CN1816623A - 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 alpha-1,4-glucopyranoside bond in the maltose to reversively undergo phosphorolysis; a novel trehalose phosphorylase which acts only on trehalose to cause the alpha-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

Novel microorganism, maltose phosphorylase and trehalose phosphorylase and manufacture method thereof

Technical field

The present invention relates to the manufacture method of novel microorganism, novel enzyme and novel enzyme.Specifically, the series bacillus that the present invention relates to have the throughput of necessary maltose phosphorylase and trehalose phosphorylase with the Production by Enzymes trehalose time belong to novel microorganism, the gene of the aminoacid sequence of the novel maltose phosphorylase that obtains by this microorganism and trehalose phosphorylase, encoding such enzymes and the manufacture method of these enzymes.

Background technology

Trehalose is expected to be widely used in fields such as pharmaceuticals, makeup and food, has carried out many trials in order to carry out industrial production in the past.These technology can roughly be divided into following three classes.The first from trehalose can be accumulated in endobacillary microorganism extract and the method for refining this material (for example, with reference to J.Am.Chem.Soc.72 volume, 2059 pages, nineteen fifty; No. 266584 communique of German Patent; Te Kaiping 3-130084 communique; Te Kaiping 5-91890 communique; Te Kaiping 5-184353 communique and spy open flat 5-292986 communique).This method comprises the cultivation operation, separation circuit of microorganism, extracts the refining crystallization operation of the trehalose of the operation of trehalose, extraction from microorganism, and the manufacturing process of trehalose is quite complicated.And not only the productivity of trehalose is low, and has also produced a large amount of microorganism extraction residues as waste, so can not be called economical efficiency method preferably.

And, also developed produce trehalose fermentation method (for example, open flat 5-211882 communique with reference to the spy), in this method, the microorganism of cultivating brevibacterium sp (Brevibacterium) and Corynebacterium (Corynebacterium) etc. is as the microorganism that produces trehalose outside thalline.Yet in this method, the rate of accumulation of trehalose in substratum is lower, only is about 3% (w/v), at industrial mass production trehalose, needs big volumetrical fermentor tank and the purification apparatus that matches of scale with it, therefore has problems at economic aspect.And, in this method, not only need to carry out sterilization, but also need remove when cultivating numerous and diverse operation by inclusion that uses bacterial strain to produce or medium component etc. in order to obtain the purified trehalose.

On the other hand, also developed enzyme process in order to solve the variety of issue that exists in these fermentation methods.Promptly, orthophosphoric acid salt β-D-glucanotransferase) and derive from the trehalose phosphorylase (α of algae in the presence of phosphoric acid, will derive from the maltose phosphorylase (maltose: of microorganism, α-trehalose: orthophosphoric acid salt β-D-glucanotransferase) act on maltose and produce the method for trehalose (for example, with reference to No. 1513517 communique of Japanese Patent; Agric.Biol.Chem., 49 volumes, 2113 pages, 1985), orthophosphoric acid salt β-D-glucanotransferase) and derive from the trehalose phosphorylase (α of basidiomycetes with the sucrose phosphorylase that in the presence of phosphoric acid, will derive from bacterium (sucrose:, α-trehalose: orthophosphoric acid salt β-D-glucanotransferase) act on the method (for example, with reference to putting down into 6 years Japanese chemurgy meeting conference lecture summary collection 3Ra14) that sucrose obtains trehalose.Can generate trehalose with 60%～70% higher yields from maltose or sucrose according to these methods.And, owing to used high purity sugar after refining,, to compare with additive method so the trehalose that obtains by enzyme reaction also is easy to make with extra care as raw material, this method is considered to industrial useful method.Yet, employed enzyme in these methods, particularly the source of trehalose phosphorylase only limits to euglena (Euglena) or fine and soft (Grifola ffondosa) such algae or the basidiomycetes of dance, in order stably to produce enzyme not only in the economic aspect existing problems, also has any problem at technical elements.And, the dimensional discrepancy of optimal pH separately of the trehalose phosphorylase that obtains, the sucrose phosphorylase that is mated use, maltose phosphorylase is bigger, and also very low to the stability of temperature, can only under 25 ℃～37 ℃ cold condition, carry out the formation reaction of trehalose.The problem of Cun Zaiing is like this, not only when being used two kinds of enzymes, make the control of pH become difficult, and, when using opening reactive tank to carry out enzyme reaction, can cause the pollution of assorted bacterium because temperature of reaction is low, for the side reaction that prevents to bring thus also needs strict hygiene control.And, when being used these known enzymes,, thereby can't use the raw material of high density because these enzymes have the concentration of substrate dependency.Therefore, these methods can not be called as the good method of economical efficiency.

