JP2004187674A - New amylomaltase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an amylomaltase of which the reactivity to a substrate is increased and/or the hydrolysis reaction is reduced or eliminated. <P>SOLUTION: Amino acid residues existing in a region interactive with an acarbose inhibitor which is thought to be important in enzyme reaction are modified in an amino acid sequence of the amylomaltase. The amylomaltase of which the reactivity to the substrate is increased and/or the hydrolysis reaction is decreased or eliminated is provided by the modification. Further, various uses in which an amylomaltase modified product is used, a glucan, a food, and the like obtained by the same are provided, respectively. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a novel amylomaltase, a gene encoding the amylomaltase, and a method for producing the amylomaltase. The present invention also relates to a method for producing cyclic glucan using the novel amylomaltase, and a method for producing and modifying foods and the like. The detailed description of the invention is described below.

  Amylomaltase (EC 2.4.1.25) is an enzyme called amylomaltase, 4-α-glucanotransferase and the like, and plant-derived amylomaltase is also called D-enzyme. This enzyme catalyzes a reaction of transferring an α-glucan chain from one α-glucan molecule to another α-glucan molecule (or glucose). Amilomartase is widely distributed in microorganisms such as Escherichia coli and Thermus bacteria, and plant tissues such as potato tubers, malt barley, sweet potatoes, and spinach. Microbial-derived amylomaltase and plant-derived amylomaltase have similarity in amino acid sequence.

  In recent years, it has been found that amylomaltase can catalyze the cyclization reaction of α-glucan. For example, it has been reported that potato tuber-derived D-enzyme can synthesize cycloamylose, which is a cyclic α-glucan having a polymerization degree of about 17 or more, by catalyzing the intramolecular transfer reaction (cyclization reaction) of amylose. Yes. It has been reported that amylomaltase can also synthesize a branched cyclic glucan by catalyzing a cyclization reaction of an α-glucan containing a branched structure having an α-1,6-linkage such as amylopectin (Patent Document 1). See). By utilizing this reaction, it is possible to synthesize branched cyclic glucan having various structures such as inner branched cyclic glucan and outer branched cyclic glucan.

  Such a cyclic glucan has an ability to form an inclusion compound and has a very high solubility in water, and therefore is expected to be applied to foods, pharmaceuticals and the like. Cyclic glucan is particularly useful for raw materials in the starch processing industry, food and drink compositions, food additive compositions, infusion solutions, adhesive compositions, inclusions or adsorbents, starch anti-aging agents, or biodegradable plastics. It is useful as a substitute for starch (see Patent Document 1).

  To date, plant-derived amylomaltase (D-enzyme) has been recognized in various plant tissues such as potato tubers, germinated barley, sweet potatoes, and spinach green leaves. For potato-derived D-enzyme, cDNA has been isolated and the nucleotide sequence has been determined (Non-patent Document 1). Potato-derived D-enzyme is expressed in Escherichia coli in an active state and used for the production of cycloamylose on a laboratory scale (Non-patent Document 2).

  On the other hand, as microorganism-derived amylomaltase, an enzyme derived from Escherichia coli, which is a mesophilic bacterium, has been well studied (Non-patent Document 3). The base sequences of the amylomaltase genes of Haemophilus influenzae, Streptococcus, and Clostridium bacteria, which are also mesophilic bacteria, have been determined (GenBank accession numbers U32760, JO1796, L37874). Recently, thermostable amylomaltase has also been identified from thermostable bacteria such as Thermus genus bacteria (see Patent Document 2).

  Amylomaltase can be applied in various industrial applications. As one example, amylomaltase can be utilized in alpha-glucan processing such as the production of cyclic glucan. When an enzyme is used industrially, it is desirable to carry out the reaction at as high a temperature as possible, preferably at about 60 ° C. or higher. This is to prevent aging of the substrate α-glucan and prevent contamination of the reaction system by various bacteria. As another example of applying amylomaltase to industrial applications, amylomaltase can be used directly to modify foods rich in starch. The use of glucanotransferase, such as amylomaltase, as an enzyme for modifying foods has also been studied (see, for example, Patent Document 3). This is because amylomaltase lowers the molecular weight of starch by a cyclization reaction to produce a cyclic glucan having an antiaging effect. For such applications, the thermostable amylomaltase derived from the Thermus genus has been found to be useful (see Patent Document 2).

  As described above, amylomaltase is used in various applications, but its reactivity with respect to a substrate is not so high, and there is also a disadvantage that hydrolysis activity remains. Therefore, when using a conventional enzyme, it has been necessary to devise considerable measures in order to suppress the side reaction of hydrolysis activity.

  Even the amylomaltase derived from the above-mentioned Thermus genus is not very high in substrate reactivity, and the hydrolysis activity still remains.

Therefore, there is a need for an amylomaltase that does not substantially catalyze or reduce the hydrolysis reaction and / or has increased reactivity.
JP-A-8-311103 JP 11-46780 A International Application Publication WO 97/41735 J. et al. Biol. Chem. vol. 268, pp. 1391-1396 (1993) J. et al. Biol. Chem. vol. 271, pp. 2902-2908 (1996) Eur. J. et al. Biochem. vol. 69, pp. 105-115 (1976)

  An object of the present invention is to provide an amylomaltase having increased reactivity to a substrate and / or reduced or eliminated hydrolysis reaction.

  The present inventors have elucidated the three-dimensional structure of amylomaltase discovered so far, and modified amino acid residues in the region that interacts with acabose, which is considered to be important for enzymatic reactions based on the elucidated information. By unexpectedly finding that the enzyme activity is improved, in particular, a dramatic modification of the yield of cycloamylose and a decrease in hydrolysis activity are preferably achieved simultaneously. completed. In addition, with a wild-type enzyme, the yield is lowered when the substrate concentration is increased, and the production can be performed only at a low concentration, so that the productivity is low. Therefore, in such a low productivity state, industrial use is difficult. The present invention can also solve such problems.

  Accordingly, the present invention provides the following.

(1) an amylomaltase polypeptide,
A) comprising at least one substitution, addition or deletion in the amino acid sequence of the wild-type amylomaltase polypeptide,
The substitution, addition or deletion includes substitution in the amino acid sequence of the wild-type amylomaltase polypeptide with an amino acid other than the amino acid of the wild-type sequence at the position of the amino acid residue that interacts with abose.
Amylomaltase polypeptide.

  (2) The amylomaltase polypeptide according to item 1, having at least one characteristic selected from the group consisting of an increase in enzyme activity and a decrease in hydrolysis activity, as compared to the wild-type amylomaltase polypeptide.

  (3) The amylomaltase according to item 1, wherein the property includes a decrease in the hydrolysis activity, and the decrease in the hydrolysis activity includes a decrease of at least 10% or more compared to the wild-type amylomaltase polypeptide. Polypeptide.

  (4) The amylomaltase according to item 1, wherein the characteristics include a decrease in the increase in the enzyme activity, and the increase in the enzyme activity includes an increase of at least 10% as compared with the wild-type amylomaltase polypeptide. Polypeptide.

(5) an amylomaltase polypeptide,
A) comprising at least one substitution, addition or deletion in the amino acid sequence of the wild-type amylomaltase polypeptide,
The above substitution, addition, or deletion is performed by the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine of the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2, Amino acid at each residue position shown in SEQ ID NO: 1 at the residue position of the wild-type amylomaltase polypeptide corresponding to at least one residue selected from the group consisting of position tyrosine and position 153 glycine Including having an amino acid other than
Amylomaltase polypeptide.

(6) The amylomaltase polypeptide according to item 1,
The substitution, addition, or deletion is performed at the position of the 54-position tyrosine of the wild-type amylomaltase polypeptide at the residue corresponding to the 54-position tyrosine of the amylomaltase from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2. Including having an amino acid other than the amino acid of the corresponding residue,
Amylomaltase polypeptide.

(7) The amylomaltase polypeptide according to item 1,
A) in the sequence shown in SEQ ID NO: 2 comprising at least one substitution, addition or deletion, wherein said substitution, addition or deletion comprises a substitution to an amino acid other than tyrosine at position 54 tyrosine,
An amylomaltase polypeptide.

  (8) The amylomaltase polypeptide according to item 7, wherein amino acids other than tyrosine are substituted by non-conservative substitution.

  (9) The amylomaltase polypeptide according to item 7, wherein the amino acid other than tyrosine is selected from the group consisting of natural amino acids other than tyrosine and phenylalanine.

  (10) The amino acid other than tyrosine is selected from the group consisting of alanine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, arginine, serine and threonine, amylomaltase polysal peptide.

  (11) The amylomaltase polypeptide according to item 7, wherein the amino acid other than tyrosine is an amino acid other than tyrosine, phenylalanine and lysine.

  (12) The amylomaltase polypeptide according to item 7, wherein the amino acid other than tyrosine is selected from the group consisting of glycine, proline, threonine and tryptophan.

  (13) The amylomaltase polypeptide according to item 7, wherein the amino acid other than tyrosine is threonine or glycine.

  (14) The amylomaltase polypeptide according to item 7, wherein the amino acid other than tyrosine is glycine.

  (15) A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide according to any one of items 1 to 14.

  (16) A vector comprising the nucleic acid molecule according to item 15.

  (17) A cell comprising the nucleic acid molecule according to item 15.

  (18) A biological tissue comprising the nucleic acid molecule according to item 15.

  (19) A transgenic organism comprising the nucleic acid molecule according to item 15.

  (20) A step of adding the amylomaltase polypeptide according to any one of items 1 to 14 to a food material before or immediately after the heat treatment of the material, wherein the amylomaltase polypeptide is the food. A method for producing a food comprising a step of producing cyclic glucan from starch in a material.

  (21) The above-mentioned food is cooked rice, Japanese confectionery, snack confectionery, bakery, noodles, dumplings and shumai peels, fishery products, frozen or refrigerated processed foods, baby food, baby food, pet food, animal feed, Item 21. The method of item 20, wherein the method is selected from the group consisting of beverages, sports foods, and nutritional supplements.

  (22) A food material, food additive, or food modifier containing the polypeptide according to any one of items 1 to 14.

  (23) α-1,4-glucoside, comprising a step of reacting linear α-1,4-glucan or a saccharide containing these with the polypeptide according to any one of items 1 to 14. A method for producing glucan having one cyclic structure formed by a bond in the molecule or a derivative thereof.

  (24) A food material, a food additive, comprising a step of reacting linear α-1,4-glucan or a saccharide containing these with the polypeptide according to any one of items 1 to 14, A method for producing a food modifier, a food-drinking composition, an infusion solution or an adhesive composition.

  (25) A computer-readable recording medium comprising information on the amino acid sequence of the amylomaltase polypeptide according to any one of items 1 to 14 or a nucleic acid sequence encoding the amino acid sequence.

  (26) A cyclic glucan obtained by allowing the amylomaltase polypeptide according to any one of items 1 to 14 to act on a linear α-1,4-glucan or a saccharide containing these.

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  The present invention provides a modified amylomaltase having increased enzyme activity and / or decreased undesired hydrolysis activity as compared to conventional wild-type amylomaltase. The present invention also provides an effect of mass production of cycloamylose and cyclic glycans for industrial use by providing a mutant enzyme capable of maintaining a high yield even at a high substrate concentration.

  Embodiments of the present invention will be described below. Throughout this specification, it is to be understood that the terms used in this specification are used in the meaning normally used in the art unless otherwise specified.

(the term)
In the present specification, “amylomaltase” and “amylomaltase enzyme” are used interchangeably unless otherwise indicated, and mean an enzyme having amylomaltase activity. As amylomaltase, for example, derived from plants (potato tubers, germinated barley, sweet potato, spinach green leaves), microorganisms (E. coli, Haemophilus influenzae, Streptococcus, Clostridium bacteria, Thermus bacterium (eg, Thermus aquaticus, Thermus flavus, etc.) These amylomaltases have similar amino acid sequences not only between microorganisms and between plants but also between plants and microorganisms, and particularly their substrate interaction sites are not limited thereto. The sequence is well conserved (see FIG. 1. In particular, the amino acid sequence of amylomaltase from Thermus aquaticus ATCC 33923 is aspartic acid at position 293, glutamic acid at position 340, aspartic acid at position 395. The position of the formic acid and the corresponding amino acid residue position is included.) Thus, the properties of the enzyme due to the sequence responsible for the substrate interaction site in one amylomaltase are This is similar to the properties of the enzyme due to the corresponding sequence responsible for the site of action, and this is also apparent from the results of three-dimensional structural analysis and computer modeling (Przylas I. et al. J). Mol.Biol.296,873-886 (2000), Przylas et al.Eur.J.Biochem.267, 6903-6913 (2000)).

  In the present specification, “α-glucan” means α-1,4-glucan (polysaccharide having a chain structure having maltose as a constituent disaccharide unit), or α-, having an α-1,6-branched structure. 1,4-glucan, and includes amylose, amylopectin, starch and glycogen, as well as waxy starch, high amylose starch, soluble starch, dextrin, starch hydrolysate, phosphorylase and enzymatically synthesized amylopectin.

  In this specification, “cyclic glucan” means cyclic α-1,4-glucan having only α-1,4-glucoside bond, and α-1,4-glucoside bond and α-1,6-glucoside bond. A branched cyclic glucan having both of the above. “Branched” means having at least one glucoside bond other than α-1,4-bond. Examples of the branched cyclic glucan include an inner branched cyclic glucan having a branched structure having an α-1,6-link in the cyclic structure, and an outer branched cyclic glucan having a non-cyclic structure portion in addition to the cyclic structure. Etc.

  In the present specification, “cycloamylose (CA)” is a cyclic α-1,4-glucan having a degree of polymerization of about 17 or more.

  In this specification, “amylomaltase activity” means that amylomaltase is allowed to act at 70 ° C. for 10 minutes in a composition solution consisting of 50 mM sodium acetate (pH 5.5), 10% (w / v) maltotriose. It refers to the activity measured by quantifying the glucose produced at the time. The activity that generates 1 μmol of glucose per minute is defined as 1 unit.

  In the present specification, “substantially does not catalyze the hydrolysis reaction” means a buffer solution (100 mM sodium acetate (pH 5.5)) containing enzymatically synthesized amylose AS-110 having a final concentration of 0.2% (w / v) 9% (v / v) DMSO), when 0.07 unit / ml amylomaltase was allowed to act at 70 ° C. for 24 hours, the yield of cycloamylose was observed to be lower than the maximum yield. It means not being able to.

  In the present specification, “quantification of cycloamylose” refers to quantification of cycloamylose in the reaction product obtained by allowing amylomaltase to act on amylose. The amount of cycloamylose is measured as the amount of glucan that is not degraded by glucoamylase when the reaction product is digested with glucoamylase.

  In the present specification, the “yield of cycloamylose” refers to a value calculated from the quantified amount of cycloamylose as a weight ratio to the substrate.

In this specification, for example, “maintain activity” for 10 minutes or more at 60 ° C. means that amylomaltase is added at 60 ° C. in buffer A (10 mM KH ₂ PO ₄ -Na ₂ HPO ₄ , pH 7.0). It means that no decrease in activity is observed when treated for 10 minutes. For example, “inactivate” in 100 minutes at 100 ° C. means that amylomaltase is treated at 100 ° C. for 15 minutes in buffer A (10 mM KH ₂ PO ₄ —Na ₂ HPO ₄ , pH 7.0). This means that no activity is observed. The specific temperature and time for maintaining and deactivating the activity may be appropriately changed.

  As used herein, “optimum reaction pH” refers to amylo-acid for 10 minutes at 70 ° C. at each pH in the presence of 10% (w / v) maltotriose in Britton-Rhobinson buffer. It means the pH with the highest activity when maltase is allowed to act.

