same enzyme was designated as pKT22. . FIG. 27 shows the means. In the fragment containing the novel transferase gene inserted into pKT22, the base sequence excluding the multicloning site was the sequence of base numbers 1 to 2578 of SEQ ID NO: 1.

  The novel transferase activity of the transformant containing this plasmid was examined as follows. First, the transformant was cultured overnight at 37 ° C. in an LB medium containing 100 μg / ml ampicillin. The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 10 g / liter.

  10 g of the microbial cells obtained as described above are suspended in 40 ml of 50 mM sodium acetate buffer (pH 5.5) containing 5 mM EDTA, lysed by sonication at 0 ° C. for 3 minutes, and then centrifuged. A supernatant solution was obtained. This was heat-treated at 75 ° C. for 30 minutes, centrifuged again, concentrated with an ultrafiltration membrane (molecular weight cut 13000), and finally the maltotriose as a substrate was obtained as a crude enzyme solution (6 Unit / ml). It added so that it might become 10%. After reacting at pH 5.5 (50 mM sodium acetate) at 60 ° C. for 24 hours, the reaction was stopped by heating at 100 ° C. for 5 minutes. The produced glucosyl trehalose was measured by the same HPLC analysis method as in Example I-1.

  The result of HPLC analysis was as shown in FIG. The main reactant appeared as a single peak without anomer on the HPLC chart with a slight delay from the unreacted substrate. Moreover, this main product was fractionated by TSK-gel amide-80 HPLC column, and as a result of analysis by 1H-NMR and 13C-NMR, it was confirmed that it was glucosyl trehalose. As a result, it was found that the transformant had a novel transferase activity derived from the Sulfolobus solfaricus KM1 strain. Further, no novel transferase activity was detected from the transformant in which only pUC119 was added to JM109.

Example I-15 Determination of partial amino acid sequence of novel transferase derived from Sulfolobus acidocaldarius strain ATCC 33909 The partial amino acid sequence of the novel transferase obtained in Example I-4 was determined according to the method described in Example I-9. It was. The partial amino acid sequence determined below is shown.
Achromobacter protease digested peptide fragment AP-6: Arg Asn Pro Glu Ala Tyr Thr Lys (SEQ ID NO: 30)
AP-8: Asp His Val Phe Gln Glu Ser His Ser (SEQ ID NO: 31)
AP-10: Ile Thr Leu Asn Ala Thr Ser Thr (SEQ ID NO: 32)
AP-12: Ile Ile Ile Val Glu Lys (SEQ ID NO: 33)
AP-13: Leu Gln Gln Tyr Met Pro Ala Val Tyr Ala Lys (SEQ ID NO: 34)
AP-14: Asn Met Leu Glu Ser (SEQ ID NO: 35)
AP-16: Lys Ile Ser Pro Asp Gln Phe His Val Phe Asn Gln Lys (SEQ ID NO: 36)
AP-18: Gln Leu Ala Glu Asp Phe Leu Lys (SEQ ID NO: 37)
AP-19: Lys Ile Leu Gly Phe Gln Glu Glu Leu Lys (SEQ ID NO: 38)
AP-20: Ile Ser Val Leu Ser Glu Phe Pro Glu Glu (SEQ ID NO: 39)
AP-23: Leu Lys Leu Glu Glu Gly Ala Ile Tyr (SEQ ID NO: 40)
AP-28: Glu Val Gln Ile Asn Glu Leu Pro (SEQ ID NO: 41)
Asp-N digested peptide fragment DN-1: Asp His Ser Arg Ile (SEQ ID NO: 42)
DN-5: Asp Leu Arg Tyr Tyr Lys (SEQ ID NO: 43)
DN-6: Asp Val Tyr Arg Thr Tyr Ala Asn Gln Ile Val Lys Glu Cys (SEQ ID NO: 44)

Chromosomal DNA Cloning Sulfolobus acidocaldarius ATCC 33909 strain in Example I-16 Sulfolobus acidocaldarius ATCC33909 strain - derived novel transferase was obtained according to the method in from the cells Example I-10 was obtained according to the method of Example I-4. The chromosomal DNA was partially digested with Sau3AI, and then ligated to EMBL3 BamHI cleavage arm (manufactured by STRATAGENE) with T4 DNA ligase. Packaging was performed using Gigapack II Gold manufactured by STRATAGENE. The library was infected with E. coli LE392 at 37 ° C. for 15 minutes, and then inoculated on a NZY agar plate medium and cultured at 37 ° C. for about 8 to 12 hours to form plaques. After storage at 4 ° C. for about 2 hours, DNA was adsorbed onto a nylon membrane (Amersham, Hybond N +). After lightly washing with 2 × SSPE, baking was performed at 80 ° C. for 2 hours. The probe used was the EcoRI-XbaI fragment of pKT22 obtained in Example I-14 (corresponding to SEQ ID NO: 1 from 824 to 2578) and labeled with ³² P using the Amersham Mega Prime DNA labeling system. .

  Hybridization conditions were 6 × SSPE, 0.5% SDS at 60 ° C. overnight. Washing was performed twice with 2 × SSPE and 0.5% SDS at room temperature for 10 minutes.

Screening was started from about 5000 clones to obtain 8 positive clones. An approximately 7.6 kbp BamHI fragment was obtained from this clone and inserted into the above site of pUC118. This plasmid is referred to as “p09T3”. Further, the inserted fragment of the above clone was partially digested with Sau3AI, and the obtained fragment of about 6.7 kbp was inserted into the BamHI site of pUC118. This plasmid is referred to as “p09T2”. About 3.8 kbp XbaI fragment from this plasmid was inserted into the above site of pUC118. This plasmid is referred to as “p09T1”. A restriction enzyme map of the inserted fragment of this plasmid is shown in FIG. The creation method is shown in FIG. The nucleotide sequence of p09T1 was determined according to the method of Example I-13 centering on the region encoding a novel transferase. The determined nucleotide sequence and the deduced amino acid sequence are as shown in SEQ ID NOs: 3 and 4. All sequences corresponding to the partial amino acid sequences obtained in Example I-15 were found in this amino acid sequence. This amino acid sequence was 680 in length and was thought to encode a protein with an estimated molecular weight of 80.1 kDa. This molecular weight substantially coincided with the molecular weight value obtained by subjecting a purified enzyme of a novel transferase derived from Sulfolobus acidocaldarius ATCC 33909 to SDS-PAGE. Moreover, the novel transferase activity was confirmed about the transformant containing plasmid p09T1 according to the method of Example I-14.

Example I-17 Hybridization test of a novel transferase gene derived from Sulfolobus solfataricus KM1 and chromosomal DNA of other species Sulfolobus solfataricus DSM 5833, Sulfolobus shibatae DSM 5389, E. coli Chromosomal DNA of E. coli strain JM109 was obtained by the method according to Example I-10 and digested with restriction enzymes PstI and EcoRI.

  This digest was separated by 1% agarose gel electrophoresis and Southern blotted on Hybond-N manufactured by Amersham Japan. About 2.6 kbp SphI and XbaI fragments of pKT21 obtained in Example I-12 (corresponding to the sequence shown in SEQ ID NO: 1 and corresponding to the range between A and B in FIG. 26) were added to this membrane. Labeling was carried out using a DIG system kit manufactured by the company, and hybridization was performed using this.

  Conditions are: hybridization: 5 × SSC, 40 ° C., 2 hours, washing: 2 × SSC (including 0.1% SDS), 40 ° C., 5 minutes, 2 times 0.1 × SSC (including 0.1% SDS), 40 ° C. 5 minutes, 2 times.

  As a result, the SphI and XbaI fragments hybridized with the fragment of about 5.9 kbp for the Sulfobus sofatalicus DSM 5833 strain and the fragments of about 5.0 kbp and about 0.8 kbp for the Sulfobus shibatae DSM 5389 strain, respectively. On the other hand, E. is a negative control. No hybrid formation was observed for E. coli strain JM109.

  Furthermore, Sulfobus solfataricus KM1 strain, DSM5354 strain, DSM5833 strain, ATCC35091 strain, ATCC35092 strain, Sulfolobus acidocaldarius ATCC33909 strain, ATCC49426 strain, Sulfolobus shibata strain DSc89DSDS8989 strain Chromosomal DNA of E. coli strain JM109 was obtained according to the method of Example I-10 and digested with restriction enzymes HindIII, XbaI and EcoRV.

This digest was separated by 1% agarose gel electrophoresis and blotted to Amersham Japan, Hybond-N +. The region from 1880 to 2257 (378 bp) of SEQ ID NO: 1 was amplified by PCR on this membrane, labeled with ³² P according to the method of Example I-16, and hybridized using this.

  The conditions were hybridization: 6 × SSPE, 0.5% SDS at 60 ° C., overnight, washing: 2 × SSPE, 0.1% SDS at room temperature for 10 minutes, twice.

  As a result, Sulfobus solfataricus KM1 strain, DSM5354 strain, DSM5833 strain, ATCC35091 strain, ATCC35092 strain, Sulfolobus acidocaldarius ATCC33909 strain, ATCC49426 strain, Sulfolobus shibata strain. It was found that a hybrid was formed at sites of 0.7 kbp, 0.8 kbp, 3.9 kbp, 0.8 kbp, 0.8 kbp, 4.4 kbp, and 2.1 kbp. On the other hand, no binding was observed for the genomic DNA of JM109.

  In addition, the amino acid sequence data bank (Swiss proto and NBRF-PDB), the base sequence data bank indicates that there is no amino acid sequence included in the range of SEQ ID NOs: 1, 2, 3 and 4 and a sequence having homology with the base sequence. (EMBL) was confirmed using sequence analysis software genetics (software development). Therefore, it was found that this novel transferase gene was highly conserved specifically in archaea belonging to the order of Sulfolobales.

Example I-18 Comparison of the nucleotide sequence and amino acid sequence of a novel transferase from Sulfobus sofataricus KM1 strain and Sulfobus acidocaldariusu ATCC 33909 Strain Using the sequence analysis software, Genetics (software development), the amino acid sequence of Fella, the nucleotide sequence of the novel transferase derived from the KM1 strain of SEQ ID NO: 1 and the nucleotide sequence of the novel transferase derived from the ATCC 33909 strain of SEQ ID NO: 3 The analysis was performed considering the gap. The results for the amino acid sequence are shown in FIG. 31, and the results for the base sequence are shown in FIG. The upper row of each figure shows the ATCC33909 strain sequence, the lower row the KM1 strain sequence, the middle (*) is the same sequence in both, and the (.) Is an amino acid sequence with similar properties in both. Indicates. The homology was 49% at the amino acid sequence level and 57% at the base sequence level.

Example I-19 Production of Trehalose Oligosaccharide from Malto-oligosaccharide Mixture Using Recombinant Novel Transferase Derived from Transformant Soluble starch ( manufactured by Nacalai Tesque, special grade) α-amylase degradation product and iodine starch reaction A substrate that was not decomposed to oligosaccharide (α-amylase derived from A-0273 Aspergillus oryzae manufactured by Sigma) was used as a substrate.

Using the crude enzyme solution obtained in Example I-14 and the above substrate, production of glucosyl trehalose and various maltooligosyl trehaloses was attempted according to the reaction conditions of Example I-14. The analysis of the reaction solution was performed by HPLC analysis under the following conditions.
Column: BIORAD AMINEX HPX-42A (7.8 x 300mm)
Solvent: Water Flow rate: 0.6ml / min
Temperature: 85 ° C
Detector: Differential refractometer FIG. 33 (A) shows the results of HPLC analysis, and (B) shows the results of HPLC analysis when no recombinant novel transferase is added. As a result, each of the oligosaccharides of the reaction product had a shorter retention time than the control amylase alone product. In addition, trisaccharide, tetrasaccharide and pentasaccharide, which are main products from the substrates maltotriose (G3), maltotetraose (G4) and maltopentaose (G5) (all manufactured by Hayashibara Biochemical Co., Ltd.) are converted into TSK- The fraction was collected on a gel amide-80 HPLC column and analyzed by 1H-NMR and 13C-NMR. As a result, each showed a structure in which one glucose residue at the reducing end was linked by α-1 and α-1, which were respectively glucosyl trehalose (α-D-maltosyl α-D-glucopyranoside) and maltosyl trehalose (α- D-maltotriosyl α-D-glucopyranoside) and maltotriosyl trehalose (α-D-maltotetraosyl α-D-glucopyranoside).

Example I-20 Production of Glucosyl Trehalose and Maltooligosyl Trehalose Using Recombinant Novel Transferase Derived from Transformant 100 mM maltotriose (G3) to maltoheptaose (G7) (all from Hayashibara Biochemicals) The crude enzyme solution obtained in Example I-14 was lyophilized and then suspended in a 50 mM sodium acetate solution (pH 5.5) to obtain a concentrated enzyme. This concentrated enzyme 12.7 Unit / ml (enzyme activity when maltotriose was used as a substrate) was allowed to act on each substrate to produce the corresponding α-1, α-1 transferant. Each product was analyzed according to the method of Example I-1, and the yield and enzyme activity were examined. The results were as shown in Table 38. The enzyme activity in Table 38 is shown as 1 Unit for the enzyme activity for converting maltooligosaccharide to 1 μmol of the corresponding α-1, α-1 transferant per hour.

Example II-15 Determination of partial amino acid sequence of a novel amylase derived from Sulfobus sofaraticus KM1 The partial amino acid sequence of the purified enzyme obtained in Example II-2 was determined by Iwamatsu et al. (Biochemistry 63, 139 (1991)). The N-terminal amino acid sequence was determined by the method of Matsudaira T (J. Biol. Chem. 262, 10033-10038 (1987)).

  First, the purified new amylase is suspended in a buffer for electrophoresis (10% glycerol, 2.5% SDS, 2% 2-mercaptoethanol, 62 mM Tris-HCl buffer (pH 6.8)), and then subjected to SDS polyacrylamide electrophoresis. It was used for. After the electrophoresis, the enzyme was transferred from a gel to a polyvinylidene difluoride (PVDF) membrane (ProBlot, manufactured by Applied Biosystems) by electroblotting at 160 mA for 1 hour (Zalto Blot IIs, manufactured by Sartorius). .

  After the transfer, the membrane of the transferred portion of the enzyme is cut out and about 300 μl reducing buffer (6M guanidine hydrochloride, 0.5M Tris-HCl buffer (pH 3.5), 0.3% EDTA, 2% acetonitrile) 1 mg of dithiothreitol was added thereto, and reduction was performed at 60 ° C./about 1 hour under argon. To this was added 2.4 mg monoiodoacetic acid dissolved in 10 μl of 0.5N sodium hydroxide solution, and the mixture was stirred for 20 minutes under light shielding. The PVDF membrane was taken out and thoroughly washed with 2% acetonitrile, and then stirred in 0.1% SDS for 5 minutes. Next, the PVDF membrane was lightly washed with water, then immersed in 100 mM acetic acid containing 0.5% polyvinylpyrrolidone-40 and left for 30 minutes. Thereafter, the PVDF membrane was lightly washed with water and cut into about 1 mm square. For the N-terminal amino acid sequence, the cut membrane was directly analyzed by a gas phase sequencer. For the partial amino acid sequence, the cut membrane was immersed in a digestion buffer (8% acetonitrile, 90 mM Tris-HCl buffer (pH 9.0)), 1 pmol of Achromobacter Protease I (manufactured by Wako Pure Chemical Industries, Ltd.) was added at room temperature. Digested for 15 hours. This digest was separated by reverse phase HPLC using a C8 column (manufactured by Nihon Millipore Ltd., μ-Bondashare 5C8, 300A, 2.1 × 150 mm) to obtain 10 types of peptide fragments. As an elution solvent for peptides, A solvent (0.05% trifluoroacetic acid) and B solvent (2-propanol / acetonitrile 7: 3 containing 0.02% trifluoroacetic acid) were used. Elution was carried out for 40 minutes at a flow rate of 0.25 ml / min with a linear concentration gradient of 50%. The amino acid sequence of the obtained peptide fragment was determined by an automated Edman degradation method using a gas phase peptide sequencer (Applied Biosystems, model 470).

The determined N-terminal amino acid sequence and partial amino acid sequence were as follows.
N-terminal amino acid sequence
Thr Phe Ala Tyr Lys Ile Asp Gly Asn Glu (SEQ ID NO: 45)
Partial amino sequence P-6: Leu Gly Pro Tyr Phe Ser Gln (SEQ ID NO: 46)
P-7: Asp Val Phe Val Tyr Asp Gly (SEQ ID NO: 47)
P-10: Tyr Asn Arg Ile Val Ile Ala Glu Ser Asp Leu (SEQ ID NO: 48)
Asn Asp Pro Arg Val Val Asn Pro

Example II-16 Preparation of Chromosomal DNA from Sulfolobus solfaticalus KM1 Sulfolobus solfatricus KM1 strain was published by American Type Culture Collection (ATCC) containing 2 g / liter of soluble starch and 2 g / liter of yeast extract.
The cells were cultured at 75 ° C. for 3 days in the medium of medium number 1304 described in Bacteria and Pages 18th edition, 1992. The cells were collected by centrifugation and stored at -80 ° C. The yield of the microbial cells was 3.3 g / liter.

  To 1 g of the cells, 10 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 25% sucrose, 1 mg / ml lysozyme, 1 mM EDTA and 150 mM NaCl was added and suspended, and left at room temperature for 30 minutes. To this, 0.5 ml of 10% SDS and 0.2 ml of 10 mg / ml proteinase K (manufactured by Wako Pure Chemical Industries, Ltd.) were added and left at 37 ° C. for 2 hours. The solution was then extracted with phenol / chloroform and ethanol precipitated. The precipitated DNA was taken up with a sterilized glass rod, washed with 70% ethanol, and dried under reduced pressure. Finally, 1.5 mg of chromosomal DNA was obtained.

Example II-17 Expression cloning by activity staining method of a novel amylase gene derived from Sulfolobus solfariticus KM1 100 μg of chromosomal DNA of Sulfolobus solfaricus KM1 prepared in Example II-16 was partially digested with the restriction enzyme Sau3AI. The reaction solution was fractionated using a sucrose density gradient centrifugation, and a DNA fragment of 5 to 10 kb was separated and purified. On the other hand, plasmid vector pUC118 (Takara Shuzo Co., Ltd.) was digested with BamHI, purified by dephosphorylating the end with alkaline phosphatase, and the chromosomal DNA fragment of 5 to 10 kb was ligated (ligated) with T4 DNA ligase. Escherichia coli JM109 cells (manufactured by Takara Shuzo Co., Ltd.) were transformed with the mixed solution containing the pUC118 plasmid vector containing the inserted fragment. This was seeded on an LB agar plate medium containing 50 μg / ml ampicillin to form colonies to prepare a DNA library.

  Screening of transformants having a recombinant plasmid containing a novel amylase gene derived from Sulfolobus solfaricus KM1 was performed by an activity staining method.

  First, the obtained transformant was replicated on a filter paper, colonies were formed on an LB agar medium, and this filter paper was added with 1 mg / ml lysozyme (manufactured by Seikagaku Corporation), 50 mM Tris-HCl buffer (1 mM EDTA) ( pH 7.5) It was immersed in the solution and allowed to stand for 30 minutes. Subsequently, it was immersed in 1% Triton-X100 solution for 30 minutes to lyse, and heat treated at 60 ° C. for 1 hour to inactivate the host-derived enzyme. The treated filter paper was placed on an agar plate containing 0.2% soluble starch and reacted at 60 ° C. overnight. The reacted plate was placed under iodine vapor to develop starch and colonies that formed halos were defined as positive clones. As a result, 5 positive clones were obtained from 6000 transformants. When the plasmid was extracted therefrom, the shortest insert fragment had an insert fragment of about 4.3 kb.

Therefore, this insert fragment was further digested with the restriction enzyme BamHI, and the insert fragment was further reduced in the same manner as described above. As a result, a transformant carrying a plasmid having a 3.5 kb insert was obtained. This plasmid was designated “pKA1”.
The restriction enzyme map of the inserted fragment of this plasmid was as shown in FIG.