In addition, also delivered the method for making trehalose with the amylolysis thing as substrate.According to this method, to be used for generating malt oligosaccharide based mycose that end has the non-reducing sugar of trehalose structure generates enzyme (Maltooligosyl-Trehalose synthase) and is used for acting on the amylolysis thing from this non-reducing sugar specificity dissociate trehalose resolvase of trehalose, high yield with 80% generates trehalose by starch and (for example, opens flat 7-143876 communique with reference to the spy; European patent application 628630A2 specification sheets; Japan's chemurgy can be given a lecture summary collection, p31 (1995)).Yet, in this method,, being difficult to make described enzyme immobilization and flowing through substrate owing to use starch as substrate, the further manufacturing of reactor etc. is difficult to oversimplify and efficient activity.And the operation of the purging liquid that obtains behind the trehalose crystallization being carried out saccharification more also is difficult to construct.

From the above mentioned, as long as can find to make and make with extra care easy, have higher thermostability and do not have dependent novel maltose phosphorylase of concentration of substrate and trehalose phosphorylase, be that the high yield of raw material is made trehalose simply efficiently with regard to being expected to the maltose that can obtain simply in a large number.

So, first purpose of the present invention is to solve the variety of issue in the described conventional art, providing can the High-efficient Production maltose phosphorylase and the novel microorganism of trehalose phosphorylase, and described maltose phosphorylase and trehalose phosphorylase are the novel enzymes that can satisfy above-mentioned various requirement.Second purpose of the present invention be to provide make and refining easy, have higher thermostability and do not have dependent novel maltose phosphorylase of concentration of substrate and trehalose phosphorylase.And the 3rd purpose of the present invention is to provide the manufacture method of maltose phosphorylase and/or trehalose phosphorylase, in the method, uses described microorganism can obtain above-mentioned two kinds of enzymes simple and effective.

Summary of the invention

The present inventor has carried out retrieval widely for the microorganism that obtains having the ability that can produce following enzyme at nature, and this enzyme has and can be used in above-mentioned many character that industrial maltose phosphorylase and trehalose phosphorylase possesses.Found that the series bacillus microorganism belonging to genus can possess two kinds of enzymes of described condition by mass production, thereby finished the present invention.

That is, summary of the present invention is a content as described below.

(1) the invention provides the series bacillus microorganism belonging to genus with the ability that can produce maltose phosphorylase and trehalose phosphorylase, specifically providing preserving number is the series bacillus sp.SH-55 (paenibacillus sp.SH-55) of FERM BP-8420.

(2) the invention provides maltose phosphorylase with physico-chemical property shown below.

(I) effect: to the α in the maltose-1,4-Glucopyranose glycosidic bond carries out reversibility and adds the phosphoric acid decomposition, generates glucose and β-D-glucose 1-phosphoric acid;

(II) substrate specificity (decomposition reaction): act on maltose, but do not act on trehalose, sucrose, lactose and cellobiose etc.;

(III) scope of action pH, optimal pH and stable pH range: the scope of action pH is 4.5～9.5; The optimal pH of decomposition reaction is 7.0～8.0, and the optimal pH of building-up reactions is 5.5～6.5; Under 10 minutes heating condition of 50 ℃ of heating, stable in the scope of pH5.5～7.5;

(IV) scope of operative temperature and optimum temperuture: the operative temperature scope is 20 ℃～60 ℃; The optimum temperuture of decomposition reaction is near 45 ℃～55 ℃, and the optimum temperuture of building-up reactions is 50 ℃～55 ℃;

(V) temperature stability: under 15 minutes heating condition of pH6.0 heating, in extremely stable below 50 ℃ or 50 ℃; In 70 ℃ of complete deactivations;

(VI) inhibitor: be subjected to copper, mercury, N-bromosuccinimide, to the inhibition of chlorine mercury M-nitro benzoic acid (respectively being 1mM), Sodium dodecylbenzene sulfonate (1%);

(VII) iso-electric point: in the scope of pH4.8～5.0;

(VIII) molecular weight of measuring with SDS-polyacrylamide gel electrophoresis method is about 89000～90000 dalton, is about 190000 dalton with the molecular weight of gel filteration determining; This maltose phosphorylase is made of the homotype dipolymer.

(3) the invention provides as described the maltose phosphorylase of (2) record, it has following aminoacid sequence: so that the sequence of one or more aminoacid deletion, replacement, inversion, increase or insertion to be arranged in the represented aminoacid sequence of the represented aminoacid sequence of sequence number 1 or sequence number 1.

(4) the invention provides as described the maltose phosphorylase that (2) are put down in writing, aminoacid sequence that it had and the homology that has with the represented aminoacid sequence of sequence number 1 more than or equal to 52%, preferably have homology, more preferably have homology more than or equal to 90% more than or equal to 70%.

