WO2005003343A1 - Novel microorganism, maltose phosphorylase, trehalose phosphorylase, and processes for producing these - Google Patents

Abstract

A novel microorganism belonging to the genus Paenibacillus which has the ability to produce a maltose phosphorylase and a trehalose phosphorylase; a novel maltose phosphorylase which acts only on maltose to cause the α-1,4-glucopyranoside bond in the maltose to reversively undergo phosphorolysis; a novel trehalose phosphorylase which acts only on trehalose to cause the α-1,4-glucopyranoside bond in the trehalose to reversively undergo phosphorolysis; and processes for producing these enzymes. The enzymes are more easily obtained than in conventional processes. A considerable reduction in incubation time can also be attained. The enzymes have high stability to temperatures and have almost the same optimum-pH range.

Description

 Description Ifi Seen Microorganism, Maltose Phosphorylase and Torayachirose Phosphorylase and Production Method Thereof

 The present invention relates to a novel microorganism, a novel enzyme, and a method for producing a novel enzyme. More specifically, a novel microorganism belonging to the genus Paenibacillus (Paeni bad 11 us) having the ability to produce maltose phosphorylase and trehalose phosphorylase necessary for enzymatic production of treoctylose, The present invention relates to novel maltose phosphorylase and trehalose phosphorylase obtained from E. coli, genes encoding the amino acid sequences of these enzymes, and methods for producing these enzymes.

Background art

Trehalose is expected to have a wide range of uses in medicines, bffi products, foods, etc., and many attempts have been made for industrial production. These technologies can be broadly divided into the following three types. One of them is a method of extracting and purifying the substance from microorganisms having the property of accumulating treoctylose in cells (for example, see J. Am. Chem. Soc. 72, 0.259, 1 950, German Patent No. 2 660 584, Japanese Patent Application Laid-Open No. 3-130804, Japanese Patent Application Laid-Open No. 5-9-1890, Japanese Patent Application Laid-Open No. 5-1843 No. 53 and ζ ^ Keihei 5—292 9 896). This method involves the steps of culturing microorganisms, separating them, and treating trehalose from microorganisms. The process consists of an extraction process and a purification and crystallization process of the extracted trehalose, and the trehalose production process is very complicated. In addition, because the amount of microbial extraction residue that is not only low in the properties of Torehachiloose is generated as waste, it was not an efficient method.

 Brevipacterium is a microorganism that produces trehalose extracellularly.

A fermentation method has been developed in which a microorganism of the genus (Brevibacterium) genus Corynebacterium or the like is cultured to produce trehalose (see, for example, Japanese Patent Application Laid-Open No. 5-218182). However, even in this method, the accumulation rate of trehalose in the culture medium is as low as about 3% (w / v) .Therefore, in order to mass-produce trehalose on an industrial scale, a large-capacity fermenter and a purification scale suitable for it are required. Equipment is necessary and there is an economic problem. In addition, in this method, in order to obtain purified trehalose, not only is it necessary to remove bacteria, but also complicated steps are required to remove contaminants or medium components produced by the used strain at the time of culture. is necessary. ,

—On the other hand, an enzymatic method has been developed as a method for solving various problems of these fermentation methods. That is, a microorganism-derived maltose phosphorylase (maltose: orthophosphate β-D-glucosyl transferase) and an algae-derived trehalose phosphorylase (α, -treha! Ose: orthophosphate 3 -D-glucosyl transferase) are converted in the presence of phosphoric acid. A method for producing trehalose by reacting with maltose (see, for example, Patent No. 1513517, Agric. Biol. Chem, 49, 2113, 1985), and Bacterial sucrose: orthophosphate α-D-glucosy I transferase (Sucrose: orthophosphate a-D-glucosyl transferase; transferase) to sucrose in the presence of phosphoric acid to obtain trehalose (See, for example, the 3rd Annual Meeting of the Japanese Society of Agricultural Chemistry 3R a14). According to these methods, trehalose can be produced from maltose or sucrose with a high yield of 60 to 70%. In addition, since purified high-condensed saccharide is used as a raw material, it is easy to purify treoctylose obtained by an enzymatic reaction, and it is an industrially advantageous method by using i: tlS with other methods. It is believed that. However, the sources of enzymes used in these methods, particularly trehalose phosphorylase, are very limited, such as euglena and maitake, which are algae and basidiomycetes. However, in order to stably produce enzymes, there were technical problems as well as economic problems. In addition, the obtained treoctylose phosphorylase, which is used in combination with the cyanose phosphorylase, and maltose phosphorylase, differ greatly in the optimal pH region of each enzyme, and also have very high temperature stability. The reaction for the production of trehalose was only possible at low temperatures of about 25 to 37 ° C. This suggests that PH control when using two enzymes in combination is extremely difficult, but also low, and that bacterial contamination may occur during the enzymatic reaction performed using an open-type reactor. However, there are problems such as the necessity of strict hygiene management in order to prevent side reactions caused by this. Furthermore, when these known enzymes were used in combination, high raw materials could not be used due to the substrate separation dependence of these enzymes. Therefore, these methods were not economically efficient either.

Furthermore, there has been reported a method of obtaining trehalose using ¾ ^ -digest as a substrate. According to this method, a maltooligosyltrehalose synthase that produces a non-reducing carbohydrate having a trehalose structure at the terminal of the degradation product of nen, and a protein that specifically releases trehalose from the non-reducing saccharide. Activated rehalose-releasing enzyme to give a high yield of 80% from ^^ (For example, Japanese Patent Application Laid-Open No. Hei 7-143 876, Japanese Patent Application No. 628663 A2, Abstracts of Japanese Society of Agricultural Chemistry, p 3 1 (1 9 9 5)). However, in this method, since the screen is used as a substrate, it is difficult to immobilize the enzyme and pass through the substrate, and it is difficult to further simplify and increase the efficiency of production of a reactor or the like. there were. Furthermore, it has been difficult to construct a process for re-saccharifying honey nectar obtained after crystallization of trehalose. From the above, if it is possible to find new maltose phosphorylase and treoctylose phosphorylase that are easy to produce and purify, have high thermostability, and are independent, it will be easy and large-scale. It is expected that highly available and easily and efficiently toretoroose can be produced from available maltose as a raw material.

 Accordingly, a first object of the present invention is to solve the various problems of the conventional techniques as described above and to provide a novel enzyme maltose phosphorylase and trehalose phosphorylase which satisfy these various requirements. It is an object of the present invention to provide a novel microorganism which can produce E. coli with high production efficiency. The second object of the present TO is to provide a new maltose phosphorylase and a pure octylose phosphorylase that are easy to produce and purify, have high thermostability, and are independent of substrate concentration. . Further, a third object of the present invention is to provide a method for producing maltose phosphorylase and / or treoctylose phosphorylase which can easily obtain the above two enzymes in high yield using the above microorganism. The idea is to provide Disclosure of the invention

The inventors of the present invention have developed a broad spectrum of microorganisms capable of producing enzymes having the above-mentioned properties, which should be possessed by maltose phosphorylase and trehalose phosphorylase for use in industrial production. Searched. As a result, no \ enibacillus (Paen i bac ill us) It has been found that a microorganism belonging to the group described above produces a significant amount of both enzymes satisfying the above requirements, thereby completing the present invention.

 That is, the present invention is an invention having the following contents as its gist.

 (1) The present invention provides a microorganism of the genus Rhizobium genus having the ability to produce masoleose phosphorylase and trehalose phosphorylase. Specifically, for example, the present invention provides a microorganism having a deposit number of FER BP-8420. It provides Paenib acillus sp. SH-55.

 (2) The present invention also provides maltose phosphorylase having the following physicochemical properties.

 (A) Action: reversibly phosphorylates a-1,4-darcoviranoside bonds in maltose to produce glucose and i8-D-glucose 1-phosphate.

 (Mouth) Substrate specificity (decomposition reaction): It acts on maltose and does not act on trehalose, sucrose, lactose, cellobiose, etc.

 (C) Range of action pH, optimum pH and stable pH range: The range of action pH is 4.5 to 9.5. The optimal pH for the degradation J¾S is 7.0 to 8.0, and the optimal pH for the synthesis reaction is 5.5 to 6.5. Under heating conditions at 50 ° C for 10 minutes, the pH is stable within the range of 5.5 to 7.5.

 (2) Range of action and maximum: The action range is 20-60 ° C. The optimum temperature for the decomposition reaction is around 45 to 55 ° C, and the optimum temperature for the synthesis reaction is 50 to 55 ° C.

 (E) Stability: Extremely stable up to 50 ° C under heating conditions of pH 6.0 and 15 minutes. It is completely deactivated at 70 ° C.

(H) Inhibitors: copper, mercury, N-prosuccinimide, 卫 -chloromerkiuribenzoic acid (1 mM each), inhibited by sodium dodecylbenzenesulfonate (1%). (G) Isoelectricity,: pH is in the range of 4.8 to 5.0.

 (H) SDS—Polyacryloleamide electrophoresis has a molecular weight of about 89,000 to 90,000 daltons, gel method has a molecular weight of about 190,000 nights, homodimer It is composed of

 (3) Further, the present invention has the amino acid sequence represented by SEQ ID NO: i, or an amino acid sequence in which one or more amino acids in the sequence are deleted, substituted, inverted, added or inserted. 2) A maltose phosphorylase according to 2).

 (4) Further, the present invention has the amino acid sequence shown in SEQ ID NO: 1 having an amino acid sequence having a homology of 52% or more, preferably 70% or more, more preferably 90% or more. And a maltose phosphorylase.