Example II-18 Determination of DNA sequence of a novel amylase gene derived from Sulfolobus solfariticus KM1 The DNA base sequence (in the region corresponding to pKA2 described later) of the insert fragment in plasmid pKA1 obtained in Example II-17 was determined. .

  First, a deletion plasmid was prepared from this plasmid DNA using Takara Shuzo Co., Ltd.'s kilosequence deletion kit. Subsequently, the DNA sequence of the inserted fragment of this plasmid was determined by using DNA Sequencer 37 using Perkin Elmer Japan, PRISM, Sequenase Dye Primer Sequencing Kit, Taq DyeDeoxy ™ Terminator Cycle Sequencing Kit and Applied Biosystems 3 using ESCA N Model A / G model N.

  The nucleotide sequence and the amino acid sequence deduced therefrom were as shown in SEQ ID NOs: 5 and 6.

  All the partial amino acid sequences obtained in Example II-15 were found in this amino acid sequence. This amino acid sequence was 558 in length and was thought to encode a protein with an estimated molecular weight of 64.4 kDa. This molecular weight almost coincided with the measured molecular weight of 61.0 kDa by SDS-PAGE of the purified amylase derived from Sulfolobus sofaraticus KM1.

Example II-19 Expression of Recombinant Novel Amylase in Transformant The pKA1 obtained in Example II-17 was partially digested with the restriction enzyme PstI was designated as pKA2. FIG. 35 shows the restriction enzyme map. The activity of the transformant containing pKA2 was examined as follows. First, the transformant was cultured overnight at 37 ° C. in an LB medium containing 100 μg / ml ampicillin. The cells collected by centrifugation are suspended in 4 ml of pH 5.5 (50 mM sodium acetate buffer) per gram, subjected to ultrasonic disruption and centrifugation, and the supernatant is heat-treated at 70 ° C. for 1 hour. Of amylase was inactivated. The precipitate was removed by centrifugation, and concentrated with an ultrafiltration membrane (molecular weight cut 13,000) as a crude enzyme solution and used in the following experiments.

(1) Substrate specificity The crude enzyme solution 35.2 Units / ml (where 1 Unit is the enzyme activity when maltotriosyl trehalose is used as a substrate and the reaction conditions are 1 hour according to Example II-1) Is defined as the activity to produce 1 μmol of α, α-trehalose from maltotriosyl trehalose) and is allowed to act on 10 mM substrate (3.0% for amylopectin and soluble starch) shown in Table 39 below. And analysis of decomposition products. For various maltooligosaccharides, amylose DP-17, amylopectin, soluble starch, various isomaltooligosaccharides, and panose, the production activity of monosaccharide + disaccharide is used as an index, and various trehalose oligosaccharides, amylose DP-17α-1, α- Α, α-trehalose production for 1-transition (oligosaccharide with α-1, α-1 bond between the first and second glucose residues on the reducing end side of amylose DP-17) Using activity as an index, maltose and α, α-trehalose were analyzed by the TSK-gel Amide-80 HPLC analysis method shown in Example II-1 using glucose production activity as an index.
In addition, the enzyme activity in a table | surface showed the enzyme activity which releases 1 micromol of monosaccharide and disaccharide in 1 hour, respectively as 1Unit.

The results were as shown in Table 39 below.

The result of analysis of the reaction product from maltotriosyltrehalose by TSK-gel Amide-80 HPLC performed under the conditions of Example II-1 was as shown in FIG. Moreover, the analysis result by AMINEX HPX-42A HPLC which performed the reaction product from soluble starch on the following conditions was as showing in FIG.36 (B).
Column: AMINEX HPX-42A (7.8 X 300mm)
Solvent: Water Flow rate: 0.6ml / min
Temperature: 85 ° C
Detector: Differential refractometer From the above results, this enzyme acts extremely well on trehalose oligosaccharides such as maltotriosyl trehalose in which the glucose residue on the reducing end side is α-1, α-1 linked, α, It was confirmed that α-trehalose and the corresponding maltooligosaccharide with a degree of polymerization decreased by two were produced. Further, it was confirmed that glucose or maltose was mainly liberated from maltose (G2) to maltopentaose (G5), amylose, and soluble starch. However, none of them reacted with α, α-trehalose, isomaltose, isomalttriose, isomalttetraose, isomaltopentose, and panose.

(2) Endo-amylase activity The crude enzyme solution 150 Unit / ml (activity unit is the same as in (1) above) was allowed to act on soluble starch. The disappearance of iodine color development was measured under the same conditions as in the method for measuring amylolytic activity shown in Example II-1, and according to the conditions of the HPLC analysis method for determining substrate specificity shown in (1) above. Monosaccharide and disaccharide production was measured. From these, the starch hydrolysis rate was determined.
The change with time was as shown in FIG. From the figure, it was confirmed that the hydrolysis rate of starch was as low as 4.5% when iodine reaction coloration disappeared by 50%, and thus the crude enzyme exhibited the properties of endo-amylase.

(3) Study Uridinediphosphoglucose glucose action mechanism ^{[glucose-6-3 H],} and maltotetraose in glycogen synthase (from rabbit skeletal muscle, Sigma Co. G-2259) by the action of, the non-reducing end glucose Maltopentaose whose residue was radioactively labeled with ³ H was synthesized and purified preparatively.

Next, 10 mM maltopentaose labeled with radioactivity was used as a substrate, and the recombinant novel transferase obtained in Example I-20 (10 Unit / ml: activity unit according to Example I-1) was added. This was allowed to act at 60 ° C. for 3 hours to synthesize maltotriosyl trehalose in which the glucose residue at the non-reducing end was radiolabeled with ³ H, and this was fractionated and purified. The product was allowed to act on glucoamylase (derived from Rhizopus, manufactured by Seikagaku Corporation) to completely decompose it into glucose and α, α-trehalose. These were fractionated by thin-phase chromatography and the radioactivity was measured with a liquid scintillation counter. As a result, no radioactivity was observed in the α, α-trehalose fraction, and radioactivity was recovered in the glucose fraction, which was not reduced. It was confirmed that the terminal glucose residue was radiolabeled.

Was prepared as described above, non-reducing end glucose residues are radiolabeled with ^{3 H} maltopentaose, and non-reducing terminal glucose residues is radioactivity labeled maltotriosyltrehalose with ^{3 H} substrate As a result, the above crude enzyme solution was allowed to act on 30 Units / ml and 10 Units / ml, respectively. The reaction was sampled before the reaction and after 3 hours at 60 ° C. This reaction product was developed by thin phase chromatography (Kieselgel 60 Merck, solvent; butanol: ethanol: water = 5: 5: 3). When the radioactivity was measured with a liquid scintillation counter after separating the portion corresponding to each of the obtained sugars, radioactivity was found in the hydrolyzate glucose and maltose fractions when maltopentaose was used as the substrate. First, radioactivity was recovered in the maltotetraose and maltotriose fractions. When maltotriosyl trehalose was used as a substrate, no radioactivity was observed in the α, α-trehalose fraction that was a hydrolyzate, and radioactivity was recovered in the maltotriose fraction.

  From the above results, it was confirmed that the mechanism of action of this recombinant novel amylase has the activity of mainly producing monosaccharides and disaccharides from the reducing end side, together with the amylase activity acting on the endo-type.

  The reagents used in the above examples were obtained from α, α-trehalose: Sigma, maltose (G2): Wako Pure Chemicals, maltotriose-maltopentaose (G3-G5): Hayashibara Bio Chemical Co., Amylose DP17: Hayashibara Biochemical Co., Ltd., Isomaltose: Wako Pure Chemical Industries, Isomalt Triose: Wako Pure Chemical Industries, Isomalt Tetraose: Seikagaku Corporation, Isomalt Pentaose: Seikagaku Corporation, Panose : Tokyo Chemical Co., Amylopectin: Nacalai Tesque.

Example II-20 Determination of partial amino acid sequence of a novel amylase derived from Sulfolobus acidocaldarius ATCC33909 The partial amino acid sequence of the purified enzyme obtained in Example II-4 was determined according to the method described in Example II-15. The partial amino acid sequence was as follows.
AP-9 Leu Asp Tyr Leu Lys (SEQ ID NO: 49)
AP-10 Lys Arg Glu Ile Pro Asp Pro Ala Ser Arg Tyr Gln Pro Leu Gly Val His
(SEQ ID NO: 50)
AP-11 Lys Asp Val Phe Val Tyr Asp Gly Lys (SEQ ID NO: 51)
AP-12 His Ile Leu Gln Glu Ile Ala Glu Lys (SEQ ID NO: 52)
AP-16 Lys Leu Trp Ala Pro Tyr Val Asn Ser Val (SEQ ID NO: 53)
AP-17 Met Phe Ser Phe Gly Gly Asn (SEQ ID NO: 54)
AP-18 Asp Tyr Try Tyr Gln Asp Phe Gly Arg Ile Glu Asp Ile Glu
(SEQ ID NO: 55)
AP-21 Lys Ile Asp Ala Gln Trp Val (SEQ ID NO: 56)

Example II-21 Sulfolobus acidocardarius ATCC 33909 New amylase Preparation of DNA probe based on partial amino acid sequence Oligonucleotide DNA primer based on partial amino acid sequence information determined in Example II-20 (Applied Biosystems) Made by model 381). The sequence was as follows.
AP-10
Amino acid sequence N-terminal Pro Ala Ser Arg Tyr Gln Pro C-terminal

DNA primer 5 'AGCTAGTAGATACATCAACC 3'
(SEQ ID NO: 57)
Base sequence A GC C G

AP-11
(Complementary strand)
Amino acid sequence N-terminal Asp Val Phe Val Tyr Asp Gly Lys C-terminal
DNA primer 5'TTTCTCCATCATAAACAAAAAACATC 3 '
(SEQ ID NO: 58)
Base sequence C A G T G T
C

  PCR was carried out using about 100 ng of each chromosomal DNA of Sulfolobus acidocaldarius ATCC33909 prepared according to the method of Example II-16 from 100 pmol of each of these primers and the cells obtained according to the method of Example II-4. The PCR apparatus was a Gene Amp PCR system model 9600 manufactured by PerkinElmer, Inc., and one cycle was performed at 94 ° C. for 30 seconds, 54 ° C. for 30 seconds, and 72 ° C. for 30 seconds, with 30 cycles and a total liquid volume of 100 μl. The amplified fragment of about 830 bp was subcloned into pT7 Blue T-Vector (manufactured by Novagen). When the nucleotide sequence of the inserted fragment of this plasmid was determined, a sequence corresponding to the amino acid sequence obtained in Example II-20 was found.

Example II-22 Cloning of a Novel Amylase Gene Derived from Sulfobus acidocaldarius ATCC33909 The chromosomal DNA of Sulfobus acidocaldarius ATCC33909 was obtained from the cells obtained according to the method of Example II-4 according to the method of Example II-16. The chromosomal DNA was partially digested with the restriction enzyme Sau3AI, and then ligated to EMBL3 BamHI cleavage arm (manufactured by STRATAGENE) by T4 DNA ligase. Packaging was performed using Gigapack II Gold manufactured by STRATAGENE. After the above library was infected with E. coli LE392 at 37 ° C. for 15 minutes, it was seeded on a NZY agar plate medium and cultured at 37 ° C. for about 8 to 12 hours to form plaques. After storage at 4 ° C. for about 2 hours, DNA was adsorbed onto a nylon membrane (Amersham, Hybond N +). After lightly washing with 2 × SSPE, baking was performed at 80 ° C. for 2 hours. The probe used was the PCR fragment of Example II-21 and labeled with ³² P using an Amersham Mega Prime DNA labeling system.

  Hybridization conditions were 6 × SSPE, 0.5% SDS at 65 ° C. overnight. Washing was performed twice with 2 × SSPE and 0.1% SDS at room temperature for 10 minutes.

Screening was started from about 8000 clones to obtain 17 positive clones. An approximately 5.4 kbp BamHI fragment was obtained from this clone and inserted into the above site of pUC118. The obtained pramide was named p09A2. The plasmid DNA further digested with the restriction enzyme SacI is designated as p09A1. FIG. 38 shows a restriction enzyme map of the insert fragment of p09A1, and FIG. 39 shows a method for producing p09A1. The p09A1 was prepared using a double-strand nested deletion kit manufactured by Pharmacia. According to the method of Example II-18, the DNA sequence was determined around the structural gene region of the novel amylase. These DNA sequences and amino acid sequences deduced therefrom were as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
In this amino acid sequence, all the sequences corresponding to the partial amino acid sequence obtained in Example II-20 were observed. This amino acid sequence was 556 in length and was considered to encode a protein with an estimated molecular weight of 64.4 kDa. This molecular weight almost coincided with the molecular weight value obtained by subjecting a purified enzyme of a novel amylase derived from Sulfolobus acidocaldarius ATCC33909 to SDS-PAGE. Moreover, the novel amylase activity was confirmed about the transformant containing plasmid p09A1 according to the method of Example II-19.

Example II-23 Amino acid sequences of the above enzymes of KM1 strain and ATCC33909 strain, amino acid sequence of novel amylase derived from KM1 strain of SEQ ID NO: 6 and amino acid of novel amylase derived from ATCC 33909 strain of SEQ ID NO: 8 The sequence of the amylase derived from the KM1 strain of SEQ ID NO: 5 and the nucleotide sequence of the novel amylase derived from the ATCC 33909 strain of SEQ ID NO: 7 using sequence analysis software, Genetics (software development) Each analysis was performed considering the gap. The results for the amino acid sequence are shown in FIG. 40, and the results for the base sequence are shown in FIG. The lower row of each figure shows the sequence of the KM1 strain, the upper row shows the sequence results of the ATCC33909 strain, the middle (*) indicates the sequence that matches both, and the middle (.) Indicates the amino acids that are similar in nature. Show. The homology was about 59% at the amino acid level and 64% at the base sequence level.

Example II-24 Sulfobus sofaraticus KM1 strain and
Sulfobus acidocaldarius ATCC 33909-derived novel amylase gene hybridization test with chromosomal DNA of other organisms The chromosomal DNA of E. coli strain JM109 was digested with restriction enzyme Hind III according to the method of Example II-16.

  This digest was separated by 1% agarose gel electrophoresis and Southern blotted on Hybond-N manufactured by Amersham Japan. About 1.9 kbp PstI fragment of plasmid pKA1 (corresponding to the sequence from No. 1 to 1845 of SEQ ID NO: 5) was labeled on this membrane with a DIG system kit manufactured by Boehringer Mannheim, and this was used for hybridization. went.

  The conditions are: hybridization: 5 × SSC, 40 ° C., 3 hours, washing: 2 × SSC (including 0.1% SDS), 40 ° C., 5 minutes, twice 0.1 × SSC (including 0.1% SDS), 40 ° C. 5 minutes, 2 times.

  As a result, the PstI fragment was about 13.0 kbp for Sulfolobus solfatricus DSM 5833 strain, about 9.8 kbp for Sulfolobus shibatae DSM 5389, and about about 9.8 kbp fragment for Acidianus brierleyi Dsp 1651 ps. And formed a hybrid. On the other hand, E. is a negative control. No hybrid formation was observed for E. coli strain JM109.

In addition, Sulfobus solfataricus KM1 strain, DSM5354 strain, DSM5833 strain, ATCC35091 strain, ATCC35092 strain, Sulfolobus acidocaldarius ATCC33909 strain, ATCC49426 strain, Sulfobus Shibatae DSc5 strain DSc89DS strain. Chromosomal DNA of E. coli strain JM109 was obtained according to the method of Example II-16 and digested with restriction enzymes XbaI, HindIII, and EcoRV. This digest was separated by 1% agarose gel electrophoresis and Southern blotted to Hybond N + manufactured by Amersham. Using this membrane as a probe, the region of SEQ ID NO: 7 from 1393 to 2121 (digested with restriction enzymes EcoT22I and EcoRV from p09A1 obtained in Example II-22 and recovered from the gel) was used as a probe. According to the method, it was labeled with ³² P, and this was used for hybridization. The conditions were hybridization 6 × SSPE, 0.5% SDS, 60 ° C. overnight. Washing was performed twice with 2 × SSPE and 0.1% SDS at room temperature for 10 minutes. As a result, Sulfobus solfataricus KM1 strain, DSM5354 strain, DSM5833 strain, ATCC35091 strain, ATCC35092 strain, Sulfolobus acidocaldarius ATCC33909 strain, ATCC49426 strain Sulfolobus shibata strain, , 0.9 kbp, 1.0 kbp, 0.9 kbp, 0.9 kbp, 1.4 kbp, and 0.9 kbp were hybridized. On the other hand E. It did not form a hybrid with the chromosomal DNA of E. coli strain JM109. Amino acid data bank (Swiss proto, NBRF-PDB), nucleotide sequence data bank that there is no amino acid sequence contained within the range of SEQ ID NOs: 5, 6, 7, and 8 and a sequence having homology with the nucleotide sequence (EMBL) was confirmed using sequence analysis software and genetics (software development). Therefore, it was found that this novel amylase gene is highly conserved specifically in archaea belonging to the order of Sulfolobales.

Example III-1 Production of α, α-trehalose using recombinant novel amylase and recombinant novel transferase Crude purified novel novel amylase obtained in Example II-19 and concentration obtained in Example I-20 An attempt was made to produce α, α-trehalose using recombinant novel transferase and 10% soluble starch (manufactured by Nacalai Tesque, special grade), supplemented with pullulanase. The reaction was performed as follows.

First, 10% soluble starch was treated with 0.5-50 Unit / ml pullulanase (derived from Klebsiella pneumoniae: Wako Pure Chemical Industries, Ltd.) for 1 hour at 40 ° C., and then the above recombinant novel transferase (10 Unit / ml) and the above Recombinant novel amylase (150 Unit / ml) was added and reacted at pH 5.5 and 60 ° C. for 100 hours. Next, the reaction solution was heated at 100 ° C. for 5 minutes to stop the reaction, and the unreacted substrate was decomposed with glucoamylase. Then, it measured by the HPLC analysis method of the conditions shown in Example II-1.
The analysis result by TSK-gel Amide-80 HPLC was as shown in FIG.

  Here, the enzymatic activity of the recombinant novel amylase is shown as 1 Unit, which is the activity of releasing 1 μmol of α, α-trehalose from maltotriosyl trehalose per hour. The enzyme activity of the recombinant novel transferase was shown as 1 Unit, the activity of converting maltotriose to 1 μmol of glucosyl trehalose per hour. As the definition of the enzyme activity of pullulanase, the amount of enzyme that produces 1 μmol of maltotriose from pullulan per minute at pH 6.0 and 30 ° C. is shown as 1 Unit.

  When the added amount of pullulanase was 50 Unit / ml, the yield of α, α-trehalose was 67%. This value indicates that the recombinant novel amylase gives almost the same yield as when the purified novel amylase derived from Sulfolobus solfatricus KM1 was allowed to act under the above conditions.

INDUSTRIAL APPLICABILITY A novel transferase capable of producing trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose by acting on sugars such as maltooligosaccharide obtained by the enzyme production method based on the novel purification method of the present invention By using this, it is possible to provide a novel method for producing trehalose oligosaccharides such as glucosyl trehalose and malto-oligosyl trehalose in an efficient and high yield using raw materials such as maltooligosaccharides.

  By using the novel amylase of the present invention in combination with the novel transferase of the present invention, α, α-trehalose can be produced efficiently and in high yield from saccharide raw materials such as starch, starch degradation products, and maltooligosaccharides. A novel method can be provided.