(5) the invention provides polynucleotide, its coding is the aminoacid sequence of the maltose phosphorylase of (2) record as described, and described polynucleotide are any one polynucleotide that are selected from the group of following (a)～(c) composition:

(a) coding has the polynucleotide of the polypeptide of the aminoacid sequence shown in the sequence number 1 of sequence table;

(b) coding has the polynucleotide of polypeptide of following aminoacid sequence, and described aminoacid sequence is the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in the sequence number 1 of sequence table;

(c) have the polynucleotide of the nucleotide sequence shown in the sequence number 2 of sequence table.

(6) the invention provides recombinant vectors and microorganism, described recombinant vectors has as described the polynucleotide of (5) record; Described microorganism transforms with described recombinant vectors.

(7) the present invention also provides the trehalose phosphorylase with physico-chemical property as follows.

(I) effect: to the α in the trehalose-1,1-Glucopyranose glycosidic bond carries out reversibility and adds the phosphoric acid decomposition, generates glucose and β-D-glucose 1-phosphoric acid;

(II) substrate specificity (decomposition reaction): act on trehalose, but do not act on maltose, sucrose, lactose and cellobiose etc.;

(III) scope of action pH, optimal pH and stable pH range: the scope of action pH is 4.5～9.5; The optimal pH of decomposition reaction is 7.0～8.0, and the optimal pH of building-up reactions is 5.8～7.8; Under 10 minutes heating condition of 50 ℃ of heating, stable in the scope of pH5.5～9.5;

(IV) scope of operative temperature and optimum temperuture: the operative temperature scope is 25 ℃～70 ℃; The optimum temperuture of decomposition reaction is near 50 ℃～65 ℃, and the optimum temperuture of building-up reactions is 45 ℃～60 ℃;

(V) temperature stability: under pH7.0,15 minutes heating condition of heating, in extremely stable below 60 ℃ or 60 ℃; In 70 ℃ of complete deactivations;

(VI) inhibitor: be subjected to copper, mercury, N-bromosuccinimide, to the inhibition of chlorine mercury M-nitro benzoic acid; Be not subjected to the inhibition of Sodium dodecylbenzene sulfonate (1%);

(VII) iso-electric point: in the scope of pH4.8～5.2;

(VIII) molecular weight of measuring with SDS-polyacrylamide gel electrophoresis method is about 89000～90000 dalton, is about 190000 dalton with the molecular weight of gel filteration determining, and this trehalose phosphorylase is made of the homotype dipolymer.

(8) the invention provides as described the trehalose phosphorylase of (7) record, it has following aminoacid sequence: so that the sequence of one or more aminoacid deletion, replacement, inversion, increase or insertion to be arranged in the represented aminoacid sequence of the represented aminoacid sequence of sequence number 3 or sequence number 3.

(9) the invention provides as described the trehalose phosphorylase that (7) are put down in writing, aminoacid sequence that it had and the homology that has with the represented aminoacid sequence of sequence number 3 more than or equal to 63%, preferably have homology, more preferably have homology more than or equal to 90% more than or equal to 75%.

(10) the invention provides polynucleotide, its coding is the aminoacid sequence of the trehalose phosphorylase of (7) record as described, and described polynucleotide are any one polynucleotide that are selected from the group of following (d)～(f) composition:

(d) coding has the polynucleotide of the polypeptide of the aminoacid sequence shown in the sequence number 3 of sequence table;

(e) coding has the polynucleotide of polypeptide of following aminoacid sequence, and described aminoacid sequence is the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in the sequence number 3 of sequence table;

(f) have the polynucleotide of the nucleotide sequence shown in the sequence number 4 of sequence table.

(11) the invention provides recombinant vectors and microorganism, described recombinant vectors has as described the polynucleotide of (10) record; Described microorganism transforms with described recombinant vectors.

(12) the present invention also provides the manufacture method of the mixture of maltose phosphorylase or trehalose phosphorylase or above-mentioned two kinds of enzymes; It is characterized in that, cultivation has the series bacillus microorganism belonging to genus of producing maltose phosphorylase and trehalose phosphorylase ability, generate and accumulate at least a in the trehalose phosphorylase of the maltose phosphorylase of (2) or (3) record as described and/or (7) or (8) record as described, and extract the enzyme that is generated.

(13) the invention provides as described the manufacture method that (12) are put down in writing, it is the manufacture method of maltose phosphorylase or maltose phosphorylase and trehalose phosphorylase mixture; It is characterized in that, carry out described cultivation containing in the presence of the carbon source of maltose, generate and accumulate maltose phosphorylase and trehalose phosphorylase.