  In the present specification, the “optimum reaction temperature” means that in each buffer at a temperature of 10% (w / v) maltotriose in a buffer solution (50 mM sodium acetate (pH 5.5)). It means the temperature at which activity is highest when amylomaltase is allowed to act for 10 minutes at pH 5.5.

  In the present specification, the “thermophilic bacterium” refers to a microorganism having an optimum growth temperature of 50 ° C. to 105 ° C. and hardly proliferating at 30 ° C. or less. Among these, a microorganism having an optimum growth temperature of 90 ° C. or higher is referred to as “super thermophilic bacterium”.

  In the present specification, the “mesophilic bacterium” refers to a microorganism having a growth temperature in a normal temperature environment, and particularly refers to a microorganism having an optimum growth temperature of 20 ° C. to 40 ° C.

  As used herein, “increased reactivity to substrate” or “enhance enzyme activity” is used interchangeably and is a buffer containing enzyme-synthesized amylose AS-110 at a final concentration of 0.2% (w / v). Obtained by reacting 0.022 units / ml amylomaltase at 70 ° C. in liquid (100 mM sodium acetate (pH 5.5), 9% (v / v) DMSO). It means that the yield of cycloamylose (CA) in the reaction product increases. In the present invention, an increase in reactivity to a substrate or an increase in enzyme activity can be said to be a favorable result because cycloamylose, which is the target product, can be obtained more efficiently. Such an increase in yield is, for example, at least about 10% or more, preferably about 30% or more, more preferably about 50% or more, and most preferably about 80% or more compared to the wild type.

  In this specification, “the hydrolysis activity decreases” means a buffer solution (50 mM sodium acetate (pH 5.5) containing cycloamylose having a final concentration of 0.5% (w / v) and a degree of polymerization of about 20-50. ), When amylomaltase is allowed to act at 70 ° C. for 1 hour, the amount of reduced sugar derived from hydrolysis is reduced by the hydrolysis of a reference enzyme (for example, wild-type amylomaltase). It means that it is decreasing compared to the amount of increase. In the present invention, the hydrolysis activity is an activity of degrading the target product, cycloamylose, and it is preferable that such activity is reduced. Such a decrease in hydrolytic activity is, for example, at least about 10% or more, preferably about 30% or more, more preferably about 50% or more, more preferably about 80% or more, and most preferably about wild-type. 90% or more (that is, the residual activity is less than about 10%).

(Amylomaltase and its purification)
One feature of the amylomaltase of the present invention is that the reactivity with respect to the substrate is increased as compared with the conventional wild-type amylomaltase. Another feature of the amylomaltase of the present invention is that its hydrolytic activity is lower than that of conventional wild-type amylomaltase. Preferably, the amylomaltase of the present invention has an increased reactivity with respect to a substrate and a reduced activity and hydrolysis activity compared to conventional wild-type amylomaltase. Preferably, the amylomaltase of the present invention is heat resistant. The amylomaltase of the present invention preferably remains active at 70 ° C. for 10 minutes, more preferably at 80 ° C. for 10 minutes, preferably at 100 ° C. for 10 minutes, more preferably at 100 ° C. for 5 minutes. The amylomaltase of the present invention preferably does not substantially catalyze the hydrolysis reaction even at 70 ° C.

  An amylomaltase (also referred to as an amylomaltase variant) comprising a modification by deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is within the scope of the present invention. The polypeptide in Such deletions, substitutions or additions of one or more amino acids are described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Biomolecules Biomolecules in Bio38. John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5562 (1984), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.

  For such modification, the amino acid sequence of amylomaltase derived from Thermus aquaticus shown in SEQ ID NO: 2 at position 53 glycine, position 54 tyrosine, position 55 glycine, position 56 aspartic acid, position 98 glycine, position 101 tyrosine and position 153 It includes that the wild-type amylomaltase polypeptide residue corresponding to at least one residue selected from the group consisting of glycine has an amino acid other than the amino acid at each position shown in SEQ ID NO: 1. . The modified amylomaltase has an activity equal to or higher than that of wild-type amylomaltase without modification, and has hydrolytic activity equivalent to or less than that of wild-type amylomaltase without modification. Have. Preferably, the amylomaltase variant exhibits the same or better or worse heat resistance as wild-type amylomaltase without modification and does not substantially catalyze the hydrolysis reaction. Such modifications may occur naturally or may be caused by the action of mutagens or artificially using the introduction of site-directed mutagenesis. Site-directed mutagenesis is preferred. This is because if site-directed mutagenesis is used, a target modification can be introduced at a target site.

  Methods for producing polypeptide variants utilized herein are well known in the art. For example, amino acid deletion, substitution or addition of the polypeptide of the present invention can be carried out by site-directed mutagenesis which is a well-known technique. Site-directed mutagenesis techniques are well known in the art. For example, Nucl. Acid Research, Vol. 10, pp. 6487-6500 (1982).

  As used herein, “deletion, substitution, or addition of one or more amino acids” or “deletion, substitution, or addition of at least one amino acid” when used for amylomaltase refers to an enzyme of amylomaltase The number of deletions, substitutions, or additions to such an extent that the activity (preferably cycloamylose synthesis activity) is not lost, and preferably the enzyme activity is equal to or higher than that of the reference (eg, wild type) Say. One skilled in the art can readily select amylomaltase variants having the desired properties. Alternatively, the polypeptide of interest may be synthesized directly from the desired sequence. Such chemical synthesis methods are well known in the art.

  The modified amylomaltase of the present invention can be obtained as follows. After mass-culturing the host organism transformed to produce the modified amylomaltase, the culture is crushed, and the culture crushed material is separated by centrifugation or filtration to obtain a supernatant. Use crude enzyme solution. Furthermore, a purified enzyme with improved specific activity can be obtained by appropriately combining the crude enzyme solution with conventional enzyme purification means such as dialysis, lyophilization, isoelectric focusing, ion exchange chromatography, and crystallization. it can.

(Amylomaltase gene)
Genes encoding amylomaltase variants of the invention are also within the scope of the invention. Such genes can be obtained using methods known in the art based on the disclosure of the present specification.

  For example, purified amylomaltase derived from Thermus aquaticus ATCC 33923, Thermus flavus, and the like is trypsinized, the resulting digested fragments are separated by HPLC, and the N-terminal sequence of the peptide fragment corresponding to the obtained peak is converted into a peptide sequencer. To identify. Then, by using a synthetic oligonucleotide probe prepared based on the identified sequence, an appropriate genomic library or cDNA library can be screened to obtain a wild-type amylomaltase gene that is the basis of the modified type. . The basic strategies for preparing oligonucleotide probes and DNA libraries and for screening them by nucleic acid hybridization are well known to those skilled in the art. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (1989); DNA Cloning, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed Hc 1984); D. Hames & S. J. Higgins (1984).

  When screening a genomic library, the resulting gene can be subcloned using methods well known to those skilled in the art. For example, a plasmid containing the target gene can be easily obtained by mixing λ phage containing the target gene, appropriate E. coli, and appropriate helper phage. Subsequently, the gene of interest can be subcloned by transforming appropriate E. coli with a solution containing the plasmid. The obtained transformant is cultured to obtain plasmid DNA by, for example, alkaline SDS method, and the base sequence of the target gene can be determined. Methods for determining the base sequence are well known to those skilled in the art. In addition, using primers synthesized based on the base sequence of the DNA fragment, the amylomaltase gene is directly amplified using polymerase chain reaction (PCR) using the genomic DNA such as Thermus flavus and Thermus aquaticus ATCC 33923 as a template. You can also.

  Based on the nucleic acid sequence or amino acid sequence information of the wild-type amylomaltase gene thus obtained, the modification of the present invention is introduced. Such introduction methods can be performed using well-known methods, by the action of mutagens, or artificially using the introduction of site-directed mutagenesis. Site-directed mutagenesis is preferred. This is because if site-directed mutagenesis is used, a target modification can be introduced at a target site. Alternatively, a nucleic acid molecule having a target sequence may be directly synthesized. Such chemical synthesis methods are well known in the art.

  A gene consisting of a DNA having the base sequence shown in SEQ ID NO: 1 in the sequence listing, and a gene consisting of a DNA hybridizing under stringent conditions with the DNA having the base sequence shown in SEQ ID NO: 1 in the sequence listing are: Within the scope of the invention. It has an activity equal to or higher than that of wild-type amylomaltase without modification, and has hydrolytic activity equivalent to or less than that of wild-type amylomaltase without modification. Preferably, the amylomaltase variant exhibits the same or better or worse heat resistance as wild-type amylomaltase without modification and does not substantially catalyze the hydrolysis reaction. Those skilled in the art can easily select a desired amylomaltase gene.

  As used herein, the term “stringent conditions” refers to conditions that hybridize to specific sequences but not to non-specific sequences. The setting of stringent conditions is well known to those skilled in the art and is described, for example, in Molecular Cloning (Sambrook et al., Supra). Specifically, for example, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized, and then 0.1 to 2 times. Polynucleotides that can be identified by washing the filter at 65 ° C. using a SSC (saline-sodium citrate) solution at a concentration (composition of a 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) Means.

(Recombinant expression of amylomaltase)
Expression of the cloned or modified gene is achieved by constructing an appropriate expression vector into which the DNA encoding it has been inserted, introducing the expression vector into a microorganism, and culturing the recombinant organism and collecting the product. Is done. These methods are well known to those skilled in the art. Biological hosts used in the present invention include prokaryotes and eukaryotes. Preferred prokaryotic hosts include mesophilic bacteria such as E. coli. Such recombinant organisms may be microorganisms, but may be plants, animals, and the like. Examples of the plant include monocotyledonous plants such as dicotyledonous plants, rice, wheat, barley, and corn, but are not limited thereto. Grains such as rice have the property of accumulating storage protein in seeds, and it is possible to express the modified product of the present invention so as to accumulate in seeds using a storage protein system (Japanese Patent Laid-Open No. 2002-2002). 58492).

  An expression vector refers to a medium that is operably linked so that a gene of interest is transcribed and translated, and further includes factors necessary for replication in a microorganism and selection of a recombinant, as necessary. When secretory production of the expression product is intended, the base sequence encoding the secretory signal peptide is bound in the correct reading frame upstream of the DNA encoding the target protein. It is well known to those skilled in the art that the type of appropriate expression vector can vary depending on the biological host used.

  In some cases, the target amylomaltase gene should be processed in order to be operably linked to factors required for transcription and translation in the expression vector. These are, for example, when the distance between the promoter and the coding region is too long and a decrease in transcription efficiency is expected, or when the interval between the ribosome binding site and the translation initiation codon is not appropriate. Examples of processing means include digestion with restriction enzymes, digestion with exonucleases such as Bal31 and ExoIII, or introduction of site-directed mutagenesis using single-stranded DNA such as M13 or PCR.

  For the culture of a transformed strain that has acquired the ability to produce amylomaltase by introducing an expression vector, an appropriate condition depends on the host organism to be used, the type of factor that regulates expression in the expression vector, and the substance to be expressed. Is selected. For example, a normal shaking culture method can be used.

The medium used for culturing the transformant is not particularly limited as long as the host to be used can grow. In addition to carbon and nitrogen sources, the medium includes inorganic salts such as phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Ni ²⁺ , Na ⁺ , K ^+, etc. The salts can be used as appropriate, or mixed alone as required. Moreover, various inorganic substances and organic substances necessary for the growth of the transformant and the production of the enzyme can be added as necessary.

  The temperature of the culture is selected so as to be suitable for the growth of the transformant used. Usually 15 ° C to 60 ° C. Incubation of the transformed strain is continued for sufficient time for the production of amylomaltase.

  When an expression vector having an inducible promoter is used, expression can be controlled by adding an inducer, changing the culture temperature, adjusting the medium components, and the like. For example, when using an expression vector having a lactose-inducible promoter, expression can be induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG).

  After the transformant is cultured in this manner, for example, when the expression product is secreted outside the cell, the cell is separated by centrifuging or filtering the culture to obtain a supernatant. Next, the supernatant containing amylomaltase is concentrated using a usual means (for example, salting-out method, solvent precipitation, ultrafiltration) to obtain a fraction containing amylomaltase. This fraction is filtered or subjected to treatment such as centrifugation and desalting to obtain a crude enzyme solution. Furthermore, a crude enzyme or a purified enzyme having an improved specific activity can be obtained by appropriately combining the crude enzyme solution with conventional enzyme purification means such as freeze-drying, isoelectric focusing, ion exchange chromatography, and crystallization. . If an enzyme that hydrolyzes α-glucan such as α-amylase is not included, the crude enzyme can be used as it is for the production of cyclic glucan.

  The recombinant expression as described above can greatly improve the productivity of the amylomaltase of the present invention. In addition, when expressed, amylomaltase can be easily purified using its heat resistance. Briefly, when the bacterial cell extract containing amylomaltase is heated at about 60 ° C., the contaminating enzyme is insolubilized. The insolubilized material is removed by centrifugation or the like, and dialysis is performed.

(Cyclization reaction with amylomaltase)
(1) Production of cyclic α-1,4-glucan The modified amylomaltase of the present invention can be used to produce cycloamylose, which is a cyclic α-1,4-glucan, using α-glucan as a substrate. In this case, amylomaltase can also be used as an immobilized enzyme.

  Cycloamylose can be produced by allowing amylomaltase to act in a buffer containing an appropriate concentration of α-glucan. As α-glucan, linear α-1,4-glucan is preferable, and amylose having a high polymerization degree of at least 20 or more is more preferable. By using the amylomaltase of the present invention, the cyclization reaction proceeds smoothly, and typically, cycloamylose having a polymerization degree of about 20 to 400 can be produced. As the main product, it is particularly preferable to produce cycloamylose having a degree of polymerization of about 20 to 200.

  In a preferred embodiment using thermostable amylomaltase, the reaction temperature is 60 ° C. or higher, preferably 60 ° C. to 80 ° C., particularly preferably 65 to 70 ° C. This prevents the aging of the substrate amylose and contamination of the reaction system by various bacteria. In another embodiment, the reaction temperature can be less than 60 ° C. Such a reaction temperature varies depending on the optimum temperature of the amylomaltase used. Therefore, the optimum temperature of amylomaltase to be used is ± 10 ° C., preferably the optimum temperature ± 5 ° C., and most preferably, the vicinity of the optimum temperature is used as the reaction temperature.

  After the cyclization reaction has sufficiently progressed, the reverse reaction in the purification step can be prevented by completely inactivating amylomaltase at a high temperature. Preferably, the deactivation temperature is 90-100 ° C.

  Cycloamylose is recovered from the reaction solution and purified. In order to facilitate removal of the acyclic glucan, glucoamylase can be further added to the reaction solution after inactivating amylomaltase to decompose the acyclic glucan into glucose. Pure cycloamylose can be obtained using conventional purification methods such as ethanol precipitation or chromatography.

  Since the enzyme of the present invention does not substantially catalyze the hydrolysis reaction, the cycloamylose yield is maintained at a high level for a long time in the cyclization reaction. Under typical reaction conditions herein, at least 6 from the start of the reaction. No decrease in yield is observed until after time. Therefore, a high yield reaction can be performed with good reproducibility. According to the method of the present invention, cycloamylose production can be achieved in a yield of at least about 70% or more, typically about 75-85%.

(2) Production of branched cyclic glucan In addition, the amylomaltase of the present invention is used to catalyze the cyclization reaction of α-glucan containing a branched structure having α-1,6-linkage such as amylopectin, and the inner branched type Branched cyclic glucans having various structures such as cyclic glucans and outer branched cyclic glucans can be produced. At this time, amylomaltase can also be used as an immobilized enzyme.