Aspects of the Invention

Hereinafter, preferred embodiments of the present invention will be described.
1. Using at least 3 sugars with 3 or more glucose residues as α-1,4 bonds from the reducing end as a substrate and transferring the α-1,4 bonds at the reducing end to α-1, α-1 bonds A novel transferase having the effect of
The molecular weight by SDS polyacrylamide gel electrophoresis is 74000-76000, and
The above enzyme having the following physicochemical properties:
(1) Optimum pH: 4.5-6.0
(2) Optimal temperature: 60-80 ° C
(3) Stable pH: 4.5-10.0
(4) Temperature stability: 90% or more remaining after treatment at 80 ° C. for 6 hours.
2. A novel transferase having an action of transferring a malto-oligosaccharide having all glucose residues α-1,4 bonds as a substrate and transferring α-1,4 bonds at the reducing end to α-1, α-1 bonds. ,
The molecular weight by SDS polyacrylamide gel electrophoresis is 74000-76000, and
The above enzyme having the following physicochemical properties:
(1) Optimum pH: 4.5-6.0
(2) Optimal temperature: 60-80 ° C
(3) Stable pH: 4.5-10.0
(4) Temperature stability: 90% or more remaining after treatment at 80 ° C. for 6 hours.
3. The enzyme according to embodiment 1 or 2, wherein the isoelectric point by isoelectric focusing is 5.3 to 6.3.
4). _4. The enzyme according to any one of aspects 1 to 3, which is 100% inhibited by 5 mM CuSO ₄ .
5. The enzyme according to any one of aspects 1 to 4, which is obtained from Archabacterium of the order of Sulfolobales.
6). The enzyme according to aspect 5, obtained from a bacterium belonging to the genus Sulfolobus.
7). The enzyme according to aspect 5, obtained from a bacterium belonging to the genus Acidianus.
8). The enzyme according to aspect 6, which is obtained from a bacterium belonging to Sulfolobus solifericus.
9. The enzyme according to aspect 8, which is obtained from Sulfolobus sofaraticus KM1 strain (FERM BP-4626).
10. The enzyme according to aspect 8, which is obtained from Sulfolobus sofaraticus DSM 5833 strain.
11. The enzyme according to aspect 6, obtained from a bacterium belonging to Sulfolobus acidocaldarius.
12 The enzyme according to aspect 11, obtained from Sulfolobus acidocaldarius ATCC 33909 strain.
13. The enzyme according to aspect 7, obtained from a bacterium belonging to Acidianus brierleyi.
14 The enzyme according to aspect 13, obtained from Acidianus brierleyi DSM 1651 strain.
15. A bacterium belonging to the genus Sulfolobus having the ability to produce a transferase according to any one of aspects 1 to 4 or a bacterium belonging to the genus Acidianus is cultured in a medium, and the activity of producing trehalose oligosaccharide from the culture using malto-oligosaccharide as a substrate is used as an indicator The method for producing a transferase according to any one of aspects 1 to 4, wherein the transferase is isolated and purified based on the activity measurement method described above.
16. 16. The production method according to aspect 15, wherein a bacterium belonging to Sulfolobus solifericus is cultured.
17. The manufacturing method of aspect 16 which culture | cultivates Sulfolobus solfaricus KM1 strain | stump | stock (FERM BP-4626).
18. The production method according to aspect 16, wherein the Sulfolobus solifericus DSM 5833 strain is cultured.
19. The production method according to aspect 15, wherein a bacterium belonging to Sulfolobus acidocaldarius is cultured.
20. The production method according to aspect 19, wherein the Sulfolobus acidocaldarius ATCC 33909 strain is cultured.
21. The production method according to aspect 15, wherein a bacterium belonging to Acidianus brierleyi is cultured.
22. The production method according to aspect 21, wherein the Acidianus brierleyi DSM 1651 strain is cultured.
23. Using the enzyme according to any one of aspects 1 to 4, at least three sugar residues having α-1,4 bonds at least three glucose residues from the reducing end are allowed to act as a substrate, and at least at the reducing end side. A trisaccharide is composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond between the second and third glucoses on the terminal side. A method for producing a sugar in which the terminal disaccharide is an α-1, α-1 bond, wherein a sugar having an α-1,4 bond is produced.
24. The production method according to embodiment 23, wherein each maltooligosaccharide alone or a mixture thereof is used as a substrate.
25. 25. The production method according to embodiment 24, wherein trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose are produced.
26. A novel amylase having an activity of freeing mainly monosaccharide and / or disaccharide by using at least 3 sugars composed of glucose units from at least the reducing terminal as a substrate and hydrolyzing from the reducing terminal side Because
The molecular weight by SDS polyacrylamide gel electrophoresis is 61,000-64,000, and
The above enzyme having the following physicochemical properties:
(1) Optimum pH: 4.5 to 5.5
(2) Optimal temperature: 60-85 ° C
(3) Stable pH: 4.0 to 10.0
(4) Temperature stability: 100% remaining after treatment at 80 ° C. for 6 hours.
27. At least the three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond. The α-1,4 bond between the second and third glucoses is hydrolyzed by using three or more sugars whose bonds between the first glucose are α-1,4 bonds as substrates, and α, α The novel amylase according to embodiment 26, which has the main activity of releasing trehalose.
28. The novel amylase according to embodiment 26 or 27, which has an activity of hydrolyzing α-1,4 bonds in the molecular chain of the substrate in an endo form.
29. Activity to release α, α-trehalose by hydrolyzing α-1,4 bonds between the second and third glucoses at the reducing end using trehalose oligosaccharides such as glucosyl trehalose and maltooligosyl trehalose as a substrate 29. A novel amylase according to embodiment 26, 27 or 28, having
30. 30. The enzyme according to any one of aspects 26 to 29, wherein the isoelectric point by isoelectric focusing is 4.3 to 5.4.
31. The enzyme according to any one of aspects 26 to 30, which is 100% inhibited by 5 mM CuSO ₄ .
32. 32. The enzyme according to any one of aspects 26 to 31, obtained from an archaea belonging to the order of Sulfolobales.
33. The enzyme according to aspect 32, obtained from a bacterium belonging to the genus Sulfolobus.
34. 34. The enzyme according to aspect 33, obtained from a bacterium belonging to Sulfolobus solifericus.
35. 35. The enzyme according to aspect 34, which is obtained from Sulfolobus solifericus KM1 strain (FERM BP-4626) or a mutant thereof.
36. 35. The enzyme according to aspect 34, which is obtained from the Sulfolobus solfaricus DSM 5833 strain or a mutant thereof.
37. 34. The enzyme according to aspect 33, obtained from a bacterium belonging to Sulfolobus acidocaldarius.
38. The enzyme according to aspect 37, obtained from the Sulfolobus acidocaldarius ATCC 33909 strain or a mutant thereof.
39. A bacterium belonging to the genus Sulfolobus having the ability to produce amylase according to any one of embodiments 26 to 31 is cultured in a medium, and the activity of producing α, α-trehalose from the culture using trehalose oligosaccharide as a substrate is used as an index. 32. The method for producing amylase according to any one of embodiments 26 to 31, wherein the amylase is isolated and purified based on an activity measurement method.
40. 40. The method for producing an amylase according to aspect 39, wherein a bacterium belonging to Sulfolobus solifericus is cultured.
41. 41. The method for producing an amylase according to aspect 40, wherein the Sulfolobus solfaricus KM1 strain (FERM BP-4626) is cultured.
42. 41. The method for producing an amylase according to aspect 40, wherein the Sulfolobus solfariticus DSM 5833 strain is cultured.
43. The method for producing amylase according to aspect 39, wherein a bacterium belonging to Sulfolobus acidocaldarius is cultured.
44. 44. The method for producing amylase according to aspect 43, wherein the Sulfolobus acidocaldarius ATCC 33909 strain is cultured.
45. The novel amylase according to any one of Embodiments 26 to 38, and at least three sugars having at least three glucose residues α-1,4 bonds from the reducing end as a substrate, α-1, A method for producing α, α-trehalose, which is used in combination with the novel transferase according to any one of embodiments 1 to 14 having an action of transferring 4 bonds to α-1 and α-1 bonds.
46. The method for producing α, α-trehalose according to embodiment 45, wherein the amylase and the transferase are allowed to act at 60 to 80 ° C.
47. 47. The method for producing α, α-trehalose according to embodiment 45 or 46, wherein the concentrations of the amylase and the transferase in the reaction solution are 1.5 Units / ml and 0.1 Unit / ml or more, respectively.
48. The α according to embodiment 45 or 46, wherein the concentration of the amylase and the transferase in the reaction solution is 1.5 Units / ml and 1 Unit / ml or more, respectively, and the concentration ratio of amylase to transferase is 0.075 to 100. , Α-trehalose production method.
49. The production of α, α-trehalose according to aspect 48, wherein the concentration of the amylase and the transferase in the reaction solution is 15 Units / ml and 1 Unit / ml or more, respectively, and the concentration ratio of amylase to transferase is 3 to 40. Law.
50. 50. The method for producing α, α-trehalose according to any of embodiments 45 to 49, wherein at least three or more glucose residues having α-1,4 bonds from the reducing end are used as a substrate.
51. The method for producing α, α-trehalose according to any one of embodiments 45 to 49, wherein starch or starch degradation product is used as a substrate.
52. 52. The method for producing α, α-trehalose according to aspect 51, wherein the starch degradation product is a starch degradation product produced by acid degradation or enzymatic degradation.
53. 53. The method for producing α, α-trehalose according to aspect 52, wherein the starch degradation product is obtained using a debranching enzyme.
54. 54. The method for producing α, α-trehalose according to embodiment 53, wherein the debranching enzyme is pullulanase or isoamylase.
55. 50. The method for producing α, α-trehalose according to any one of embodiments 45 to 49, wherein malto-oligosaccharides in which all glucose residues are α-1,4 bonds are used as substrates, either alone or as a mixture thereof.
56. The method for producing α, α-trehalose according to embodiment 45 or 46, further using a debranching enzyme.
57. 57. The method for producing α, α-trehalose according to aspect 56, wherein the debranching enzyme is pullulanase or isoamylase.
58. 58. The method for producing α, α-trehalose according to embodiment 57, wherein pullulanase or isoamylase is used in combination at least once in any stage of the production of α, α-trehalose.
59. The method for producing α, α-trehalose according to aspect 58, wherein pullulanase or isoamylase is used in combination at least once in the initial stage of production of α, α-trehalose.
60. The method for producing α, α-trehalose according to any one of embodiments 56 to 59, wherein starch or a starch degradation product is used as a substrate.
61. The method for producing α, α-trehalose according to aspect 60, wherein the starch degradation product is a starch degradation product produced by acid degradation or enzymatic degradation.
62. The method for producing α, α-trehalose according to embodiment 61, wherein the starch degradation product is obtained using a debranching enzyme.
63. 63. The method for producing α, α-trehalose according to aspect 62, wherein the debranching enzyme is pullulanase or isoamylase.
64. The method for producing α, α-trehalose according to any one of aspects 45 to 63, wherein an enzyme derived from archaea belonging to the order of Sulfolobales is used as the transferase.
65. The method for producing α, α-trehalose according to aspect 64, wherein an enzyme obtained from a bacterium belonging to the genus Sulfolobus is used as the transferase.
66. The method for producing α, α-trehalose according to aspect 64, wherein an enzyme obtained from a bacterium belonging to the genus Acidianus is used as the transferase.
67. 68. The method for producing α, α-trehalose according to embodiment 65, wherein an enzyme obtained from a bacterium belonging to Sulfobus sofataricus is used as the transferase.
68. 68. The method for producing α, α-trehalose according to aspect 67, using an enzyme obtained from Sulfolobus solifericus KM1 strain (FERM BP-4626) or a mutant thereof.
69. 68. The method for producing α, α-trehalose according to aspect 67, using an enzyme obtained from Sulfolobus solfariticus DSM 5833 strain or a mutant thereof.
70. The method for producing α, α-trehalose according to aspect 65, wherein an enzyme obtained from a bacterium belonging to Sulfolobus acidocaldarius is used as the transferase.
71. The method for producing α, α-trehalose according to aspect 70, using an enzyme obtained from Sulfolobus acidocaldarius ATCC 33909 or a mutant thereof.
72. 67. The method for producing α, α-trehalose according to aspect 66, wherein an enzyme obtained from a bacterium belonging to Acidianus brierleyi is used as the transferase.
73. The method for producing α, α-trehalose according to embodiment 72, using an enzyme obtained from Acidianus brierleyi DSM 1651 strain or a mutant thereof.
74. 27. A DNA fragment comprising a DNA sequence encoding the novel transferase according to aspect 1, which is represented by the restriction enzyme map shown in FIG.
75. A DNA fragment comprising a DNA sequence encoding the novel transferase according to Aspect 1, which is represented by the restriction enzyme map shown in FIG.
76. A DNA fragment comprising a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or an equivalent sequence thereof.
77. 77. A DNA fragment according to aspect 76, comprising the nucleotide sequence from the 335th to 2518th of the base sequence shown in SEQ ID NO: 1.
78. 77. A DNA fragment according to aspect 76, comprising the base sequence from No. 1 to No. 2578 of the base sequence shown in SEQ ID NO: 1.
79. A DNA fragment comprising a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 4 or an equivalent sequence thereof.
80. 80. A DNA fragment according to aspect 79, comprising the base sequence from number 816 to number 2855 of the base sequence shown in SEQ ID NO: 3.
81. 80. A DNA fragment according to aspect 79, comprising the base sequence of Nos. 1 to 3467 of the base sequence shown in SEQ ID NO: 3.
82. The DNA fragment according to any one of aspects 74 to 81, which is derived from an archaea belonging to the order of Sulfolobales.
83. 83. A DNA fragment according to aspect 82, which is derived from a bacterium belonging to the genus Sulfolobus.
84. 84. A DNA fragment according to aspect 83, which is derived from a bacterium belonging to Sulfolobus solfatricus.
85. 85. A DNA fragment according to aspect 84, which is derived from Sulfolobus solfatricus KM1 strain.
86. 84. A DNA fragment according to aspect 83, which is derived from a bacterium belonging to Sulfolobus acidocaldarius.
87. 87. A DNA fragment according to aspect 86, which is derived from Sulfolobus acidocaldarius ATCC 33909 strain.
88. A hybrid is formed on the base sequence from 1880 to 2257 of the base sequence shown in SEQ ID NO: 1 or its complement at 60 ° C. under an ionic strength of 6 × SSPE, and at least 3 glucose residues from the reducing end. A DNA encoding a novel transferase having the action of transferring α-1,4 bond at the reducing end to α-1, α-1 bond using a saccharide of 3 or more sugars whose group is α-1,4 bond as a substrate A fragment, and a DNA fragment encoding an amino acid sequence encoded by the DNA fragment.
89. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or an equivalent sequence thereof.
90. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 or an equivalent sequence thereof.
91. Transfer at least the α-1,4 bond of the reducing end to the α-1, α-1 bond, using as a substrate a sugar of 3 or more sugars with at least 3 glucose residues α-1,4 bond from the reducing end The polypeptide according to embodiment 89 or 90, which has an action of
92. The polypeptide according to any one of aspects 89 to 91, wherein the optimum temperature for the action is 60 to 80 ° C.
93. A recombinant DNA molecule comprising a DNA fragment according to any of aspects 74-88.
94. The recombinant DNA molecule according to aspect 93, wherein the DNA fragment according to any one of aspects 74 to 88 is incorporated into a plasmid vector.
95. 95. A host cell transformed with the recombinant DNA molecule of embodiment 93 or 94.
96. 96. The host cell according to aspect 95, wherein the host cell is a microorganism belonging to the genus Escherichia or Bacillus.
97. 99. The host cell according to aspect 96, wherein the host cell is Escherichia coli JM109 strain.
98. At least 3 or more sugar residues with α-1,4 bonds from the reducing end to α-1,4 bonds are used as substrates, and the α-1,4 bonds at the reducing ends are transferred to α-1, α-1 bonds. A method for producing a recombinant novel transferase having the action of:
98. The method as described above, comprising culturing a host cell according to any one of aspects 95 to 97, producing the recombinant novel transferase in the culture, and collecting it.
99. A method for producing a recombinant novel transferase encoded by the DNA fragment according to any one of aspects 74 to 88, or a recombinant novel transferase comprising the polypeptide according to any one of aspects 89 to 92,
98. The method as described above, comprising culturing a host cell according to any one of aspects 95 to 97, producing the recombinant novel transferase in the culture, and collecting it.
100. At least the three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond, and the second and third sugars on the terminal side are A method for producing trehalose oligosaccharide in which the first glucose bond is α-1,4 bond,
The method as described above, comprising contacting the recombinant novel transferase obtained in the embodiment 98 or 99 with three or more sugars in which at least three or more glucose residues are α-1,4 bonds from the reducing end. .
101. 35. A DNA fragment comprising a DNA sequence encoding the novel amylase according to embodiment 26, which is represented by the restriction enzyme map shown in FIG.
102. A DNA fragment comprising a DNA sequence encoding the novel amylase according to Aspect 26, which is represented by the restriction enzyme map shown in FIG.
103. 102. A DNA fragment according to aspect 101 comprising a DNA sequence encoding the novel amylase according to aspect 27.
104. 102. A DNA fragment according to aspect 102, comprising a DNA sequence encoding the novel amylase according to aspect 27.
105. 105. The DNA fragment according to any one of aspects 101 to 104, comprising a DNA sequence encoding a novel amylase having an activity of hydrolyzing α-1,4 bonds in sugar chains in an endo form.
106. The novel amylase has trehalose oligosaccharide as a substrate, hydrolyzes α-1,4 bonds between the second and third glucoses on the reducing end side, and has the activity of releasing α, α-trehalose The DNA fragment according to any one of aspects 101 to 105, wherein
107. The DNA fragment according to any one of aspects 101 to 106, wherein the optimum temperature of the novel amylase is 60 to 85 ° C.
108. A DNA fragment comprising a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 6 or an equivalent sequence thereof.
109. 109. A DNA fragment according to aspect 108, comprising the base sequence from the 642th to the 2315th base sequence of the base sequence shown in SEQ ID NO: 5.
110. 109. A DNA fragment according to aspect 108, comprising the nucleotide sequence from the 639th to the 2315th of the nucleotide sequence shown in SEQ ID NO: 5.
111. 109. A DNA fragment according to Aspect 108, comprising the nucleotide sequence from No. 1 to No. 2691 of the nucleotide sequence shown in SEQ ID NO: 5.
112. A DNA fragment comprising a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 8, or an equivalent sequence thereof.
113. 113. A DNA fragment according to aspect 112, comprising the base sequence from 1176th to 2843th of the base sequence shown in SEQ ID NO: 7.
114. 113. A DNA fragment according to the aspect 112, comprising a base sequence from the first to the 3600th of the base sequence shown in SEQ ID NO: 7.
115. 115. The DNA fragment according to any one of aspects 101 to 114, which is derived from archaea belonging to the order of Sulfolobales.
116. 116. The DNA fragment according to aspect 115, which is derived from a bacterium belonging to the genus Sulfolobus.
117. 117. The DNA fragment according to aspect 116, which is derived from a bacterium belonging to Sulfolobus solifericus.
118. 118. The DNA fragment according to aspect 117, which is derived from Sulfolobus solifericus KM1 strain.
119. 117. The DNA fragment according to aspect 116, which is derived from a bacterium belonging to Sulfolobus acidocaldarius.
120. 120. The DNA fragment according to aspect 119, which is derived from Sulfolobus acidocaldarius ATCC 33909 strain.
121. A hybrid is formed on the nucleotide sequence from 1393 to 2121 of the nucleotide sequence shown in SEQ ID NO: 7 or its complement at 60 ° C. under an ionic strength of 6 × SSPE, and at least three or more sugars are present from the reducing end. A DNA fragment encoding a novel amylase having an activity of mainly releasing monosaccharide and / or disaccharide by hydrolysis from the reducing terminal side using a saccharide of 3 or more sugars composed of glucose units as a substrate, and the DNA A DNA fragment encoding the amino acid sequence encoded by the fragment.
122. A hybrid is formed on the base sequence from No. 1393 to No. 2121 of the base sequence shown in SEQ ID NO: 7 or its complement at 60 ° C. under ionic strength of 6 × SSPE, and at least three sugars on the reducing end side are glucose units And the bond between the first and second glucoses on the terminal side is α-1, α-1 bond, and the bond between the second and third glucoses on the terminal side is Major activity of hydrolyzing α-1,4 bond between 2nd and 3rd glucose and releasing α, α-trehalose using α-1,4 bond as 3 or more sugar as substrate A DNA fragment encoding a novel amylase having a DNA, and a DNA fragment encoding an amino acid sequence encoded by the DNA fragment.
123. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 or an equivalent sequence thereof.
124. A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8 or an equivalent sequence thereof.
125. 124. A polypeptide according to embodiment 123, further comprising Met at its N-terminus.
126. At least the three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond, and the second and third sugars on the terminal side are The linkage between the first glucose is α-1,4 bond, the sugar of 3 or more sugar is used as a substrate, the α-1,4 bond between the second and third glucose is hydrolyzed, α, The polypeptide according to any one of aspects 123 to 125, which has an action of releasing α-trehalose.
127. (1) Activity to hydrolyze α-1,4 glucoside bond in sugar chain in endo form,
(2) At least three or more sugars from the reducing end are composed of glucose units, and the sugar is a monosaccharide by hydrolysis from the reducing end side using a sugar of three or more sugars whose bonds are α-1,4 bonds as a substrate. And / or activity to release disaccharides, and (3) at least the reducing end trisaccharide is composed of glucose units, and the bond between the first and second glucoses on the terminal side is α-1, α -1 and 3 or more sugars in which the bond between the second and third glucoses on the terminal side is an α-1,4 bond as a substrate, the second and third glucoses 125. The polypeptide according to any of aspects 123-125, which has a major activity of hydrolyzing the α-1,4 bond between them to release α, α-trehalose.
128. 128. The polypeptide according to any one of aspects 123 to 127, wherein the optimum temperature of action is 60 to 85 ° C.
129. A recombinant DNA molecule comprising the DNA fragment according to any one of aspects 101-122.
130. 130. A recombinant DNA molecule according to aspect 129, wherein the DNA fragment according to any of aspects 101 to 122 is incorporated into a plasmid vector.
131. 130. A host cell transformed with the recombinant DNA molecule of embodiment 129 or 130.
132. 132. The host cell according to aspect 131, wherein the host cell is a microorganism belonging to the genus Escherichia or Bacillus.
133. 135. A host cell according to aspect 132, wherein the host cell is Escherichia coli JM109 strain.
134. A method for producing a recombinant novel amylase comprising:
The recombinant novel amylase is composed of at least a reducing end trisaccharide composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond, and Α-1,4 bond between the 2nd and 3rd glucose with 3 or more sugars with α-1,4 bond as the substrate between the 2nd and 3rd glucose on the terminal side Has the main activity of hydrolyzing and releasing α, α-trehalose,
The method as described above, comprising culturing the host cell according to any one of aspects 131 to 133, producing the recombinant novel amylase in the culture, and collecting it.
135. A method for producing a recombinant novel amylase encoded by the DNA fragment according to any one of aspects 101 to 122, or a recombinant novel amylase comprising the polypeptide according to any one of aspects 123 to 128,
The method as described above, comprising culturing the host cell according to any one of aspects 131 to 133, producing the recombinant novel amylase in the culture, and collecting it.
136. A method for producing α, α-trehalose, comprising the novel transferase according to any one of embodiments 1-14 or the recombinant novel transferase obtained in embodiment 98 or 99, and the novel recombinant transferase obtained in embodiment 134 or 135. Amylase,
The above-mentioned method comprising the step of contacting with at least 3 sugars having 3 or more glucose residues which are α-1,4 bonds from at least the reducing end.
137. A method for producing α, α-trehalose, comprising:
A recombinant novel transferase obtained in aspect 98 or 99, and a novel amylase according to any of aspects 26 to 38 or a recombinant novel amylase obtained in aspect 134 or 135,
The above-mentioned method comprising the step of contacting with at least 3 sugars having 3 or more glucose residues which are α-1,4 bonds from at least the reducing end.
138. 138. The method of embodiment 136 or 137, wherein the three or more sugars having at least three or more glucose residues from the reducing end are α-1,4 bonds are starch or starch degradation products.
139. 138. The method according to aspect 138, wherein the starch degradation product is produced by acid degradation or enzymatic degradation of starch.
140. 139. The method of aspect 138, wherein the starch degradation product is a degradation product of starch by a debranching enzyme.
141. 141. The method of embodiment 140, wherein the debranching enzyme is pullulanase or isoamylase.
142. 3 or more sugars in which at least 3 glucose residues from the reducing end are α-1,4 bonds, or a maltooligosaccharide in which all glucose residues are α-1,4 bonds are single or a mixture, 138. A method according to embodiment 136 or 137.
143. 143. Method according to any of embodiments 136-142, carried out at a temperature of 50-85 [deg.] C.