(14) the invention provides as described the manufacture method that (12) are put down in writing, it is the manufacture method of trehalose phosphorylase or maltose phosphorylase and trehalose phosphorylase mixture; It is characterized in that, carry out described cultivation containing in the presence of the carbon source of trehalose, preferentially generate and accumulate trehalose phosphorylase.

(15) the present invention relates to the manufacture method of the thick enzyme of maltose phosphorylase and/or trehalose phosphorylase, described method is to be selected from following (i) any one method in (iii):

(i) cultivate series bacillus microorganism belonging to genus, directly extract isolating thalline from the nutrient solution that obtains with production maltose phosphorylase and trehalose phosphorylase ability;

(ii) cultivate and have the series bacillus microorganism belonging to genus of producing maltose phosphorylase and trehalose phosphorylase ability, separating thallus from the nutrient solution that obtains, the thick enzyme of extraction maltose phosphorylase and/or trehalose phosphorylase from isolating thalline;

(iii) cultivate and have the series bacillus microorganism belonging to genus of producing maltose phosphorylase and trehalose phosphorylase ability, separating thallus from the nutrient solution after the resulting cultivation, the supernatant liquor of extraction nutrient solution.

(16) the present invention also provides the manufacture method of trehalose, it is characterized in that, in the presence of phosphoric acid, will be as described (2) or (3) record maltose phosphorylase and as described (7) or (8) record trehalose phosphorylase act on maltose, make trehalose with this.

Description of drawings

Fig. 1 schematically illustrates the taxonomic position of the microbiology class genus bacillus sp.SH-55 that produces enzyme of the present invention.

Fig. 2 schematically illustrates with SDS-polyacrylamide gel electrophoresis method and analyzes maltose phosphorylase of the present invention and the resulting analytical results of trehalose phosphorylase.

Fig. 3 schematically illustrate the optimal pH of the decomposition reaction of maltose phosphorylase of the present invention and trehalose phosphorylase and building-up reactions and the scope of action pH (zero is decomposition reaction, ● be building-up reactions).

Fig. 4 schematically illustrate the enzymic activity of maltose phosphorylase of the present invention and trehalose phosphorylase and the relation of pH (zero is maltose phosphorylase, ● be trehalose phosphorylase).

Fig. 5 schematically illustrate enzymic activity and operative temperature in the decomposition reaction of maltose phosphorylase of the present invention (A) and trehalose phosphorylase (B) and the building-up reactions relation (zero is decomposition reaction, ● be building-up reactions).

Fig. 6 schematically illustrate maltose phosphorylase of the present invention and trehalose phosphorylase thermotolerance (zero for maltose phosphorylase, ● be trehalose phosphorylase).

In addition, the latin name of each bacterium kind generic name correspondence is as follows among Fig. 1:

Curved spore series bacillus: Paenibacillus campinasensis

Paenibacillus polymyxa: Paenibacillus polymyxa

Ke Shi series bacillus: Paenibacillus kribbensis

Glucan genus bacillus: Paenibacillus glucanolyticus

Magnificent series bacillus: Paenibacilluslautus

Vortex genus bacillus: Bacillus vortex

Tough and tensile series bacillus: Paenibacillus durus

Bacillus circulans: Bacillus circulans

Northern series bacillus: Paenibacillus borealis

Layering series bacillus: Paenibacillus hibebsis

Separate the starch based genus bacillus: Paenibacillus amylolyticus

Series bacillus in the Ma Kuo: Paenibacillus macquariensis

Subtilis: Bacillus subtilus

Embodiment

Below the present invention will be described in more detail.

The present inventor is a purpose can be used for industrial production, for obtain having can produce maltose phosphorylase and trehalose phosphorylase (manufacturing of described enzyme and refining easy, operative temperature is higher, have thermostability and do not have the concentration of substrate dependency) the microorganism of ability carried out retrieval widely at nature, found that series bacillus belongs to two kinds of enzymes that novel microorganism can mass production possesses described condition.

That is, novel strain of the present invention be deep-sea that use ocean research such as contriver exploitation mechanism holds detect undersea boat " deep-sea 2000 " in phase mould gulf just in the bottom silt of ocean surface, the southeast, island depth of water 1174m new the separation obtain.

As an example that belongs to the novel microorganism of series bacillus genus of the present invention, can exemplify series bacillus sp.SH-55.The various bacteriology character of series bacillus sp.SH-55 are as follows.

＜morphological properties 〉

The form of cell: bacillus

The size of cell: 0.7 μ m～0.9 μ m * 2.0 μ m～4.0 μ m

Mobility: have

Flagellum: (polar flagellum) arranged

Sporozoite forms: have

＜growth conditions 〉

The form of bacterium colony: the form irregular periphery slightly is wavy, glossy, is milk yellow (inhomogeneous).

Growth temperature:, do not grow for 50 ℃ in 15 ℃～45 ℃ growths.