  Branched cyclic glucan can be produced by acting amylomaltase in a buffer solution containing α-glucan at an appropriate concentration. As α-glucan, α-glucan having a branched structure having an α-1,6-linkage is preferable, and amylopectin, glycogen, starch, waxy starch and the like are particularly preferable. By using the amylomaltase of the present invention, the cyclization reaction proceeds smoothly, and typically, a branched cyclic glucan having a polymerization degree of about 17 to 5000 can be produced. In particular, it is preferable to produce a branched cyclic glucan having a degree of polymerization of about 21 to 3000.

  In addition, the amylomaltase of the present invention can be used with an additional enzyme to produce a branched cyclic glucan having a more complicated structure. For example, a branching enzyme (EC 2.4.1.18) can be used with amylomaltase. The branching enzyme is also called α-1,4-glucan branching enzyme, and is an enzyme that catalyzes a reaction of transferring a part of α-1,4-glucan chain to the 6-position to produce a branch ( For example, see European Patent Application No. 418945). By using two or more kinds of enzymes in combination, more complicated branched cyclic glucan can be produced.

  When thermostable amylomaltase is used, the reaction temperature is 60 ° C or higher, preferably 60 ° C to 80 ° C, particularly preferably 65 to 70 ° C. This prevents the aging of the substrate amylose and contamination of the reaction system by various bacteria. In another embodiment, the reaction temperature can be less than 60 ° C. Such a reaction temperature varies depending on the optimum temperature of the amylomaltase used. Therefore, the optimum temperature of amylomaltase to be used is ± 10 ° C., preferably the optimum temperature ± 5 ° C., and most preferably, the vicinity of the optimum temperature is used as the reaction temperature.

  When the amylomaltase of the present invention is allowed to act on α-glucan such as amylopectin, a mixture of branched cyclic glucan and cyclic α-1,4-glucan is obtained. As a method for purifying only the branched cyclic glucan from this mixture, any method known to those skilled in the art can be used. For example, a branched cyclic glucan can be purified by the following procedure. From the reaction solution after inactivating amylomaltase, the branched cyclic glucan and the cyclic α-1,4-glucan are separated by, for example, gel filtration chromatography. The branched cyclic glucan can then be obtained using conventional purification methods such as ethanol precipitation.

  Since the amylomaltase of the present invention does not substantially catalyze the hydrolysis reaction or has a reduced hydrolysis activity, the cyclic glucan yield is maintained at a high level for a long time in the cyclization reaction.

  Properties of the cyclic glucan produced in the above (1) and (2) such as aging property, viscosity, anti-aging effect of starch, clathrate formation ability, digestibility, and energy conversion efficiency are known to those skilled in the art. Any method can be used to evaluate. For example, a test method described in JP-A-8-311103 can be used.

(Use of amylomaltase to modify food)
The modified amylomaltase of the present invention acts on starch in food to catalyze starch cyclization. As a result, starch in food is reduced in molecular weight to produce cyclic glucan. As described above, the cyclic glucan produced by this reaction has a very low viscosity, excellent solubility, has an anti-aging effect on starch, and has the ability to include various substances. Therefore, like the wild type, the modified amylomaltase of the present invention can be used to modify food. Typically, by causing amylomaltase to act on starch in foods to accumulate cyclic glucan, the physical properties (especially stability during storage) and texture of food are improved.

  Furthermore, the cyclic glucan produced by the cyclization reaction of starch has a structure having an α-1,4-glucoside bond and an α-1,6-glucoside bond. This cyclic glucan is easily decomposed into glucose by an enzyme present in the organism. Therefore, the cyclic glucan also has the characteristics of excellent digestibility and high energy conversion efficiency.

  As described above, the modified amylomaltase of the present invention has a significantly increased reactivity with respect to the substrate as compared with the wild type, and has substantially no hydrolytic activity. In a preferred form, the modified amylomaltase of the present invention is very excellent in heat resistance. Therefore, when using a modified amylomaltase to modify food, it is possible to obtain an equivalent effect in a small amount compared to the conventional amylomaltase, and to obtain cycloamylose efficiently. Have advantages. Moreover, since it also has heat resistance in a preferable form, it has the advantage that it is possible to add an enzyme before heat cooking of a foodstuff material or immediately after heat cooking. Furthermore, there is an advantage that unnecessary accumulation of low-molecular reducing sugar does not occur.

  The amylomaltase variants of the present invention can be used to modify a wide range of foods (ie, any food containing starch).

  Examples of foods high in starch include rice (eg, rice balls, sushi rice, and bento rice), Japanese confectionery (eg, bracken, steamed buns, rice cakes, corn, and rice balls), snacks (eg, rice crackers, rice crackers, potatoes). Chips and other snacks), bakery items (eg bread, pie, pizza, cakes, cookies, biscuits and crackers), noodles (eg udon, buckwheat noodles and ramen), and pasta such as spaghetti and macaroni ), Dumplings and shumai skin, marine products (eg Chikuwa and kamaboko), processed foods frozen or refrigerated, baby food, baby food, pet food, animal feed, beverages (eg sports drinks), sports foods , And nutritional supplements.

  Cooked rice, Japanese confectionery, snack confectionery, bakery, and noodles that contain a particularly large amount of starch are examples of foods that are preferred as the subject of the present invention. Frozen or refrigerated processed foods where stability during low temperature storage is important are other examples of preferred foods.

  In the present invention, the food material refers to any material for producing the above-mentioned food and any preparation in the process of processing the food.

  The modification of the food that has been treated with the modified amylomaltase of the present invention can be confirmed by examining that the starch in the food has been reduced in molecular weight to produce cyclic glucan. In the present invention, in the food to which the enzyme of the present invention is added, the food is modified when significant production of cyclic glucan is observed compared to the food to which the enzyme is not added.

  The food material, food additive, and food modifier containing the modified amylomaltase of the present invention are useful for producing a food modified as described above. The amount of the enzyme to be added can be appropriately selected by those skilled in the art.

  Examples of the food additive include seasonings (for example, soy sauce, sauce, sauce, soup stock, stew base, curry base, soup base, mayonnaise, dressing, ketchup, and complex seasoning).

  Examples of food modifiers include starch aging inhibitors and cooked rice modifiers.

(General description of genetic engineering)
As used herein, “homology” of genes (eg, polypeptides, polynucleotides, etc.) refers to the degree of identity of two or more gene sequences with each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% identical, the genes are homologous.

  In the present specification, comparison of sequence identity, homology and similarity is calculated using BLAST, which is a tool for sequence analysis, and using default parameters.

  Polypeptides or nucleic acids of the invention are also not identical, but homologous to sequences specifically described herein for wild-type amino acid or nucleic acid sequences (eg, SEQ ID NOs: 1 and 2, etc.). Some can also be used. The polypeptide or nucleic acid molecule of the present invention having homology to such wild-type polypeptide or nucleic acid is a sequence to be compared in the case of a nucleic acid, for example, when compared using the default parameters of Blast. At least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about In the case of a nucleic acid molecule or polypeptide comprising a nucleic acid sequence having 85%, about 90%, about 95%, about 99% identity or similarity, or at least about 30%, about 35%, about 40%, about 45% About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% identity or similarity And a polypeptide having an amino acid sequence having That include, but are not limited to.

  In the present specification, “expression” of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form. Preferably, a gene, polynucleotide or the like is transcribed and translated into a polypeptide form, but transcription to produce mRNA can also be a form of expression. More preferably, such polypeptide forms may be post-translationally processed.

  In the present specification, the “amino acid” may be natural or non-natural. “Derivative amino acid” or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art. The term “natural amino acid” refers to the L-isomer of a natural amino acid. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine , And lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention. The term “unnatural amino acid” refers to an amino acid that is not normally found in proteins in nature. Examples of non-natural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homo-arginine D-form or L-form and D-phenylalanine. “Amino acid analog” refers to a molecule that is not an amino acid but is similar to the physical properties and / or function of an amino acid. Examples of amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

  Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally accepted single letter code.

  In the present specification, the “corresponding” amino acid (or residue) means the same action as a predetermined amino acid (or residue) in a reference protein or polypeptide in a protein molecule or polypeptide molecule. An amino acid (or residue) that has or is predicted to have, especially in an enzyme molecule, an amino acid that is present at the same position in the active site and contributes similarly to catalytic activity. For example, the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2 in the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine, 101-position tyrosine and 153-position glycine The corresponding amino acid sequence will be apparent to those skilled in the art as shown in FIG. Moreover, such a corresponding amino acid can also be determined by conducting a three-dimensional structure analysis. Such a three-dimensional structure analysis technique is well known to those skilled in the art. Such a corresponding amino acid can also be identified by analyzing a complex with a substance that interacts with the target polypeptide such as a substrate. Such identification methods are also well known to those skilled in the art, and examples of such methods are also described herein.

  In the present specification, the “corresponding” nucleic acid (or base) has the same action as a predetermined nucleic acid (or base) in a polynucleotide to be compared or in a polynucleotide or nucleic acid molecule, or A nucleic acid (or base) that is predicted to have, especially in an enzyme molecule, a nucleic acid that encodes an amino acid that is present at the same position in the active site and contributes similarly to the catalytic activity. For example, the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2 in the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine, 101-position tyrosine and 153-position glycine The nucleic acid sequence encoding the corresponding amino acid sequence will be apparent to those skilled in the art as in the amino acid alignment shown in FIG. Such a corresponding nucleic acid (or base) can also be determined by identifying an amino acid by conducting a three-dimensional structure analysis. Such a three-dimensional structure analysis technique is well known to those skilled in the art. Such a corresponding nucleic acid (or base) can also be identified by analyzing a complex with a substance that interacts with the polypeptide of interest such as a substrate. Such identification methods are also well known to those skilled in the art, and examples of such methods are also described herein.

  In the present specification, a “corresponding” gene (for example, amylomaltase gene) is a gene that has or is predicted to have the same action as a predetermined gene in a species as a reference for comparison in a certain species. In the case where there are a plurality of genes having such an action, the genes having the same origin in evolution are said. Thus, the corresponding gene of a gene can be an ortholog of that gene.

  In the present specification, the “nucleotide” may be natural or non-natural. “Derivative nucleotide” or “nucleotide analog” refers to a nucleotide that is different from a naturally occurring nucleotide but has the same function as the original nucleotide. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). .

  In the present specification, the “fragment” refers to a polypeptide or a polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg 11 etc.) are also suitable as lower limits obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as an adjustment is intended to include a number above and below the number (or, for example, 10% above and below). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.

  As used herein, “biological activity” refers to activity that a certain factor (eg, polypeptide or protein) may have in vivo, and includes activity that exhibits various functions. For example, if a factor is an enzyme (eg, amylomaltase), the biological activity includes that enzyme activity (eg, amylomaltase activity). In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art. Moreover, when amylomaltase activity is biological activity, such activity can be measured according to description of this specification.

  An amino acid in a protein introduces a modification of interest in the present invention (for example, substitution in the interaction region with the inhibitor Acarbose), and without an apparent reduction or loss of interaction binding capacity, for example Other amino acids can be substituted in protein structures such as cationic regions or epitope sites. It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in a protein that still retains its original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein equivalent in enzyme activity). It is well known. In such amino acid substitution, for the purpose of conservative substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. . Therefore, in non-conservative substitutions, substitutions that deviate from these values are preferably performed. Thus, it is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  When considering conservative substitutions, the hydrophilicity index can also be considered. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and still can provide a biological equivalent. In such amino acid substitutions, for the purpose of conservative substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. . Therefore, for non-conservative substitution purposes, substitutions that deviate from these values are preferred.

  In the present invention, “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid to be replaced with the original amino acid is similar as described above. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine (or / and histidine); glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; Phenylalanine; and valine, leucine, and isoleucine, and the like.

  Therefore, in the present invention, the “non-conservative substitution” is not a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid substituted with the original amino acid is similar as described above in the amino acid substitution. Refers to substitution. Examples of non-conservative substitutions are well known to those skilled in the art, for example, substitutions between members belonging to one group and members belonging to other groups: arginine and lysine (or / and histidine); glutamic acid and Aspartic acid; serine and threonine; glutamine and asparagine; tyrosine and phenylalanine; and valine, leucine, and isoleucine, and the like.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Since orthologs are useful for the estimation of molecular phylogenetic trees, orthologs of the amylomaltase of the present invention may also be useful in the present invention.

  In the present specification, the “wild type” of amylomaltase refers to a naturally occurring amylomaltase that is most widely present in the species from which it is derived. Usually, the first amylomaltase identified in a species is wild type. The wild type is also referred to as “natural standard type”. Such a wild-type amylomaltase has the sequences shown in SEQ ID NOs: 1 and 2 in the case of Thermus aquaticus ATCC 33923 strain. Also, for example, potato, E.I. In the case of E. coli, Synechocystis PCC6803, Clostridium butyricum, and Chlamydia pneumoniae, the sequences shown in FIG. 1 (SEQ ID NOs: 12, 14, 16, 18, 20 (nucleic acid sequences), 13, 15, 17, 19, 21 (amino acid sequences), respectively)) Have

  One of the characteristics of the modified amylomaltase of the present invention is that the modification intended by the present invention is introduced. In the present specification, such “target modification” refers to a modification in an amino acid sequence of amylomaltase or a site or an amino acid residue that interacts with an amylomaltase inhibitor, Acabose. Domains responsible for such interaction with Abbose are described in Przylas et al. Eur. J. et al. Biochem. 267, 6903-6913 (2000). Therefore, such targeted modification includes, for example, the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartic acid of the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2, 98 In the wild-type amylomaltase polypeptide residue corresponding to at least one residue selected from the group consisting of position glycine, position 101 tyrosine and position 153 glycine, other than the amino acid at each position shown in SEQ ID NO: 1 Having the amino acid of

  Domains responsible for the interaction with Akabose are also known in the art for X-ray crystal structure analysis techniques (for example, X-ray analysis using BW7b beamline (DESY / EMBL, Hamburg, Germany), MarResearch imaging plate detector) Data measurement using data processing, data processing using programs such as DENZO and SCLAEPACK), and computer modeling techniques (for example, CNS program, XPLO2D program, program O, PROCHECK, WHATCHECK, WHATIF, etc.) Can be used to identify. Thus, any amylomaltase can introduce the alterations of interest of the present invention by introducing alterations in the Acarbose domain identified using the methods described above. Such modification preferably involves substituting amino acids in the wild type with other amino acids. For example, in the case of amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2, substitution of the 54th tyrosine with another amino acid is mentioned. In this case, preferably the other amino acid is other than phenylalanine, preferably selected from the group consisting of alanine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, arginine, serine and threonine. There can be one. Alternatively, the other amino acids are other than phenylalanine and lysine, more preferably glycine, proline, threonine and tryptophan. More preferred is glycine or threonine, and most preferred is glycine. In the amylomaltase of the present invention, by modifying the amino acid residue in the domain interacting with Akabose, the enzyme activity is increased and the side-reaction hydrolysis activity is decreased. In particular, it was unpredictable in the prior art that increased enzyme activity and decreased hydrolytic activity were achieved by modification of the same domain, more preferably by modification of the same residue (eg amino acid substitution) In this sense, the present invention can be said to have a remarkable effect.