[Sequence Listing]
SEQ ID NO: 1
Sequence length: 2578
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Genomic DNA
Origin: Sulfolobus solfataricus
Stock name: KM1
Array
GCATGCCATT AAAAGATGTA ACATTTTACA CTCCAGACGG TAAGGAGGTT GATGAGAAAG 60
CATGGAATTC CCCAACGCAA ACTGTTATTT TCGTGTTAGA GGGGAGCGTA ATGGATGAGA 120
TTAACATCTA TGGAGAGAGA ATTGCGGATG ATTCATTCTT GATAATTCTT AACGCAAATC 180
CCAATAACGT AAAAGTGAAG TTCCCAAAGG GTAAATGGGA ACTAGTTGTT GGTTCTTATT 240
TGAGAGAGAT AAAACCAGAA GAAAGAATTG TAGAAGGTGA GAAGGAATTG GAAATTGAGG 300
GAAGAACAGC ATTAGTTTAT AGGAGGACAG AACT ATG ATA ATA GGC ACA TAT AGG 355
Met Ile Ile Gly Thr Tyr Arg
1 5
CTG CAA CTC AAT AAG AAA TTC ACT TTT TAC GAT ATA ATA GAA AAT TTG 403
Leu Gln Leu Asn Lys Lys Phe Thr Phe Tyr Asp Ile Ile Glu Asn Leu
10 15 20
GAT TAT TTT AAA GAA TTA GGA GTA TCA CAC CTA TAT CTA TCT CCA ATA 451
Asp Tyr Phe Lys Glu Leu Gly Val Ser His Leu Tyr Leu Ser Pro Ile
25 30 35
CTT AAG GCT AGA CCA GGG AGC ACT CAC GGC TAC GAT GTA GTA GAT CAT 499
Leu Lys Ala Arg Pro Gly Ser Thr His Gly Tyr Asp Val Val Asp His
40 45 50 55
AGT GAA ATT AAT GAG GAA TTA GGA GGA GAA GAG GGG TGC TTT AAA CTA 547
Ser Glu Ile Asn Glu Glu Leu Gly Gly Glu Glu Gly Cys Phe Lys Leu
60 65 70
GTT AAG GAA GCT AAG AGT AGA GGT TTA GAA ATC ATA CAA GAT ATA GTG 595
Val Lys Glu Ala Lys Ser Arg Gly Leu Glu Ile Ile Gln Asp Ile Val
75 80 85
CCA AAT CAC ATG GCG GTA CAT CAT ACT AAT TGG AGA CTT ATG GAT CTG 643
Pro Asn His Met Ala Val His His Thr Asn Trp Arg Leu Met Asp Leu
90 95 100
TTA AAG AGT TGG AAG AAT AGT AAA TAC TAT AAC TAT TTT GAT CAC TAC 691
Leu Lys Ser Trp Lys Asn Ser Lys Tyr Tyr Asn Tyr Phe Asp His Tyr
105 110 115
GAT GAT GAC AAG ATA ATC CTC CCA ATA CTT GAG GAC GAG TTG GAT ACC 739
Asp Asp Asp Lys Ile Ile Leu Pro Ile Leu Glu Asp Glu Leu Asp Thr
120 125 130 135
GTT ATA GAT AAG GGA TTG ATA AAA CTA CAG AAG GAT AAT ATA GAG TAC 787
Val Ile Asp Lys Gly Leu Ile Lys Leu Gln Lys Asp Asn Ile Glu Tyr
140 145 150
AGA GGG CTT ATA TTA CCT ATA AAT GAT GAA GGA GTT GAA TTC TTG AAA 835
Arg Gly Leu Ile Leu Pro Ile Asn Asp Glu Gly Val Glu Phe Leu Lys
155 160 165
AGG ATT AAT TGC TTT GAT AAT TCA TGT TTA AAG AAA GAG GAT ATA AAG 883
Arg Ile Asn Cys Phe Asp Asn Ser Cys Leu Lys Lys Glu Asp Ile Lys
170 175 180
AAA TTA CTA TTA ATA CAA TAT TAT CAG CTA ACT TAC TGG AAG AAA GGT 931
Lys Leu Leu Leu Ile Gln Tyr Tyr Gln Leu Thr Tyr Trp Lys Lys Gly
185 190 195
TAT CCA AAC TAT AGG AGA TTT TTC GCA GTA AAT GAT TTG ATA GCT GTT 979
Tyr Pro Asn Tyr Arg Arg Phe Phe Ala Val Asn Asp Leu Ile Ala Val
200 205 210 215
AGG GTA GAA TTG GAT GAA GTA TTT AGA GAG TCC CAT GAG ATA ATT GCT 1027
Arg Val Glu Leu Asp Glu Val Phe Arg Glu Ser His Glu Ile Ile Ala
220 225 230
AAG CTA CCA GTT GAC GGT TTA AGA ATT GAC CAC ATA GAT GGA CTA TAT 1075
Lys Leu Pro Val Asp Gly Leu Arg Ile Asp His Ile Asp Gly Leu Tyr
235 240 245
AAC CCT AAG GAG TAT TTA GAT AAG CTA AGA CAG TTA GTA GGA AAT GAT 1123
Asn Pro Lys Glu Tyr Leu Asp Lys Leu Arg Gln Leu Val Gly Asn Asp
250 255 260
AAG ATA ATA TAC GTA GAG AAG ATA TTG TCA ATC AAC GAG AAA TTA AGA 1171
Lys Ile Ile Tyr Val Glu Lys Ile Leu Ser Ile Asn Glu Lys Leu Arg
265 270 275
GAT GAT TGG AAA GTA GAT GGG ACT ACT GGA TAT GAT TTC TTG AAC TAC 1219
Asp Asp Trp Lys Val Asp Gly Thr Thr Gly Tyr Asp Phe Leu Asn Tyr
280 285 290 295
GTT AAT ATG CTA TTA GTA GAT GGA AGT GGT GAG GAG GAG TTA ACT AAG 1267
Val Asn Met Leu Leu Val Asp Gly Ser Gly Glu Glu Glu Leu Thr Lys
300 305 310
TTT TAT GAG AAT TTC ATT GGA AGG AAA ATC AAT ATA GAC GAG TTA ATA 1315
Phe Tyr Glu Asn Phe Ile Gly Arg Lys Ile Asn Ile Asp Glu Leu Ile
315 320 325
ATA CAA AGT AAA AAA TTA GTT GCA AAT CAG TTA TTT AAA GGT GAC ATT 1363
Ile Gln Ser Lys Lys Leu Val Ala Asn Gln Leu Phe Lys Gly Asp Ile
330 335 340
GAA AGA TTA AGC AAG TTA CTG AAC GTT AAT TAC GAT TAT TTA GTA GAT 1411
Glu Arg Leu Ser Lys Leu Leu Asn Val Asn Tyr Asp Tyr Leu Val Asp
345 350 355
TTT CTA GCA TGT ATG AAA AAA TAC AGG ACT TAT TTA CCA TAT GAG GAT 1459
Phe Leu Ala Cys Met Lys Lys Tyr Arg Thr Tyr Leu Pro Tyr Glu Asp
360 365 370 375
ATT AAC GGA ATA AGG GAA TGC GAT AAG GAG GGA AAG TTA AAA GAT GAA 1507
Ile Asn Gly Ile Arg Glu Cys Asp Lys Glu Gly Lys Leu Lys Asp Glu
380 385 390
AAG GGA ATC ATG AGA CTC CAA CAA TAC ATG CCA GCA ATC TTC GCT AAG 1555
Lys Gly Ile Met Arg Leu Gln Gln Tyr Met Pro Ala Ile Phe Ala Lys
395 400 405
GGC TAT GAG GAT ACT ACC CTC TTC ATC TAC AAT AGA TTA ATT TCC CTT 1603
Gly Tyr Glu Asp Thr Thr Leu Phe Ile Tyr Asn Arg Leu Ile Ser Leu
410 415 420
AAC GAG GTT GGG AGC GAC CTA AGA AGA TTC AGT TTA AGC ATC AAA GAC 1651
Asn Glu Val Gly Ser Asp Leu Arg Arg Phe Ser Leu Ser Ile Lys Asp
425 430 435
TTT CAT AAC TTT AAC CTA AGC AGA GTA AAT ACC ATA TCA ATG AAC ACT 1699
Phe His Asn Phe Asn Leu Ser Arg Val Asn Thr Ile Ser Met Asn Thr
440 445 450 455
CTT TCC ACT CAT GAT ACT AAA TTC AGT GAA GAC GTT AGA GCT AGA ATA 1747
Leu Ser Thr His Asp Thr Lys Phe Ser Glu Asp Val Arg Ala Arg Ile
460 465 470
TCA GTA CTA TCT GAG ATA CCA AAG GAG TGG GAG GAG AGG GTA ATA TAC 1795
Ser Val Leu Ser Glu Ile Pro Lys Glu Trp Glu Glu Arg Val Ile Tyr
475 480 485
TGG CAT GAT TTG TTA AGG CCA AAT ATT GAT AAA AAC GAT GAG TAT AGA 1843
Trp His Asp Leu Leu Arg Pro Asn Ile Asp Lys Asn Asp Glu Tyr Arg
490 495 500
TTT TAT CAA ACA CTT GTG GGA AGT TAC GAG GGA TTT GAT AAT AAG GAG 1891
Phe Tyr Gln Thr Leu Val Gly Ser Tyr Glu Gly Phe Asp Asn Lys Glu
505 510 515
AGA ATT AAG AAC CAC ATG ATT AAG GTC ATA AGA GAA GCT AAG GTA CAT 1939
Arg Ile Lys Asn His Met Ile Lys Val Ile Arg Glu Ala Lys Val His
520 525 530 535
ACA ACG TGG GAA AAT CCT AAT ATA GAG TAT GAA AAG AAG GTT CTG GGT 1987
Thr Thr Trp Glu Asn Pro Asn Ile Glu Tyr Glu Lys Lys Val Leu Gly
540 545 550
TTC ATA GAT GAA GTG TTC GAG AAC AGT AAT TTT AGA AAT GAT TTT GAA 2035
Phe Ile Asp Glu Val Phe Glu Asn Ser Asn Phe Arg Asn Asp Phe Glu
555 560 565
AAT TTT GAA AAG AAA ATA GTT TAT TTC GGT TAT ATG AAA TCA TTA ATC 2083
Asn Phe Glu Lys Lys Ile Val Tyr Phe Gly Tyr Met Lys Ser Leu Ile
570 575 580
GCA ACG ACA CTT AGG TTC CTT TCG CCC GGT GTA CCA GAT ATT TAT CAA 2131
Ala Thr Thr Leu Arg Phe Leu Ser Pro Gly Val Pro Asp Ile Tyr Gln
585 590 595
GGA ACT GAA GTT TGG AGA TTC TTA CTT ACA GAC CCA GAT AAC AGA ATG 2179
Gly Thr Glu Val Trp Arg Phe Leu Leu Thr Asp Pro Asp Asn Arg Met
600 605 610 615
CCG GTG GAT TTC AAG AAA CTA AAG GAA TTA TTA AAT AAT TTG ACT GAA 2227
Pro Val Asp Phe Lys Lys Leu Lys Glu Leu Leu Asn Asn Leu Thr Glu
620 625 630
AAG AAC TTA GAA CTC TCA GAT CCA AGA GTC AAA ATG TTA TAT GTT AAG 2275
Lys Asn Leu Glu Leu Ser Asp Pro Arg Val Lys Met Leu Tyr Val Lys
635 640 645
AAA TTG CTA CAG CTT AGA AGA GAG TAC TCA CTA AAC GAT TAT AAA CCA 2323
Lys Leu Leu Gln Leu Arg Arg Glu Tyr Ser Leu Asn Asp Tyr Lys Pro
650 655 660
TTG CCC TTT GGC TTC CAA AGG GGA AAA GTA GCT GTC CTT TTC TCA CCA 2371
Leu Pro Phe Gly Phe Gln Arg Gly Lys Val Ala Val Leu Phe Ser Pro
665 670 675
ATA GTG ACT AGG GAG GTT AAA GAG AAA ATT AGT ATA AGG CAA AAA AGC 2419
Ile Val Thr Arg Glu Val Lys Glu Lys Ile Ser Ile Arg Gln Lys Ser
680 685 690 695
GTT GAT TGG ATC AGA AAT GAG GAA ATT AGT AGT GGA GAA TAC AAT TTA 2467
Val Asp Trp Ile Arg Asn Glu Glu Ile Ser Ser Gly Glu Tyr Asn Leu
700 705 710
AGT GAG TTG ATT GGG AAG CAT AAA GTC GTT ATA TTA ACT GAA AAA AGG 2515
Ser Glu Leu Ile Gly Lys His Lys Val Val Ile Leu Thr Glu Lys Arg
715 720 725
GAG TGAACTACCT ACATAGATTT ATTCTTGAAC TACTCTGGTC AGAAATGTAT 2568
Glu
TACGCAGATC 2578