Common salt concn: in 5% salt, grow, in more than or equal to 7% salt, do not grow.

Growth under the anaerobic condition: do not grow.

＜physiological characteristics 〉

Gramstaining: feminine gender

O-F tests (glucose): feminine gender; Can't help glucose generates acid and gas.

Catalase test: the positive

Oxydase test: the positive

Gel decomposition performance: feminine gender

Casein decomposability: the positive

Amylolysis performance: the positive

Hippurate resolution: feminine gender

ONPG test: the positive

Urease produces: the positive

Ornithine decarboxylase produces: feminine gender

Lysine decarboxylase produces: feminine gender

Hydrogen sulfide generates: feminine gender

Indoles generates: feminine gender

Nitrate reductase performance: feminine gender

Hydrogen sulfide produces: feminine gender

The 3-oxobutanol produces (VP test): feminine gender

Utilize characteristic: have to glycerine, L-arabinose, ribose, D-wood sugar, semi-lactosi, glucose, fructose, seminose, N.F,USP MANNITOL, arbutin, polychrom, salicyl alcohol glucoside, N-acetyl-glucosamine, lactose, melibiose, trehalose, sucrose, cellobiose, maltose, raffinose, starch, glycogen utilize characteristic.

Adopt CLUSTAL X multisequencing contrast program (version 1.81) that the taxonomic position of the 16SrDNA sequence of bacterial strain of the present invention is resolved.Adopt contiguous connection method to put down in writing the genealogical tree of analysis result, this genealogical tree is as shown in table 1.Finding from its result, though bacterial strain of the present invention is nearer with glucan genus bacillus (Paenibacillus glucanolyticus) position on taxonomy, is different in genealogical tree, and obviously there is branch in the two.Therefore judge that bacterial strain of the present invention is the novel kind that series bacillus belongs to.So with its called after series bacillus sp.SH-55, this bacterial strain is deposited in Independent Administrative Leged Industrial Technology Complex Inst and speciallys permit biological preservation center (postcode 305-8566 in putting down into 15 years (2003) June 27,1 kind of ground of 1 fourth order, east, ripple city, 1 central authorities the 6th are built in the Hitachinaka County, Japan) carry out international preservation, preserving number is FERM BP-8420.

Novel strain of the present invention screens in the following way and obtains.At first, the bottom silt that extracts is suspended in the physiological saline, gets 1 this suspension and spread upon on the nutrient agar with following composition.The Agar Plating that uses contains 2% (w/v) agar, 1% trehalose or maltose, 0.5% polyprotein peptone, 0.5% yeast extract, 0.1% Rhodiaphos DKP and 0.02% magnesium sulfate heptahydrate, and pH is 7.Like this, Agar Plating is carried out aerobic in 37 ℃ cultivate, collect dull and stereotyped each bacterium colony that occurs of going up, each bacterium colony velocity fluctuation with 180rpm in the liquid nutrient medium identical with described nutrient agar composition was cultivated 24 hours～72 hours in 37 ℃.Speed with 12000 * g is carried out centrifugation at 4 ℃ to each nutrient solution, and each nutrient solution is separated into thalline and supernatant liquor.The thalline that so obtains is suspended in a spot of 0.1M phosphoric acid buffer (pH7.0), measures active with the aftermentioned method.So be separated to bacterial strain with described all bacteriology characteristics.

The novel microorganism of finding of the present invention like this, promptly the microorganism of series bacillus genus is the novel bacterium that produces maltose phosphorylase and trehalose phosphorylase.

In order to obtain enzyme of the present invention (being described maltose phosphorylase and trehalose phosphorylase) from novel microorganism of the present invention, for example can this microbial inoculant be cultivated in suitable substratum according to ordinary method, from culture, reclaim then.Consider the growth temperature of microorganism self, culture condition is preferably 25 ℃～42 ℃ temperature range, and preferred aerobic was cultivated 8 hours～70 hours.

For obtaining enzyme of the present invention, the substratum that is used for microorganism culturing is not limited only to material shown below, so long as microorganism can grow and can to produce the nutritional medium of enzyme of the present invention just passable, and synthetic medium or natural medium all can.

As the carbon source of substratum so long as the material that microorganism can utilize is just passable, for example, can use saccharics such as glucose, fructose, seminose, trehalose, sucrose, N.F,USP MANNITOL, sorbyl alcohol, molasses, or organic acids such as citric acid, succsinic acid, preferably use trehalose or maltose and contain trehalose or the saccharic of maltose.If use trehalose or the saccharic that contains this material as carbon source, microorganism then of the present invention preferentially produces trehalose phosphorylase.If use maltose or the saccharic that contains this material as carbon source, microorganism then of the present invention produces maltose phosphorylase and trehalose phosphorylase simultaneously.And, if use trehalose and maltose the two or the saccharic that contains these two kinds of materials as carbon source, then trehalose phosphorylase and maltose phosphorylase can be produced simultaneously, and the generation ratio of trehalose phosphorylase and maltose phosphorylase can also be controlled by the amount of control trehalose and maltose.