 (5) Further, the present invention relates to a polynucleotide encoding the amino acid sequence of the mas- terolose phosphorylase described in (2) above, which comprises a group consisting of the following (a) to (c):

(a) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing,

 (b) a polynucleotide encoding a polypeptide having an amino acid sequence having Ί or a plurality of amino ^^ deletions, substitutions, inversions, additions or insertions in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing , and

 (c) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, and a polynucleotide selected from the group consisting of:

(6) The present invention also provides a recombinant vector having the polynucleotide of (5) and a microorganism transformed by the recombinant vector. (7) Further, the present invention provides treoctylose phosphorylase having the following physicochemical properties.

 (A) Action: Reversibly phosphorolytically decomposes (¾-1,1-Darcovyranoside bond) in trehalose to produce glucose and / 8-D-Darcose 1-phosphate.

 (Mouth) Substrate specificity (decomposition reaction): Acts on trehalose and does not act on maltose, sucrose, lactose, cellobiose, etc.

 (C) Range of action pH, optimum pH and stable pH range: The range of action pH is 4.5-9.5. The optimal pH of the decomposition J¾S is 7.0 · 8.0, and the optimal pH of the synthesis reaction is 5.8 ~ 7.8. Under heating conditions of 50 ° C for 10 minutes, it is stable within the range of ρΗ5.5 to 9.5.

 (2) Range of action and maximum: The action range is 25 to 0. C. The optimum temperature for the decomposition reaction is around 50-65 ° C, and the optimum temperature for the synthesis reaction is 45-60 ° C.

 (E) Temperature stability: H7 is extremely stable up to 60 ° C under heating conditions of 7.0 and 15 minutes. It is completely deactivated at 70 ° C.

 (H) P and harmful agents: Inhibited by copper, 7k silver, N-prosuccinimide, ク ロ ロ -chloromerkiuribenzoic acid. Not inhibited by sodium dodecylbenzenesulfonate (1%).

 (U) Isoelectric point ρΗ4 · 8-5.2.

 (H) The molecular weight determined by SDS-polyacrylamide electrophoresis is about 89,000 to 900,000 taletons, and the molecular weight obtained by removal is about 190,000 taletones, and is composed of homodimers. .

(8) Further, the present invention relates to the amino acid sequence represented by SEQ ID NO: 3 or one or more amino acid deletion, substitution, inversion, addition or insertion in this sequence. 6

 8

The present invention provides the trehalose phosphorylase according to (7).

 (9) The invention according to (7), wherein the present invention has an amino acid sequence having 63% or more, more preferably 75% or more, and preferably 90% or more homology with the amino acid sequence represented by SEQ ID NO: 3. And the like.

 (10) The present invention also relates to a polynucleotide encoding the amino acid sequence of trehalose phosphorylase according to (7), wherein the polynucleotide comprises the following groups (d) to (f):

(d) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing,

 (e) a polynucleotide encoding a polypeptide having one or more amino acid deletion, substitution, inversion, addition or insertion in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, and

 (f) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, and a polynucleotide selected from the group consisting of:

 (11) The present invention also relates to a recombinant vector having the polynucleotide according to the above (10) and a microorganism transformed with the recombinant vector. .

 (12) The present invention further provides a maltose phosphorylase according to the above (2) or (3), comprising culturing a microorganism belonging to the genus Baenibacillus having maltose phosphorylase and treoctylose phosphorylase-producing ability. , And Z or at least one of the trehalose phosphorylases according to (7) or (8) above, which is collected and collected. The present invention provides a method for producing octylose phosphorylase or a mixture of both.

(13) In addition, the present invention provides that the culturing is performed in the presence of a carbon source containing maltose, 4009606

 (9) A method for producing maltose phosphorylase or a mixture of maltose phosphorylase and torrehose phosphorylase according to the above (12), wherein the method produces and accumulates l-tulose phosphorylase and tol-lactose phosphorylase. It is.

 (14) In addition, the present invention provides the method according to (12), wherein the culturing is performed in the presence of a carbon source containing trehalose, and trehalose phosphorylase is preferentially produced and accumulated. An object of the present invention is to provide a method for producing a ribose phosphorylase or a mixture of maltose phosphorylase and trenopenulose phosphorylase.

 (15) Further, the present invention is a method for producing a crude enzyme of maltose phosphorylase and / or trehalose phosphorylase selected from any one of the following methods (i) to (iii).

 (i) cultivating a microorganism belonging to the genus Baenibacillus having the ability to produce mal I sphosphorylase and trehalose phosphorylase, and directly collecting the cells isolated from the resulting culture;

 (ii) Culture of Paenibacillus microorganisms capable of producing mal I ^ -monophosphorylase and trehalose phosphorylase, and extraction of maltose phosphorylase and / or trehalose phosphorylase crude enzyme from cells isolated from the resulting culture solution. Or

(iii) A phase capable of producing malt phosphorylase and trehalose phosphorylase: z A microorganism of the genus Bacillus is cultured, the cells are separated from the resulting culture, and the culture supernatant is collected. .

(16) The present invention further provides a maltose phosphorylase according to the above (2) or (3), and a treoctylose phosphorylase according to the above (7) or (8) in the presence of phosphoric acid. Another object of the present invention is to provide a method for producing trehalose, which is characterized by allowing zeto act on maltose. Brief Description of Drawings

 FIG. 1 is a diagram showing the taxonomic position of the microorganism producing the enzyme of the present invention, Baenibacillus sp. SH-55.

 FIG. 2 is a diagram showing the results of analysis of malt phosphorylase and tolactose phosphorylase of the present invention by SDS-polyacrylamide electrophoresis.

 FIG. 3 is a diagram showing the pH and the action pH range of the decomposition reaction (open circles) of maltose phosphorylase and trehalose phosphorylase of the present invention (open circles) and the synthesis J¾¾ (closed circles).

 FIG. 4 is a diagram showing the relationship between the enzyme activity and pH of maltose phosphorylase (open circles) and trehalose phosphorylase (black circles) of the present invention.

 FIG. 5 shows the relationship between the maleose phosphorylase (A) and the trehalose phosphorylase of the present invention.

(A) Diagram showing the relationship between the enzyme activity and action of decomposition J¾5 (open circles) and the synthesis reaction (closed circles) of (B).

FIG. 6 is a diagram showing the heat resistance of malt phosphorylase (open circles) and toreohose phosphorylase (solid circles) of the present invention. JP2004 / 009606

 11

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail.

 SUMMARY OF THE INVENTION The present inventors have proposed that, for the purpose of being usable for industrial production, it is easy to produce and purify, has a high working temperature of ±±, has high thermal stability, and has no substrate dependence. We searched the natural world extensively to obtain microorganisms capable of producing phosphorylase and trehalose phosphorylase, and as a result, a novel microorganism belonging to Paenibacillus identified two enzymes with the above requirements. It was found that the production was extremely large.

 That is, the novel strain of the present invention was obtained by the present inventors using a purple submarine `` Deep Sea 2000 '', which is owned by the Japan Agency for Marine-Earth Science and Technology, from a southern offshore Hatsushima, Sagami Bay, at a depth of 7 l at a depth of 117 m. It is newly isolated.

 An example of such a novel microorganism belonging to Paenibacillus of the present invention is Paenibacillus sp. SH-55. The bacteriological properties of Paenibacillus SP H-55 are shown below.

<Morphological properties>

 Cell morphology: Bacillus

 Cell size: (0, 7-0.9 x2. 0-4.0 tm)

 Mobility: Yes

 Flagella: Yes (extreme flagella)

 Sporulation: Yes

<Growth status>

Colony morphology: irregular and slightly wavy around. Glossy recolor (Non-uniform).

Growth temperature: Growing at 15-45 ° C. Does not grow at 50 ° C.

Salt concentration: grows on 5% salt. Does not grow on more than 7% salt.

Anaerobic growth: Does not grow.

 Physical properties>

Gram ¾fe: negative

 O-F test (glucose): P Does not produce acid and gas from glucose. Catalase test: positive.

Oxidase test: positive.

Gelatin resolution: Yin! ^

Casein resolution: positive.

Starch resolution: enteric.

Hippurate resolution; negative

 O NP G test: positive.

ゥ Rease production: positive.

Orditin decarboxylase production: P-type.

 Lysine decarboxylase production: negative.

Sulfuric acid production: negative.

Indori: negative.

Nitrification reducing ability: negative.

Sulfuric acid production: negative.

Casein production (VP test): Assimilation: glycerol, L-arabinose, ribose, D-xylose, galactose, glucose, fruc I ^ -is, mannose, mannii! ^-L, arbutin, esculin, salicin, N-acetyldarcosamine , Lactose, melibio X, trehalose, sucrose, cellobiose, marille! ^ There is a lifetime of capital, laffinose, starch, and glycogen. The 16S rDNA sequence of the bacterium of the present invention was analyzed for its chemical position using CLUSTAL X Muitiple Ie Sequence AI ig nment Promram (version 1.81). Figure 1 shows a phylogenetic tree describing the results of analysis based on neighbor-joining. From these results, it was found that the fungus of the present invention is ^ 1 close to (Eenibacillus glucanolyticus), but clearly present at a different branch in the phylogenetic tree.