SEQ ID NO: 2
Sequence length: 728
Sequence type: Number of amino acid chains: Single chain Topology: Linear sequence type: Protein origin Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Met Ile Ile Gly Thr Tyr Arg Leu Gln Leu Asn Lys Lys Phe Thr Phe
1 5 10 15
Tyr Asp Ile Ile Glu Asn Leu Asp Tyr Phe Lys Glu Leu Gly Val Ser
20 25 30
His Leu Tyr Leu Ser Pro Ile Leu Lys Ala Arg Pro Gly Ser Thr His
35 40 45
Gly Tyr Asp Val Val Asp His Ser Glu Ile Asn Glu Glu Leu Gly Gly
50 55 60
Glu Glu Gly Cys Phe Lys Leu Val Lys Glu Ala Lys Ser Arg Gly Leu
65 70 75 80
Glu Ile Ile Gln Asp Ile Val Pro Asn His Met Ala Val His His Thr
85 90 95
Asn Trp Arg Leu Met Asp Leu Leu Lys Ser Trp Lys Asn Ser Lys Tyr
100 105 110
Tyr Asn Tyr Phe Asp His Tyr Asp Asp Asp Lys Ile Ile Leu Pro Ile
115 120 125
Leu Glu Asp Glu Leu Asp Thr Val Ile Asp Lys Gly Leu Ile Lys Leu
130 135 140
Gln Lys Asp Asn Ile Glu Tyr Arg Gly Leu Ile Leu Pro Ile Asn Asp
145 150 155 160
Glu Gly Val Glu Phe Leu Lys Arg Ile Asn Cys Phe Asp Asn Ser Cys
165 170 175
Leu Lys Lys Glu Asp Ile Lys Lys Leu Leu Leu Ile Gln Tyr Tyr Gln
180 185 190
Leu Thr Tyr Trp Lys Lys Gly Tyr Pro Asn Tyr Arg Arg Phe Phe Ala
195 200 205
Val Asn Asp Leu Ile Ala Val Arg Val Glu Leu Asp Glu Val Phe Arg
210 215 220
Glu Ser His Glu Ile Ile Ala Lys Leu Pro Val Asp Gly Leu Arg Ile
225 230 235 240
Asp His Ile Asp Gly Leu Tyr Asn Pro Lys Glu Tyr Leu Asp Lys Leu
245 250 255
Arg Gln Leu Val Gly Asn Asp Lys Ile Ile Tyr Val Glu Lys Ile Leu
260 265 270
Ser Ile Asn Glu Lys Leu Arg Asp Asp Trp Lys Val Asp Gly Thr Thr
275 280 285
Gly Tyr Asp Phe Leu Asn Tyr Val Asn Met Leu Leu Val Asp Gly Ser
290 295 300
Gly Glu Glu Glu Leu Thr Lys Phe Tyr Glu Asn Phe Ile Gly Arg Lys
305 310 315 320
Ile Asn Ile Asp Glu Leu Ile Ile Gln Ser Lys Lys Leu Val Ala Asn
325 330 335
Gln Leu Phe Lys Gly Asp Ile Glu Arg Leu Ser Lys Leu Leu Asn Val
340 345 350
Asn Tyr Asp Tyr Leu Val Asp Phe Leu Ala Cys Met Lys Lys Tyr Arg
355 360 365
Thr Tyr Leu Pro Tyr Glu Asp Ile Asn Gly Ile Arg Glu Cys Asp Lys
370 375 380
Glu Gly Lys Leu Lys Asp Glu Lys Gly Ile Met Arg Leu Gln Gln Tyr
385 390 395 400
Met Pro Ala Ile Phe Ala Lys Gly Tyr Glu Asp Thr Thr Leu Phe Ile
405 410 415
Tyr Asn Arg Leu Ile Ser Leu Asn Glu Val Gly Ser Asp Leu Arg Arg
420 425 430
Phe Ser Leu Ser Ile Lys Asp Phe His Asn Phe Asn Leu Ser Arg Val
435 440 445
Asn Thr Ile Ser Met Asn Thr Leu Ser Thr His Asp Thr Lys Phe Ser
450 455 460
Glu Asp Val Arg Ala Arg Ile Ser Val Leu Ser Glu Ile Pro Lys Glu
465 470 475 480
Trp Glu Glu Arg Val Ile Tyr Trp His Asp Leu Leu Arg Pro Asn Ile
485 490 495
Asp Lys Asn Asp Glu Tyr Arg Phe Tyr Gln Thr Leu Val Gly Ser Tyr
500 505 510
Glu Gly Phe Asp Asn Lys Glu Arg Ile Lys Asn His Met Ile Lys Val
515 520 525
Ile Arg Glu Ala Lys Val His Thr Thr Trp Glu Asn Pro Asn Ile Glu
530 535 540
Tyr Glu Lys Lys Val Leu Gly Phe Ile Asp Glu Val Phe Glu Asn Ser
545 550 555 560
Asn Phe Arg Asn Asp Phe Glu Asn Phe Glu Lys Lys Ile Val Tyr Phe
565 570 575
Gly Tyr Met Lys Ser Leu Ile Ala Thr Thr Leu Arg Phe Leu Ser Pro
580 585 590
Gly Val Pro Asp Ile Tyr Gln Gly Thr Glu Val Trp Arg Phe Leu Leu
595 600 605
Thr Asp Pro Asp Asn Arg Met Pro Val Asp Phe Lys Lys Leu Lys Glu
610 615 620
Leu Leu Asn Asn Leu Thr Glu Lys Asn Leu Glu Leu Ser Asp Pro Arg
625 630 635 640
Val Lys Met Leu Tyr Val Lys Lys Leu Leu Gln Leu Arg Arg Glu Tyr
645 650 655
Ser Leu Asn Asp Tyr Lys Pro Leu Pro Phe Gly Phe Gln Arg Gly Lys
660 665 670
Val Ala Val Leu Phe Ser Pro Ile Val Thr Arg Glu Val Lys Glu Lys
675 680 685
Ile Ser Ile Arg Gln Lys Ser Val Asp Trp Ile Arg Asn Glu Glu Ile
690 695 700
Ser Ser Gly Glu Tyr Asn Leu Ser Glu Leu Ile Gly Lys His Lys Val
705 710 715 720
Val Ile Leu Thr Glu Lys Arg Glu
725

SEQ ID NO: 3
Sequence length: 3467
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Genomic DNA
Origin: Sulfolobus acidocaldarius
Strain name: ATCC 33909 strain sequence
GCTAATAAAC TGAACAATGA GGACGGAATG AATGAAAATT ATAGCTGGAA TTGTGGAGTA 60
GAAGGAGAAA CTAACGATTC TAATATTCTT TATTGTAGAG AAAAACAAAG AAGAAATTTT 120
GTAATAACAT TATTTGTTAG CCAAGGTATA CCAATGATCT TAGGGGGAGA CGAAATAGGA 180
AGAACACAAA AAGGCAACAA TAATGCTTTT TGTCAGGATA ATGAGACAAG TTGGTATGAT 240
TGGAACCTTG ATGAAAATCG TGTAAGGTTT CATGATTTTG TGAGGAGACT TACCAATTTT 300
TATAAAGCTC ATCCGATATT TAGGAGGGCT AGATATTTTC AGGGTAAGAA GTTACACGGT 360
TCCCCATTAA AGGATGTGAC GTGGCTAAAA CCTGACGGCA ATGAAGTTGA TGATTCAGTG 420
TGGAAATCTC CAACAAATCA TATTATTTAT ATATTAGAGG GAAGTGCTAT CGATGAAATA 480
AATTATAATG GAGAAAGGAT AGCTGACGAC ACTTTTCTAA TTATTTTGAA TGGAGCAAGT 540
ACTAATCTTA AGATAAAAGT ACCTCATGGA AAATGGGAGT TAGTGTTACA TCCTTATCCA 600
CATGAGCCAT CTAACGATAA AAAGATAATA GAAAACAACA AAGAAGTAGA AATAGATGGA 660
AAGACTGCAC TAATTTACAG GAGGATAGAG TTCCAGTGAT ATCAGCAACC TACAGATTAC 720
AGTTAAATAA GAATTTTAAT TTTGGTGACG TAATCGATAA CCTATGGTAT TTTAAGGATT 780
TAGGAGTTTC CCATCTCTAC CTCTCTCCTG TCTTA ATG GCT TCG CCA GGA AGT AAC 836
Met Ala Ser Pro Gly Ser Asn
1 5
CAT GGG TAC GAT GTA ATA GAT CAT TCA AGG ATA AAC GAT GAA CTT GGA 884
His Gly Tyr Asp Val Ile Asp His Ser Arg Ile Asn Asp Glu Leu Gly
10 15 20
GGA GAG AAA GAA TAC AGG AGA TTA ATA GAG ACA GCT CAT ACT ATT GGA 932
Gly Glu Lys Glu Tyr Arg Arg Leu Ile Glu Thr Ala His Thr Ile Gly
25 30 35
TTA GGT ATT ATA CAG GAC ATA GTA CCA AAT CAC ATG GCT GTA AAT TCT 980
Leu Gly Ile Ile Gln Asp Ile Val Pro Asn His Met Ala Val Asn Ser
40 45 50 55
CTA AAT TGG CGA CTA ATG GAT GTA TTA AAA ATG GGT AAA AAG AGT AAA 1028
Leu Asn Trp Arg Leu Met Asp Val Leu Lys Met Gly Lys Lys Ser Lys
60 65 70
TAT TAT ACG TAC TTT GAC TTT TTC CCA GAA GAT GAT AAG ATA CGA TTA 1076
Tyr Tyr Thr Tyr Phe Asp Phe Phe Pro Glu Asp Asp Lys Ile Arg Leu
75 80 85
CCC ATA TTA GGA GAA GAT TTA GAT ACA GTG ATA AGT AAA GGT TTA TTA 1124
Pro Ile Leu Gly Glu Asp Leu Asp Thr Val Ile Ser Lys Gly Leu Leu
90 95 100
AAG ATA GTA AAA GAT GGA GAT GAA TAT TTC CTA GAA TAT TTC AAA TGG 1172
Lys Ile Val Lys Asp Gly Asp Glu Tyr Phe Leu Glu Tyr Phe Lys Trp
105 110 115
AAA CTT CCT CTA ACA GAG GTT GGA AAT GAT ATA TAC GAC ACT TTA CAA 1220
Lys Leu Pro Leu Thr Glu Val Gly Asn Asp Ile Tyr Asp Thr Leu Gln
120 125 130 135
AAA CAG AAT TAT ACC CTA ATG TCT TGG AAA AAT CCT CCT AGC TAT AGA 1268
Lys Gln Asn Tyr Thr Leu Met Ser Trp Lys Asn Pro Pro Ser Tyr Arg
140 145 150
CGA TTC TTC GAT GTT AAT ACT TTA ATA GGA GTA AAT GTC GAA AAA GAT 1316
Arg Phe Phe Asp Val Asn Thr Leu Ile Gly Val Asn Val Glu Lys Asp
155 160 165
CAC GTA TTT CAA GAG TCC CAT TCA AAG ATC TTA GAT TTA GAT GTT GAT 1364
His Val Phe Gln Glu Ser His Ser Lys Ile Leu Asp Leu Asp Val Asp
170 175 180
GGC TAT AGA ATT GAT CAT ATT GAT GGA TTA TAT GAT CCT GAG AAA TAT 1412
Gly Tyr Arg Ile Asp His Ile Asp Gly Leu Tyr Asp Pro Glu Lys Tyr
185 190 195
ATT AAT GAC CTG AGG TCA ATA ATT AAA AAT AAA ATA ATT ATT GTA GAA 1460
Ile Asn Asp Leu Arg Ser Ile Ile Lys Asn Lys Ile Ile Ile Val Glu
200 205 210 215
AAA ATT CTG GGA TTT CAG GAG GAA TTA AAA TTA AAT TCA GAT GGA ACT 1508
Lys Ile Leu Gly Phe Gln Glu Glu Leu Lys Leu Asn Ser Asp Gly Thr
220 225 230
ACA GGA TAT GAC TTC TTA AAT TAC TCC AAC TTA CTG TTT AAT TTT AAT 1556
Thr Gly Tyr Asp Phe Leu Asn Tyr Ser Asn Leu Leu Phe Asn Phe Asn
235 240 245
CAA GAG ATA ATG GAC AGT ATA TAT GAG AAT TTC ACA GCG GAG AAA ATA 1604
Gln Glu Ile Met Asp Ser Ile Tyr Glu Asn Phe Thr Ala Glu Lys Ile
250 255 260
TCT ATA AGT GAA AGT ATA AAG AAA ATA AAA GCG CAA ATA ATT GAT GAG 1652
Ser Ile Ser Glu Ser Ile Lys Lys Ile Lys Ala Gln Ile Ile Asp Glu
265 270 275
CTA TTT AGT TAT GAA GTT AAA AGA TTA GCA TCA CAA CTA GGA ATT AGC 1700
Leu Phe Ser Tyr Glu Val Lys Arg Leu Ala Ser Gln Leu Gly Ile Ser
280 285 290 295
TAC GAT ATA TTG AGA GAT TAC CTT TCT TGT ATA GAT GTG TAC AGA ACT 1748
Tyr Asp Ile Leu Arg Asp Tyr Leu Ser Cys Ile Asp Val Tyr Arg Thr
300 305 310
TAT GCT AAT CAG ATT GTA AAA GAG TGT GAT AAG ACC AAT GAG ATA GAG 1796
Tyr Ala Asn Gln Ile Val Lys Glu Cys Asp Lys Thr Asn Glu Ile Glu
315 320 325
GAA GCA ACC AAA AGA AAT CCA GAG GCT TAT ACT AAA TTA CAA CAA TAT 1844
Glu Ala Thr Lys Arg Asn Pro Glu Ala Tyr Thr Lys Leu Gln Gln Tyr
330 335 340
ATG CCA GCA GTA TAC GCT AAA GCT TAT GAA GAT ACT TTC CTC TTT AGA 1892
Met Pro Ala Val Tyr Ala Lys Ala Tyr Glu Asp Thr Phe Leu Phe Arg
345 350 355
TAC AAT AGA TTA ATA TCC ATA AAT GAG GTT GGA AGC GAT TTA CGA TAT 1940
Tyr Asn Arg Leu Ile Ser Ile Asn Glu Val Gly Ser Asp Leu Arg Tyr
360 365 370 375
TAT AAG ATA TCG CCT GAT CAG TTT CAT GTA TTT AAT CAA AAA CGA AGA 1988
Tyr Lys Ile Ser Pro Asp Gln Phe His Val Phe Asn Gln Lys Arg Arg
380 385 390
GGA AAA ATC ACA CTA AAT GCC ACT AGC ACA CAT GAT ACT AAG TTT AGT 2036
Gly Lys Ile Thr Leu Asn Ala Thr Ser Thr His Asp Thr Lys Phe Ser
395 400 405
GAA GAT GTA AGG ATG AAA ATA AGT GTA TTA AGT GAA TTT CCT GAA GAA 2084
Glu Asp Val Arg Met Lys Ile Ser Val Leu Ser Glu Phe Pro Glu Glu
410 415 420
TGG AAA AAT AAG GTC GAG GAA TGG CAT AGT ATC ATA AAT CCA AAG GTA 2132
Trp Lys Asn Lys Val Glu Glu Trp His Ser Ile Ile Asn Pro Lys Val
425 430 435
TCA AGA AAT GAT GAA TAT AGA TAT TAT CAG GTT TTA GTG GGA AGT TTT 2180
Ser Arg Asn Asp Glu Tyr Arg Tyr Tyr Gln Val Leu Val Gly Ser Phe
440 445 450 455
TAT GAG GGA TTC TCT AAT GAT TTT AAG GAG AGA ATA AAG CAA CAT ATG 2228
Tyr Glu Gly Phe Ser Asn Asp Phe Lys Glu Arg Ile Lys Gln His Met
460 465 470
ATA AAA AGT GTC AGA GAA GCT AAG ATA AAT ACC TCA TGG AGA AAT CAA 2276
Ile Lys Ser Val Arg Glu Ala Lys Ile Asn Thr Ser Trp Arg Asn Gln
475 480 485
AAT AAA GAA TAT GAA AAT AGA GTA ATG GAA TTA GTG GAA GAA ACT TTT 2324
Asn Lys Glu Tyr Glu Asn Arg Val Met Glu Leu Val Glu Glu Thr Phe
490 495 500
ACC AAT AAG GAT TTC ATT AAA AGT TTC ATG AAA TTT GAA AGT AAG ATA 2372
Thr Asn Lys Asp Phe Ile Lys Ser Phe Met Lys Phe Glu Ser Lys Ile
505 510 515
AGA AGG ATA GGG ATG ATT AAG AGC TTA TCC TTG GTC GCA TTA AAA ATT 2420
Arg Arg Ile Gly Met Ile Lys Ser Leu Ser Leu Val Ala Leu Lys Ile
520 525 530 535
ATG TCA GCC GGT ATA CCT GAT TTT TAT CAG GGA ACA GAA ATA TGG CGA 2468
Met Ser Ala Gly Ile Pro Asp Phe Tyr Gln Gly Thr Glu Ile Trp Arg
540 545 550
TAT TTA CTT ACA GAT CCA GAT AAC AGA GTC CCA GTG GAT TTT AAG AAA 2516
Tyr Leu Leu Thr Asp Pro Asp Asn Arg Val Pro Val Asp Phe Lys Lys
555 560 565
TTA CAC GAA ATA TTA GAA AAA TCC AAA AAA TTT GAA AAA AAT ATG TTA 2564
Leu His Glu Ile Leu Glu Lys Ser Lys Lys Phe Glu Lys Asn Met Leu
570 575 580
GAG TCT ATG GAC GAT GGA AGA ATT AAG ATG TAT TTA ACA TAT AAG CTT 2612
Glu Ser Met Asp Asp Gly Arg Ile Lys Met Tyr Leu Thr Tyr Lys Leu
585 590 595
TTA TCC CTA AGA AAA CAG TTG GCT GAG GAT TTT TTA AAG GGC GAG TAT 2660
Leu Ser Leu Arg Lys Gln Leu Ala Glu Asp Phe Leu Lys Gly Glu Tyr
600 605 610 615
AAG GGA TTA GAT CTA GAA GAA GGA CTA TGT GGG TTT ATT AGG TTT AAC 2708
Lys Gly Leu Asp Leu Glu Glu Gly Leu Cys Gly Phe Ile Arg Phe Asn
620 625 630
AAA ATT TTG GTA ATA ATA AAA ACC AAG GGA AGT GTT AAT TAC AAA CTG 2756
Lys Ile Leu Val Ile Ile Lys Thr Lys Gly Ser Val Asn Tyr Lys Leu
635 640 645
AAA CTT GAA GAG GGA GCA ATT TAC ACA GAT GTA TTG ACA GGA GAA GAA 2804
Lys Leu Glu Glu Gly Ala Ile Tyr Thr Asp Val Leu Thr Gly Glu Glu
650 655 660
ATT AAA AAA GAG GTA CAG ATT AAT GAG CTA CCT AGG ATA CTA GTT AGA 2852
Ile Lys Lys Glu Val Gln Ile Asn Glu Leu Pro Arg Ile Leu Val Arg
665 670 675
ATG TAAGTTATAA TAATCCGATT TTTATGTGAC AAGATTTACG CTTACGAAAA 2905
Met
680
GGACTGTTAA ATCAACTTTT ATGTGAATTA TGAAACGTAA ATTATAAGTT TCCTGAGGAT 2965
AAACATATAT ATCTCTATCT CTCATTGATA TCACATGAGT ATTAGATTAA GGGGAAGTAA 3025
TTCTTACGGA CATTCAGGCT GGTTTACAGT ATACTGTAGA ATATGTAATA GGAAAATAAG 3085
AATAGGAACG GACTTAGTCT ACAAATGCCC TAAATGTGAA AAGAAGTATA ACGCATTCTT 3145
CTGTGAAGCA GATGCTAGGG GATTAAAGAA AAAGTGCCCA TACTGTGGTA CTGAACTTGT 3205
CAGTGCAATT TAAGACTCAA ATAGAAGGTA AAAATATTTT TATACTGAAT AATGAGTTGT 3265
TTTACGCTGA TACGGATATA GTTATTCGAA ATCAAGATTT TATTAAGAAA CTCACCTTTA 3325
CACAATATAA TAAGATTGCC TATATTGACA TGGACATAGA AACGACAGAA TTTAAGATAT 3385
TAAGATTAGT AGTGTGTAAA ACTAGAATAA ATATTTATGT TTGCAACGTA ATTGGTAAAT 3445
TGAAAGAAAC TAATTTTGAA AA 3467