All kinds of organic or inorganic nitrogen compounds can be contained in the substratum as nitrogenous source, all kinds of inorganic salt can also be contained.As nitrogenous source, can use the compound that generally is used for microorganism culturing, for example organic nitrogen source such as corn steep liquor, soybean meal or various peptones and sulphur ammonium, ammonium nitrate, phosphorus ammonium, urea etc. are inorganic nitrogen-sourced etc.Urea and organic nitrogen source etc. also can be as carbon sources needless to say.

In addition, as inorganic components, for example, suitable calcium salt, magnesium salts, sylvite, sodium salt, phosphoric acid salt, manganese salt, zinc salt, molysite, mantoquita, molybdenum salt, the cobalt salt etc. of using.Can also suit to use amino acid, VITAMIN etc. as required.

Appropriate media as the culturing micro-organisms that is used to obtain enzyme of the present invention, for example, when preferential production trehalose phosphorylase, the pH that suitable use contains 0.5%～3% (w/v) trehalose, 0.5%～2% yeast extract, 0.15% ammonium phosphate, 0.1%～0.2% urea, 0.5%～1.5% salt, 0.05%～0.3% Rhodiaphos DKP, 0.01%～0.05% magnesium sulfate heptahydrate and 0.1%～0.3% lime carbonate is 7.0～7.5 liquid nutrient medium.In addition, when producing maltose phosphorylase and trehalose phosphorylase at the same time, can use the liquid nutrient medium that contains 0.5%～3% (w/v) maltose, 1%～3% polyprotein peptone S (Japanese pharmacy manufacturing), 0.1%～0.3% ammonium phosphate, 0.05%～0.3% urea, 0.5%～1.5% salt, 0.05%～0.25% Rhodiaphos DKP, 0.01%～0.05% magnesium sulfate heptahydrate and 0.1%～0.3% lime carbonate.The concentration of these substratum is restriction not, can suitably change according to the kind and the concentration of carbon source and nitrogenous source etc.Not only produce maltose phosphorylase when using maltose to make carbon source, also produce a certain amount of trehalose phosphorylase.Therefore, when production is used for producing the thick enzyme (mixture of maltose phosphorylase and trehalose phosphorylase) of trehalose, use maltose more economically as carbon source.

Cultivation is generally the aerobic that is selected from following condition and cultivates: temperature is 20 ℃～45 ℃, is preferably 25 ℃～42 ℃; PH5～9, be preferably pH6～8.As long as it is just passable that incubation time is not less than the time that microorganism begins to breed, preferred 8 hours to 70 hours.And, there is not particular restriction for the dissolved oxygen concentration of nutrient solution, usually preferred 0.5ppm～20ppm.For this reason, can regulate air flow, stirring or in ventilation, append oxygen.And training method can be selected batch culture and cultured continuously.

Cultivate thus after the microorganism of the present invention, to the enzyme of the present invention that is generated, promptly maltose phosphorylase and trehalose phosphorylase reclaim.The enzyme major part that is generated is accumulated in the thalline, and a part is accumulated in outside the thalline.Therefore, be extracted in maltose phosphorylase and/or the trehalose phosphorylase that thalline is interior or thalline generates and accumulates outward.

The recovery of enzyme of the present invention can be carried out according to the usual method of extracting enzyme.Recovery method is not limited only to following method, can exemplify into, obtain the bacterial cell disruption thing with bacterial cell disruption methods such as ultrasonic disruption method, French press filtration (French press) method, granulated glass sphere crush method, Dino mill methods, operation by this bacterial cell disruption thing of centrifuging or culture etc., be separated into thalline and nutrient solution supernatant liquor, the nutrient solution supernatant liquor that obtains with separation is used as crude enzyme liquid.

This crude enzyme liquid both can directly use, also can be as required, be used in combination separation means such as salting-out process, the precipitator method, ultrafiltration process, for example, be used in combination well-known methods such as ion exchange chromatography, iso-electric point chromatography, hydrophobicity chromatography method, gel-filtration chromatography, adsorption chromatography, affinity chromatography, reverse-phase chromatography method, further the enzyme liquid behind the separation and purification uses.

In addition, as the additive method that obtains enzyme of the present invention, can also exemplify into, from described bacterial strain of the present invention, extract the gene of code book invention enzyme after, make recombinant microorganism with genetic engineering technique, cultivate this recombinant microorganism then.Specifically be exactly, from described bacterial strain, extract the nucleotide sequence of the aminoacid sequence of code book invention enzyme, afterwards this nucleotide sequence is imported suitable carriers, use hosts such as this carrier transformed into escherichia coli or Bacillus subtilus then, cultivate this cell to produce enzyme of the present invention, from culture, extract enzyme of the present invention again.