(PaenibaciNus) was determined to be a new species. This was named PaenibaciNus sp. SH-55, and on June 27, 2003, the Independent Research Institute for Past and Future Technology 去 Licensed Biological Deposit Center (Postal Service) International Deposit No. 305-8566, Deposit No. FERM BP-8420, at 1-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan The new strain of the present invention was screened as follows. First, the collected seabed mud was suspended in physiological saline, and a drop of the suspension was applied to an agar medium having the following composition. The agar plate medium used was agar 2% (w / v), trehalose or maltose 1%, polypeptone 0.5%, yeast extract 0.5%, diammonium phosphate 0.1%, magnesium sulfate. Seven 7jd 0. It contains 0 2% and has a pH of 7. Thus, the agar plate medium was cultured aerobically at 37 ° C to obtain each colony that appeared on the plate, and each colony was placed in a liquid medium having the same composition as that used for the above agar medium ( The cells were cultured with shaking at 180 rpm at 37 ° C for 24 to 72 hours at pH 7). Next, each culture was centrifuged at 12, 00 and 10 minutes at 4 ° C to separate cells and supernatant. The cells thus obtained were suspended in a small amount of 0.1 M phosphate buffered night (pH 7.0), and the activity was measured by a key method. As a result, the strain having the above-mentioned mycological characteristics was able to be isolated.

 The microorganisms belonging to the genus Lactobacillus, which are the novel microorganisms of the present invention thus found, are new microorganisms that produce malt phosphorylase and treoctylose phosphorylase.

 In order to obtain the enzymes of the present invention, maltose phosphorylase and trehalose phosphorylase, from the novel microorganism of the present invention, for example, the microorganism is inoculated into an appropriate medium according to a conventional method, cultured, and then cultured. What is necessary is just to collect | recover from a thing. Culture conditions are preferably in the range of ^ J of 25 to 42 ° C from the viewpoint of the growth ^ J of the microorganism itself, and it is 'preferable' that the aerobic culture is carried out for 8 to 70 hours.

 The culture medium used for culturing the microorganism for obtaining the enzyme of the present invention is not particularly limited to the following, and may be a nutrient medium capable of growing the microorganism and producing the enzyme of the present invention. Any of isolated ground and natural medium may be used.

The medium may be any carbon source as long as the microorganism can assimilate it. Examples of such a medium include sugars such as glycerose, fruc I-I-is, mannose, trehalose, sucrose, mannitol, sorbitol, and molasses. And organic acids such as citric acid and co-octanoic acid can be used, but using trehalose, maltose, and saccharides containing these. Preferably. When treoctylose or a saccharide containing the substance is used as a carbon source, the microorganism of the present invention preferentially produces treoctylose phosphorylase. When maltose or a saccharide containing the substance is used as a carbon source, the microorganism of the present invention simultaneously produces maltose phosphorylase and tolactose phosphorylase. Furthermore, when both trehalose and maltose or a saccharide containing these substances are used as the carbon source, trehalose phosphorylase and maltose phosphorylase can be produced simultaneously, and trenoperose and By controlling the amount of maltose, the production ratio of trehalose phosphorylase and maltose phosphorylase can also be controlled.

 As the nitrogen source, various kinds of organic and inorganic nitrogen can be used. Further, the medium may contain various kinds of nitrogen. Examples of the nitrogen source include organic nitrogen sources such as corn starch, soybean meal, and various peptones, and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, phosphorous ammonium, and urea. Can be used. It goes without saying that urea and organic nitrogen sources can also be carbon sources.

 As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphorus, manganese salt, Sift salt, iron salt, the same salt, molybdenum salt, conoreto salt and the like are appropriately used. Furthermore, amino acids, vitamins, and the like are appropriately used as needed.

As a medium suitable for culturing microorganisms to obtain the enzyme of the present invention, specifically, for example, when preferentially producing trehalose phosphorylase, 0.5 to 3% of trehalose is used. (w / v), yeast extract 0.5-2%, ammonium phosphate 0.15%, urea 0.1-0.2%, salt 0.5-1.5%, dipotassium phosphate 0 0 5 to 0.3%, sulfuric acid It is 'suitable' to use a humid site with a pH of 7.0-7.5, containing 0.01% to 0.05% of calcium carbonate and 0.1% to 0.3% of calcium carbonate. In addition, when maltose phosphorylase and treoctylose phosphorylase are produced simultaneously, maltose 0.5 to 3% (w / v), polypeptone S (Nippon Pharmaceutical) 1-3%, ammonium phosphate 0% 1-0.3%, urea 0.05-0.3%, salt 0.5-1.5%, dipotassium phosphate 0.05-0.25%, magnesium sulfate-77K1 ^ 0.01. A liquid containing 0.05% and 0.1-0.3% potassium carbonate can be used. These mediums U can be appropriately changed depending on the type of fiber such as carbon source or nitrogen source without limitation. When maltose is used as a carbon source, not only maltose phosphorylase but also some amount of trehalose phosphorylase is produced. Therefore, it is more economical to use maltose as a carbon source in order to produce a crude enzyme (a mixture of maltose phosphorylase and torachirose phosphorylase) for production of toreja mouth. It is.

 The cultivation is performed aerobically at a temperature of 20 to 45 ° C, preferably 25 to 42 ° C, and a pH of 5 to 9, preferably 6 to 8. The fermentation time may be any time longer than the time when the microorganisms start to proliferate, and is preferably 8 hours to 70 hours. There is no particular limitation on the separation of the culture solution, but usually 0.5 to 2 Oppm is preferred. For this purpose, the ventilation rate may be adjusted, agitated, or added to the ventilation. The culture method may be either batch culture or continuous culture.

After culturing the microorganism of the present invention in this way, the produced enzymes of the present invention, maltophosphorylase and treoctylose phosphorylase, are recovered. Most of the enzymes produced are accumulated inside the cells, and some are accumulated outside the cells. Therefore, maltose phosphorylase and / or trerose enzyme produced and accumulated inside or outside the cells. 4 009606

 17 Collect the relyase. The enzyme recovery method of the present invention can be carried out according to general means for collecting enzymes. The method shown below is not particularly limited, but for example, a crushed cell obtained by a cell crushing method such as an ultrasonic crushing method, a French press method, a glass bead crushing method, a dynomill crushing method, or a cultured product may be used. The culture supernatant obtained by separating cells and culture supernatant by operations such as centrifugation and filtration can be used as a crude enzyme solution.

 This crude enzyme solution can be used as it is, but if necessary, separation methods such as salting-out, sedimentation, and ultrafiltration; 1® removal, for example, ion-exchange chromatography, isoelectric chromatography Chromatography, hydrophobic chromatography, gel filtration chromatography,

Those separated and purified by a combination of known methods such as P-sorption chromatography, affinity chromatography, reverse phase chromatography, etc. can also be used. In addition, as another method for obtaining the enzyme of the present invention, a gene encoding the enzyme of the present invention is extracted from the above-described strain of the present invention, and then a recombinant microorganism is transformed using genetic engineering techniques. And a method of culturing the recombinant microorganism. Specifically, a nucleotide sequence encoding the amino acid sequence of the enzyme of the present invention is obtained from the above strain, then this nucleotide sequence is inserted into an appropriate vector, and the vector is used to transform a host such as Escherichia coli or Bacillus subtilis. The enzyme may be produced by transforming and culturing the enzyme, and the enzyme of the present invention may be searched for from the culture.

Hereinafter, a method for producing the enzyme of the present invention using a specific genetic engineering technique will be described. Both enzymes of the present invention, ie, maltose phosphorylase and trehalose phosphorylase, have the amino acid sequences represented by SEQ ID NOs: 1 and 3 in the sequence listing, respectively. Since one or more amino acids in the sequence of the above are deleted, substituted, inverted, added or inserted amino acid sequences, it is necessary to use nucleotide sequences corresponding to these. is necessary.

 Examples of the nucleotide sequence encoding the amino acid sequence of the enzyme of the present invention include, in the case of maltose phosphorylase, a polynucleotide selected from the group consisting of the following (a) to (c). Can be

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing,

 (b) a polynucleotide encoding a polypeptide having one or more amino acid deletion, substitution, inversion, addition or insertion in the amino acid sequence shown in SEQ ID NO: の in the sequence listing,

 (c) a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 2 in Sequence Listing; In addition, as an example of the nucleotide sequence encoding the amino acid sequence of the enzyme of the present invention, in the case of trehalose phosphorylase, specifically, a polynucleotide selected from the group consisting of the following (d) to (f) is mentioned. .

 (d) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 3 in the sequence listing,

 (e) a polynucleotide encoding a polypeptide having one or more amino acid deletions, substitutions, inversions, additions or insertions in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing ,

(f) A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. The production of the recombinant microorganism producing the enzyme of the present invention can be performed by combining known means. PT / JP2004 / 009606

 19 That is, for example, the acquisition and amplification of a nucleotide sequence encoding the enzyme of the present invention from the above-mentioned Geneva sp. SH-55, insertion of a nucleotide sequence into a vector, transformation of a host with the l 当 S gene, etc. This is done by appropriately using the methods described in books in this field. Among these, an example of the production of the recombinant microorganism is not particularly limited, but the following method can be used. That is

The two corresponding enzyme genes are obtained from Shota cloning, PCR amplification using a specific primer, etc., from Baenibacillus sp. 5 1 "1-55. These genes are transformed into EK Escherichia coli. Recombinants are obtained by introducing them into gram-negative bacteria such as (Escherichia coli) or gram-positive bacteria such as BS-based Bacillus subtilis. For the transformation, a method in which extranuclear genes such as plasmids are used all at once, or a method in which the ability of the host bacterium to originally take up DNA or the like can be used can also be used.