  “Conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. It should be understood herein that within the scope of conservative variants, even if they contain non-conservative substitutions, so long as they have the enzymatic activity of amylomaltase. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. All nucleic acid sequences herein that encode a polypeptide also include all possible silent modifications of the nucleic acid. Silent modification includes “silent substitution” in which the encoded nucleic acid does not change and the case where the nucleic acid does not encode an amino acid in the first place. When a nucleic acid encodes an amino acid, silent modification is synonymous with silent substitution. As used herein, “silent substitution” refers to substituting a nucleic acid sequence encoding a certain amino acid with another nucleic acid sequence encoding the same amino acid in the nucleic acid sequence. Based on the phenomenon of degeneracy in the genetic code, when there are a plurality of nucleic acid sequences encoding a certain amino acid (for example, glycine), substitution with such a siren is possible. Thus, a polypeptide having an amino acid sequence encoded by a nucleic acid sequence generated by silent substitution has the same amino acid sequence as the original polypeptide. Therefore, in the amylomaltase of the present invention, the intended modification of the present invention (substitution of amino acid residues that interact with Acarbose, an inhibitor of amylomaltase polypeptide, to amino acid residues other than wild-type amino acid residues ( For example, the amino acid sequence of amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2 from the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine, 101-position tyrosine and 153-position glycine In addition to having an amino acid other than the amino acid at each position shown in SEQ ID NO: 1 in the residue of the wild-type amylomaltase polypeptide corresponding to at least one residue selected from the group consisting of: Nucleic acid sequence In Le, it is also possible to include silent substitutions. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon that encodes methionine, and TGG, which is usually the only codon that encodes tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide.

  In this specification, in order to produce a polypeptide functionally equivalent to the variant of amylomaltase of the present invention, in addition to the purpose of the present invention, in addition to amino acid substitution, amino acid addition, deletion, Or modifications can also be made. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. Addition of an amino acid means adding 1 or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the terms “peptide analog” or “polypeptide analog” are used interchangeably and are different compounds from a peptide or polypeptide, but the peptide or polypeptide and at least one chemical function or organism. Refers to an equivalent scientific function. Thus, peptide analogs include those in which one or more amino acid analogs are added or substituted to the original peptide. Peptide analogs have the same function as the original peptide (eg, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog. In the present specification, “polypeptide” includes this peptide analog unless otherwise specified.

  In the present specification, when referring to a gene, a “vector” refers to one that can transfer a target polynucleotide sequence into a target cell. Such a vector can be autonomously replicated in a host cell such as an individual animal or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the polynucleotide of the present invention. Are illustrated. As used herein, a vector can be a plasmid.

  As used herein, “expression vector” refers to a nucleic acid sequence in which various regulatory elements are operably linked in a host cell in addition to a structural gene and a promoter that regulates its expression. Regulatory elements can preferably include terminators, selectable markers such as drug resistance genes, and enhancers. It is well known to those skilled in the art that the type of expression vector of an organism (eg, animal) and the type of regulatory elements used can vary depending on the host cell.

  As used herein, “recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence into a target cell. Examples of such vectors include those that can autonomously replicate in a host cell such as an individual animal or can be integrated into a chromosome and contain a promoter at a position suitable for transcription of the polynucleotide of the present invention. Is done.

  As used herein, “terminator” is a sequence that is located downstream of a protein-coding region of a gene and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly A sequence. It is known that the terminator is involved in the stability of mRNA and affects the expression level of the gene.

  As used herein, the term “promoter” refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is a base sequence that initiates transcription upon binding of RNA polymerase. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic base sequence is predicted using DNA analysis software, The promoter region can be estimated. The putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is present within about 2 kbp upstream from the first exon translation start point.

  In the present specification, an “enhancer” can be used to increase the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of enhancers may be used, but one may be used or may not be used.

  As used herein, “operably linked” refers to a transcriptional translational regulatory sequence (eg, promoter, enhancer, etc.) or a translational regulatory sequence that has the expression (operation) of a desired sequence. That means. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but need not necessarily be adjacent.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like.

  In the case of plants, methods for redifferentiating transformants into tissues or plants are well known in the art. Such methods include Rogers et al. , Methods in Enzymology 118: 627-640 (1986); Tabata et al. , Plant Cell Physiol. , 28: 73-82 (1987); Shaw, Plant Molecular Biology: A practical approach. IRL press (1988); Shimamoto et al. , Nature 338: 274 (1989); Maliga et al. , Methods in Plant Molecular Biology: A laboratory course. Cold Spring Harbor Laboratory Press (1995). The woody plant is described in Molecular Biology of Woody Plants (Vol. I, II) (ed. S. Mohan Jain, Subhash C. Minocha), Kluwer Academic Publishers, (2000). For woody plants, it is described in detail, for example, in Plant Cell Reports (1999) 19: 106-110. Therefore, those skilled in the art can regenerate by appropriately using the above-described well-known methods according to the transgenic plant. The transgenic plant thus obtained is introduced with the gene of interest, and the introduction of such a gene can be performed by a method described herein such as Northern blot, Western blot analysis or other methods. This can be confirmed using well-known conventional techniques.

(Various amylomaltase)
Although amylomaltase was first discovered from potato, it has been found to be present in various plants and microorganisms such as Escherichia coli and Thermus bacteria. Therefore, the origin of the wild-type amylomaltase for producing the modified amylomaltase of the present invention is not limited. Therefore, a gene encoding an enzyme derived from a plant is modified according to the present invention (for example, the 53-position glycine, the 54-position tyrosine, the 55-position glycine of the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2, 56 A residue corresponding to at least one residue selected from the group consisting of position aspartic acid, position 98 glycine, position 101 tyrosine and position 153 glycine has an amino acid other than the amino acid at each position shown in SEQ ID NO: 1 Modified) and expressed using a host such as Escherichia coli. Here, a method for purifying amylomaltase from potato and Escherichia coli is disclosed as an example, but not limited thereto.

  A method for purifying amylomaltase from potato is described in Takaha et al. Biol. Chem. vol. 268, 1391-1396 (1993). First, potato tubers are homogenized in an appropriate buffer containing 5 mM mercaptoethanol, centrifuged, passed through a 0.45 μm membrane, applied to a Q-Sepharose column, and containing, for example, 5 mM 2-mercaptoethanol. Wash with a buffer containing 150 mM NaCl in 20 mM Tris-HCl (pH 7.5) (buffer A). The amylomaltase elutes in buffer A containing 450 mM NaCl. This was dialyzed against buffer A, loaded with a solution containing 500 mM ammonium sulfate onto a Phenyl Toyopearl 650M (Tosoh) column, and eluted by changing the ammonium sulfate concentration in buffer A from 500 mM to 0 mM. The maltase active fraction is collected and dialyzed against buffer A. The dialyzed internal solution was loaded onto a PL-SAX column (Polymer Laboratory UK) equilibrated with buffer A, and the NaCl concentration in buffer A was changed to 150 mM-400 mM and eluted, and the amylomaltase active fraction was eluted. Collect. Amylomaltase can be purified from potato by the above method. The enzyme activity was measured by reacting 100 mM Tris-HCl (pH 7.0), 5 mM 2-mercaptoethanol, 1% (W / V) maltotriose, and 100 μl of a reaction mixture containing the enzyme at 37 ° C. for 10 minutes. The reaction solution is heated in boiling water for 3 minutes to stop the reaction, and glucose released by the reaction can be quantified by a method using glucose oxidase (Barham et al. (1972) Analyst 97: 142). In this specification, the amount of enzyme that produces 1 μmol of glucose per minute is defined as 1 unit. Such a technique can be applied when purifying and quantifying the amylomaltase of the present invention.

  Takaha et al., J., supra. Biol. Chem. vol. 268, 1391-1396 (1993) discloses a cDNA sequence of potato amylomaltase (p. 1394, FIG. 3). Such sequence information can be obtained by considering the conserved region and the like. Naturally it can be used to introduce modifications of As a method for purifying amylomaltase from E. coli, for example, first, Escherichia coli TG-1 which is a production strain of amylomaltase is cultured at 37 ° C. to the logarithmic growth phase using LB liquid medium, and then maltose is added at a final concentration of 1% ( w / v), and further cultured at 37 ° C. for 2 hours. The bacterial cells collected by centrifugation are suspended in the buffer A and subjected to sonication and centrifugation to obtain a bacterial cell extract. Next, for example, a Q-Sepharose Fast Flow (Pharmacia) column equilibrated with buffer A is loaded, elution is carried out by changing the NaCl concentration in buffer A from 0 mM to 500 mM, and the amylomaltase activity fraction is obtained. Gather. To the active fraction, ammonium sulfate was added to a final concentration of 1M, left to stand, insoluble precipitate was removed by centrifugation, and the supernatant was equilibrated with buffer A containing 1M ammonium sulfate Phenyl Toyopearl 650M (Tosoh) Load into the column. Elution is carried out by changing the ammonium sulfate concentration in buffer A from 1 M to 0 mM, and the amylomaltase active fraction is collected. This fraction is dialyzed against buffer A and then loaded onto a Resource Q column (Pharmacia) in which the dialysis internal solution is equilibrated with buffer A, and the NaCl concentration in buffer A is changed from 0 mM to 500 mM, for example. Elution is carried out by changing to purify amylomaltase. The biochemical properties of the amylomaltase into which the modification of the present invention has been introduced may have changed. In such a case, such a modified amylomaltase can be purified by appropriately modifying the purification method described above. Such modifications in the purification method are well known to those skilled in the art, and those skilled in the art can easily carry out based on the disclosure of the present specification with appropriate consideration of well-known techniques.

  The modified amylomaltase of the present invention can be purified as described above in the same manner as in the wild type. However, if endo-type amylases acting on α-1,4-glucoside bonds in the starch molecule are not detected, Any crude enzyme from any of the above purification steps can be advantageously used for glucan synthesis.

  In addition, the modified amylomaltase used in the present invention may be used for the reaction regardless of whether it is a purified enzyme or a crude enzyme, and the reaction may be performed in a batch or continuous manner. As the immobilization method, a carrier bonding method (for example, a covalent bonding method, an ionic bonding method, or a physical adsorption method), a crosslinking method, or a comprehensive method (lattice type or microcapsule type) can be used.

(Immunochemistry)
The production of antibodies that recognize the polypeptides of the present invention is also well known in the art. For example, a polyclonal antibody can be prepared by administering a full-length or partial fragment purified preparation of the obtained polypeptide or a peptide having a partial amino acid sequence of the protein of the present invention to an animal as an antigen. .

  Rabbits, goats, rats, mice, hamsters and the like can be used as animals to be administered. The dose of the antigen is preferably 50 to 100 μg per animal. In the case of using a peptide, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin. The peptide used as an antigen can be synthesized with a peptide synthesizer. The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. On the 3rd to 7th day after each administration, blood was collected from the fundus venous plexus, and the serum reacted with the antigen used for immunization. Enzyme immunoassay [Enzyme immunoassay (ELISA): 1976, published by Medical Shoin. Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

  Serum can be obtained from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and polyclonal antibodies can be separated and purified from the serum using well-known techniques. The production of monoclonal antibodies is also well known in the art. For the preparation of antibody-producing cells, first, a rat whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the polypeptide of the present invention used for immunization was used as a source of antibody-producing cells. A hybridoma is prepared by fusion with myeloma cells. Thereafter, a hybridoma that specifically reacts with the partial fragment polypeptide of the polypeptide of the present invention is selected by enzyme immunoassay or the like. The monoclonal antibody produced from the hybridoma thus obtained can be used for various purposes.

  Such an antibody can be used, for example, in the immunological detection method of the polypeptide of the present invention. As the immunological detection method of the polypeptide of the present invention using the antibody of the present invention, a microtiter plate is used. Examples of the ELISA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method that can be used.

It can also be used in an immunological quantification method of the polypeptide of the present invention. As a method for quantifying the polypeptide of the present invention, sandwich ELISA method using two kinds of monoclonal antibodies having different epitopes among antibodies that react with the polypeptide of the present invention in a liquid phase, labeled with a radioactive isotope such as ¹²⁶ I Examples thereof include a radioimmunoassay method using the protein of the present invention and an antibody that recognizes the protein of the present invention.

  Methods for quantifying mRNA of the polypeptide of the present invention are also well known in the art. For example, the expression level of the DNA encoding the polypeptide of the present invention can be quantified at the mRNA level by the Northern hybridization method or the PCR method using the oligonucleotide prepared from the polynucleotide or the DNA of the present invention. Such techniques are well known in the art and are also described in the literature listed herein.

(Crystal structure analysis)
Protein crystal structure analysis can be performed using methods well known in the art. Such methods are described in, for example, X-ray crystal structure analysis of proteins (Springer Fairlark Tokyo), Introduction to crystal analysis for life science (Maruzen), and the like. Such a method can be arbitrarily used in the book.

In general, it is known that protein crystals are considerably damaged by X-rays. Therefore, in order to succeed in X-ray crystal structure analysis, it is important to obtain crystals that are not easily damaged. In recent years, it is often performed to obtain high quality and high resolution diffraction data by freezing crystals and measuring diffraction data in a frozen state (Methods in ENZYMOLOGY Vol. 276, Macromolecular Crystallography, Part). A, C. W. Carter, Jr. and RM Sweet, Practical Crystallography (DW Rodgers)). In general, in order to prevent protein crystals from freezing by freezing, the protein crystals are frozen by treatment with a solution containing a freeze stabilizer such as glycerol. In the present invention, the native crystals of amylomaltase and the frozen crystals of heavy atom derivatives are obtained by taking out the crystallized droplets or the crystals in the immersion solution without adding a freeze stabilizer and immersing them directly in liquid nitrogen. It can be prepared by freezing. Frozen crystals can also be prepared by performing the instant freezing operation on crystals immersed in a storage solution to which a freeze stabilizer is added.
Heavy atom isomorphous substitution method (Methods in ENZYMOLOGY Vol. 115, Diffractiven Methods for Biological Macromolecules, Part B, H. W. Wyckoff, C. H. Mo., G. H., and S. N. Tim. Volume, Macromolecular Crystallography, Part A, CW Carter, Jr. and RM Sweet Edition) or multiwavelength anomalous dispersion (S.N. Timasheff, and Methods in ENZYMOLOGY Volume 276, Macromolecular Pullet. A, C.W. Carter, Jr. Fine R.M.Sweet eds) also it is possible to perform structural analysis by using. Here, the three-dimensional structure is obtained by obtaining the initial phase for calculating the electron density from the diffraction intensity difference between the diffraction data of the native crystal and the heavy atom derivative crystal, or the diffraction intensity difference between the diffraction data measured at different wavelengths. Can be determined. In determining the three-dimensional structure of amylomaltase to which the heavy atom isomorphous substitution method or the multiwavelength anomalous dispersion method is applied, a heavy atom derivative crystal containing, for example, mercury, gold, platinum, uranium, selenium atoms, etc. as heavy metal atoms can be used. Preferably, a heavy atom derivative crystal obtained by an immersion method using a mercury compound EMTS or potassium dicyanogold (I) is used.

  Diffraction data of native crystals and heavy atom derivative crystals can be measured using, for example, a protein crystal structure analysis beam line of R-AXIS IIc (Rigaku Denki) or SPring-8 (Nishiharima Large Synchrotron Radiation Facility). Diffraction data measurement at a plurality of wavelengths for applying the multi-wavelength anomalous dispersion method can be performed using, for example, a SPring-8 protein crystal structure analysis beam line. The measured diffraction image data is processed into reflection intensity data using, for example, a data processing program attached to R-AXIS IIc or a program DENZO (Mac Science) or a similar image processing program (or single crystal analysis software). . From the reflection intensity data of native crystals, the reflection intensity data of heavy atom derivative crystals at multiple wavelengths, the position of heavy metal atoms bound to proteins in the crystal using the difference Patterson diagram After obtaining, for example, using the program PHASES (W.Furey, University of Pennsylvania or CCP4 (British Biotechnology & Biological Science Research Counil, SERC) or the same diffraction data analysis program using the same diffraction data analysis program using the same diffraction data analysis program) The determined initial phase is, for example, the program DM (CCP4 package) or a similar phase improvement program (electron density modification). Highly reliable by performing phase expansion calculation gradually from low resolution to high resolution with the solvent region in the amylomaltase crystal as 30-50%, for example, according to the solvent smoothing method and histogram matching method using the good program) In the present specification, 30-60% of the volume of protein crystals is occupied by solvent molecules other than protein (mainly water molecules). In general, when the obtained protein crystal has non-crystallographic symmetry, an electron density averaging called non-crystallographic symmetry (NCS) averaging is performed to further It is possible to increase the reliability of the phase, and the amylomaltase crystal contains two molecules in the asymmetric unit based on the result of the crystal density measurement. It is estimated that the amylomaltase molecule in the crystal has a non-crystallographic two-fold axis, an electron density map calculated using the phase after applying the solvent smoothing method, and a refined heavy atom. From the coordinates, a non-crystallographic symmetry matrix, including translation and rotation, is calculated, and at the same time, from the electron density map obtained by the solvent smoothing method, a region where protein molecules called masks exist can be identified. By performing NCS averaging calculation using a program DM or the like using a geometric symmetry matrix and a mask, the phase is improved to a highly reliable phase, and an electron density diagram used to construct a three-dimensional structure model is obtained.