SEQ ID NO: 4
Sequence length: 680
Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Protein origin Organism: Sulfolobus acidocaldarius
Strain name: ATCC 33909 strain sequence
Met Ala Ser Pro Gly Ser Asn His Gly Tyr Asp Val Ile Asp His Ser
1 5 10 15
Arg Ile Asn Asp Glu Leu Gly Gly Glu Lys Glu Tyr Arg Arg Leu Ile
20 25 30
Glu Thr Ala His Thr Ile Gly Leu Gly Ile Ile Gln Asp Ile Val Pro
35 40 45
Asn His Met Ala Val Asn Ser Leu Asn Trp Arg Leu Met Asp Val Leu
50 55 60
Lys Met Gly Lys Lys Ser Lys Tyr Tyr Thr Tyr Phe Asp Phe Phe Pro
65 70 75 80
Glu Asp Asp Lys Ile Arg Leu Pro Ile Leu Gly Glu Asp Leu Asp Thr
85 90 95
Val Ile Ser Lys Gly Leu Leu Lys Ile Val Lys Asp Gly Asp Glu Tyr
100 105 110
Phe Leu Glu Tyr Phe Lys Trp Lys Leu Pro Leu Thr Glu Val Gly Asn
115 120 125
Asp Ile Tyr Asp Thr Leu Gln Lys Gln Asn Tyr Thr Leu Met Ser Trp
130 135 140
Lys Asn Pro Pro Ser Tyr Arg Arg Phe Phe Asp Val Asn Thr Leu Ile
145 150 155 160
Gly Val Asn Val Glu Lys Asp His Val Phe Gln Glu Ser His Ser Lys
165 170 175
Ile Leu Asp Leu Asp Val Asp Gly Tyr Arg Ile Asp His Ile Asp Gly
180 185 190
Leu Tyr Asp Pro Glu Lys Tyr Ile Asn Asp Leu Arg Ser Ile Ile Lys
195 200 205
Asn Lys Ile Ile Ile Val Glu Lys Ile Leu Gly Phe Gln Glu Glu Leu
210 215 220
Lys Leu Asn Ser Asp Gly Thr Thr Gly Tyr Asp Phe Leu Asn Tyr Ser
225 230 235 240
Asn Leu Leu Phe Asn Phe Asn Gln Glu Ile Met Asp Ser Ile Tyr Glu
245 250 255
Asn Phe Thr Ala Glu Lys Ile Ser Ile Ser Glu Ser Ile Lys Lys Ile
260 265 270
Lys Ala Gln Ile Ile Asp Glu Leu Phe Ser Tyr Glu Val Lys Arg Leu
275 280 285
Ala Ser Gln Leu Gly Ile Ser Tyr Asp Ile Leu Arg Asp Tyr Leu Ser
290 295 300
Cys Ile Asp Val Tyr Arg Thr Tyr Ala Asn Gln Ile Val Lys Glu Cys
305 310 315 320
Asp Lys Thr Asn Glu Ile Glu Glu Ala Thr Lys Arg Asn Pro Glu Ala
325 330 335
Tyr Thr Lys Leu Gln Gln Tyr Met Pro Ala Val Tyr Ala Lys Ala Tyr
340 345 350
Glu Asp Thr Phe Leu Phe Arg Tyr Asn Arg Leu Ile Ser Ile Asn Glu
355 360 365
Val Gly Ser Asp Leu Arg Tyr Tyr Lys Ile Ser Pro Asp Gln Phe His
370 375 380
Val Phe Asn Gln Lys Arg Arg Gly Lys Ile Thr Leu Asn Ala Thr Ser
385 390 395 400
Thr His Asp Thr Lys Phe Ser Glu Asp Val Arg Met Lys Ile Ser Val
405 410 415
Leu Ser Glu Phe Pro Glu Glu Trp Lys Asn Lys Val Glu Glu Trp His
420 425 430
Ser Ile Ile Asn Pro Lys Val Ser Arg Asn Asp Glu Tyr Arg Tyr Tyr
435 440 445
Gln Val Leu Val Gly Ser Phe Tyr Glu Gly Phe Ser Asn Asp Phe Lys
450 455 460
Glu Arg Ile Lys Gln His Met Ile Lys Ser Val Arg Glu Ala Lys Ile
465 470 475 480
Asn Thr Ser Trp Arg Asn Gln Asn Lys Glu Tyr Glu Asn Arg Val Met
485 490 495
Glu Leu Val Glu Glu Thr Phe Thr Asn Lys Asp Phe Ile Lys Ser Phe
500 505 510
Met Lys Phe Glu Ser Lys Ile Arg Arg Ile Gly Met Ile Lys Ser Leu
515 520 525
Ser Leu Val Ala Leu Lys Ile Met Ser Ala Gly Ile Pro Asp Phe Tyr
530 535 540
Gln Gly Thr Glu Ile Trp Arg Tyr Leu Leu Thr Asp Pro Asp Asn Arg
545 550 555 560
Val Pro Val Asp Phe Lys Lys Leu His Glu Ile Leu Glu Lys Ser Lys
565 570 575
Lys Phe Glu Lys Asn Met Leu Glu Ser Met Asp Asp Gly Arg Ile Lys
580 585 590
Met Tyr Leu Thr Tyr Lys Leu Leu Ser Leu Arg Lys Gln Leu Ala Glu
595 600 605
Asp Phe Leu Lys Gly Glu Tyr Lys Gly Leu Asp Leu Glu Glu Gly Leu
610 615 620
Cys Gly Phe Ile Arg Phe Asn Lys Ile Leu Val Ile Ile Lys Thr Lys
625 630 635 640
Gly Ser Val Asn Tyr Lys Leu Lys Leu Glu Glu Gly Ala Ile Tyr Thr
645 650 655
Asp Val Leu Thr Gly Glu Glu Ilu Lys Lys Glu Val Gln Ile Asn Glu
660 665 670
Leu Pro Arg Ile Leu Val Arg Met
675 680

SEQ ID NO: 5
Sequence length: 2691
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Genomic DNA
Origin: Sulfolobus solfataricus
Stock name: KM1
Array
CTGCAGTAAC TAGCGCTATC GAAGACGTTA TAAAGAGAAG GATAAATAGA GTTCCAGTGA 60
GTCTAGAAGA CCTTTTTGAA TAAGGACTTT AATATCATTT AAATTTATTT TTTGGAACAT 120
GCAGAGGTAA ACCCATGAAT GTCATTTTCG ACGTATTAAA CGAGATCCAT GGGTTTTTTG 180
GTGCATTGTG GGCGGGAGCA GCTCTACTTA ACTACTTAGT TAAGCCTCAA GATAAGAGGC 240
AATTTGAGAG AATAGGGAAA TTCTTCATGA TAAACTCAGT CATTACAGTA ATAACTGGGA 300
TAATAATTTT CGCCTACATT TACCTAGCCC CTTATCAAGG GAATTTATTT CTAGTAGCGG 360
CAATTCTACG TTCAAGCCTT GACATTAGGT TAAGGGCCTT ACTAAACTTA ATAGGAGGAG 420
CGTTTGGGTT ATTGGCTTTT GGGGCAGGGA TAGTTATAAG CAATAGGATA AGGCTTATGG 480
TACGTGTTAA GGAAGGTGAC GCTACAATCC TAGAGTTGAG GAATAGTATT GCCAATTTAT 540
CTAAAATTAG TTTAATCTTC TTATTACTTT CCTTAGCCAT GATGATACTT GCTGGTTCCA 600
TAGCACAAGT TATAAGTTAG AGTTGAAAGA AAAATTTA ATG ACG TTT GCT TAT AAA 656
Met Thr Phe Ala Tyr Lys
0 1 5
ATA GAT GGA AAT GAG GTA ATC TTT ACC TTA TGG GCA CCT TAT CAA AAG 704
Ile Asp Gly Asn Glu Val Ile Phe Thr Leu Trp Ala Pro Tyr Gln Lys
10 15 20
AGC GTT AAA CTA AAG GTT CTA GAG AAG GGA CTT TAC GAA ATG GAA AGA 752
Ser Val Lys Leu Lys Val Leu Glu Lys Gly Leu Tyr Glu Met Glu Arg
25 30 35
GAT GAA AAA GGT TAC TTC ACC ATT ACC TTA AAC AAC GTA AAG GTT AGA 800
Asp Glu Lys Gly Tyr Phe Thr Ile Thr Leu Asn Asn Val Lys Val Arg
40 45 50
GAT AGG TAT AAA TAC GTT TTA GAT GAT GCT AGT GAA ATA CCA GAT CCA 848
Asp Arg Tyr Lys Tyr Val Leu Asp Asp Ala Ser Glu Ile Pro Asp Pro
55 60 65
GCA TCC AGA TAC CAA CCA GAA GGT GTA CAT GGG CCT TCA CAA ATT ATA 896
Ala Ser Arg Tyr Gln Pro Glu Gly Val His Gly Pro Ser Gln Ile Ile
70 75 80 85
CAA GAA AGT AAA GAG TTC AAC AAC GAG ACT TTT CTG AAG AAA GAG GAC 944
Gln Glu Ser Lys Glu Phe Asn Asn Glu Thr Phe Leu Lys Lys Glu Asp
90 95 100
TTG ATA ATT TAT GAA ATA CAC GTG GGG ACT TTC ACT CCA GAG GGA ACG 992
Leu Ile Ile Tyr Glu Ile His Val Gly Thr Phe Thr Pro Glu Gly Thr
105 110 115
TTT GAG GGA GTG ATA AGG AAA CTT GAC TAC TTA AAG GAT TTG GGA ATT 1040
Phe Glu Gly Val Ile Arg Lys Leu Asp Tyr Leu Lys Asp Leu Gly Ile
120 125 130
ACG GCA ATA GAG ATA ATG CCA ATA GCT CAA TTT CCT GGG AAA AGG GAT 1088
Thr Ala Ile Glu Ile Met Pro Ile Ala Gln Phe Pro Gly Lys Arg Asp
135 140 145
TGG GGT TAT GAT GGA GTT TAT TTA TAT GCA GTA CAG AAC TCT TAC GGA 1136
Trp Gly Tyr Asp Gly Val Tyr Leu Tyr Ala Val Gln Asn Ser Tyr Gly
150 155 160 165
GGG CCA GAA GGT TTT AGA AAG TTA GTT GAT GAA GCG CAC AAG AAA GGT 1184
Gly Pro Glu Gly Phe Arg Lys Leu Val Asp Glu Ala His Lys Lys Gly
170 175 180
TTA GGA GTT ATT TTA GAC GTA GTA TAC AAC CAC GTT GGA CCA GAG GGA 1232
Leu Gly Val Ile Leu Asp Val Val Tyr Asn His Val Gly Pro Glu Gly
185 190 195
AAC TAT ATG GTT AAA TTG GGG CCA TAT TTC TCA CAG AAA TAC AAA ACG 1280
Asn Tyr Met Val Lys Leu Gly Pro Tyr Phe Ser Gln Lys Tyr Lys Thr
200 205 210
CCA TGG GGA TTA ACC TTT AAC TTT GAC GAT GCT GAA AGC GAT GAG GTT 1328
Pro Trp Gly Leu Thr Phe Asn Phe Asp Asp Ala Glu Ser Asp Glu Val
215 220 225
AGG AAG TTC ATC TTA GAA AAC GTT GAG TAC TGG ATT AAG GAA TAT AAC 1376
Arg Lys Phe Ile Leu Glu Asn Val Glu Tyr Trp Ile Lys Glu Tyr Asn
230 235 240 245
GTT GAT GGG TTT AGA TTA GAT GCG GTT CAT GCA ATT ATT GAC ACT TCT 1424
Val Asp Gly Phe Arg Leu Asp Ala Val His Ala Ile Ile Asp Thr Ser
250 255 260
CCT AAG CAC ATC TTG GAG GAA ATA GCT GAC GTT GTG CAT AAG TAT AAT 1472
Pro Lys His Ile Leu Glu Glu Ile Ala Asp Val Val His Lys Tyr Asn
265 270 275
AGG ATT GTC ATA GCC GAA AGT GAT TTA AAC GAT CCT AGA GTC GTT AAT 1520
Arg Ile Val Ile Ala Glu Ser Asp Leu Asn Asp Pro Arg Val Val Asn
280 285 290
CCC AAG GAA AAG TGT GGA TAT AAT ATT GAT GCT CAA TGG GTT GAC GAT 1568
Pro Lys Glu Lys Cys Gly Tyr Asn Ile Asp Ala Gln Trp Val Asp Asp
295 300 305
TTC CAT CAT TCT ATT CAC GCT TAC TTA ACT GGT GAG AGG CAA GGC TAT 1616
Phe His His Ser Ile His Ala Tyr Leu Thr Gly Glu Arg Gln Gly Tyr
310 315 320 325
TAT ACG GAT TTC GGT AAC CTT GAC GAT ATA GTT AAA TCG TAT AAG GAC 1664
Tyr Thr Asp Phe Gly Asn Leu Asp Asp Ile Val Lys Ser Tyr Lys Asp
330 335 340
GTT TTC GTA TAT GAT GGT AAG TAC TCC AAT TTT AGA AGA AAA ACT CAC 1712
Val Phe Val Tyr Asp Gly Lys Tyr Ser Asn Phe Arg Arg Lys Thr His
345 350 355
GGA GAA CCA GTT GGT GAA CTA GAC GGA TGC AAT TTC GTA GTT TAT ATA 1760
Gly Glu Pro Val Gly Glu Leu Asp Gly Cys Asn Phe Val Val Tyr Ile
360 365 370
CAA AAT CAC GAT CAA GTC GGA AAT AGA GGC AAA GGT GAA AGA ATA ATT 1808
Gln Asn His Asp Gln Val Gly Asn Arg Gly Lys Gly Glu Arg Ile Ile
375 380 385
AAA TTA GTC GAT AGG GAA AGC TAC AAG ATC GCT GCA GCC CTT TAC CTT 1856
Lys Leu Val Asp Arg Glu Ser Tyr Lys Ile Ala Ala Ala Leu Tyr Leu
390 395 400 405
CTT TCC CCC TAT ATT CCA ATG ATT TTC ATG GGA GAG GAA TAC GGT GAG 1904
Leu Ser Pro Tyr Ile Pro Met Ile Phe Met Gly Glu Glu Tyr Gly Glu
410 415 420
GAA AAT CCC TTT TAT TTC TTT TCT GAT TTT TCA GAT TCA AAA CTG ATA 1952
Glu Asn Pro Phe Tyr Phe Phe Ser Asp Phe Ser Asp Ser Lys Leu Ile
425 430 435
CAA GGT GTA AGG GAA GGG AGA AAA AAG GAA AAC GGG CAA GAT ACT GAC 2000
Gln Gly Val Arg Glu Gly Arg Lys Lys Glu Asn Gly Gln Asp Thr Asp
440 445 450
CCT CAA GAT GAA TCA ACT TTT AAC GCT TCC AAA CTG AGT TGG AAG ATT 2048
Pro Gln Asp Glu Ser Thr Phe Asn Ala Ser Lys Leu Ser Trp Lys Ile
455 460 465
GAC GAG GAA ATC TTT TCA TTT TAC AAG ATT TTA ATA AAA ATG AGA AAG 2096
Asp Glu Glu Ile Phe Ser Phe Tyr Lys Ile Leu Ile Lys Met Arg Lys
470 475 480 485
GAG TTG AGC ATA GCG TGT GAT AGG AGA GTA AAC GTC GTG AAT GGC GAA 2144
Glu Leu Ser Ile Ala Cys Asp Arg Arg Val Asn Val Val Asn Gly Glu
490 495 500
AAT TGG TTG ATC ATC AAG GGA AGA GAA TAC TTT TCA CTC TAC GTT TTC 2192
Asn Trp Leu Ile Ile Lys Gly Arg Glu Tyr Phe Ser Leu Tyr Val Phe
505 510 515
TCT AAA TCA TCT ATT GAA GTT AAG TAC AGT GGA ACT TTA CTT TTG TCC 2240
Ser Lys Ser Ser Ile Glu Val Lys Tyr Ser Gly Thr Leu Leu Leu Ser
520 525 530
TCA AAT AAT TCA TTC CCT CAG CAT ATT GAA GAA GGT AAA TAT GAG TTT 2288
Ser Asn Asn Ser Phe Pro Gln His Ile Glu Glu Gly Lys Tyr Glu Phe
535 540 545
GAT AAG GGA TTT GCT TTA TAT AAA CTT TAGGACA GGAGAGTTTA AAAATTTCTA 2342
Asp Lys Gly Phe Ala Leu Tyr Lys Leu
550 555
TGAATGATTA TACTTTAGAT GATGAGTAAA AGCAAGATCG ATGAGGAAGA GAAAAGGAGA 2402
AGAGAAGAAG TCAAAAAGTT AGTAATGCTC TTAGCAATGT TAAGATAATG TTTTTTTAAA 2462
CTCAAATAAT AATAAATACC ATCATGTCAA TATTCTTCAG AACTAGAGAT AGACCTTTAC 2522
GTCCCGGAGA TCCGTATCCA TTAGGTTCAA ATTGGATAGA AGATGAGGAT GGCGTAAATT 2582
TTTCCTTGTT CTCAGAGAAT GCAGACAAAG TGGAGTTGAT TCTTTATTCA CAAACAAATC 2642
AAAAGTATCC AAAGGAGATA ATAGAGGTTA AGAATAGAAC GGGGGATCC 2691

SEQ ID NO: 6
Sequence length: 558
Sequence type: Number of amino acid chains: Single chain Topology: Linear sequence type: Protein origin Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Thr Phe Ala Tyr Lys Ile Asp Gly Asn Glu Val Ile Phe Thr Leu Trp
1 5 10 15
Ala Pro Tyr Gln Lys Ser Val Lys Leu Lys Val Leu Glu Lys Gly Leu
20 25 30
Tyr Glu Met Glu Arg Asp Glu Lys Gly Tyr Phe Thr Ile Thr Leu Asn
35 40 45
Asn Val Lys Val Arg Asp Arg Tyr Lys Tyr Val Leu Asp Asp Ala Ser
50 55 60
Glu Ile Pro Asp Pro Ala Ser Arg Tyr Gln Pro Glu Gly Val His Gly
65 70 75 80
Pro Ser Gln Ile Ile Gln Glu Ser Lys Glu Phe Asn Asn Glu Thr Phe
85 90 95
Leu Lys Lys Glu Asp Leu Ile Ile Tyr Glu Ile His Val Gly Thr Phe
100 105 110
Thr Pro Glu Gly Thr Phe Glu Gly Val Ile Arg Lys Leu Asp Tyr Leu
115 120 125
Lys Asp Leu Gly Ile Thr Ala Ile Glu Ile Met Pro Ile Ala Gln Phe
130 135 140
Pro Gly Lys Arg Asp Trp Gly Tyr Asp Gly Val Tyr Leu Tyr Ala Val
145 150 155 160
Gln Asn Ser Tyr Gly Gly Pro Glu Gly Phe Arg Lys Leu Val Asp Glu
165 170 175
Ala His Lys Lys Gly Leu Gly Val Ile Leu Asp Val Val Tyr Asn His
180 185 190
Val Gly Pro Glu Gly Asn Tyr Met Val Lys Leu Gly Pro Tyr Phe Ser
195 200 205
Gln Lys Tyr Lys Thr Pro Trp Gly Leu Thr Phe Asn Phe Asp Asp Ala
210 215 220
Glu Ser Asp Glu Val Arg Lys Phe Ile Leu Glu Asn Val Glu Tyr Trp
225 230 235 240
Ile Lys Glu Tyr Asn Val Asp Gly Phe Arg Leu Asp Ala Val His Ala
245 250 255
Ile Ile Asp Thr Ser Pro Lys His Ile Leu Glu Glu Ile Ala Asp Val
260 265 270
Val His Lys Tyr Asn Arg Ile Val Ile Ala Glu Ser Asp Leu Asn Asp
275 280 285
Pro Arg Val Val Asn Pro Lys Glu Lys Cys Gly Tyr Asn Ile Asp Ala
290 295 300
Gln Trp Val Asp Asp Phe His His Ser Ile His Ala Tyr Leu Thr Gly
305 310 315 320
Glu Arg Gln Gly Tyr Tyr Thr Asp Phe Gly Asn Leu Asp Asp Ile Val
325 330 335
Lys Ser Tyr Lys Asp Val Phe Val Tyr Asp Gly Lys Tyr Ser Asn Phe
340 345 350
Arg Arg Lys Thr His Gly Glu Pro Val Gly Glu Leu Asp Gly Cys Asn
355 360 365
Phe Val Val Tyr Ile Gln Asn His Asp Gln Val Gly Asn Arg Gly Lys
370 375 380
Gly Glu Arg Ile Ile Lys Leu Val Asp Arg Glu Ser Tyr Lys Ile Ala
385 390 395 400
Ala Ala Leu Tyr Leu Leu Ser Pro Tyr Ile Pro Met Ile Phe Met Gly
405 410 415
Glu Glu Tyr Gly Glu Glu Asn Pro Phe Tyr Phe Phe Ser Asp Phe Ser
420 425 430
Asp Ser Lys Leu Ile Gln Gly Val Arg Glu Gly Arg Lys Lys Glu Asn
435 440 445
Gly Gln Asp Thr Asp Pro Gln Asp Glu Ser Thr Phe Asn Ala Ser Lys
450 455 460
Leu Ser Trp Lys Ile Asp Glu Glu Ile Phe Ser Phe Tyr Lys Ile Leu
465 470 475 480
Ile Lys Met Arg Lys Glu Leu Ser Ile Ala Cys Asp Arg Arg Val Asn
485 490 495
Val Val Asn Gly Glu Asn Trp Leu Ile Ile Lys Gly Arg Glu Tyr Phe
500 505 510
Ser Leu Tyr Val Phe Ser Lys Ser Ser Ile Glu Val Lys Tyr Ser Gly
515 520 525
Thr Leu Leu Leu Ser Ser Asn Asn Ser Phe Pro Gln His Ile Glu Glu
530 535 540
Gly Lys Tyr Glu Phe Asp Lys Gly Phe Ala Leu Tyr Lys Leu
545 550 555