As described above, the present invention is obtained by culturing a microorganism belonging to the genus Baenibacillus of the present invention or a recombinant microorganism into which a gene encoding the amino acid sequence of the enzyme of the present invention has been incorporated, and recovering it from the culture medium. Enzyme can be obtained. As described above, the above-mentioned maltose phosphorylase and trehalose phosphorylase, which are enzymes of the present invention, accumulate in the culture supernatant of these cultured orchids or extracellular cells, so that they are usually used. It can be obtained by isolation according to the method. First, the enzymes in the cells can be used as crude enzymes together with the cells. Furthermore, the crude enzyme can be recovered by extracting the enzyme from the cells. Further, the enzyme is also contained in the culture supernatant outside the cells, and the remaining culture solution obtained by separating the cells can be used as a crude enzyme-containing solution. In addition, these The crude enzyme can be purified using a conventional method such as solvent precipitation with ethanol, acetone or isopropano, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like. Further, the separation of maltose phosphorylase and treoctylose phosphorylase can be performed by, for example, anion exchange chromatography using the difference in isoelectric point between both enzymes.

 The enzyme of the present invention obtained in this manner is, as will be described in detail in Examples below, a type 1 enzyme having physicochemical properties as described above, namely, malrethose phosphorylase and Trehalose phosphorylase.

 The measurement of the activity of these enzymes was carried out using the method of Paenibacillus sp. SH-55 of the present invention in which α-darcosidase (maltase), dalcoamylase, and Therefore, in order to measure the activity in the case of phosphorolysis, maltose or treoctylose is used as a substrate and dulose produced by the enzyme J¾S in the presence of phosphorus is used for the dulose-based oxidase method. A simple method of measuring can be used. In the case of a synthetic reaction, the mixture of j8-D-glyrecose 1-phosphate and grecos is used as a substrate, and the inorganic phosphate generated by the enzyme is measured by a conventional method. be able to.

 Hereinafter, a method for measuring the activity of the enzyme of the present invention will be described.

(In the case of 0-decomposition reaction: 50 mM phosphoric acid solution (pH 7) dissolved in 20 mM maltose or treoctylose solution 0.5 mL, enzyme solution 0. Add lml, react at 50 ° C for 15 minutes, and stop the enzymatic reaction by heating in a boiling water bath for 3 minutes.After cooling in ice water, generate glucose and remove glucose. Method (Wakotai Kogyo Co., Ltd., Glucose C-II test 'ヮ Koichi'), where 1 min. 4009606

 twenty one

The amount of enzyme that produces AtmoIe glucose was defined as one unit of enzyme activity.

 (ii) In the case of synthetic J¾¾: 70 mM HES (27 mM) dissolved at night (ρΗ7,0) 3-D-glucose 1-phosphate Na salt and the same ^ J Add 0.05 mL of the enzyme solution to 0.15 mL of a mixed solution with glucose, react at 50 ° C for 15 minutes, and heat in a boiling water bath for 2 minutes to stop the enzyme activity. After cooling in a flask, the generated inorganic phosphoric acid is measured using Pitest Co., Ltd. manufactured by Wako Tokuhyo Co., Ltd. Here, the amount of the enzyme that generates Ίm 0 Ie of inorganic phosphoric acid per minute under these measurement conditions was defined as Ί unit of enzyme activity.

As described above, the novel enzymes maltose phosphorylase and trehalose-phosphorylase of the present invention are the novel microorganisms of the genus Paenibacillus of the present invention (for example, Paenibacil sp SH-55 (Paenibaci Ilus). sp.S H-55)) or a recombinant microorganism into which a gene encoding the amino acid sequence of the enzyme of the present invention has been incorporated, and the culture product can be easily recovered. . Then, these two enzymes of the present invention reversibly phosphorylate the α-1,4-darcopyranoside bond in maltose and the α-1,1,1-darcoviranoside bond in trehalose, respectively. It has the characteristic of producing 3-D-glucose 1-phosphate with glucose. Therefore, by combining maltose with these two enzymes in the presence of phosphoric acid, trehalose can be produced extremely efficiently. Moreover, the action ρΗ range of these two enzymes is as wide as 4.5 to 9.5, and the optimal ρΗ range of the two enzymes overlaps, making it very easy to handle in the enzyme reaction. Can be used at a relatively high temperature of 20-60 ° C, so that enzymatic reaction at a high temperature is possible, and there is no risk of contamination by bacteria as in the conventional method. Can be produced. Since maltose, which is easily available in large quantities, can be used as a raw material, and purified high-purity material can be used, contamination of the product after the enzyme J¾¾ with impurities is small, and thus It also has the disadvantage that it is easy to purify the product and it is possible to produce trehalose very efficiently.

 These enzymes of the present invention can be utilized as ma enzymes or purified enzymes. Furthermore, cells having the activity of both enzymes and immobilized cells adsorbed or chemically bound to the appropriate carriers may be used for the production of Torehachiguchi. it can. Furthermore, both enzymes of the present invention can also be used as immobilized enzymes immobilized by known methods. Examples Hereinafter, the present invention will be described in more detail with reference to Examples. Example 1:

Production and purification of intracellular and extracellular maltose phosphorylase

No. Enibacillus sp. SH-55 (Paenibacillus sp. SH-55) (FERM BP-8420) was converted to Maltose 1% (w / v), Polypeptone S (Nippon Pharmaceutical) 2.5%, ammonium phosphate 0 PH 7.0 solution containing 15%, urea 0.15%, salt 1%, dipotassium phosphate 0.1%, magnesium sulfate77] salt 0.02% and calcium carbonate 0.2% Inoculated on the surgical site. Next, this liquid medium was aerobically cultured at 37 ° C for 24 hours. The obtained culture was centrifuged at 12,000 presentations at 4 ° C for 15 minutes for 15 minutes to separate the cells from the cells. T / JP2004 / 009606

 twenty three

The cells obtained here were suspended in a small amount of 20 mM phosphoric acid at night (pH 7.0) and then disrupted by ultrasonication. Ammonium sulfate was added to the crushed cell suspension to make it 30% saturated, and placed at 4 ° C. Subsequently, the supernatant liquid obtained by removing the precipitate by centrifugation was further added with ammonium sulfate to 70% saturation. The resulting precipitate was left overnight at 4 ° C., collected by centrifugation, dissolved in 20 mM phosphate (pH 7), and dialyzed thoroughly in the same buffer overnight.

 Next, the enzyme was adsorbed on a DEAE-fract gel (manufactured by Merck) force ram equilibrated with 20 mM phosphoric acid (pH 7). The adsorbed enzyme was eluted by a gradient method from 0 M to 0.5 M salt contained in the above buffer night, and then concentrated by a UF membrane (YM-30, manufactured by Amicon). The enzymatic enzyme was gel-purified on a Cefacryl S-300 (Pharmacia) column containing 0.2 M salt and equilibrated at night as described above. The obtained maltose phosphorylase-active fractions were collected, dialyzed against the same buffer containing 1.5 M ammonium sulfate, and dialyzed in the same buffer containing 1.5 M ammonium sulfate. Enzyme was adsorbed to the column. After the adsorbed enzyme was eluted in the same buffer with a concentration gradient of 1.5 M to 0 M ammonium sulfate, the obtained maltose phosphorylase-active fractions were collected, and the same buffer containing 0.2 M salt was added to the buffer. I prayed. After concentration using the UF membrane described above, gel filtration chromatography was performed again using Superdex 200 (manufactured by Pharmacia) equilibrated with the same buffer containing 0.2 M salt and the same buffer as above. The one phosphorylase active fraction was concentrated by the method described above.

Here, the active fraction is a fraction obtained by separation by gel chromatography or the like, and is a fraction that has been found to have an activity when the activity is measured using maltose or treoctylose as a substrate. Ari means a maltose phosphorylase activity fraction and a trehalose phosphorylase activity fraction, respectively. In other words, when the activity on maltose or treoctylose is observed, when the DEAE-fract gel column is used, the salt concentration PT / JP2004 / 009606

 twenty four

There is a maltose phosphorylase active fraction at around 0.3 M, and there is a tresco and oral phosphorylase active fraction at 0.335 M.

 As a result, uniform maltose phosphorylase (activity yield: about 30%) was obtained as an enzyme in the orchid in polyacrylamide gel slab electrophoresis and SDS-polyacrylamide electrophoresis. The extracellular enzyme was also purified using the culture supernatant as a starting material in the same manner as described above to obtain a purified enzyme of maltose phosphorylase with an activity yield of about 25%. When the molecular weight of the purified enzyme obtained by the SDS-polyacrylamide electrophoresis method was measured, the molecular weight was in the vicinity of 89,000 to 90,000 tallileton, and was determined from the deduced amino acid sequence determined in Example 3 described later. It was in good agreement with the calculated 87,762 Dalton. When the molecular weight of the purified enzyme was measured by cefacryl S-200 gel-ti method, the amount was approximately 200,000 daltons, indicating that the enzyme was composed of a homodimer. Example 2:

Production and purification of intracellular and extracellular trehalose phosphorylase