  The three-dimensional structure model of amylomaltase can be constructed from the electron density map displayed on the three-dimensional graphics by the program O (O) (A. Jones, Uppsala Universitet, Sweden) by the following procedure. First, a plurality of regions having a characteristic amino acid sequence (such as a partial sequence containing a tryptophan residue) are searched for on an electron density map. Next, a partial structure of amino acid residues suitable for the electron density is constructed on the three-dimensional graphics using the program O while referring to the amino acid sequence starting from the found region. By sequentially repeating this operation, all amino acid residues of amylomaltase are adapted to the corresponding electron density, and an initial three-dimensional structure model of the whole molecule is constructed. The constructed three-dimensional structure model is used as a starting model structure, and the three-dimensional coordinates describing the three-dimensional structure are refined according to the refinement protocol of the structure refinement program XPLOR (AT Brunger, Yale University). The Further, the three-dimensional structure of native crystals of amylomaltase (for example, amylomaltase containing the amino acid sequence shown in SEQ ID NO: 2) and amylomaltase variants (eg, amylomaltase variants having the sequence shown in SEQ ID NO: 2) native crystals The three-dimensional structure can be determined by determining the initial phase by the molecular replacement method using the three-dimensional structure of the obtained EMTS derivative crystal and following the above-described electron density improvement, model construction, and structure refinement procedures. . This completes the determination of the three-dimensional structure of the amylomaltase of the present invention. Regarding the determined three-dimensional structure of amylomaltase, from the viewpoint of topology, various proteins (including enzymes similar in function to amylomaltase) registered in Protein Data Bank (PDB), which is an official data bank of protein three-dimensional structure ) Can be compared with the three-dimensional structure.

  As used herein, “topology” refers to the arrangement or spatial arrangement of protein secondary structural units.

  In the present specification, the “α helix” is one of secondary structures of proteins or polypeptides, and has an energetic structure having a helical structure with a pitch of 5.4 A with one rotation of every 3.6 residues of amino acids. Refers to one of the most stable structures. Examples of amino acids that are likely to form an α helix include glutamic acid, lysine, alanine, and leucine. Conversely, amino acids that are less likely to form an α helix include valine, isoleucine, proline, and glycine. In the present specification, the “β sheet” is one of secondary structures of proteins or polypeptides, and two or more polypeptide chains having a zigzag conformation are arranged in parallel. A structure in which the amide group and carbonyl group are combined in an energetically stable sheet form by forming a hydrogen bond with the carbonyl group and amide group of the adjacent peptide chain, respectively. “Parallel β sheet” refers to a β sheet having the same direction of arrangement of amino acid sequences of adjacent polypeptide chains, and “anti-parallel β sheet” refers to an adjacent poly sheet of β sheets. A peptide chain whose amino acid sequence is reversed. Furthermore, in this specification, “β strand” refers to a single peptide chain having a zigzag conformation that forms a β sheet.

  Therefore, when preparing the modified amylomaltase of the present invention, the above topology may be taken into consideration.

  In the present specification, “three-dimensional structure data” of a protein refers to data relating to the three-dimensional structure of the protein. The three-dimensional structure data of proteins typically includes atomic coordinate data, topology, and molecular force field constant. The atomic coordinate data is typically data obtained from X-ray crystal structure analysis or NMR structure analysis, and such atomic coordinate data is obtained by newly performing X-ray crystal structure analysis or NMR structure analysis. Or can be obtained from a known database (eg, Protein Data Bank (PDB)). Atomic coordinate data can also be data created by modeling or calculation.

  The topology can be calculated using a commercially available or freeware tool program, but a self-made program may also be used. Further, a molecular topology calculation program attached to a commercially available molecular force field calculation program (for example, prepar program attached to PRESTO, Protein Engineering Laboratory Co., Ltd.) can be used. The molecular force field constant (or molecular force field potential) can also be calculated using a commercially available or freeware tool program, but self-made data may also be used. Further, molecular force field constant data attached to a commercially available molecular force field calculation program (for example, AMBER, Oxford Molecular) can be used.

  In addition, the specific coordinate data of the three-dimensional structure of the wild type amylomaltase of Thermus Aquaticus is a protein data bank (PDB, Protein Data Bank, The Research Collaborative For Structural Bioinformatics (registered by W registered as CSB and registered by C as the number 1)) And this data is incorporated herein. In addition to this, 1ESW (Thermus aquaticus), 1 LWH (Thermomoto maritima), and 1 LWJ (Thermomoto maritima) are registered in the PDB as the three-dimensional structure of wild-type amylomaltase, and these data are also incorporated herein.

  By completing the determination of the three-dimensional structure of the amylomaltase of the present invention, it is possible to estimate the residues involved in the catalytic activity of the enzyme. The three-dimensional structure model of the combined complex is converted into a molecular modeling technique (Swiss-PDBViewer (supra), Autodock (Oxford Molecular), Guex, N. and Peitsch, MC (1997) SWISS-MODEL and the Swiss- PDBViewer: An environmental for competitive protein modeling, Electrophoresis 18, 2714-2723; Morris, GM et al., J. Computation al Chemistry, 19: 1639-1662, 1998; Morris, GM, et al., J. Computer-Aided Molecular Design, 10, 294-304, 1996; Goodsell, DS, et al., J. Mol. : 1-5, 1996). From this three-dimensional structure and three-dimensional structure model, knowledge on the structure of the amino acid residue of the catalytic reaction group and the reaction mechanism in the active site can be obtained.

  The active site of amylomaltase is the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine, 101-position tyrosine of the amino acid sequence of Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2. And 153 glycine. Since position 54 tyrosine is known to interact with the second abose, in one embodiment, the targeted modification of the present invention is that the residue corresponding to position 54 tyrosine is replaced by another amino acid. Preferably it has been modified.

  Based on the knowledge obtained from these three-dimensional structures, modification design for the purpose of improving the stability or enzyme activity of amylomaltase can be performed. In the present specification, “thermostability” of an enzyme means that the enzyme activity is at least 10 as compared with that before heat denaturation even after denaturation of the enzyme at a temperature higher than the normal biological environment (for example, 90 ° C.). %, Preferably at least 25%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90%. The improvement in stability can be measured, for example, by ΔTm (denaturation temperature difference). In this specification, “enzyme activity” is used synonymously with “amylomaltase activity” unless otherwise specified, and is composed of 100 mM sodium acetate (pH 5.5), 0.2% (w / v) maltotriose. The activity measured by quantifying glucose produced when amylomaltase is allowed to act in a liquid at 70 ° C. for 10 minutes.

  In the present specification, the design of a variant molecule is made by analyzing the amino acid sequence and the three-dimensional structure of a protein or polypeptide molecule (for example, a wild-type molecule) before mutation to determine what characteristics (for example, a catalyst Activity, interaction with other molecules, etc.) and calculate the appropriate amino acid mutations to bring about the desired property modification (eg, improved catalytic activity, improved protein stability, etc.) Is done. The design method is preferably performed using a computer. Examples of the computer program used in such a design method include the following as mentioned in this specification: As a program for analyzing the structure, DENZO (Mac Science) is a processing program for X-ray diffraction data. ); As a processing program for determining the phase, PHASES (Univ. Of Pennsylvania, PA, USA); As a program for improving the initial phase, the program DM (CCP4 package, SERC); to obtain three-dimensional graphics Program O (Uppsala University, Uppsala, Sweden); three-dimensional structure refinement program, XPLOR (Yale University, CT, USA); As a program for the ring, Swiss-PDBViewer (supra).

  In the present invention, based on the disclosure of the present invention, it is also intended to provide a drug (for example, an inhibitor, an activator, etc.) by computer modeling.

(Composition)
The amylomaltase of the present invention can be provided as a composition (for example, a pharmaceutical composition, an agrochemical composition, an edible composition). Such a composition may contain additional components such as carriers, depending on its purpose (medicine, agrochemical, edible, etc.).

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include pH 7.0-8.5 Tris buffer or pH 4.0-5.5 acetate buffer, which may further include sorbitol or a suitable substitute thereof. . The pH of the solution should also be selected based on the relative solubility of the factors of the invention at various pHs.

  The solvent in the composition can have either aqueous or non-aqueous properties. In addition, the vehicle can be used to modify or maintain the pH, osmolarity, viscosity, clarity, color, sterility, stability, isotonicity, disintegration rate, or odor of the formulation. Material may be included. Similarly, the compositions of the present invention may include other formulation materials to modify or maintain the release rate of the active ingredient or to promote absorption or permeation of the active ingredient.

(Sugar)
Examples of the linear α-1,4-glucan that can be used in the present invention or a saccharide having the same include malto-oligosaccharides such as maltotriose, maltotetraose, maltopentaose, amylose, amylopectin, glycogen, starch, waxy starch , High amylose starch, soluble starch, dextrin, starch residue, partially hydrolyzed starch, enzymatically synthesized starch using phosphorylase, and derivatives thereof. These may be used alone or in combination. Here, the starch branch material refers to a product obtained by enzymatically cleaving all or part of the bonds other than α-1,4-glucoside bonds in starch. Further, the partial starch hydrolyzate refers to a product obtained by enzymatically or chemically cleaving a part of α-1,4-glucoside bond in starch. For example, the degree of polymerization is about 100 or more. Amylopectin, amylose having a degree of polymerization of about 20 or more can be used as a raw material.

  Moreover, it has one linear structure in a molecule | numerator comprised by the linear (alpha) -1, 4- glucan which can be used for this invention, or a saccharide | sugar which has this, and at least 14 (alpha) -1, 4- glucoside bond. The reaction with an enzyme capable of producing glucan can be carried out in the presence of phosphorylase and glucose-1-phosphate. Phosphorylase catalyzes the elongation reaction of α-1,4-glucan chains when glucose-1-phosphate is present in excess. For this reason, phosphorylase and glucose-1-phosphate coexist when the α-1,4-glucan chain of the raw material does not have a sufficient length to undergo the cyclization reaction of the enzyme. Thus, the yield of the glucan of the present invention can be increased.

  Furthermore, a derivative of the starch or a partially decomposed product of starch can also be used as a raw material. For example, a derivative in which at least one of the alcoholic hydroxyl groups of the starch is etherified (carboxymethylated, hydroxyalkylated, etc.), esterified (phosphorylated, acetylated, sulfated, etc.), crosslinked or grafted. Etc. can also be used. Furthermore, a mixture of two or more of these may be used as a raw material.

  As the reaction between the raw material and the enzyme that can be used in the present invention, any of the conditions such as pH and temperature at which the glucan of the present invention is generated can be used.

  Taking amylomaltase as an example, the pH of the reaction is usually from 3 to 10, preferably from 4 to 9, more preferably from 6 to 8, from the viewpoints of reaction rate, efficiency, enzyme stability, and the like. The temperature is preferably about 10 ° C. to 90 ° C., preferably about 20 ° C. to 60 ° C., more preferably 30 ° C. to 40 ° C. in terms of reaction rate, efficiency, enzyme stability, and the like. When an enzyme obtained from a thermostable microorganism is used, it can be used at a high temperature of 50 ° C to 90 ° C. The concentration of the raw material (substrate concentration) can also be determined in consideration of the degree of polymerization of the substrate used and the reaction conditions. Usually, from the viewpoint of 0.1% to 30%, reaction rate, efficiency, ease of handling of the substrate solution, etc., preferably 0.1% to 10%, more preferably 0.5% from the viewpoint of solubility. 5%. The amount of the enzyme to be used is determined in relation to the reaction time and the concentration of the substrate, and it is usually preferable to select the amount of the enzyme so that the reaction is completed in about 1 to 48 hours. 50 to 10,000 units, preferably 70 to 2,500 units, more preferably 400 to 2,000 units.

  The glucan of the present invention can be separated and purified by applying a method known per se. For example, after completion of the above reaction, the glucan of the present invention can be separated and purified by precipitation with a solvent, separation with a membrane, separation by chromatography, and the like. Preferably, the reaction solution is heated or purified as it is. The cyclic glucan having only the α-1,4-glucoside bond of the present invention is obtained by adding exo-type β-amylase or glucoamylase and decomposing the remaining linear α-1,4-glucan. . When a branched polysaccharide such as starch is used as a raw material, an exo-type β-amylase or glucoamylase and an enzyme that cleaves an α-1,6-glucoside bond are used in combination to form a cyclic α-1,4. -React so that only glucan remains. Thereafter, separation and purification means such as precipitation using a solvent, membrane separation, and chromatographic separation can be applied.

  Moreover, the glucan obtained by the polypeptide of the present invention can be easily etherified, esterified, crosslinked and grafted, can be made into a derivative, and can be used for the above purpose and application. As a derivatization method, a method usually used for modification of starch can be used (Biochemical Experimental Method 19, “Starch / Related Sugar Experimental Method”: Nakamura et al., Academic Publishing Center, 1986, pp. 273-303. ). As an example of phosphorylation, the glucan of the present invention is reacted with phosphorus oxychloride in dimethylformamide to obtain a phosphorylated glucan derivative of the present invention.

  When the amylomaltase of the present invention is used, a glucan having greatly improved solubility in water as compared with existing starch, amylose, and amylopectin can be produced efficiently and / or while reducing by-products. The aqueous glucan solution obtained using the amylomaltase of the present invention has an excellent property that the aging observed in the existing aqueous solutions of starch, amylose, and amylopectin does not occur.

  It was not possible with conventional enzymes to produce glucans having such superior properties more efficiently than wild-type amylomaltase and / or with significantly reduced by-products (hydrolysates). It can be said that this is an exceptional effect. Moreover, such a manufacturing method can be used for all the foodstuffs which can use starch, dextrin, etc. conventionally, and can be used for the composition for almost all food-drinks or food additives. This eating and drinking composition is a general term for human food, animal feed, and pet food. In other words, liquid and powdered beverages such as coffee, tea, Japanese tea, oolong tea, juice and sports drinks, bakery items such as bread, cookies, crackers, biscuits, cakes, pizzas and pies, pasta items such as spaghetti and macaroni , Noodles such as udon, soba, ramen, confectionery such as caramel, gum, chocolate, snack confectionery such as rice cake, potato chips, snacks, frozen confectionery such as ice cream, sorbet, cream, margarine, cheese, powdered milk, condensed milk, Dairy products such as dairy drinks, Western confectionery such as jelly, pudding, mousse, yogurt, Japanese confectionery such as buns, uro, mochi, and hagi, soy sauce, sauce, soup of noodles, sauce, dashi-mochi, stew-mochi, soup Nomoto, compound seasoning, curry base, mayonnaise, dressing Seasonings such as ketchup, retort or canned foods such as curry, stew, soup and bowl, chilled and frozen foods such as ham, hamburger, meatballs, croquettes, pilaf, rice balls, and marine products such as chikuwa and kamaboko It can also be used effectively for food, rice balls, bento rice, sushi rice and other cooked rice, as well as dumpling skin and shumai skin. Furthermore, it can be used for infant milk, baby food, baby food, pet food, animal feed, sports drinks, sports foods, dietary supplements and the like by utilizing its high digestibility.