Below the method for using genetic engineering technique to make enzyme of the present invention is specifically described.Two kinds of enzymes of the present invention, be that maltose phosphorylase and trehalose phosphorylase are respectively the polypeptide with following aminoacid sequence, described aminoacid sequence is respectively with the sequence number 1 of sequence table and the aminoacid sequence shown in the sequence number 3 or is respectively the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in described sequence number 1 and the sequence number 3, therefore must use and the corresponding nucleotide sequence of these aminoacid sequences.

For the nucleotide sequence of the aminoacid sequence of code book invention enzyme, be example with the maltose phosphorylase, can specifically be exemplified as selected polynucleotide from the group that following (a)～(c) forms:

(a) coding has the polynucleotide of the polypeptide of the aminoacid sequence shown in the sequence number 1 of sequence table;

(b) coding has the polynucleotide of polypeptide of following aminoacid sequence, and described aminoacid sequence is the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in the sequence number 1 of sequence table;

(c) have the polynucleotide of the nucleotide sequence shown in the sequence number 2 of sequence table.

For the nucleotide sequence of the aminoacid sequence of code book invention enzyme, be example with the trehalose phosphorylase, can specifically be exemplified as selected polynucleotide from the group that following (d)～(f) forms:

(d) coding has the polynucleotide of the polypeptide of the aminoacid sequence shown in the sequence number 3 of sequence table;

(e) coding has the polynucleotide of polypeptide of following aminoacid sequence, and described aminoacid sequence is the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in the sequence number 3 of sequence table;

(f) have the polynucleotide of the nucleotide sequence shown in the sequence number 4 of sequence table.

The recombinant microorganism of production enzyme of the present invention can be made in conjunction with multiple generally acknowledged mode.That is, for example from described series bacillus sp.SH-55, extract the nucleotide sequence of code book invention enzyme, this nucleotide sequence that increases, nucleotide sequence is imported carrier, can be undertaken by the method for being put down in writing in the existing publication in this area with processes such as this gene pairs host transform.Wherein, as the recombinant microorganism manufacture method, can use method as follows, but be not particularly limited.Promptly, with the shotgun cloning method or use specific primer to carry out pcr amplification, from series bacillus sp.SH-55, extract the gene of described two kinds of enzymes, these genes are imported intestinal bacteria E.coli with EK system) be that the Gram-negative bacteria of representative or the Bacillus subtilus (B.subtilis) that is they are the gram-positive microorganism of representative with BS, obtain recombinant chou.When transforming, can be with extranuclear genes such as plasmids as carrier, or utilize the ability of the absorption DNA that host bacteria just had originally to transform.

As mentioned above,, perhaps cultivate the recombinant microorganism of the gene of the code book invention enzyme amino acid sequence of having recombinated, and from the gained culture, reclaim, can access enzyme of the present invention by cultivating series bacillus microorganism belonging to genus of the present invention.

As previously mentioned, because enzyme of the present invention, promptly aforesaid maltose phosphorylase and trehalose phosphorylase are accumulated in cultivating thalline or in the nutrient solution supernatant outside the thalline, can be separated obtaining by ordinary method.At first, can be with endobacillary enzyme together with whole thalline as thick enzyme.Can reclaim thick enzyme by from thalline, extracting enzyme then.And, also contain enzyme in the outer nutrient solution supernatant of thalline, can utilize separating thallus after remaining nutrient solution contain liquid as thick enzyme.Then, can utilize refining these thick enzymes of ordinary methods such as solvent precipitation, sulphur ammonium partition method, ion exchange chromatography, hydrophobicity chromatography method, gel-filtration chromatography of ethanol, acetone, Virahol etc.And the difference of separating the iso-electric point that for example can utilize two kinds of enzymes of maltose phosphorylase and trehalose phosphorylase waits by anion exchange chromatography carries out.

The enzyme of the present invention that obtains thus promptly is that these also will be elaborated in the embodiment of back as maltose phosphorylase and trehalose phosphorylase with novel enzyme of described physico-chemical property.

The determination of activity of these enzymes can be carried out with following short-cut method.Because series bacillus sp.SH-55 of the present invention does not produce the alpha-glycosidase (maltin), glucoamylase, trehalase of hydrolysis maltose or trehalose etc., when the activity when adding the phosphoric acid decomposition reaction is measured, can be substrate with maltose or trehalose respectively, exist the glucose that carries out the enzyme reaction generation down to measure to phosphoric acid salt with glucose oxidase method.When the activity during to building-up reactions was measured, can adopt the mixed solution of β-D-glucose 1-phosphoric acid and glucose was substrate, with ordinary method the inorganic phosphate that enzyme reaction generates was measured, thus calculated activity.