Paenibacillus sp. 31 ~ 1-55, Trehalose 1% (w / v), Yeast Ex. 2%, Ammonium Phosphate 0.15%, Urea 0.15%. Salt 1%, Dipotassium Phosphate The plant was planted in a PH 7.0 solution containing 0.1% of magnesium, 0.02% of magnesium sulfate, and 0.2% of calcium carbonate. This liquid medium was cultured in the same manner as in Example 1 and post-treated to obtain a cell lysate and an upper night. The obtained cell lysate and purified night were purified in the same manner as in Example 1, respectively. Furthermore, the purified solution obtained from the cell lysate and the supernatant was subjected to polyacrylamide gel slab electrophoresis and SDS-polyacrylamide gel separation, respectively. 2004/009606

 twenty five

Purification was performed using the acrylamide electrophoresis method to obtain uniform intracellular torehylose phosphorylase and extracellular torehylose phosphorylase. The respective activity yields were 30% and 35%. When the molecular weights of both purified enzymes were measured by SDS-polyacrylamide electrophoresis, the molecular weights were in the vicinity of 89,000 to 90,000 daltons. From the estimated amino acid sequence determined in Example 3 described below, It was in good agreement with the calculated values of 87 and 151 Daltons. When the amount of the purified enzyme was measured by the Cefacryl S-200 gel method, the molecular weight was about 190,000 daltons. Therefore, it was considered that this enzyme was composed of a homodimer and was t \. Example 3:

Cloning and sequencing of maltose phosphorylase and trehalose phosphorylase genes

Cloning of the maltose phosphorylase gene was performed by the following method. When the N-terminal amino acid sequence of the purified enzyme obtained in Example 1 was determined by a conventional method, it had a sequence of MKQYLK L DEW. The purified enzyme was digested in a gel with V8 (Sigma), a kind of protease. In-gel digestion was performed by subjecting 5 ^ g of the purified enzyme to an SDS PAGE gel, simultaneously overlaying 1 tg of the V8 protease solution on the purified enzyme solution, and performing electrophoresis to decompose the target enzyme in the gel. Two types of N-terminal amino acid sequences of the obtained protein fragment were determined by a conventional method. As a result, from fragment 1, AI a-Ty rS e rG I y—Ser—Ser—L e uG I n—GI y—Ser-Ty r—Me t-AI a—GI y—Val—Ty r—Ty r—Pro—Asp—Lys, fragment 2 to Gly-Asp-VaI-AIaAIa—Gln-GIn-AIa-IIe-Arg Profit I was able to. There are ~ 6 types of DNA sequences encoding one type of amino acid. Thus, a mixed primer and an antisense mixed primer shown below based on the DNA sequence were prepared from the 1st to 9th amino acid sequences of fragment 1 and the 1st to 9th amino acid sequences of fragment 2.

 Primers based on the amino acid sequence of Fragment 1:

 5 '-CNTARAGRGGNTCNTCNCTNCAYG-3'

 Antisense primer based on the amino acid sequence of Fragment 2:

 5 '-ATNGCYTGYTGNGCNGCNACYTCG-3'

 Using the above mixed primers, PCR was performed using Ex Taq (Takara Bio) using the ¾fe DNA as a template, and the DNA fragment was amplified. The reaction conditions were as follows: After heating at 96 ° C for 2 minutes, a cycle of 96 ° C for 20 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute was repeated 30 times, and finally 10 ° C at 72 ° C. Incubated for minutes. When J¾ & solution was electrophoresed on agarose gel. * A DNA fragment of about 0.8 kb base pair was detected. The DNA fragment amplified from the reaction solution was cloned using TACl in Kit (Invitrogen) to prepare a plasmid for nucleotide sequence determination. Using this plasmid as a template, a fluorescent label RiS was performed using d Rod amine Dye Terminator Cycling Sequencing Reac- tion Reac- tion Kit (PerkinElmer I), and the DNA sequencer 377 (Applied B) was used. iosystems), and the nucleotide sequence at positions 477 to 1304 of the nucleotide sequence shown in SEQ ID NO: 5 was determined.

Based on the above base sequence, primer MF 1 represented by 5'-CAGTTGGTGCTGTTCAACACTTTG-3 ', and 5'-ATGGCGATGTAAAGAAT Primer MR 1 represented by AAAG-3 ′ was prepared. On the other hand, after cutting the body DNA with the restriction enzyme Xh0I, ligation was carried out at 16 ° C for 1 hour using Ligation Height (Toyobo), and this DNA was used as a template and as a primer. Inverse PCR was performed using LA Taq (Takara Bio) using MF1 and MR1. When the reaction mixture was subjected to agarose gel electrophoresis, a DNA fragment of about 7 kb base pair was detected. The amplified DNA fragment was purified from the reaction solution to prepare DNA for nucleotide sequence determination. Using this DNA fragment as a template, a fluorescent label J¾5 using dR 0 d amine Dye Terminator Cle c ue seq u enci nng Rea d y Reac tion Kit (PerkinElmer I) was performed, and DNA sequencer 377 was performed. (Applied Biosystems), and a maltose hoflylase gene represented by the amino acid sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2 was obtained.

 Next, cloning of the gene for trehalose phosphorylase was performed by the following method. When the N-terminal amino acid sequence of the purified enzyme obtained in Example 2 was determined by a conventional method, it was found that Met—Thr—Lys—Met—IIe—Ser—Asn—Pro—Asp—Le was u. From this, a mixed primer having a sequence represented by the following primer 11 was designed. Furthermore, a sequence with high homology was searched from the amino acid sequence of known trehalose phosphorylase, and the amino acid sequence of GIyTyr-GIu-GIyHis—Tyr—Phe—Trp—Asp was found. . From this, an antisense primer was designed having the sequence represented by Primer 2 below.

Primer 1: ₅ '-ATGACNTGGATGATHAGCAAYC-3' Primer 2: 5 '-CCAYAAYTAYTGNCCRTCYTANCC-3' T / JP2004 / 009606

 28 Using the above-mentioned mixed primers, PCR was performed by ExTaq (Yu-Kara-Bai) using the above-mentioned primers 1 and 2 with the ¾fe-form DNA as a template, and the DNA fragment was amplified. The reaction conditions were as follows: After heating at 96 ° C for 2 minutes, a cycle of 96 ° C for 20 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute was repeated 30 times, and finally at 72 ° C for 1 second. It was kept warm for 0 minutes. When the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.0 kb base pair was detected. The DNA fragment amplified from the reaction solution was cloned using TA Cloning Kit (Invitrogen) to prepare a plasmid for nucleotide sequence determination. Using this plasmid as a template, a fluorescent labeling reaction was carried out using dRodamineDineTeterminatorCycleSequencInngReadyReactionKit (Non-Kjelmer), and DNA sequencing was carried out. Analysis was performed using AppI ied B ios ystems), and the nucleotide sequence from the 113th to the 2168th nucleotide in the nucleotide sequence shown in SEQ ID NO: 7 was determined.

Based on the above £ 1 ^ base sequence, a primer TF 1 represented by 5'-ACGATGACCAGCTCCAGGAAG-3 'and a primer TR 1 represented by 5, -TCAGATAGGTACCGCGAATGG-3' were prepared. On the other hand, after chromosomal DNA was cut with restriction enzyme Xh0I, ligation was performed using Ligation on high (Toyobo), and this DNA was used as a template, and TF1 and TR1 were used as primers. And inverse PCR was performed using LA Taq (Takara Bay). When the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 6 kb base pair was detected. The amplified DNA fragment was purified from the reaction solution to prepare DNA for nucleotide sequencing. Using this DNA fragment as a template, dRod amine Dye Term inator Cycle Seq ue nc ing Ready Reac ti on Kit (PerkinElmer) PT / JP2004 / 009606

 The fluorescent labeling reaction was performed and analyzed by DNA Sequencer 377 (Applied Biosystems), and the amino acid sequence represented by SEQ ID NO: 3 and the nucleotide sequence represented by 12 We obtained the gene for Torrehachi-Rose Hofholylase.

 The molecular weight (by SDS-polyacrylamide electrophoresis) and isoelectric point estimated from SEQ ID NO: 1 and SEQ ID NO: 3 were as follows: maltose phos-tree lyase was '87, 762 tareton and pH 4.98,ヽ Loose tree sporylase was 87,151 daltons and pH 5.13. Example 4:

 Transformation of bacteria, cultivation and purification of transformants

 5'-GTGAAACAATATTTAAAGC TTG-3 'from the 343rd base, which is the female codon of the DNA sequence encoding the mal I ^ -phosphorylase of SEQ ID NO: 5 obtained in Example 3 A restriction enzyme Xh0I cleavage site at the 5 'end of the

The primer represented by 3 is MF2, and the antisense sequence from the 2649th base that is the termination codon 5 '— TTATTTTGAAGCTGCTGTG-The restriction enzyme Kpn I cleavage site is added to the 3' end of the lignonucleotide represented by 3 ' The 5'-TTATTTTGAA GCTGCTGTGGGTACCCCG—3, primer was MR2, MRfe DNA was used as template, and PCR was performed using Pyr0 vest (Takara Bay). A DNA fragment of about 2.3 kbp containing DNA was obtained. The reaction conditions were as follows: After heating at 96 ° C for 2 minutes, a cycle of 96 at 20 seconds, 55 at 30 seconds, and 72 ° C for 2 minutes was repeated 30 times, and finally incubated at 72 ° C for 10 minutes. . Obtained DN After purifying the A fragment, it was digested with restriction enzymes XhoI and KpnI. This was obtained by digesting the plasmid vector pRSETA (Invitrogen) with restriction enzymes Xh0I and KpnI, using 5 ng and Ligationhgh (Toyobo) at 16 ° C. and time La I gain one of Chillon, to create the recombinant plasmid p RSMP 1, ^ iiMB L 21 (DE3) pLy s S (οπιρϊ oSB (rB- mB-) / dcm (DE3) pLysS (Cam R)) Ltd. Transformation was performed using the competent cell method to obtain transformed Escherichia coli RSMP1.