  The glucan obtained by the polypeptide of the present invention can be used for a pharmaceutical composition such as infusion.

  The glucan obtained by the polypeptide of the present invention can be used for an agrochemical composition or an animal pharmaceutical composition.

  The glucan obtained by the polypeptide of the present invention can be used for an adhesive composition. The glucan of the present invention has adhesiveness and tackiness. Therefore, it can be used in the adhesive field where starch, dextrin or derivatives thereof are conventionally used. Adhesive compositions include, for example, surface sizing agents in the papermaking and paper processing industries, coating agents, interlayer adhesives, binders in the food industry, fibers, glues in the building materials industry, binders, and the like. .

  The glucan obtained by the polypeptide of the present invention includes or adsorbs various substances. Cyclodextrin and existing amylose also have inclusion and adsorption ability, but the glucan of the present invention has a significantly higher solubility in water than amylose and cyclodextrin, so a wider application range can be expected. Furthermore, since the degree of polymerization is significantly higher than that of cyclodextrin, it is considered that it has a guest specificity different from that of cyclodextrin. Substances can change their properties or acquire new properties by inclusion or adsorption in amylose or cyclodextrin. For example, improvement of solubility, non-volatility of volatile substances, stabilization of unstable substances, masking of unpleasant odors, etc. are well known. The substance adsorbed or included in the glucan of the present invention is not particularly limited, and examples thereof include foods such as wasabi, soy sauce, tea, sansho, yuzu, fragrance, seasoning, and pigment, and pharmaceuticals such as menthol and linoleic acid. The clathrate or adsorbate thus produced can be used as a food or a medicine. For example, it can be used for foods such as those described above, and pharmaceuticals such as bathing agents, swallowing drugs, and powder drugs.

  The glucan obtained by the polypeptide of the present invention focuses on the low viscosity of the paste liquid, in the starch processing industry, such as raw materials for biodegradable plastics and intermediates for producing cyclodextrins from starch. Can be used as a raw material.

(Description of Preferred Embodiment)
In one aspect, the present invention provides amylomaltase variants. This amylomaltase variant or amylomaltase polypeptide is substituted in the amino acid sequence with an amino acid other than the amino acid residue of wild-type amylomaltase at the position of the amino acid residue that interacts with Acarbose, an inhibitor of amylomaltase polypeptide. It is characterized by being. By substituting amino acid residues that interact with Akabose with different amino acid residues from the wild-type amino acid residues, the present invention significantly reduces the enzymatic activity and / or side-reaction hydrolysis activity. I was able to. In particular, simultaneous increase in enzyme activity and decrease in hydrolytic activity by the same modification cannot be predicted by the conventional technique and can be said to be a remarkable effect.

  Preferably, the region that interacts with Acabose is the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartic acid, 98-position glycine of the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2. A residue corresponding to at least one residue selected from the group consisting of position 101 tyrosine and position 153 glycine. Such residues can be easily identified by using techniques such as X-ray structural analysis, computer modeling, etc. so that those skilled in the art can easily identify the corresponding amino acid residues in amylomaltase from sources other than Thermus aquaticus ATCC 33923. Can do.

  In one embodiment, the modification involves substituting one amino acid for another amino acid other than the wild-type amino acid. Any other amino acid may be used as long as it exhibits a desired effect (for example, an increase in enzyme activity or a decrease in hydrolysis activity). Preferably, such modifications at the same location achieve both increased enzyme activity and decreased hydrolytic activity.

  In a preferred embodiment, the substitution, addition or deletion introduced in the amylomaltase polypeptide of the present invention is in the residue corresponding to position 54 tyrosine of amylomaltase from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2. It includes having an amino acid other than tyrosine. This amino acid substitution is preferably a non-conservative substitution. Such non-conservative substitutions can be made by considering the hydrophilicity index and / or hydrophobicity index as described herein. As for amylomaltase derived from Thermus aquaticus ATCC 33923, the tyrosine at position 54 has been shown to interact with acabose by crystal structure analysis as described in Examples. It has been clarified that changing such a tyrosine to another amino acid is responsible for an increase in enzyme activity and a decrease in hydrolysis activity, as shown in Examples and the like. By substituting the amino acid corresponding to tyrosine at position 54 with another amino acid (other than tyrosine in the sequence of SEQ ID NO: 2), amylomaltase having increased enzyme activity and decreased hydrolysis activity can be produced.

  In a preferred embodiment utilizing amylomaltase comprising the amino acid sequence set forth in SEQ ID NO: 2, it is preferable that amino acids other than tyrosine for substituting position 54 tyrosine are non-conservative amino acids. In another preferred embodiment, the amino acid other than tyrosine can be selected from the group consisting of alanine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, arginine, serine and threonine. In another preferred embodiment, the amino acid other than tyrosine is an amino acid other than tyrosine, phenylalanine and lysine. In another preferred embodiment, the amino acid other than tyrosine is selected from the group consisting of glycine, proline, threonine and tryptophan. In particular, amylomaltase in which position 54 tyrosine is substituted with either threonine or glycine achieves the effect that the hydrolysis activity is remarkably lost in addition to the increase in the enzyme activity. Such an effect is an unexpected and exceptional effect. Most preferably, the amino acid other than tyrosine that replaces the 54th tyrosine is glycine. The amylomaltase in which the 54th tyrosine is replaced with threonine achieves the effect that the hydrolysis activity is almost lost in addition to the increase in the enzyme activity, which can be correctly achieved by conventional techniques. It can be said that this is a remarkable effect that did not exist.

  The amylomaltase polypeptide of the present invention may comprise only natural amino acids, but may contain non-natural amino acids or amino acid analogs as long as they exhibit the desired activity (amylomaltase activity and the improved activity described above). Good.

  In another aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding an amylomaltase polypeptide of the present invention. Methods for producing and using nucleic acid molecules are described in detail herein, and those skilled in the art can produce and use this nucleic acid molecule according to its purpose.

  In another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. In addition to the nucleic acid molecule of the present invention, such a vector may contain regulatory elements such as a promoter, an enhancer, a terminator, a translation termination sequence, a translation initiation sequence, and an origin of replication.

  In another aspect, the present invention provides a cell comprising the nucleic acid molecule of the present invention. Such cells may be microbial cells. Such cells can be eukaryotic or prokaryotic. E. from the point of simplicity. It is preferable to use E. coli. Preferably, the cell may be transformed with the vector of the present invention. Alternatively, the nucleic acid molecule of the present invention may be integrated into the genome.

  In another aspect, the present invention provides a biological tissue comprising the nucleic acid molecule of the present invention. Such biological tissue can be regenerated from cells containing the nucleic acid molecules of the present invention. Examples of such biological tissues include, but are not limited to, plant tissues and animal tissues. Such tissue regeneration methods are well known in the art.

  In another aspect, the present invention provides a transgenic organism comprising the nucleic acid molecule of the present invention. Such a transgenic organism is the same as the cell in the case of a unicellular organism, but refers to the whole organism in the case of a multicellular organism. Examples of such transgenic organisms include, but are not limited to, plants and animals.

  In another aspect, the present invention is a step of adding the amylomaltase polypeptide of the present invention to a food material before or immediately after the heat treatment of the material, wherein the amylomaltase is derived from starch in the food material. A method for producing a food product comprising the step of producing a cyclic glucan is provided. Such a cyclic glucan production process itself is well known, but by using the amylomaltase polypeptide of the present invention instead of the conventional one, a food containing cyclic glucan can be produced more efficiently than the conventional method. be able to. Moreover, if this method is used, the foodstuff from which the undesirable contaminant called a hydrolyzate was reduced significantly can be manufactured.

  In a preferred embodiment, the food is cooked rice, Japanese confectionery, snack confectionery, bakery, noodles, gyoza and shumai peel, fish paste product, frozen or refrigerated processed food, baby food, baby food, pet food, animal use. Selected from the group consisting of feed, beverages, sports food, and nutritional supplements.

  In another aspect, the present invention provides a food material, a food additive and / or a food modifier characterized by containing the amylomaltase polypeptide of the present invention. The process itself of including such an amylomaltase polypeptide is well known, but by using the amylomaltase polypeptide of the present invention in place of the conventional one, it is more efficient and / or compared with conventional food materials and the like. Alternatively, it can provide a reduced undesirable reaction.

  In another aspect, the present invention includes an α-1,4-glucoside bond comprising a step of reacting a linear α-1,4-glucan or a saccharide containing them with the amylomaltase polypeptide of the present invention. A method for producing glucan having one cyclic structure constituted by or a derivative thereof is provided. The step of reacting the linear α-1,4-glucan or saccharide with amylomaltase polypeptide itself is well known, but by using the amylomaltase polypeptide of the present invention, compared to the conventional method, A cyclic glucan or a derivative thereof can be efficiently produced. Moreover, if this method is used, a glucan or a derivative thereof in which undesirable impurities such as hydrolysis products are remarkably reduced can be produced.

  In another aspect, the present invention also includes a step of reacting a linear α-1,4-glucan or a saccharide containing these with the amylomaltase polypeptide of the present invention, a food material, a food additive, Provided is a method for producing a food modifier, a food-drinking composition, an infusion solution or an adhesive composition. The process itself of reacting linear α-1,4-glucan or a saccharide containing these with amylomaltase polypeptide is well known, but by using the amylomaltase polypeptide of the present invention, compared to the conventional method. A food material, a food additive, a food modifier, a food-drinking composition, an infusion solution or an adhesive composition containing cyclic glucan or a derivative thereof can be efficiently produced. Moreover, if this method is used, a glucan or a derivative thereof in which undesirable impurities such as hydrolysis products are remarkably reduced can be produced.

  In another aspect, the present invention provides a computer-readable recording medium containing information on the amino acid sequence of the polypeptide of the present invention or the nucleic acid sequence encoding the amino acid sequence. In yet another aspect, the present invention provides a computer-readable recording medium containing information on the three-dimensional structure data of the amylomaltase polypeptide of the present invention. Examples of the computer-readable recording medium include a magnetic tape, a magnetic disk (for example, floppy (registered trademark) disk), a magneto-optical disk (for example, MO), and an optical disk medium (for example, CD-ROM, CD-R). CD-RW, DVD-RAM, DVD-R, DVD-RW, DVD + RW, DVD-ROM, memory card, etc.). The recording medium may be a hard disk. A recording medium recording such useful information may further include data relating to enzyme activity and hydrolysis activity. By including such data, these recording media are useful for producing further preferred amylomaltases.

  In another preferred embodiment, the present invention provides a glucan obtained by the amylomaltase polypeptide of the present invention. The composition containing glucan obtained by the amylomaltase of the present invention is useful because the hydrolyzate is remarkably reduced.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

(Example 1: Purification of amylomaltase from the extract of Thermus aquaticus ATCC 33923 strain)
(1) Purification of amylomaltase Thermus aquaticus ATCC 33923 strain was prepared at 70 ° C. in 33 liters of medium (1% maltose, 0.4% yeast extract, 0.8% polypeptone, 0.2% NaCl, pH 7.5). Shake for 18 hours. The bacterial cells were collected by centrifugation, washed with 10 mM KH ₂ PO ₄ -Na ₂ HPO ₄ (pH 7.5) (buffer A), suspended again in this buffer solution, and disrupted by ultrasonic waves. This was centrifuged and the supernatant was used as a crude enzyme solution. Ammonium sulfate was added to the crude enzyme solution to a final concentration of 1 M, left overnight at 4 ° C., and then loaded onto a phenyl Toyopearl column (manufactured by TOSOH) equilibrated with buffer A containing 1 M ammonium sulfate. After washing with buffer A containing 300 mM ammonium sulfate, the enzyme was eluted by changing the ammonium sulfate concentration in buffer A from 300 mM to 0 mM, and the active fraction was collected. The resulting enzyme solution was dialyzed against buffer A.

  The dialyzed enzyme solution was loaded onto a Source 15Q column (Pharmacia) equilibrated with buffer A. After washing with buffer A, the enzyme was eluted by changing the NaCl concentration in the buffer from 0 mM to 400 mM, and the active fraction was collected. The resulting enzyme solution was dialyzed against buffer A.

The dialyzed enzyme solution was loaded onto a Superdex 75 pg column (manufactured by Pharmacia) equilibrated with 50 mM KH ₂ PO ₄ -Na ₂ HPO ₄ (pH 7.5) containing 150 mM NaCl, and the enzyme was eluted with buffer A. The obtained enzyme solution was dialyzed against buffer A to obtain a purified enzyme solution. The purified enzyme showed a single band by SDS polyacrylamid gel electrophoresis. The molecular weight of amylomaltase determined from the mobility in SDS-PAGE was 57,000.

(2) Activity measurement of amylomaltase The activity of amylomaltase is obtained when amylomaltase is allowed to act on 10% (w / v) maltotriose at 70 ° C. for 10 minutes in a 50 mM sodium acetate buffer (pH 5.5). Glucose produced in was measured by quantification.

  The amount of enzyme that liberates 1 μmol of glucose per minute was defined as 1 unit, and the activity unit was calculated.

(Example 2: Partial amino acid sequence determination of Thermus aquaticus ATCC 33923 strain amylomaltase)
(1) C4 column for HPLC (Vydac 214TP54 (0.46 × 25 cm)) in which the enzyme solution obtained in Example 1 was equilibrated with 48.4% acetonitrile eluent containing 0.1% trifluoroacetic acid (TFA). The enzyme was eluted by loading the enzyme solution into Vydac) and changing the acetonitrile concentration in the eluent from 48.4% to 52.4%. The obtained enzyme solution was concentrated under reduced pressure. The N-terminal amino acid sequence of the enzyme was determined using a peptide sequencer (MELP-RA- (SEQ ID NO: 3)).

(2) Determination of amino acid sequence of purified amylomaltase trypsin digested fragment The enzyme was eluted from the HPLC C4 column in the same manner as in (1) above. The obtained enzyme solution was dried under reduced pressure, dissolved in a solution containing 8 M urea, 0.4 M NH ₄ HCO ₃ , 4.5 mM dithiothreitol (DTT), 10 mM iodoacetamide (Iodeacetoamide), and trypsin was added. And allowed to react at 37 ° C. for 24 hours. The trypsin digested fragment thus obtained was loaded onto an HPLC ODS column (Vydac 218TP54 (0.46 × 25 cm), manufactured by Vydac) equilibrated with 1.6% acetonitrile eluent containing 0.06% TFA. Then, elution was performed by changing the acetonitrile concentration in the eluent from 1.6% to 78.4%. Three peak fractions well separated from other peaks were collected and concentrated under reduced pressure. The N-terminal amino acid sequences of these peptide fragments were determined by a peptide sequencer (S-V-A-R-L-A-V-Y-P-V-Q-D-V-L-A-; M- N-Y-P-G-R-P-S-G-N-?-A-; I-I-G-D-M-P-I-F-V-A-E-D- (each sequence Numbers 4-6)).

(Example 3: Isolation of Thermus aquaticus ATCC 33923 strain amylomaltase gene)
(A) Preparation of a genomic library of Thermus aquaticus ATCC 33923 (A1) Preparation of genomic DNA A 1000 ml medium containing 0.89% tryptone, 0.4% yeast extract and 0.2% sodium chloride In a 2 L Sakaguchi flask, the cells were cultured with shaking at 70 ° C. for 10 to 16 hours. The cells were collected by centrifugation, and genomic DNA was prepared by the phenol method (Saito and Miura, Biochimica et Biophysica Acta, 72, 619 (1963)).