SEQ ID NO: 7
Sequence length: 3600
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Genomic DNA
Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
ATTCGTTTTG AGTCACTCGG CGTAGGTCTG TAGTCTTTCT TGGCGAGGGC TAATAAGTTG 60
AGATAATGCT TGCCAAGAAT CGAAGAAGGC GTCCTGCCCT GCATGAAATC GATTACCTCG 120
GCACTAACTC CGAGCTCCGC GAGTTTAGTA GTCACGAATT TGCGTACATA TTTCGGCGCT 180
ATCCCTTTCT CATGCAATAA ATTCTTCGCG TAGTTGTACG TTATATCAGT CTTAGCTATA 240
GACGAAATGT GAAAGACATA GAACACTTTC TTTGGCCCTC TAGTCCAGTT GAGCGTGTAT 300
ACGTAGAAGC CGTCCTCTTT CACGTTGTTC TTCTCGTCAT ACTCATTGAG AACCTTTACA 360
GCCTCCCTAA GCCTTATACC GCTCTCAAGG AGGAGCTTGA AGACTAGCTC TACCTCAATA 420
CCTCTAACAG CCTCCAACCA CCTCCCTATC TCGTCAGCTC CTGGAACCTT AAGATCAACA 480
CCAGACTTTT TCGTTTTCAG CTTTTTCCAT GCCTCAAGAT CCCCTTTCCA CTTGTAGAAC 540
TTCTTCCAGG CTAGGATAGA GTTCTTAGCA TTACTAGGGG GCTTCTTCAG ATAATTGATA 600
TACTGCCTGC AAGTTTCCTC ACTGGCCATT TTCAAACAAT ATTCATAAAA TTCAATTAAT 660
TCCTTTTCCG TGAGACCATT TTTGCCCTCC CTAGAAGTAA GGGAGTTTAG GGCAAATCCC 720
TTACTCTCTT CATCATTTGA AAGAGGGGTT TTAGGGGATT CCTCCCCTAA CCAGGGCTTT 780
GGCCCCTGGG ACCAGGGTTC GAGTCCCTGC CCGGCTACCT TTGAAAGGTT AGGGGGATAC 840
ACCCTAATAC CCCACTTCTA TCTTACAATT TCAGGTAAGT CTTTACTAGG TCAACTAAAG 900
CACCAACGTA AGTCTCCTTC GTCTTACCAC CTTGACTCTT CTTGATAAAG TAAACATAAT 960
ATCATCCATA GACTTACCTT ATTCTTATAT TACCATATGA TTTTATTATT TTGTATTTCT 1020
ATTAGATAAG TCCCACTCAT AGAACAAATG ATGGTTTTAA CTTATATACT AAATACTCTA 1080
ATAACTCAAC AATAATAAGA ATTTAATCAG TTCTGATAAG TATTTTCACT CGAAAACATT 1140
TAAATATATT AAGACATAAT TTCTATTTAA ACAGC ATG TTT TCG TTC GGT GGA AAT 1196
Met Phe Ser Phe Gly Gly Asn
1 5
ATT GAA AAA AAT AAA GGT ATC TTT AAG TTA TGG GCA CCT TAT GTT AAT 1244
Ile Glu Lys Asn Lys Gly Ile Phe Lys Leu Trp Ala Pro Tyr Val Asn
10 15 20
AGT GTT AAG CTG AAG TTA AGC AAA AAA CTT ATT CCA ATG GAA AAA AAC 1292
Ser Val Lys Leu Lys Leu Ser Lys Lys Leu Ile Pro Met Glu Lys Asn
25 30 35
GAT GAG GGA TTT TTC GAA GTA GAA ATA GAC GAT ATC GAG GAA AAT TTA 1340
Asp Glu Gly Phe Phe Glu Val Glu Ile Asp Asp Ile Glu Glu Asn Leu
40 45 50 55
ACC TAT TCT TAT ATT ATA GAA GAT AAG AGA GAG ATA CCT GAT CCC GCA 1388
Thr Tyr Ser Tyr Ile Ile Glu Asp Lys Arg Glu Ile Pro Asp Pro Ala
60 65 70
TCA CGA TAT CAA CCT TTA GGA GTT CAT GAC AAA TCA CAA CTT ATA AGA 1436
Ser Arg Tyr Gln Pro Leu Gly Val His Asp Lys Ser Gln Leu Ile Arg
75 80 85
ACA GAT TAT CAG ATT CTT GAC CTT GGA AAA GTA AAA ATA GAA GAT CTA 1484
Thr Asp Tyr Gln Ile Leu Asp Leu Gly Lys Val Lys Ile Glu Asp Leu
90 95 100
ATA ATA TAT GAA CTC CAC GTT GGT ACT TTT TCC CAA GAA GGA AAT TTC 1532
Ile Ile Tyr Glu Leu His Val Gly Thr Phe Ser Gln Glu Gly Asn Phe
105 110 115
AAA GGA GTA ATA GAA AAG TTA GAT TAC CTC AAG GAT CTA GGA ATC ACA 1580
Lys Gly Val Ile Glu Lys Leu Asp Tyr Leu Lys Asp Leu Gly Ile Thr
120 125 130 135
GGA ATT GAA CTG ATG CCT GTG GCA CAA TTT CCA GGG AAT AGA GAT TGG 1628
Gly Ile Glu Leu Met Pro Val Ala Gln Phe Pro Gly Asn Arg Asp Trp
140 145 150
GGA TAC GAT GGT GTT TTT CTA TAC GCA GTT CAA AAT ACT TAT GGC GGA 1676
Gly Tyr Asp Gly Val Phe Leu Tyr Ala Val Gln Asn Thr Tyr Gly Gly
155 160 165
CCA TGG GAA TTG GCT AAG CTA GTA AAC GAG GCA CAT AAA AGG GGA ATA 1724
Pro Trp Glu Leu Ala Lys Leu Val Asn Glu Ala His Lys Arg Gly Ile
170 175 180
GCC GTA ATT TTG GAT GTT GTA TAT AAT CAT ATA GGT CCT GAG GGA AAT 1772
Ala Val Ile Leu Asp Val Val Tyr Asn His Ile Gly Pro Glu Gly Asn
185 190 195
TAC CTT TTA GGA TTA GGT CCT TAT TTT TCA GAC AGA TAT AAA ACT CCA 1820
Tyr Leu Leu Gly Leu Gly Pro Tyr Phe Ser Asp Arg Tyr Lys Thr Pro
200 205 210 215
TGG GGA TTA ACA TTT AAT TTT GAT GAT AGG GGA TGT GAT CAA GTT AGA 1868
Trp Gly Leu Thr Phe Asn Phe Asp Asp Arg Gly Cys Asp Gln Val Arg
220 225 230
AAA TTC ATT TTA GAA AAT GTC GAG TAT TGG TTT AAG ACC TTT AAA ATC 1916
Lys Phe Ile Leu Glu Asn Val Glu Tyr Trp Phe Lys Thr Phe Lys Ile
235 240 245
GAT GGT CTG AGA CTG GAT GCA GTT CAT GCA ATT TTT GAT AAT TCG CCT 1964
Asp Gly Leu Arg Leu Asp Ala Val His Ala Ile Phe Asp Asn Ser Pro
250 255 260
AAG CAT ATC CTC CAA GAG ATA GCT GAA AAA GCC CAT CAA TTA GGA AAA 2012
Lys His Ile Leu Gln Glu Ile Ala Glu Lys Ala His Gln Leu Gly Lys
265 270 275
TTT GTT ATT GCT GAA AGT GAT TTA AAT GAT CCA AAA ATA GTA AAA GAT 2060
Phe Val Ile Ala Glu Ser Asp Leu Asn Asp Pro Lys Ile Val Lys Asp
280 285 290 295
GAT TGT GGA TAT AAA ATA GAT GCT CAA TGG GTT GAC GAT TTC CAC CAC 2108
Asp Cys Gly Tyr Lys Ile Asp Ala Gln Trp Val Asp Asp Phe His His
300 305 310
GCA GTT CAT GCA TTC ATA ACA AAA GAA AAA GAT TAT TAT TAC CAG GAT 2156
Ala Val His Ala Phe Ile Thr Lys Glu Lys Asp Tyr Tyr Tyr Gln Asp
315 320 325
TTT GGA AGG ATA GAA GAT ATA GAG AAA ACT TTT AAA GAT GTT TTT GTT 2204
Phe Gly Arg Ile Glu Asp Ile Glu Lys Thr Phe Lys Asp Val Phe Val
330 335 340
TAT GAT GGA AAG TAT TCT AGA TAC AGA GGA AGA ACT CAT GGT GCT CCT 2252
Tyr Asp Gly Lys Tyr Ser Arg Tyr Arg Gly Arg Thr His Gly Ala Pro
345 350 355
GTA GGT GAT CTT CCA CCA CGT AAA TTT GTA GTC TTC ATA CAA AAT CAC 2300
Val Gly Asp Leu Pro Pro Arg Lys Phe Val Val Phe Ile Gln Asn His
360 365 370 375
GAT CAA GTA GGA AAT AGA GGA AAT GGG GAA AGA CTT TCC ATA TTA ACC 2348
Asp Gln Val Gly Asn Arg Gly Asn Gly Glu Arg Leu Ser Ile Leu Thr
380 385 390
GAT AAA ACG ACA TAC CTT ATG GCA GCC ACA CTA TAT ATA CTC TCA CCG 2396
Asp Lys Thr Thr Tyr Leu Met Ala Ala Thr Leu Tyr Ile Leu Ser Pro
395 400 405
TAT ATA CCG CTA ATA TTT ATG GGC GAG GAA TAT TAT GAG ACG AAT CCT 2444
Tyr Ile Pro Leu Ile Phe Met Gly Glu Glu Tyr Tyr Glu Thr Asn Pro
410 415 420
TTT TTC TTC TTC TCT GAT TTC TCA GAT CCC GTA TTA ATT AAG GGT GTT 2492
Phe Phe Phe Phe Ser Asp Phe Ser Asp Pro Val Leu Ile Lys Gly Val
425 430 435
AGA GAA GGT AGA CTA AAG GAA AAT AAT CAA ATG ATA GAT CCA CAA TCT 2540
Arg Glu Gly Arg Leu Lys Glu Asn Asn Gln Met Ile Asp Pro Gln Ser
440 445 450 455
GAG GAA GCG TTC TTA AAG AGT AAA CTT TCA TGG AAA ATT GAT GAG GAA 2588
Glu Glu Ala Phe Leu Lys Ser Lys Leu Ser Trp Lys Ile Asp Glu Glu
460 465 470
GTT TTA GAT TAT TAT AAA CAA CTG ATA AAT ATC AGA AAG AGA TAT AAT 2636
Val Leu Asp Tyr Tyr Lys Gln Leu Ile Asn Ile Arg Lys Arg Tyr Asn
475 480 485
AAT TGT AAA AGG GTA AAG GAA GTT AGG AGA GAA GGG AAC TGT ATT ACT 2684
Asn Cys Lys Arg Val Lys Glu Val Arg Arg Glu Gly Asn Cys Ile Thr
490 495 500
TTG ATC ATG GAA AAA ATA GGA ATA ATT GCA TCG TTT GAT GAT ATT GTA 2732
Leu Ile Met Glu Lys Ile Gly Ile Ile Ala Ser Phe Asp Asp Ile Val
505 510 515
ATT AAT TCT AAA ATT ACA GGT AAT TTA CTT ATA GGC ATA GGA TTT CCG 2780
Ile Asn Ser Lys Ile Thr Gly Asn Leu Leu Ile Gly Ile Gly Phe Pro
520 525 530 535
AAA AAA TTG AAA AAA GAT GAA TTA ATT AAG GTT AAC AGA GGT GTT GGG 2828
Lys Lys Leu Lys Lys Asp Glu Leu Ile Lys Val Asn Arg Gly Val Gly
540 545 550
GTA TAT CAA TTA GAA TGAAAGATCG ACCATTAAAG CCTGGTGAAC CTTATCCTTT 2883
Val Tyr Gln Leu Glu
555
AGGGGCAACT TGGATAGAGG AAGAAGATGG AGTTAATTTT GTACTATTCT CTGAGAACGC 2943
CACAAAAGTA GAACTGTTAA CGTACTCTCA GACTAGACAA GATGAGCCAA AGGAAATAAT 3003
AGAACTTAGA CAGAGAACCG GAGATCTCTG GCATGTTTTT GTACCTGGTT TAAGACCAGG 3063
TCAGTTGTAT GGGTACAGGG TGTATGGTCC ATATAAACCA GAGGAAGGGT TAAGGTTTAA 3123
TCCTAATAAA GTACTGATAG ATCCTTATGC AAAAGCTATA AACGGATTAT TACTATGGGA 3183
TGATTCGGTC TTTGGATATA AAATTGGAGA TCAGAACCAG GATCTCAGTT TCGATGAGAG 3243
AAAAGACGAT AAATTTATAC CTAAAGGGGT CATAATAAAT CCTTATTTTG ATTGGGAGGA 3303
CGAGCATTTC TTCTTTAGAA GAAAGATACC TTTTAAGGAT AGTATAATTT ATGAGACACA 3363
TATAAAAGGA ATAACTAAAT TAAGGCAAGA TTTACCGGAG AACGTTAGAG GCACTTTTTT 3423
GGGTTTAGCA TCAGATACTA TGATTGATTA CCTAAAAGAT TTAGGAATTA CAACCGTTGA 3483
GATAATGCCT ATTCAGCAAT TTGTAGATGA GAGATTCATT GTCGATAAAG GGTTAAAGAA 3543
CTACTGGGGT TACAATCCGA TAAATTATTT CTCTCCTGAA TGTAGATACT CAAGCTC 3600

SEQ ID NO: 8
Sequence length: 556
Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Protein Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Met Phe Ser Phe Gly Gly Asn Ile Glu Lys Asn Lys Gly Ile Phe Lys
1 5 10 15
Leu Trp Ala Pro Tyr Val Asn Ser Val Lys Leu Lys Leu Ser Lys Lys
20 25 30
Leu Ile Pro Met Glu Lys Asn Asp Glu Gly Phe Phe Glu Val Glu Ile
35 40 45
Asp Asp Ile Glu Glu Asn Leu Thr Tyr Ser Tyr Ile Ile Glu Asp Lys
50 55 60
Arg Glu Ile Pro Asp Pro Ala Ser Arg Tyr Gln Pro Leu Gly Val His
65 70 75 80
Asp Lys Ser Gln Leu Ile Arg Thr Asp Tyr Gln Ile Leu Asp Leu Gly
85 90 95
Lys Val Lys Ile Glu Asp Leu Ile Ile Tyr Glu Leu His Val Gly Thr
100 105 110
Phe Ser Gln Glu Gly Asn Phe Lys Gly Val Ile Glu Lys Leu Asp Tyr
115 120 125
Leu Lys Asp Leu Gly Ile Thr Gly Ile Glu Leu Met Pro Val Ala Gln
130 135 140
Phe Pro Gly Asn Arg Asp Trp Gly Tyr Asp Gly Val Phe Leu Tyr Ala
145 150 155 160
Val Gln Asn Thr Tyr Gly Gly Pro Trp Glu Leu Ala Lys Leu Val Asn
165 170 175
Glu Ala His Lys Arg Gly Ile Ala Val Ile Leu Asp Val Val Tyr Asn
180 185 190
His Ile Gly Pro Glu Gly Asn Tyr Leu Leu Gly Leu Gly Pro Tyr Phe
195 200 205
Ser Asp Arg Tyr Lys Thr Pro Trp Gly Leu Thr Phe Asn Phe Asp Asp
210 215 220
Arg Gly Cys Asp Gln Val Arg Lys Phe Ile Leu Glu Asn Val Glu Tyr
225 230 235 240
Trp Phe Lys Thr Phe Lys Ile Asp Gly Leu Arg Leu Asp Ala Val His
245 250 255
Ala Ile Phe Asp Asn Ser Pro Lys His Ile Leu Gln Glu Ile Ala Glu
260 265 270
Lys Ala His Gln Leu Gly Lys Phe Val Ile Ala Glu Ser Asp Leu Asn
275 280 285
Asp Pro Lys Ile Val Lys Asp Asp Cys Gly Tyr Lys Ile Asp Ala Gln
290 295 300
Trp Val Asp Asp Phe His His Ala Val His Ala Phe Ile Thr Lys Glu
305 310 315 320
Lys Asp Tyr Tyr Tyr Gln Asp Phe Gly Arg Ile Glu Asp Ile Glu Lys
325 330 335
Thr Phe Lys Asp Val Phe Val Tyr Asp Gly Lys Tyr Ser Arg Tyr Arg
340 345 350
Gly Arg Thr His Gly Ala Pro Val Gly Asp Leu Pro Pro Arg Lys Phe
355 360 365
Val Val Phe Ile Gln Asn His Asp Gln Val Gly Asn Arg Gly Asn Gly
370 375 380
Glu Arg Leu Ser Ile Leu Thr Asp Lys Thr Thr Tyr Leu Met Ala Ala
385 390 395 400
Thr Leu Tyr Ile Leu Ser Pro Tyr Ile Pro Leu Ile Phe Met Gly Glu
405 410 415
Glu Tyr Tyr Glu Thr Asn Pro Phe Phe Phe Phe Ser Asp Phe Ser Asp
420 425 430
Pro Val Leu Ile Lys Gly Val Arg Glu Gly Arg Leu Lys Glu Asn Asn
435 440 445
Gln Met Ile Asp Pro Gln Ser Glu Glu Ala Phe Leu Lys Ser Lys Leu
450 455 460
Ser Trp Lys Ile Asp Glu Glu Val Leu Asp Tyr Tyr Lys Gln Leu Ile
465 470 475 480
Asn Ile Arg Lys Arg Tyr Asn Asn Cys Lys Arg Val Lys Glu Val Arg
485 490 495
Arg Glu Gly Asn Cys Ile Thr Leu Ile Met Glu Lys Ile Gly Ile Ile
500 505 510
Ala Ser Phe Asp Asp Ile Val Ile Asn Ser Lys Ile Thr Gly Asn Leu
515 520 525
Leu Ile Gly Ile Gly Phe Pro Lys Lys Leu Lys Lys Asp Glu Leu Ile
530 535 540
Lys Val Asn Arg Gly Val Gly Val Tyr Gln Leu Glu
545 550 555

SEQ ID NO: 9
Sequence length: 6
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Val Ile Arg Glu Ala Lys
1 5

SEQ ID NO: 10
Sequence length: 6
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Ile Ser Ile Arg Gln Lys
1 5

SEQ ID NO: 11
Sequence length: 5
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Ile Ile Tyr Val Glu
1 5

SEQ ID NO: 12
Sequence length: 5
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Met Leu Tyr Val Lys
1 5

SEQ ID NO: 13
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Ile Leu Ser Ile Asn Glu Lys
1 5

SEQ ID NO: 14
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Val Val Ile Leu Thr Glu Lys
1 5

SEQ ID NO: 15
Sequence length: 10
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asn Leu Glu Leu Ser Asp Pro Arg Val Lys
1 5 10

SEQ ID NO: 16
Sequence length: 12
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Met Ile Ile Gly Thr Tyr Arg Leu Gln Leu Asn Lys
1 5 10

SEQ ID NO: 17
Sequence length: 9
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Val Ala Val Leu Phe Ser Pro Ile Val
1 5 9

SEQ ID NO: 18
Sequence length: 11
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Ile Asn Ile Asp Glu Leu Ile Ile Gln Ser Lys
1 5 10

SEQ ID NO: 19
Sequence length: 12
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Glu Leu Gly Val Ser His Leu Tyr Leu Ser Pro Ile
1 5 10

SEQ ID NO: 20
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Glu Val Phe Arg Glu Ser
1 5