 Similarly, it is represented by 5′-ATGACGTGGATGAT AAGCAATC-3 ′ from the base 113, which is the start codon of the DNA sequence encoding torerose phosphorylase of SEQ ID NO: 7 obtained in Example 3. Restriction enzyme BamH at the 5 'end of the oligonucleotide

Primer represented by 5'-CGCGGATTCATGACGTGGATGATAAGC AATC-3 'with I cleavage site is TF2, antisense sequence from 3rd base 341 which is a stop codon 5'-TTTTTTGAAGCTGCTGTG-3 5'-TTAT with a restriction enzyme Eco RI cleavage site at the 3 'end of the primer, TR 2 as primer, chromosome DNA as a template, PCR using Pyrobest An approximately 2.3 kbp DNA fragment containing lyase was obtained. J¾5 conditions are as follows: after heating at 96 ° C for 2 minutes, repeat the cycle at 96 ° C for 20 seconds, 55 ° C for 30 seconds, 72 ° C for 2 minutes 30 times, and finally at 72 ° C. Incubated for 10 minutes. After purifying the obtained DNA fragment, the restriction enzymes BamHI and Ec

Cut at 0 RI. This was the plasmid vector pRSETA (Invitrogen) cut with restriction enzymes BamHI and EcoI. 5 ng and Ligationoh

Ligation was performed at 16 ° C for 2 hours using 1 gh (Toyobo) to obtain the recombinant plasmid p. 6

 31

RSTP l was created. This was introduced into the E. coli BL 21 (DE 3) p Lys S (F-'ompT hsdS (rB--) gal dcm (OE3) pLysS (Cam ^R )) strain using the competent cell method. Transformed S. cerevisiae R STP1 was obtained.

 Next, the transformed bacteria RSM P1 and R STP t were each cultured for tli to prepare a recombinant enzyme.

First, a single colony of RSMP1 or RSTP1 of the obtained transformant was placed in an Erlenmeyer flask having a capacity of 30 OmL in an LB medium (1% bactopeptone, 0,5% yeast extract, 0,5% sodium chloride). ) With ampicillin solution

The chloramphenicol solution was inoculated into 3 OmL of a solution added to a final volume of 34 g / mL, and incubated at 37 C, 180 rpm T16 for 6 hours to obtain SMP1 or RSTP1. A seed culture was prepared.

Next, the transformant was cultured as follows. That is, for each of the transformants RSM P1 or R STP1, 0.5 L of LB medium (1% of bactopeptone, 0.5% of yeast extract, 1% of sodium chloride) was placed in a 2 L Erlenmeyer flask. After sterilization, add the ampicillin solution to a final concentration of 50 tg / m, add the chloramphenicol solution to a final concentration of 34 g / ml, and transfer the seed culture using RSMP1 or RSTP1. Inoculate each to 1%; C, OD ₆₀ at 180 rpm. (Turbidity at 600 nm) was 0.5. Thereafter, isopropylthiogalacto viranoside (IPTG) was added to a final concentration of 1 mM at 1 J, and the cells were further cultured for 3 hours. After completion of the culture, the culture was centrifuged at 5000 × 10 minutes for 10 minutes to obtain bacterial cells. The obtained Mi orchid was ultrasonically disrupted and centrifuged at 12,000 X directly for 15 minutes. The obtained crude extract is chromatographed twice with an affinity column (TALON Resin, Clontech). The procedure was followed by t \ (washing, 5 mM imidazole; elution, 100 mM imidazole) to obtain homogeneously purified maltose phosphorylase (MP) and pure octylose phosphorylase (TP). . Table 1 shows the results of purification of both enzymes by affinity columns. table 1

As shown in Table 1, the specific activities of purified malt phosphorylase (MP) and trehalose phosphorylase (TP) were about 50 and 42 units / mg, respectively. The molecular weights of these recombinant malt phosphorylase and trehalose phosphorylase determined by SDS-polyacrylamide gel electrophoresis were about 90,000 to 92,000 daltons (see FIGS. 2A and 2B, respectively). The calculated value was 92,233 daltons (Fig. 2A) and about 89,000-91,000 talliletons (calculated value, 91,280 tallileton) (Fig. 2B) The molecular weights calculated from the sequence were in good agreement with the respective molecular weights of 92,233 daltons and 91,280 daltons. Example 5 Transformation of Bacillus subtilis, culture and purification of transformants

 Expression in Bacillus subtilis was performed by the following method. That is, the DNA sequences of maltose phosphorylase and trehalose phosphorylase obtained in Example 3 were ligated into PHY300 PLK (Yukara Co., Ltd.), an expression vector for Bacillus subtilis, respectively. was IS W1224 (/ e "A8 me hsm) to obtain transformants BSMP1 and BSTP1.

 In the same manner as in Example 4, a Syndal colony of BSMP1 or B STP1 of the obtained transformant was placed in a test tube in a PM medium (polypeptone S4%, maltose 4%, yeast mash 0. 1%, LAB L EMCO POWDER (Oxoid) 0.2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.02%, calcium chloride 0,02%, tetracycline 15 tg / mL ) Was planted in 5 mL, and cultured at 30 C and 120 rpm for 24 hours to prepare a seed culture solution of BSMP1 or BSTP1.

Next, for each of the obtained transformants BSMP 1 and BSTP 1, a PM medium (polypeptone S 4%, maltose 4%, yeast extract 0.1%, LAB LEMCO POWDER (Oxo id) 0.2 30%, 0.1% rhodium dihydrogen phosphate, 0.02% magnesium sulfate, 0.02% calcium chloride, 15 tg / mL tetracycline), and inoculate 1% of the above seed culture with 30% Culture was performed at 120 ° C. for 64 hours at 120 ° C. After completion of the culture, the culture solution was centrifuged at 10,000 × 10 minutes for 10 minutes to obtain a culture supernatant and cells of BSMPMP and BSTP1. The cells were ultrasonically disrupted and centrifuged at Ί2000 × for 15 minutes to prepare a crude extract. When the respective activities were measured, it was 0.2 single & / mg for malt phosphorylase and 0.1 0.1 ii / mg for the bacterial cells, and 0.1 ha ii / mg for trehalose phosphorylase. 0.4 single & / mg, 0.2 single i / m g. Example 6:

Deduced amino acid sequences of malt phosphorylase and tolactose phosphorylase and homology of amino acid sequences of other enzymes

 For the malt phosphorylase gene and the trehalose phosphorylase gene obtained in Example 3, FAS TA homology search of the amino acid sequences encoded by these genes (http: //ddbj.nig. .jp). The phosphorylase of the present invention includes Bacillus sp. RK-1 (Bacillus sp. RK-1, AB 0084460), Entelococcus hirae (Enterococcus hirae, E21769), and Lactobacillus brevis.

Lactobaci 1 lus brevis. And only 51.4%, 51.4%, 48.3%, 48.9%, 45.1%, and 48.4%, respectively, across all amino acids. In addition, the trehalose phosphorylase of the present invention may be a Bacillus stearosa morphophilus S K1

(Bad I lus stearothermophilus SK-1, AB 0796 10), Samoaerobium block ATCC35047 (Thertnoanaerobium brocki i ATCC35047, A B073930), and a force that matches only 62.Ί% and 44.2% through all amino acids, respectively. I got it. Example 7 T / JP2004 / 009606

 35 Enzyme-chemical properties of male I-phosphorylase and toreylose phosphorylase produced by Paenibatilile sp SH-55

 Purification was carried out in the same manner as in Examples 1, 2, 4 and 5 for the general enzymatic chemical properties of the novel malt phosphorylase and torehylose phosphorylase of the present invention produced by Paenibatilile sp SH-55. It examined using the purified enzyme obtained by the above. As a result of the experiment, both maltose phosphorylase and trehalose phosphorylase, both intracellular and extracellular enzymes of Paenibacillus sp. Since both enzymes had almost the same physicochemical and enzymatic properties, they were obtained here in Example 4; various properties of the recombinant enzyme expressed in the fungus are shown.

 (A) Action

A 1% (w / v) solution of malIs and trehalose in 1 OmM phosphate buffer (pH 7.0) was added to the solution of mal! ^-Phosphorylase and trehalose phosphorylase as substrates. After adding 5 units (decomposition reaction activity) to 1 g each and reacting at 50 ° C for 5 hours, the enzyme was inactivated by heating in a boiling water bath for 3 minutes. The sugar in the obtained saccharified solution was measured by high performance liquid chromatography, and as a result, glucose and glucose monophosphate were respectively detected. In addition, 1% (w / v) glucose and] 8-D-glucose 1-phosphate Na salt or en-D-dal are dissolved in 1 OmM tris-hydrochloride at night (pH 7.0). Using a mixed solution of course 1 and a phosphate Na salt as a substrate, maltose phosphorylase and trehalose phosphorylase were added in 5 units each to 1 g of the substrate, and reacted at 50 for 5 hours. As a result of the measurement of glucose produced by the treatment as described above, maltose and trehalose were detected from -D-glucose 1-phosphate with glucose, and maltose phosphorylase and treoctylose phosphorylase were detected. The synthesis reaction was confirmed by No synthesis reaction of disaccharide from glucose and a-D-glucose 1-phosphate was detected, and the production was analyzed by the following method. First, the insolubles in the saccharified solution obtained by heat inactivation were separated by filtration through a 0.45 μπι pore size membrane filter. The obtained filtrate was used as a test sugar solution, and measurement was performed by a high performance liquid chromatography method using a YMC-Pack, ODS-AQ (AQ-304, manufactured by YMC) column. Water was used for the mobile phase, the column temperature was 30 ° C, and a differential refractometer was used for detection.