(A2) Insertion of genomic DNA into λ-ZAP express The genomic DNA obtained in (A1) above is partially digested with the restriction enzyme Sau3AI, and then subjected to sodium chloride density gradient centrifugation to give a DNA fragment of 5 to 10 Kbp. A fraction having was obtained. The obtained DNA fragment fraction was ligated with BamHI-treated λ-ZAP express vector (Stratagene) using T4 DNA ligase. This reaction solution was treated with an in vitro packaging kit, Lambdyne (Nippon Gene) to obtain a recombinant λ phage suspension.

(A3) Introduction of Recombinant λ Phage into Escherichia coli E. coli VCS257 strain was prepared using L medium (10 g / l tryptone (Difco), 5 g / l yeast extract (Difco), 10 g / l NaCl, pH 7) containing 10 mM magnesium chloride. 0.0) and suspended in 20 ml of 10 mM magnesium chloride solution. Next, it was mixed with recombinant λ phage and incubated at 37 ° C. for 20 minutes. Thereafter, L upper agar was added onto L agar medium (10 g / l tryptone (Difco), 5 g / l yeast extract (Difco), 10 g / l NaCl, pH 7.0, 1.5% (w / v) Agar). Overlay with medium (10 g / l tryptone (Difco), 5 g / l yeast extract (Difco), 10 g / l NaCl, pH 7.0, 0.8% (w / v) Agar) and mix at 37 ° C. Incubate overnight to obtain a plate dotted with lysis spots.

(B) Screening of the genomic library of Thermus aquaticus ATCC 33923 with an oligonucleotide probe
(B1) Preparation of oligonucleotide probe for isolation of amylomaltase gene Partial amino acid sequence of thermus aquaticus ATCC 33923 amylomaltase determined in Example 2 (AVYPVVQDV) A synthetic oligo DNA (5′-GCIGITAYCCIGTICARGAYGT-3 ′ (SEQ ID NO: 7) (nucleic acid description conforms to the IUPAC nucleic acid code)) was prepared. This synthetic oligo DNA was radiolabeled and used as a probe for amylomaltase gene isolation.

(B2) Selection of recombinant λ phage containing amylomaltase gene Nylon filter (Amersham) was closely attached to the plate prepared in (A3) above, and alkali treatment was performed to denature the recombinant λ phage DNA in the lysis spots. And fixed to the filter. This filter was immersed in the probe solution prepared in (B1) for hybridization. Recombinant λ phage containing the amylomaltase gene was selected by contacting the X-ray film and detecting the signal.

(B3) Subcloning of recombinant λ phage containing amylomaltase gene into plasmid vector Recombinant λ phage obtained in (B2) above, E. coli (XLI-Blue MRF strain), and helper phage (ExAssist) are mixed And kept at 37 ° C. for 15 minutes. Thereafter, 5 ml of L medium was added, and cultured with shaking at 37 ° C. for 4 to 6 hours. The culture solution was heat treated at 70 ° C. for 20 minutes, and then centrifuged to obtain a supernatant to obtain a phage solution. 20 μl of this phage solution and 200 μl of E. coli (XLOLR strain) were mixed and incubated at 37 ° C. for 15 minutes. Then, a part or the whole amount thereof was plated on an L agar medium containing 50 μg / μl of kanamycin, and kept at 37 ° C. for 10 to 18 hours. Plasmid DNA was extracted from the colonies that appeared by the alkaline-SDS method (Sambrook et al., Molecular Cloning, supra) to obtain plasmid DNA containing the amylomaltase gene.

(C) Determination of nucleotide sequence of Thermus aquaticus ATCC 33923 amylomaltase gene The nucleotide sequence of the plasmid DNA obtained above was determined using a DNA sequencer (manufactured by ABI). The entire base sequence of the amylomaltase gene of Thermus aquaticus ATCC 33923 is shown in SEQ ID NO: 1 in the sequence listing. Sequence number 2 of a sequence table is a corresponding amino acid sequence.

(Example 4: Properties of Thermus aquaticus ATCC 33923 amylomaltase)
The enzymatic properties of the amylomaltase purified in Example 1 were analyzed using methods well known to those skilled in the art. The optimum reaction temperature of this enzyme was 65 to 70 ° C., and the optimum reaction pH was 5.5. Further, even when the enzyme was subjected to a heat treatment at 80 ° C. for 10 minutes, the enzyme had a residual activity of almost 100% and almost no inactivation was observed. However, it was completely deactivated by heat treatment at 100 ° C. for 10 minutes.

(Example 5: Production of recombinant amylomaltase from Thermus aquaticus ATCC 33923 strain)
Recombinant amylomaltase was produced using the nucleic acid sequence information of the amylomaltase gene derived from Thermus aquaticus ATCC 33923 obtained in Example 3.

(A) Amplification of amylomaltase gene by PCR Using the Thermus aquaticus ATCC 33923-derived genomic DNA or plasmid obtained in the above example as a template, two types of oligonucleotides (P1: 5'-TTCATCATGGAGCTTCCCCCGCGCTTTCGGTCTGCTTT-3 '(SEQ ID NO: 8) ), P2: 5′-TTTTATTATTGGGGCTGGTCCCACCTAGAGCCGTTCGT-3 ′) (SEQ ID NO: 9) as a primer and PCR (98 ° C. for 10 seconds, 68 ° C. for 1 minute for 25 cycles) under conditions containing glycerol at a final concentration of 10%. went. By this amplification, an amylomaltase gene was obtained in which a restriction enzyme NdeI site was added to the N-terminal side of the structural gene of amylomaltase and a restriction enzyme EcoRI site was added to the C-terminal side. The amplified DNA was completely digested with restriction enzymes NdeI and EcoRI and then subjected to agarose electrophoresis to recover a DNA fragment of about 1.5 kb containing the amylomaltase gene.

  (B) Preparation of Expression Vector The following sequence is located at the BamHI site of the commercially available E. coli expression vector pGEX-5X-3 (Pharmacia):

(SEQ ID NOs: 10 and 11)
Was inserted to obtain plasmid pGEX-Nde (see FIG. 2).

  An approximately 1.5 kb DNA fragment containing the amylomaltase gene prepared in (A) was introduced into the NdeI-EcoRI site of the newly prepared expression plasmid pGEX-Nde to obtain an amylomaltase expression plasmid pFQG8 (FIG. 2). See).

  (C) Expression of thermostable amylomaltase in Escherichia coli 1 liter of LB medium (1% tryptone, 0.5%) containing E. coli TG-1 strain transformed with amylomaltase expression plasmid pFQG8 containing ampicillin at a final concentration of 100 μg / ml Yeast extract, 1% NaCl, pH 7.5) was cultured at 37 ° C. until the late logarithmic growth phase (about 6 hours), and then IPTG with a final concentration of 0.1 mM was added. After further culturing at 37 ° C. for 16 hours, the cells were collected by centrifugation. The obtained microbial cells were washed twice with 300 ml of buffer A and then dispersed in 60 ml of buffer A. The bacterial cells were crushed by ultrasonic waves, and the centrifuged supernatant was used as a crude enzyme solution. The crude enzyme solution thus obtained had an amylomaltase activity of 30 units / ml, which was about 500 times (per 1 ml of culture solution) the activity obtained when the Thermus aquaticus ATCC 33923 strain was cultured.

(Example 6: Crystal structure analysis of recombinant amylomaltase derived from Thermus aquaticus ATCC 33923 strain)
Purification of amylomaltase for crystal structure analysis is described in Przylas I. et al. J. et al. Mol. Biol. 296, 873-886 (2000). Specifically, it is as follows.

  The amylomaltase is an E. coli harboring the recombinant plasmid pFQG8. E. coli strain MC1062 (Terada et al. (1999) Appl. Environ. Microbiol. 65, 910-915.). The expressed amylomaltase is described in Terada et al. Purified by making the following modifications according to the method of (1999).

  After hydrophobic interaction chromatography, amylomaltase was dialyzed against 20 mM Tris-HCl (pH 7.2). A single band was observed by SDS-PAGE. This band was separated by a linear gradient from 2 mM to 200 mM sodium chloride by high resolution anion exchange chromatography (HR 10/10 MonoQ, Amersham Pharmacia Biotech, Uppsala, Sweden), and appeared as three peaks. The first peak, which is the main peak, is pooled, dialyzed against 5 mM Tris-HCl (pH 7.6), 1 mM DTT, 100 mM NaCl, concentrated to a concentration of 10 mg / ml, and the following crystal analysis is performed. It was. Although the thing obtained from the 2nd peak was similarly pooled and crystallized and analyzed, the crystal structure was the same.

The purified amylomaltase was crystallized using the hanging drop vapor diffusion method. The best crystals are mixed at 291 K with equal amounts of amylomaltase solution and 17% (w / v) PEG 8000, 100 mM Hepes (pH 7.5) and 10% (v / v) ethylene glycol reservoir solution. Obtained when 3 weeks passed. For data collection, the crystals were transferred to a mother liquor containing 30% ethylene glycol, and cryoprotectant was added stepwise. Crystals belong to space group P6 _4, the unit lattice constant was a = b = 154Å and c = 64 Å.

The heavy atom derivative crystals are 20 mM PCMBS (4 days), 1 mM HgCl ₂ (2 days), 5 mM K ₂ PtCl (2 days), 5 mM KAu (CN) ₂ (4 days), 1 mM K ₂ Pt (SCN) ₆ ( A total of six were made by immersing crystals in mother liquor containing 2 days) and 5 mM (CH ₃ ) ₃ PbAc (7 days).

High-resolution raw data was generated by performing crystal structure analysis at 100K using a BW7a beamline from DESY / EMBL (Hamburg, Germany). Raw and derivative data sets for phasing were collected at room temperature using Cu- _Kα radiation from a rotating anode source. In all cases, measurements were made with a MarResearch imaging plate detector and data processed with Denzo / Scalepack (Otwinski, Z. (1993). Pp. 56-62, SERC, Daresbury Laboratory, Warrington, UK.). . High resolution data of native enzyme with 3.4 redundancy was used for structure refinement. The I / σ _I ratio was 15.1 for the entire data set and the 87.5% reflection was I / σ _I > 1 (72.9% for the outer shell).

The MIR phase was derived from heavy atom derivatives in combination with a room temperature raw data set. Experiments with heavy atom isomorphous substitution Patterson maps of derivatives using the CCP4 package (CCP4, 1994) revealed a single heavy atom site for each derivative. These sites were refined and the phase was calculated using a program called SHARP (De La Fortelle, E. & Bricogne, G. (1997). Methods Enzymol. 276, 472-494.). The phase was improved by performing solvent smoothing by using SOLOMON (CCP 4, 1994). The resulting map was of high quality and was sufficient to create an initial model using program O (Jones, TA, Zou, JY, Cowan, SW. & Kjelgaard, M. (1991) Crystallographic Computing (Moras, D., Podany, AD & Thierry, JC, eds), pp. 413-432, Oxford University Press, Ox. The data obtained is shown in the table below.

Crystal data refinement was performed on a room temperature data set (native-2), and then refinement was continued using a high resolution frozen data set (native-1). The refinement procedure included simulation annealing and minimizing conjugate gradient energy against the maximum likelihood target. This is a program called CNS (Brünger, AT, Adams, PD, Clore, GM, Delano, WL, Gros, PO, Grose-Kunstlev, RW. , Jiang, JS, Kuszewski, J., Nilses, M., Pannu, NS, Read, RJ, Rice, LM, Simonson, T. & Waren, GL. (1998). Acta Crystallog.sect.D, 54, 905-921.). Bulk solvent correction was performed. The final model included all 500 amino acid residues and 739 water molecules. Water molecules were automatically picked up from the Fo-Fc difference electron density map and checked by a human on a graphics station. Individual power coefficients were refined for all non-hydrogen atoms. Final R value _{(R free)} value was 19.6% (22.6%). The coordinate error was estimated to be 0.23 cm according to the Luzzati plot. Of the total amino acid residues, 90.6% were in the most preferred region and 1.0% were generally acceptable (Asp370, Glu253) or were not allowed (Phe251, Ser252) (program PROCHECK (Laskowski, RA, MacArthur, MW, Moss, D. & Thornton, JM (1993) J. Appl. Crystallog. 26, 283-291.435-446. Schmidt, A. K., Cottaz, S., Draguez, H. & Schulz, GE (1998))). The data obtained is shown in the table below.

  The coordinate data thus obtained was registered in the RCSB protein data bank (registration number 1CWY).

  The coordinate data thus obtained is shown below in Przylas et al. (2000) Eur. J. et al. Biochem. 267, 6903-6913, a three-dimensional diagram of the binding mode was generated for the conjugate of wild-type amylomaltase and the inhibitor Acabose. The procedure is shown below.

Using the atomic coordinates of raw amylomaltase as a starting model, we refined the three-dimensional structure of the conjugate of amylomaltase and Acabose. After performing rigid body and simulation annealing using the program CNS (Bruinger, AT et al. (1998), Acta Crystallogr. D54, 905-921), continuous density was observed for both of two Acarbose. The topology and parameters for the Acarbose inhibitor were determined using the XPLO2D program (Kleywegt, GA & Jones, TA (1997), Methods Enzymol. 277, 208-230) and the structure of porcine amylase and the Acarbose complex (1PPI). ). All parameters were manually checked to make sure they were consistent with the expected chair conformation for the glucose residue and the expected half chair conformation for the cyclitol ring. To test for deviations from these standard shapes, refinements were made without constraints on dihedral angles. No significant deviation was observed from the results of constrained refinement. This indicates that acarbiocin units and maltose units are in a standard conformation. The dihedral angle defining the linkage between sugar units was not constrained. Several cycles of refinement (program CNS) and manual reconstruction (program O (Jones, TA, Zou, JY, Cowan, SW & Kjelgaard, M. (1991) Improved methods) for the building of protein models. In Crystallographic Computing. (Moras, D., Podany, AD & Thierry, JC, eds), pp. 413-432, Oxford Prev. 500 amino acid residues, two Akabosu molecule, to produce a final model that includes all two water molecules ethylene glycol molecule and 603 to. final R value is 19.2% (R _{f ee} are shown in Table 3 below those of 21.9%) was. More detailed data collection and refinement.

The average B coefficient of the Abbose molecules bound to the active site was 37 ² . This that bound to the protein side chains around through hydrogen bonds (a 28 Å ² for the main chain atoms, 28 Å 2 for the side chain ^atoms) greater than the average B factor. Only Gln256 residue also showed a somewhat higher average B factor of 38 ² . Aka Dose units A to D had approximately the same B coefficient (39 ² , 37 ² , 35 ² and 37 ^{2 respectively} ). The cause of the high B-factor of Acarbose compared to surrounding residues may be due to the high degree of freedom of Acarbose in the binding groove, but the binding site may not be fully occupied. A similar situation existed at the second Acabose binding site. At the second Acabose binding site, a value of 49 ² was observed for all four Acabose units. Programs PROCHECK (Laskowski, RA, MacArthur, MW, Moss, D. & Thornton, J.M. (1993) J. Appl. Crystallogr. 26, 283-291) and WHATCHECK (Vriend.G. (1990) J. Mol. Graph. 8.52-56), the structure was evaluated, and the structure was analyzed using programs O and WHATIF. The results obtained are shown in FIGS. FIG. 3 is a schematic diagram relating to the first Aca Bose, and FIG. 4 is a schematic diagram relating to the second Aca Bose.

  Based on this, as typical residues predicted to interact with the substrate, 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine, 101-position tyrosine and 153-position glycine Was selected (see FIG. 4).