SEQ ID NO: 21
Sequence length: 4
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Tyr Phe Lys
1

SEQ ID NO: 22
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Gly Leu Tyr Asn Pro Lys
1 5

SEQ ID NO: 23
Sequence length: 8
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Ile Asn Gly Ile Arg Glu Cys
1 5

SEQ ID NO: 24
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Phe Glu Asn Phe Glu Lys
1 5

SEQ ID NO: 25
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Leu Leu Arg Pro Asn Ile
1 5

SEQ ID NO: 26
Sequence length: 5
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Ile Ile Glu Asn
1 5

SEQ ID NO: 27
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Asn Ile Glu Tyr Arg Gly
1 5

SEQ ID NO: 28
Sequence length: 18
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Array
YTCWCKRAAW ACYTCATC 18

SEQ ID NO: 29
Sequence length: 20
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Array
GATAAYATWG ARTAYAGRGG 20

SEQ ID NO: 30
Sequence length: 8
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Arg Asn Pro Glu Ala Tyr Thr Lys
1 5

SEQ ID NO: 31
Sequence length: 9
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Asp His Val Phe Gln Glu Ser His Ser
1 5

SEQ ID NO: 32
Sequence length: 8
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Ile Thr Leu Asn Ala Thr Ser Thr
1 5

SEQ ID NO: 33
Sequence length: 6
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Ile Ile Ile Val Glu Lys
1 5

SEQ ID NO: 34
Sequence length: 11
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Leu Gln Gln Tyr Met Pro Ala Val Tyr Ala Lys
1 5 10

SEQ ID NO: 35
Sequence length: 5
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Asn Met Leu Glu Ser
1 5

SEQ ID NO: 36
Sequence length: 13
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Lys Ile Ser Pro Asp Gln Phe His Val Phe Asn Gln Lys
1 5 10

SEQ ID NO: 37
Sequence length: 8
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Gln Leu Ala Glu Asp Phe Leu Lys
1 5

SEQ ID NO: 38
Sequence length: 10
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Lys Ile Leu Gly Phe Gln Glu Glu Leu Lys
1 5 10

SEQ ID NO: 39
Sequence length: 10
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Ile Ser Val Leu Ser Glu Phe Pro Glu Glu
1 5 10

SEQ ID NO: 40
Sequence length: 9
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Leu Lys Leu Glu Glu Gly Ala Ile Tyr
1 5

SEQ ID NO: 41
Sequence length: 8
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Glu Val Gln Ile Asn Glu Leu Pro
1 5

SEQ ID NO: 42
Sequence length: 5
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Asp His Ser Arg Ile
1 5

SEQ ID NO: 43
Sequence length: 6
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Asp Leu Arg Tyr Tyr Lys
1 5

SEQ ID NO: 44
Sequence length: 14
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Asp Val Tyr Arg Thr Tyr Ala Asn Gln Ile Val Lys Glu Cys
1 5 10

SEQ ID NO: 45
Sequence length: 10
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of N-terminal fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Thr Phe Ala Tyr Lys Ile Asp Gly Asn Glu
1 5 10

SEQ ID NO: 46
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Leu Gly Pro Tyr Phe Ser Gln
1 5

SEQ ID NO: 47
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Asp Val Phe Val Tyr Asp Gly
1 5

SEQ ID NO: 48
Sequence length: 19
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Origin of middle fragment Organism: Sulfolobus solfataricus
Stock name: KM1
Array
Tyr Asn Arg Ile Val Ile Ala Glu Ser Asp Leu Asn Asp Pro Arg Val
1 5 10 15
Val Asn Pro

SEQ ID NO: 49
Sequence length: 5
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Leu Asp Tyr Leu Lys
1 5

SEQ ID NO: 50
Sequence length: 17
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Lys Arg Glu Ile Pro Asp Pro Ala Ser Arg Tyr Gln Pro Leu Gly Val
1 5 10 15
His
17

SEQ ID NO: 51
Sequence length: 9
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Lys Asp Val Phe Val Tyr Asp Gly Lys
1 5

SEQ ID NO: 52
Sequence length: 9
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
His Ile Leu Gln Glu Ile Ala Glu Lys
1 5

SEQ ID NO: 53
Sequence length: 10
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Lys Leu Trp Ala Pro Tyr Val Asn Ser Val
1 5 10

SEQ ID NO: 54
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Met Phe Ser Phe Gly Gly Asn
1 5

SEQ ID NO: 55
Sequence length: 14
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Asp Tyr Try Tyr Gln Asp Phe Gly Arg Ile Glu Asp Ile Glu
1 5 10

SEQ ID NO: 56
Sequence length: 7
Sequence type: Number of amino acid chains: Single chain topology: Linear sequence type: Peptide fragment type: Intermediate fragment Origin: Sulfolobus acidocaldarius
Stock name: ATCC33909
Array
Lys Ile Asp Ala Gln Trp Val
1 5

SEQ ID NO: 57
Sequence length: 18
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Array
AGCWAGKAGM TAYCARCC 18

SEQ ID NO: 58
Sequence length: 24
Sequence type: Number of nucleic acid strands: Single-stranded topology: Linear sequence type: Other nucleic acid Synthetic DNA
Array
YTTHCCATCR TAWACRAAWA CATC 24

It is a graph which shows the analysis result by TSK-gel Amide-80 HPLC of the product by the microbial cell extract derived from Sulfolobus solfataricus KM1 strain obtained in Example I-1. It is a graph which shows the temperature stability of this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the pH stability of this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the reactivity in each temperature of this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows reaction optimal pH of this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the pattern of the reaction product from maltotriose by this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the pattern of the reaction product from maltotetraose by this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the pattern of the reaction product from maltopentaose by this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the analysis result by AMINEX HPX-42A HPLC of the reaction product from a maltooligosaccharide mixture by this enzyme transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example I-2. It is a graph which shows the analysis result by TSK-gel Amide-80 HPLC of the reaction product from maltotriosyl trehalose which made the crude enzyme liquid of Sulfolobus solfataricus KM1 strain | stump | stock obtained in Example II-1 act. It is a graph which shows the analysis result by AMINEX HPX-42A HPLC of the reaction product from the soluble starch which made the crude enzyme liquid of Sulfolobus solfataricus KM1 strain | stump | stock obtained in Example II-1 act. It is a graph which shows the temperature stability of this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2. It is a graph which shows the pH stability of this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2. It is a graph which shows the reactivity in each reaction temperature of this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2. It is a graph which shows reaction optimal pH of this enzyme amylase derived from Sulfolobus solfataricus KM1 strain | stump | stock obtained in Example II-2. It is a graph which shows the reactivity with respect to various substrates of this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2. The graph which shows the analysis result by AMINEX HPX-42A HPLC of the reaction product from maltopentaose, amylose DP-17, and soluble starch which made this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2 act. is there. It is a graph which shows the analysis result by TSK-gel Amide-80 HPLC of the reaction product from maltotriosyl trehalose which made this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2 act. It is a graph which shows the analysis result by TSK-gel Amide-80 HPLC of the reaction product from maltopentaosyl trehalose which made this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2 act. It is a graph which shows the time-dependent change of the disappearance of iodine coloring, and a starch hydrolysis rate when this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2 is made to act on soluble starch. It is a graph which shows the time-dependent change of the radioactivity of the reaction product from the radiolabeled maltopentaose which made this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2 act. It is a graph which shows the time-dependent change of the radioactivity of the reaction product from the radiolabeled maltotriosyl trehalose which made this enzyme amylase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2 act. It is a graph which shows the reactivity with respect to various substrates of (alpha) -amylase derived from a porcine pancreas. It is a graph which shows the analysis result by TSK-gel Amide-80 HPLC of the reaction product from maltopentaosyl trehalose which made the alpha pancreatic-derived alpha-amylase act. It is a graph which shows the analysis result by AMINEX HPX-42A HPLC of the reaction product from soluble starch which made this enzyme amylase and transferase derived from Sulfolobus solfataricus KM1 strain obtained in Example II-2. It is the figure which showed the restriction enzyme map of the insertion fragment of pKT1, pKT11, and pKT21 containing the novel transferase gene derived from Sulfolobus solfaricus KM1 obtained in Example I-12. It is the figure which showed the construction method of pKT22 plasmid. It is the figure which showed the analysis result by TSK-gel Amide-80 HPLC of the product when a recombinant novel transferase is made to act on maltotriose. It is the figure which showed the restriction enzyme map of the insertion fragment of p09T1 containing the novel transferase gene derived from Sulfolobus acidocaldarius ATCC33909 obtained in Example I-16. It is the figure which showed the construction method of p09T1 plasmid. It is the figure which showed the homology of the amino acid sequence of the novel transferase derived from Sulfolobus solfataricus KM1 strain and Sulfolobus acidocaldarius ATCC33909 strain. It is the figure which showed the homology of the base sequence of the novel transferase gene derived from Sulfolobus solfaricus KM1 strain and Sulfolobus acidocaldarius ATCC33909 strain. It is the figure which showed the homology of the base sequence of the novel transferase gene derived from Sulfolobus solfaricus KM1 strain and Sulfolobus acidocaldarius ATCC33909 strain. It is the figure which showed the analysis result by AMINEX HPX-42A HPLC of the product when a recombinant novel transferase is made to act on a maltooligosaccharide mixture. It is the figure which showed the restriction enzyme map of the insertion fragment of pKA1 containing the novel amylase gene derived from Sulfolobus sofaraticus KM1 strain. It is the figure which showed the restriction enzyme map of pKA2. (A) is the figure which showed the analysis result of the product when the recombinant novel amylase by this invention was made to act on maltotriosyl trehalose, and (B) is the recombinant novel amylase by this invention to soluble starch. It is the figure which showed the analysis result of the product when it was made to act. It is a graph which shows the time-dependent change of the loss | disappearance of iodine coloring when the recombinant novel amylase by this invention is made to act on soluble starch, and a starch hydrolysis rate. It is a restriction map of the insert fragment of p09A1 containing a novel amylase gene derived from Sulfolobus acidocaldarius ATCC33909. It is the figure which showed the preparation method of p09A1 from p09A2. It is the figure which compared the homology about the amino acid sequence of the novel amylase derived from Sulfolobus acidocaldarius ATCC33909 strain and Sulfolobus solfataricus KM1 strain. It is the figure which compared the homology about the base sequence of the novel amylase gene derived from Sulfolobus acidocaldarius ATCC33909 strain and Sulfolobus solfataricus KM1 strain. It is the figure which compared the homology about the base sequence of the novel amylase gene derived from Sulfolobus acidocaldarius ATCC33909 strain and Sulfolobus solfataricus KM1 strain. It is the figure which showed the analysis result of the product when the recombinant novel amylase of Example II-19 and the recombinant novel transferase of Example I-20 were made to act on 10% soluble starch.

Claims (25)

Three or more sugars obtained from Sulfolobus solfataricus KM1 (FERM BP-4626) strain having one or more sequences selected from SEQ ID NOs: 45 to 48, wherein at least three or more sugars are composed of glucose units from the reducing end. Is a novel amylase having the activity of hydrolyzing from the reducing end side to mainly release monosaccharides and / or disaccharides, and has a molecular weight of 61,000-64,000 by SDS polyacrylamide gel electrophoresis And the enzyme having the following physicochemical properties:
(1) Optimum pH: 4.5 to 5.5
(2) Optimal temperature: 70 to 85 ° C
(3) Stable pH: 3.5-10.0
(4) Temperature stability: 100% remaining after treatment at 85 ° C. for 6 hours (5) Isoelectric point by isoelectric focusing method: 4.8
(6) Reaction inhibition: 100% inhibition with 5 mM CuSO ₄ . The reducing end is obtained from a Sulfolobus acidocaldarius ATCC33909 strain having one or more sequences selected from SEQ ID NOs: 49 to 56, using at least three sugars composed of glucose units as a substrate. A novel amylase having an activity of hydrolyzing from the side to mainly release monosaccharides and / or disaccharides, having a molecular weight of 61,000 to 64,000 as determined by SDS polyacrylamide gel electrophoresis, The above enzyme having the following physicochemical properties:
(1) Optimum pH: 5.0 to 5.5
(2) Optimal temperature: 60-80 ° C
(3) Stable pH: 4.0 to 13.0
(4) Temperature stability: 100% remaining after treatment at 80 ° C. for 6 hours (5) Isoelectric point by isoelectric focusing method: 5.4
(6) Reaction inhibition: 100% inhibition with 5 mM CuSO ₄ . At least the reducing terminal trisaccharide obtained from Sulfolobus solfataricus KM1 (FERM BP-4626) strain having one or more sequences selected from SEQ ID NOs: 45 to 48 is composed of glucose units; Substrates of three or more sugars in which the bond between the second glucose is α-1, α-1 bond and the bond between the second and third glucose at the terminal is α-1,4 bond The novel amylase according to claim 1, which has a main activity of hydrolyzing the α-1,4 bond between the second and third glucose and releasing α, α-trehalose, The enzyme having a molecular weight of 61,000-64,000 as determined by polyacrylamide gel electrophoresis and having the following physicochemical properties:
(1) Optimum pH: 4.5 to 5.5
(2) Optimal temperature: 70 to 85 ° C
(3) Stable pH: 3.5-10.0
(4) Temperature stability: 100% remaining after treatment at 85 ° C. for 6 hours (5) Isoelectric point by isoelectric focusing method: 4.8
(6) Reaction inhibition: 100% inhibition with 5 mM CuSO ₄ . At least the trisaccharide on the reducing end side obtained from Sulfolobus acidocaldarius ATCC33909 strain having one or more sequences selected from SEQ ID NOs: 49 to 56 is composed of glucose units, between the first and second glucose at the end Are α-1 and α-1 linkages, and the linkage between the second and third glucoses at the terminal is α-1,4 linkages, and a saccharide of three or more sugars is used as a substrate. 3. The novel amylase according to claim 2 , which has a main activity of hydrolyzing an α-1,4 bond between the third glucose and releasing α, α-trehalose, wherein the polyacrylamide gel electrophoresis method is SDS. The above enzyme having a molecular weight of 61,000 to 64,000 and having the following physicochemical properties:
(1) Optimum pH: 5.0 to 5.5
(2) Optimal temperature: 60-80 ° C
(3) Stable pH: 4.0 to 13.0
(4) Temperature stability: 100% remaining after treatment at 80 ° C. for 6 hours (5) Isoelectric point by isoelectric focusing method: 5.4
(6) Reaction inhibition: 100% inhibition with 5 mM CuSO ₄ .   The Sulfolobus solfataricus KM1 (FERM BP-4626) strain having the ability to produce the amylase according to claim 1 or 3 is cultured in a medium, and α, α-trehalose is produced from the culture using trehalose oligosaccharide as a substrate. 4. The method for producing amylase according to claim 1 or 3, wherein the amylase is isolated and purified based on an activity measurement method using activity as an index.   The activity which uses as an index the activity which produces | generates Sulforobus acidocaldarius ATTCC33909 strain bacteria which have the capability to produce the amylase of Claim 2 or 4 to a culture medium, and uses a trehalose oligosaccharide as a substrate from a culture. The method for producing amylase according to claim 2 or 4, wherein the amylase is isolated and purified based on a measurement method. The amino acid sequence shown in SEQ ID NO: 6, or a sequence in which some amino acids are inserted, substituted, or deleted, or added to both ends in the sequence , and at least three or more sugars from the reducing end Contains a DNA sequence that encodes a sequence that still retains a novel amylase activity that uses 3 or more sugars composed of glucose units as a substrate and hydrolyzes from the reducing end side to release mainly monosaccharides and / or disaccharides A DNA fragment consisting of The DNA fragment according to claim 7 , comprising the base sequence from the 642nd to the 2315th of the base sequence shown in SEQ ID NO: 5. The DNA fragment according to claim 7 , comprising a base sequence from 639th to 2315th of the base sequence shown in SEQ ID NO: 5. The DNA fragment according to claim 7 , comprising the nucleotide sequence from the 1st to the 2691th of the nucleotide sequence shown in SEQ ID NO: 5. The amino acid sequence shown in SEQ ID NO: 8, or a sequence in which some amino acids are inserted, substituted, or deleted, or added to both ends in the sequence , and at least three or more sugars from the reducing end Contains a DNA sequence that encodes a sequence that still retains a novel amylase activity that uses 3 or more sugars composed of glucose units as a substrate and hydrolyzes from the reducing end side to release mainly monosaccharides and / or disaccharides A DNA fragment consisting of The DNA fragment according to claim 11 , comprising the base sequence from 1176th to 2843th of the base sequence shown in SEQ ID NO: 7. The DNA fragment according to claim 11 , comprising the base sequence from the 1st to the 3600th of the base sequence shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 6, or a sequence in which some amino acids are inserted, substituted, or deleted, or added to both ends in the sequence , and at least three or more sugars from the reducing end A polypeptide comprising a sequence that still retains a novel amylase activity that is hydrolyzed from the reducing end side to liberate mainly monosaccharides and / or disaccharides using a saccharide of three or more sugars composed of glucose units as a substrate . The amino acid sequence shown in SEQ ID NO: 8, or a sequence in which some amino acids are inserted, substituted, or deleted, or added to both ends in the sequence , and at least three or more sugars from the reducing end A polypeptide comprising a sequence that still retains a novel amylase activity that is hydrolyzed from the reducing end side to liberate mainly monosaccharides and / or disaccharides using a saccharide of three or more sugars composed of glucose units as a substrate . The polypeptide according to claim 14 , further comprising Met at its N-terminus. At least the three sugars on the reducing terminal side are composed of glucose units, and the bond between the first and second glucoses on the terminal side is an α-1, α-1 bond. The linkage between the first glucose is α-1,4 bond, and a saccharide of three or more sugars is used as a substrate to hydrolyze the α-1,4 bond between the second and third glucose, α, The polypeptide according to any one of claims 14 to 16 , which has an action of releasing α-trehalose. (1) an activity of hydrolyzing an α-1,4 glucoside bond in a sugar chain in an endo form,
(2) At least three or more sugars from the reducing end are composed of glucose units, and the sugar is a monosaccharide by hydrolysis from the reducing end side using a sugar of three or more sugars whose bonds are α-1,4 bonds as a substrate. And / or the activity of releasing disaccharides, and (3) at least the reducing end trisaccharide is composed of glucose units, and the bond between the first and second glucoses on the terminal side is α-1, α -1 bond, and the second and third glucoses using as a substrate a saccharide of three or more sugars in which the bond between the second and third glucoses on the terminal side is an α-1,4 bond The polypeptide according to any one of claims 14 to 16 , which has a main activity of hydrolyzing an α-1,4 bond therebetween to release α, α-trehalose. Comprising a DNA fragment according to any one of claims 7 to 13, the recombinant DNA molecule. The recombinant DNA molecule according to claim 19 , wherein the DNA fragment according to any one of claims 7 to 13 is incorporated into a plasmid vector. A host cell transformed with the recombinant DNA molecule according to claim 19 or 20 . The host cell according to claim 21 , wherein the host cell is a microorganism belonging to the genus Escherichia or Bacillus. The host cell according to claim 22 , wherein the host cell is Escherichia coli JM109 strain. A method for producing a recombinant novel amylase, wherein the recombinant novel amylase comprises at least a trisaccharide on the reducing end side composed of glucose units, and a bond between the first and second glucoses on the terminal side is α- 1 and α-1 linkages, and the second and third sugars are composed of 3 or more sugars whose linkages between the second and third glucoses on the terminal side are α-1,4 linkages. Hydrolyze the α-1,4 bond between glucose in the eye and have the main activity of releasing α, α-trehalose,
24. A method comprising culturing a host cell according to any one of claims 21 to 23 , producing the recombinant novel amylase in the culture and collecting it. A recombinant novel amylase encoded by the DNA fragment according to any one of claims 7 to 13 , or a recombinant novel amylase comprising the polypeptide according to any one of claims 14 to 18. Law,
24. A method comprising culturing a host cell according to any one of claims 21 to 23 , producing the recombinant novel amylase in the culture and collecting it.

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