 (Mouth) Substrate specificity (Degradation

 (i) In the case of malt phosphorylase:

 In the enzyme activity measurement method (degradation reaction) described above, instead of maltose, the substrate to be used is torehose, isomale! ^ -Isose, neotrehalose, sucrose, lactose, or cellobiose. Degradation activity of these substrates was examined, but no enzymatic activity was observed for these substrates.

 (ii) Trehalose phosphorylase:

 Similarly, in the enzyme activity measurement method (decomposition reaction), the substrate to be used is replaced with maltose, isoma! Instead of trehalose, neat trehalose, sucrose, lactose, or cellobiose. Degradation activities of these substrates as substrates were examined, but no enzymatic activity was observed for these substrates.

 (C) Action pH range, optimal pH and stable pH range

Using the purified enzyme obtained in Example 4, the optimal pH for the decomposition and synthesis reactions was measured. Figure 3 shows the results. As shown in Fig. 3A, the optimal pH for the maltose phosphorylase degradation reaction (open circles) was 7.0 to 8.0, and the working pH range was 4.5 to 9.5. . same 9606

 37

The optimal pH for the synthesis reaction of black maltose phosphorylase (black circle) was 5.5-6.5, and the range of action ρΗ was 4.5-9.5.

 In addition, as shown in Fig. 3, で は, the optimum PH is ρΗ7.0-8.0 for the degradation of Torehachirose phosphorylase J¾5 (open circles), and the synthesis reaction (black circle) is ρΗ5.8-7.8. Was optimal. The effect range was 4.5 to 9.5 for both the decomposition reaction and the synthetic J 反 応 &.

 In the case of the decomposition reaction, 5 OmM monophosphate monophosphate 锾 night (ρΗ4.5 to 8.0 ·), phosphorus ^ ¾ΒΙ¾ΒΙacid buffer (ρΗ8.0 · 9.5) and glycine-NaOH buffer A solution obtained by adding 25 mM potassium phosphate to (ρΗ9.0 to 12.0) was used. In the case of a synthesis reaction, MES (pH 5.5 to 6.5), MOPS (pH 6.5 to 7.0), HEPES (pH 7.0 to 8.0), tris-hydrochloric acid (pH 7 5-9. 0) Each night was used.

 Both purified enzymes were treated in a sac for 10 minutes at 50 ° C., and their residual enzyme activities were measured by a decomposition reaction. As shown in Fig. 4, maltose phosphorylase (open circles) is stable in the pH range of 5.5 to 7.5, and trehalose phosphorylase (solid circles) is stable up to pH 5.5 to 9.5. Was. The pH was adjusted with 5 OmM monophosphate phosphate (pH 4.5-8.0), phosphate-ii acid buffer (ρΗ8.0-9.5) and glycine-NaOH buffer (PH9. Each night of 0-1 2.0) was used.

 (2) Range of action and maximum ^ ¾

As shown in Fig. 5A, in the case of the decomposition reaction of mul! ^-Phosphorylase (open circles), 45-55. Optimum action Sit around C (50 mM phosphoric acid, pH 7.0), action temperature range is 20-60 ° C, and in the case of synthesis reaction (black circle), it is optimal around 50-55 ° C It has an operating temperature and an operating temperature range of 20-60 ° C. Also, as shown in Fig. 5B, JP2004 / 009606

 38

The decomposition temperature of trehalose phosphorylase (open circle) has an optimum working temperature around 50 to 65 ° C (50 mM phosphoric acid, pH 7.0, pH 7.0), and the working temperature range is 25 to 70 ° C. In the case of (black circle), the optimum working temperature was around 45-60 ° C, and the working temperature range was 25-70 ° C.

 (E) Stability

 Heat resistance of mal I ^ -phosphorylase and trehalose phosphorylase was treated with various ^^ for 15 minutes under conditions of pH 6.0 and 7.0 (50mM phosphate Hffij at night), respectively. The remaining activity was determined by a conventional method. Figure 6 shows the results. As can be seen from Fig. 6, malt phosphorylase (open circles) was extremely stable up to 50 ° C and completely inactivated at 70 ° C. In addition, Torehachirose phosphorylase (black circle) was extremely stable up to 60 ° C and completely inactivated at 70 ° C.

 (H) Inhibitor

 Degradation activity of maltose tree sporolase and trehalose phosphorylase was measured in the presence of various inorganic ions and inhibitors. As a result, the activities of both enzymes were strongly inhibited by copper, 7k silver, N-prosuccinimide, and chloromethyluribenzoic acid (1 mM each). Maltose phosphorylase was strongly inhibited by SDS (1%), but trehalose phosphorylase was not.

 (U) Isoelectric point

The isoelectric point of maltos-phosphorylase and trehalose phosphorylase was measured by iso-elect mouth focusing gel (manufactured by FMC BioProducto). As a result, maltose phosphorylase was ρΗ4.8-5.0 (calculated value, ρΗ4 98), and the value of trehalose phosphorylase was ρΗ4.8-5.2 (calculated value, ρΗ5.13). (H) Molecular weight by gel filtration

 The molecular weights of malt phosphorylase and tolactose phosphorylase were measured by gel filtration using Cefacryl S-200. As a result, the amount of both enzymes was about 190,000 daltons in the gel filtration method, but about 89,000 to 90,000 tariles of mal! Phosphorylase in the SDS-polyacrylamide electrophoresis method. Tons (calculated, 92,233 tallileton) and trenodulose phosphorylase were approximately 89,000 to 90,000 tallileton (calculated, 91,280 tallileton). It was expected to be composed of homodimers. The molecular weight of both recombinases is slightly larger than that of the wild type because the His tag and the 23 amino acid sequence of the spacer are added at the N-terminus. Because it is. Example 8:

 Production of intracellular and extracellular trehalose phosphorylase

Trehalose 1% (w / v), yeast extract (Difco) 2%, ammonium phosphate 0.25%, urea 0.15%, salt 1%, dipotassium phosphate 0.1%, Paenibacillus sp. SH —55 previously cultured overnight in the same medium in 10 L (liter) of a pH 7.5 liquid containing 0.02% of magnesium sulfate and 7% of jjdg and 0.5% of calcium carbonate. 50 OmL of the inoculum of Aspergillus was aseptically added, and the mixture was aerated and agitated for 24 hours under the conditions of 35 ° C., 300 rpm, and an air flow of 1 vvm [air flow (L) / medium (L) / min]. . As a result of measuring the trehalose phosphorylase activity of the nutrient solution, it was 1.8 units / mL of the culture solution. Similarly, maltose phosphorylase activity was measured, but the activity was trace. Then, Was centrifuged at 12,000 X for 10 minutes at 4 ° C to obtain about 75 g of cells (wet amount) and about 10 L (liter) of supernatant. . The supernatant was concentrated on a UF membrane (YM-30) manufactured by Amicon to obtain about 50 OmL of extracellular concentrated crude enzyme. As a result of measuring the enzymatic activity during the night, the activity was about 25% of the total activity (about 14 × 10 ³ units). The cells were thoroughly washed with a 10 mM phosphoric acid solution (pH 7), suspended in 240 mL of the same buffer overnight, and disrupted with an ultrasonic cell disrupter. As a result of measuring trehalose phosphorylase activity by a conventional method, about 75% of the total activity was contained in the cells.

 Therefore, by culturing Paenibacillus sp. SH-55 using torayasu as a carbon source, toretoose can be preferentially produced, in which case about 75% of the enzyme is contained in the cells, and 25-

Example 9:

 Production of intracellular and orchid extracellular enzymes containing maltose phosphorylase and trehalose phosphorylase

In the medium components used in Example 2, the amount of trehalose was replaced with maltose, and the amount of yeast extract 2% was replaced with Polypeptone FC (manufactured by Nippon Pharmaceutical Co., Ltd.) 4.5% (w / v). Other than that, Baenibacillus sp. SH-55 was cultured under the same conditions and method. As a result of measuring the activity of maltose phosphorylase and trehalose phosphorylase in this culture, 0.8 units of maltose phosphorylase and 0.5 units of trehalose phosphorylase were detected per mL of culture. Was producing. Centrifugation was carried out in the same manner as in Example 8 to obtain about 50 g of cells (wet amount) and 7 L (liter) of supernatant. Maltose phos in the obtained cells and supernatant As a result of measuring the holyrase activity, it was found that about 80% of the total activity of malt phosphorylase was contained in the simplicity (culture supernatant). In addition, about 80% of the total activity of trehalose phosphorylase is contained in the cells, and about 20% is contained outside the cells (culture supernatant). The culture supernatant was concentrated in the same manner as in Example 1 to obtain about 33 OmL of the concentrated enzyme. The enriched enzyme contained approximately 550 units of malus phosphorylase and 500 units of trehalose phosphorylase.