(Example 7: Production of modified recombinant amylomaltase derived from Thermus aquaticus ATCC 33923 strain)
In the same manner as in Example 5, a recombinant amylomaltase expression vector derived from Thermus aquaticus ATCC 33923 strain was prepared. In this example, among the groups that were found to interact with the substrate in Example 6, by way of example, all the other natural amino acids encoding tyrosine at position 54 (ie, glycine, alanine, valine, leucine, isoleucine) , Serine, methionine, threonine, phenylalanine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine). In the resulting SEQ ID NO: 2, the nucleic acid molecule having the same sequence as SEQ ID NO: 2 except that the tyrosine at position 54 was changed to the respective amino acid and encoding the variant corresponded similarly. Has a mutation. For substitution with other amino acids, site-directed mutagenesis was used. The detailed description will be described below.

  Expression of thermostable amylomaltase in Escherichia coli Using amylomaltase expression plasmid pFQG8 as a template, two types of oligonucleotide primers, except for alanine substitution: 5'-ttggggccccggggc gcc -3 ′ (SEQ ID NO: 17), 5′-ttgggccccacgggg cgg ggcgactccccctacc-3 ′ (SEQ ID NO: 12) and 5′-ggtaggggggagtcgcc ccg gcccgtggggccccagg-3 ′ (cggccg ggcgactcc cccacc-3 '(SEQ ID NO: 14) and 5'-ggtaggggggagtcgcc cgt gcccgtggggggccccagg-3' (SEQ ID NO: 15), buffer solution, DNA replication enzyme from Quick Change Site-Directed MutageneSTI PCR, pFQG8 25 ng, 2 types of oligonucleotide primers 20 pmol each, concentrated reaction buffer 5 μl, dNTP 1 μl, mixed at 95 ° C. for 30 seconds, 55 ° C. for 1 minute, 68 ° C. for 13 minutes under 16 cycles. It was. After PCR, the sample was placed in ice for 2 minutes, 1 μl of Dpn I was added and mixed, reacted at 37 ° C. for 1 hour, 1 μl was used to transform E. coli BL21 (STRATAGENE), and a final concentration of 100 μg. Cultured overnight at 37 ° C. in LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, pH 7.5) containing / ml ampicillin. An appropriate number of transformants obtained (about 10 clones) was selected, and 2 ml of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.5) containing ampicillin at a final concentration of 100 μg / ml. ) At 37 ° C. overnight, the plasmid was extracted and subjected to DNA sequencing. After confirming the mutation of the target portion and the DNA sequence of the entire amylomaltase gene, the plasmid containing the gene encoding the variant was obtained.

  Escherichia coli TG-1 strain transformed with a plasmid containing the gene encoding the variant was added to 1 liter LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) containing ampicillin at a final concentration of 100 μg / ml. , PH 7.5) until the late logarithmic growth phase (about 6 hours), and cultured at 37 ° C., IPTG having a final concentration of 0.1 mM was then added. After further culturing at 37 ° C. for 16 hours, the cells were collected by centrifugation. The obtained microbial cells were washed twice with 300 ml of buffer A and then dispersed in 60 ml of buffer A. The bacterial cells were crushed by ultrasonic waves, and the centrifuged supernatant was used as a crude enzyme solution. Then, it refine | purified by the method similar to the refinement | purification of the wild-type amylomaltase described in Example 1 (1), and the amylomaltase modified body was obtained.

(Example 8: Various properties of modified amylomaltase)
The enzymatic properties of the modified amylomaltase produced in Example 7 were analyzed using a method well known to those skilled in the art together with the natural amylomaltase of Example 5.

  First, the ability to synthesize cycloamylose was examined for the modified amylomaltase and the wild-type amylomaltase purified in Example 5. The assay for synthetic ability was performed in a buffer containing 100% sodium acetate (pH 5.5), 9% (v / v) DMSO containing enzyme-synthesized amylose AS-110 at a final concentration of 0.2% (w / v) for 1 hour. Then, 0.022 unit / ml modified amylomaltase or wild-type amylomaltase was allowed to act at 70 ° C., and the yield of cycloamylose was measured 1 hour after the start of the reaction. The results are shown in FIG.

  As is clear from FIG. 5, it is recognized that the yield of cycloamylose increased in all mutant enzymes other than phenylalanine (Y54F) compared to the wild type enzyme (Y), as is clear from these results. It was. In addition, cycloamylose was recovered in a yield twice or more that of the wild-type enzyme in the conversion to alanine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, arginine, serine and threonine. Therefore, the modification of the present invention significantly increases the activity of amylomaltase. This increase in activity is considered to be a result of an increase in reactivity to the substrate from the above-described crystal structure analysis.

  Next, the modified amylomaltase and the wild-type aminomaltase purified in Example 5 were allowed to act on cycloamylose, and the degree of hydrolysis was measured. Specifically, a modified amylomaltase or wild type in a buffer solution (50 mM sodium acetate (pH 5.5)) containing cycloamylose having a final concentration of 0.5% (w / v) and a polymerization degree of about 20-50. Hydrolysis activity was measured by allowing amylomaltase to act at 70 ° C. and measuring the increase in reducing sugar amount 1 hour after the start of the reaction. The amount of enzyme that increases the amount of 1 μmol of reducing sugar per minute was taken as 1 unit, and the specific activity per 1 mg of amylomaltase was calculated. Regarding the obtained results, FIG. 6 shows a graph of the relative activity of each variant when the wild-type hydrolysis activity is 1.

  As is clear from FIG. 6, the hydrolytic activity of all the mutant enzymes other than phenylalanine and lysine was lower than that of the wild type. In particular, the decrease in hydrolytic activity was significant in glycine, proline, threonine, and troptophan, and decreased to less than about 20%. Even if the conventional wild-type amylomaltase once synthesize | combined cycloamylose, it became a linear form by the hydrolysis action which exists simultaneously, and caused the fall of the yield. It was shown that the modified amylomaltase of the present invention has a weak action. Therefore, it has been clarified that the substituted amino acid in the mutant enzyme of the present invention is preferably an amino acid corresponding to a non-conservative substitution. Thus, virtually any amino acid substitution has been shown to be effective in improving enzyme activity and / or reducing side reactions in the mutant enzymes of the present invention. In addition, when used for cycloamylose synthesis, any modification may be used, but it has been found that the 54th tyrosine is particularly preferably substituted with threonine or glycine, more preferably glycine.

(Example 9: Production of cyclic glucan)
(A) Production of cyclic α-1,4-glucan (production of cycloamylose using amylomaltase of the present invention)
In 1 ml of a buffer (50 mM sodium acetate buffer (pH 5.5), 9% (v / v) DMSO) containing enzyme-synthesized amylose AS-110 (manufactured by Nakatsuji vinegar) at a final concentration of 0.2%, The purified amylomaltase variant 0.022 unit obtained in Example 7 was allowed to act at 70 ° C. After unreacted acyclic amylose was decomposed into glucose by glucoamylase, cycloamylose in the reaction product was quantified. The modified amylomaltase of the present invention cyclized amylose and reached the maximum reaction yield by 1 hour after the start of the reaction. For comparison, Thermus aquaticus ATCC 33923 wild type amylomaltase was allowed to act under the same conditions. In this case, the maximum yield was reached after 6 hours. Therefore, it is clear that the modified amylomaltase of the present invention has an increased reactivity to the substrate and is more suitable as an enzyme for producing cycloamylose than the conventional one.

  The sample at 24 hours after the reaction was digested with glucoamylase to decompose unreacted acyclic amylose into glucose, and then purified by precipitation of ethanol with ethanol. The thus obtained cycloamylose was analyzed by a sugar analysis system (liquid feeding system: DX300, detector: PAD-2, analysis column: CarboPacPA100) manufactured by Dionex. Elution is performed at a flow rate of 1 ml / min, NaOH concentration: 150 mM, sodium acetate concentration: 0 min-50 mM, 2 min-50 mM, 37 min-350 mM (Gradient curve NO.3), 45 min-850 mM (Gradient curve No. 7). ), 47 min-850 mM.

  The purified cycloamylose was a mixture having a polymerization degree of 21 or more. The degree of polymerization of cycloamylose was determined from the elution position of cycloamylose standard (Koizumi et al., J. Chromatography A, vol. 852, pp. 407-416, (1999)) having a degree of polymerization of 9 to 31.

  Accordingly, it was demonstrated that the modified amylomaltase of the present invention has an increased enzyme activity and / or decreased undesirable hydrolysis activity as compared with the conventional wild-type amylomaltase.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, a modified amylomaltase having increased enzyme activity and / or decreased undesired hydrolysis activity as compared with conventional wild-type amylomaltase is provided and has great utility in the sugar processing industry. The present invention also has utility in that cycloamylose and cyclic glycans can be mass-produced for industrial use by providing a mutant enzyme capable of maintaining a high yield even at a high substrate concentration.

It is a figure which shows the homology of well-known wild-type amylomaltase. Residues surrounded by boxes are homologous residues, and residues shown by diagonal lines pointing downward to the right are identical residues. Residues indicated by diagonal lines pointing downward to the left are the same residues and have a catalytic side chain. A region surrounded by a region indicated by a downward-pointing triangle is a particularly stored region (region1, region2, region3, region4, and 250s loop). It is a schematic diagram which shows the preparation process of an amylomaltase expression vector. It is a three-dimensional view showing the binding mode of wild-type amylomaltase with Akabose. It is a three-dimensional view showing the binding mode of wild-type amylomaltase in the vicinity of Tyr at position 54 with acabose. It is a figure which shows the cycloamylose synthetic ability of the modified amylomaltase of this invention and wild type amylomaltase. The conversion rate for the substrate was 100%. It is a figure which shows the hydrolyzing activity of cycloamylase of the modified amylomaltase of the present invention and wild-type amylomaltase. Wild-type hydrolysis activity was taken as 1.

(Explanation of sequence listing)
SEQ ID NO: 1: Wild-type nucleic acid sequence of Thermusaquaticus ATCC 33923 strain;
SEQ ID NO: 2: amino acid sequence as above;
SEQ ID NO: 3 partial amino acid sequence 1;
SEQ ID NOs: 4-6: partial amino acid sequences 2-4;
SEQ ID NO: 7: synthetic oligo (Example 3);
SEQ ID NOs: 8 and 9: PCR primers;
SEQ ID NOs: 10 and 11: adapter sequence;
SEQ ID NOs: 12, 14, 16, 18, 20: potato, E. coli amylomaltase nucleic acid sequence of E. coli, Synechocystis PCC 6803, Clostridium butyricum, Chlamydia pneumoniae;
SEQ ID NOs: 13, 15, 17, 19, 21: Potato, E. coli amylomaltase amino acid sequence of E. coli, Synechocystis PCC6803, Clostridium butyricum, Chlamydia pneumoniae;
SEQ ID NOs: 16-27; PCR primers used in making the variants.

Claims (26)

An amylomaltase polypeptide comprising:
A) comprising at least one substitution, addition or deletion in the amino acid sequence of the wild-type amylomaltase polypeptide,
The substitution, addition or deletion includes substitution in the amino acid sequence of the wild-type amylomaltase polypeptide with an amino acid other than the amino acid of the wild-type sequence at a position of an amino acid residue that interacts with abose.
Amylomaltase polypeptide. The amylomaltase polypeptide according to claim 1, which has at least one characteristic selected from the group consisting of an increase in enzyme activity and a decrease in hydrolysis activity as compared to the wild-type amylomaltase polypeptide. 3. The amylomaltase polyps of claim 2, wherein the property includes a decrease in the hydrolytic activity, wherein the decrease in hydrolytic activity includes a decrease of at least 10% or more compared to the wild type amylomaltase polypeptide. peptide. 3. The amylomaltase polyps of claim 2, wherein the property includes a decrease in the increase in the enzyme activity, wherein the increase in the enzyme activity comprises an increase of at least 10% or more compared to the wild type amylomaltase polypeptide. peptide. An amylomaltase polypeptide comprising:
A) comprising at least one substitution, addition or deletion in the amino acid sequence of the wild-type amylomaltase polypeptide,
The substitution, addition or deletion is carried out by the 53-position glycine, 54-position tyrosine, 55-position glycine, 56-position aspartate, 98-position glycine of the amino acid sequence of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2. Amino acid at each residue position shown in SEQ ID NO: 1 at the residue position of the wild-type amylomaltase polypeptide corresponding to at least one residue selected from the group consisting of position tyrosine and position 153 glycine Including having an amino acid other than
Amylomaltase polypeptide. The amylomaltase polypeptide of claim 1, comprising:
The substitution, addition, or deletion is performed at the position 54 corresponding to the tyrosine 54 of the wild-type amylomaltase polypeptide at the residue corresponding to position 54 tyrosine of the amylomaltase derived from Thermus aquaticus ATCC 33923 shown in SEQ ID NO: 2. Including having an amino acid other than the amino acid of the corresponding residue,
Amylomaltase polypeptide. The amylomaltase polypeptide of claim 1, comprising:
A) In the sequence shown in SEQ ID NO: 2, comprising at least one substitution, addition or deletion, wherein the substitution, addition or deletion comprises a substitution of a tyrosine at position 54 with an amino acid other than tyrosine,
An amylomaltase polypeptide. 8. The amylomaltase polypeptide of claim 7, wherein amino acids other than tyrosine are substituted by non-conservative substitutions. The amylomaltase polypeptide according to claim 7, wherein the amino acid other than tyrosine is selected from the group consisting of natural amino acids other than tyrosine and phenylalanine. The amylomaltase polypeptide according to claim 7, wherein the amino acid other than tyrosine is selected from the group consisting of alanine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, leucine, asparagine, arginine, serine and threonine. The amylomaltase polypeptide according to claim 7, wherein the amino acid other than tyrosine is an amino acid other than tyrosine, phenylalanine and lysine. The amylomaltase polypeptide according to claim 7, wherein the amino acid other than tyrosine is selected from the group consisting of glycine, proline, threonine and tryptophan. The amylomaltase polypeptide according to claim 7, wherein the amino acid other than tyrosine is threonine or glycine. The amylomaltase polypeptide according to claim 7, wherein the amino acid other than tyrosine is glycine. A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1-14. A vector comprising the nucleic acid molecule of claim 15. A cell comprising the nucleic acid molecule according to claim 15. A biological tissue comprising the nucleic acid molecule of claim 15. A transgenic organism comprising the nucleic acid molecule of claim 15. 15. A step of adding the amylomaltase polypeptide according to any one of claims 1 to 14 to a food material before or immediately after the heat treatment of the material, wherein the amylomaltase polypeptide is contained in the food material. Producing a cyclic glucan from the starch of the method. The food is cooked rice, Japanese confectionery, snack confectionery, bakery, noodles, dumplings and shumai peels, marine products, frozen or refrigerated processed foods, baby food, baby food, pet food, animal feed, beverages, sports 21. The method of claim 20, wherein the method is selected from the group consisting of a food product, as well as a dietary supplement. A food material, food additive, or food modifier containing the polypeptide according to any one of claims 1 to 14. A linear α-1,4-glucan or a saccharide containing them and a polypeptide according to any one of claims 1 to 14 are reacted with an α-1,4-glucoside bond. A method for producing glucan having one cyclic structure in the molecule or a derivative thereof. A food material, a food additive, a food modification comprising a step of reacting a linear α-1,4-glucan or a saccharide containing these with the polypeptide according to any one of claims 1 to 14. The manufacturing method of a quality agent, a composition for eating and drinking, an infusion solution, or an adhesive composition. A computer-readable recording medium comprising information on an amino acid sequence of the amylomaltase polypeptide according to any one of claims 1 to 14 or a nucleic acid sequence encoding the amino acid sequence. The cyclic glucan obtained by making the amylomaltase polypeptide of any one of Claims 1-14 act on linear alpha-1, 4- glucan or saccharides containing these.