 Therefore, by culturing Paenibacillus sp. SH-55 using mal I as a carbon source, maltose phosphorylase can be produced.At the same time, trehalose phosphorylase can also be produced. It was found to be about 80% in the body and about 20% outside the bacteria. Example 10:

Manufacture of Torre Hachirosu from Maru I-Isu

10%, 20%, 30%, and 40% (W / V) maltose solutions dissolved in 1 OmL of 1 OmM phosphoric acid fjj night (pH 6) were added to the trehalose phosphorylase of the present invention and Maltose phosphorylase was added in an amount of 5 units (degrading activity) per 1 g of substrate weight, and reacted at 55 ° C for 70 hours. After the reaction was completed, the enzyme was inactivated by heating the mixture at 100 ° C. for 5 minutes, and the content of trehalose in the saccharified solution obtained was measured. As a result, for each of the above four types of maltose solutions, 58.2%, 58.1%, 58.6%, and 57.9% of trehalose were formed with respect to the substrate weight, respectively. . In addition, quantification of Torehachilo was performed by the following method. That is, water was added to the heat-inactivated saccharified solution to about 1% (W / V), and then 0.5 ml of the saccharified solution was added to dalcoamylase (neat). 0.01 μl of Pure Grade 3 OUZmg (manufactured by Chemical Industry) was added, and the mixture was subjected to J¾S at 50 ° C. and pH 5.0 for 1 hour to completely decompose unreacted maltose into glucose. After heating in a boiling water bath at 100 ° C for 5 minutes to inactivate dalcoamylase, the insoluble protein produced is removed with a 0.45 membrane filter, and the filtrate obtained contains trehalose. The amount was measured by a liquid chromatography method (HPLC method) using a YMC-Pack ODS-AQ (AQ-304, YMC) column. The measurement was performed using water as the mobile phase, the column temperature was set at 30 ° C, and a differential refractometer was used for detection. Example 11:

Maru! ^ Manufacture of Torehachiloise from one sushi wrap

 20% (W / V) of high maltose syrup (manufactured by Nippon Shokuhin Kako Co., Ltd., trade name: MC-95, glucose: glucose 2.5) %, 95.2% of mal I ^ 1%, 0.8% of maltotriose, 1.5% of maltotetraose) and the halo-octylose phosphorylase and masolose phosphorylase of the present invention. The reaction was carried out in the same manner as in Example 10 by adding 5 liters per 1 g of the substrate weight. As a result of measuring the generated trehalose by the HPLC method, 54.3% of trehalose was generated based on the weight of the substrate used. Industrial applicability

According to the present invention, there is provided a novel microorganism capable of producing maltose phosphorylase and trehalose phosphorylase necessary for enzymatic production of trehalose in and out of cells. Since the microorganism of the present invention is a bacterium, In addition to green algae and basidiomycetes, which are known as sources of i-ze, the method for obtaining the enzyme is not only very easy, but also the cultivation time can be greatly reduced and it is economical. Furthermore, the microorganism of the present invention has practically remarkable advantages also in that it can simultaneously produce two kinds of enzymes required for the enzymatic production of trehalose using only one kind of microorganism. Further, the two enzymes of the present invention satisfy all of the requirements required for enzymatic production of trehalose, so that the effective use of the obtained trehalose is remarkably facilitated, and economically. Can also achieve significant improvements. That is, the maltose phosphorylase and trehalose phosphorylase of the present invention have high ^ stability and can be used only at high temperature of the enzyme J¾s, so that they can be free from bacterial contamination during the reaction. Furthermore, since both enzymes have almost the same optimum pH range, there is an advantage that complicated pH control during the reaction is not required. Further, as another method for obtaining the enzyme of the present invention, after extracting the gene encoding the enzyme of the present invention from the above-mentioned orchid strain, a recombinant microorganism is produced using genetic engineering techniques, and By culturing microorganisms, it became possible to produce mal I ^ -phosphorylase and trehalose phosphorylase, which are required for enzymatic production of trehalose, and to improve both enzymes in terms of protein engineering.

Claims

The scope of the claims

1. A microorganism of the genus Paenibacillus having the ability to produce malt phosphorylase and tolactose phosphorylase.

2. The malt-phosphosylylase described in claim 1 of claim 1, wherein the microorganism is Paenibacillus sp. SH-55 (Paenibacillus sp. SH-5_5) (Accession No. FERM BP-8420). A microorganism having the ability to produce torayachirose phosphorylase.

3. Maltose phosphorylase having the following physicochemical properties.

 (A) Action: Reversibly phosphorylates α-1,4-darcopyranoside bonds in maltose to produce darco, sucrose and -D-glucose monophosphate.

 (Mouth) Substrate specificity (decomposition reaction): Acts on maltose, but not on trehalose, sucrose, lactose, cellobiose, etc.

 (8) Optimal ρΗ and stable ρΗ range: The optimal ρΗ of the decomposition J¾S is 7.0 to 8.0, and the optimal ρΗ of the composite J¾5 is 5.5-6.5. It is stable within the range of pH 5.5 to 7.5 under caloric heat conditions at 50 ° C for 10 minutes.

 (2) Range of action and maximum iSJt: The action range is 20 to 60 ° C. The optimal temperature for the decomposition reaction is 45-55 ° C, and the optimal temperature for the synthesis reaction is 50-55 ° C.

(E) Temperature stability: Extremely stable up to 50 ° C under heating conditions of pH 6.0 and 15 minutes. It is completely deactivated at 70 ° C. (F) The amount by SDS-polyacrylamide electrophoresis is about 89,000-900,000 daltons, and the molecular weight by gel removal is about 190,000 daltons. It is composed of homodimers.

4. The amino acid sequence represented by SEQ ID NO: 1, or one or more amino acids in the sequence have a deleted, substituted, inverted, added or inserted amino acid sequence. Maltose phosphorylase.

5. The maltose phosphorylase according to claim 3, which has an amino acid sequence having at least 52% homology with the amino acid sequence represented by SEQ ID NO: 1.

6. A polynucleotide encoding the amino acid sequence of maltose phosphorylase according to claim 3, wherein the group comprises the following (a) to (c):

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing,

 (b) a polynucleotide encoding a polypeptide having one or more amino acid deletion, substitution, inversion, addition or insertion in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing , and

 (c) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing;

7. A recombinant vector having the polynucleotide according to claim 6.

8. A microorganism transformed by the recombinant vector according to claim 7.

9. Trehachi-rose phosphorylase having the following physicochemical properties.

 (A) Action: Reversibly phosphorolytically decomposes the α-1,1-darcoviranoside bond in Torreguchi and produces glucose and i8-D-glucose monophosphate.

 (Mouth) Substrate specificity (decomposition reaction): Acts on trehalose and does not act on maltose, sucrose, lactose, cellobiose, etc.

 (8) {ρ} and stable pH range: The optimum pH for the decomposition reaction is 7.0 to 8.0, and the optimum pH for Compound J-5 is 5.8 to 7.8. Force at 50 ° C for 10 minutes で は Under heat condition, it is stable within pH 5.5 to 9.5.

 (2) Range of action and maximum: The action ^^ range is 25 ~ 70 ° C. The optimal temperature for decomposition J¾S is 5, 0 to 65 ° C, and the optimal temperature for the synthesis reaction is 45 to 60 ° C.

 (E) Temperature stability: Extremely stable up to about 60 ° C under heating conditions of pH 7.0 and 15 minutes. It is completely deactivated at 70 ° C.

 (F) SDS—Polyacrylamide electrophoresis has a molecular weight of about 89,000-90,000 tallileton, gel {1} has a molecular weight of about 190,000 daltons, and homo 2 It is composed of monomers.

10. The claim 9 having the amino acid sequence represented by SEQ ID NO: 3, or having one or more amino acid deletion, substitution, inversion, addition or insertion in the sequence. The torayachi-rose phosphorylase described.

11. The trehalose phosphorylase according to claim 9, which has an amino amino sequence having 63% or more homology with the amino acid sequence represented by SEQ ID NO: 3.

1 2. A polynucleotide encoding the amino acid sequence of treoctylose phosphorylase according to claim 9, wherein the group comprises the following (d) to (f):

 (d) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing,

 (e) a polynucleotide encoding a polypeptide having one or more amino acid deletions, substitutions, inversions, additions or insertions in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing , and

 (f) a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, or a polynucleotide selected from the group consisting of:

1 3. A recombinant vector having the polynucleotide according to claim 12.

1 4. A microorganism transformed with the recombinant vector according to claim 13.

1 5. Culture of a microorganism of the genus Paenibacillus having maltose phosphorylase and treoctylose phosphorylase-producing ability, wherein the malt phosphorylase and / or claim according to claim 3 or 4 is cultivated. Producing, accumulating, and collecting at least one of the glucose octylose phosphorylase described in paragraph 9 or 10; A method for producing maltose phosphorylase or trehalose phosphorylase, or a mixture of both.

16. The maltose phosphoryla according to claim 15, wherein the culturing is performed in the presence of a carbon source containing maltose to produce and accumulate maltose phosphorylase and trehalose phosphorylase. A method for producing a mixture of glucose and malt phosphorylase and glucose phosphatase.

17. The trehalose phosphorylase or malt according to claim 15, wherein the culturing is performed in the presence of a trehalose-containing carbon source to preferentially generate and accumulate trehalose phosphorylase. A method for producing a mixture of phosphorylase and ureharose phosphorylase.

1 8. A method for producing a crude enzyme of maltose phosphorylase and Z or trehalose phosphorylase selected from the following methods L and L;

 (Cultivating a microorganism belonging to the genus Baenibacillus having maltose phosphorylase and trehalose phosphorylase-producing abilities, and directly collecting the cells isolated from the resulting culture solution,

(ii) Culture of a microorganism belonging to the genus Baenibacillus having maltose phosphorylase and trehalose phosphorylase-producing ability, and extraction of maltose phosphorylase and / or trehalose phosphorylase crude enzyme from cells isolated from the resulting culture solution Or (iii) has the ability to produce maltose phosphorylase and trehalose phosphorylase Culture the microorganisms of the genus Paecotilus, isolate the cells from the resulting culture, and collect the culture at night. 9. In the presence of phosphoric acid, the maltose phosphorylase according to claim 3 or 4 and the trehalose phosphorylase according to claim 9 or 10 to mariletose. A process for producing trehalose.