Below, the activation measurement of enzyme of the present invention is described.

(i) decomposition reaction: in the 20mM of 0.5mL maltose or aqueous trehalose (being dissolved in the phosphoric acid buffer (pH 7) of 50mM), add 0.01mL enzyme liquid, in 50 ℃ of reactions after 15 minutes in boiling water bath heating enzyme reaction was stopped in 3 minutes.After the cooling, (Wako Pure Chemical Industries, Ltd. makes, and glucose C-II detects-WAKO) glucose that generates measured with glucose oxidase method in frozen water.The enzyme amount that will be under this condition determination generates 1 μ mol glucose here, in 1 minute is defined as the enzymic activity of 1 unit.

(ii) building-up reactions: in 0.15mL contains the mixing solutions (being dissolved in the HEPES damping fluid (pH7.0) of 70mM) of 27mM β-D-glucose 1-sodium phosphate salt and 27mM glucose, add 0.05mL enzyme liquid, in 50 ℃ of reactions after 15 minutes in boiling water bath heating enzyme reaction was stopped in 2 minutes.After the cooling, the P-Test WAKO that makes with Wako Pure Chemical Industries, Ltd. measures the inorganic phosphate that generates in frozen water.The enzyme amount that will be under this condition determination generates 1 μ mol inorganic phosphate here, in 1 minute is defined as the enzymic activity of 1 unit.

As mentioned above, by being that series bacillus microorganism belonging to genus (for example series bacillus sp.SH-55) is cultivated or the recombinant microorganism of the gene of the code book invention enzyme amino acid sequence of having recombinated is cultivated to novel microorganism of the present invention, and reclaim this cultivation resultant, can make novel enzyme of the present invention, i.e. maltose phosphorylase and trehalose phosphorylase simply thus.These two kinds of features that enzyme had of the present invention are that respectively to the α in the maltose-1, the α in 4-Glucopyranose glycosidic bond and the trehalose-1,1-Glucopyranose glycosidic bond carry out reversibility and add the phosphoric acid decomposition, generate glucose and β-D-glucose 1-phosphoric acid.Therefore, to the maltose effect, can produce trehalose by these two kinds of enzymes of combination in the presence of phosphoric acid with very high efficient.And, because action pH wide ranges to 4.5～9.5 of these two kinds of enzymes, and the optimal pH scope of two kinds of enzymes is overlapping, and very easy to use in enzyme reaction.In addition, the operative temperature scope is 20 ℃～60 ℃, can use under comparatively high temps, so can at high temperature carry out enzyme reaction, can produce trehalose under the situation of not worrying to occur the living contaminants seen in the previous methods.Because the maltose as raw material can obtain simply in a large number, and can use the high purity substance after making with extra care, the impurity of sneaking in the resultant after enzyme reaction finishes is few, therefore the advantage that adopts described enzyme and had is, resultant refining simple and can produce trehalose extremely efficiently.

These enzymes of the present invention can use with the form of thick enzyme or refining enzyme respectively.And, can will have the thalline of two kinds of enzymic activitys and described enzyme be comprised the immobilized thallus that is adsorbed in the appropriate carriers or be used for the manufacturing of trehalose by the immobilized thallus that Chemical bond is fixed in suitable carrier with this thalline.And two kinds of enzymes of the present invention can also be to press the form use of known method fixed immobilized enzyme separately.

Embodiment

The present invention will be described in more detail below by embodiment.

Embodiment 1:

In the thalline and the manufacturing of the outer maltose phosphorylase of thalline and refining

To containing 1% (w/v) maltose, 2.5% polyprotein peptone S (Japanese pharmacy manufacturing), 0.15% ammonium phosphate, 0.15% urea, 1% salt, 0.1% Rhodiaphos DKP, 0.02% magnesium sulfate heptahydrate and 0.2% lime carbonate and pH is inoculation series bacillus sp.SH-55 (FERMBP-8420) in 7.0 the liquid nutrient medium.Cultivated this liquid nutrient medium 24 hours down in 37 ℃ of aerobic conditions then.To the nutrient solution that obtains in 4 ℃ with the speed centrifugation of 12000 * g 15 minutes, it is divided into thalline and supernatant liquor.

With the thalline that obtains outstanding turbid in a spot of 20mM phosphoric acid buffer (pH7.0) afterwards, use ultrasonic disruption.In this fragmentation thalline suspension liquid, add the sulphur ammonium and make it be saturated to 30%, place a night in 4 ℃.Further adding the sulphur ammonium then in the supernatant liquor that obtains behind the centrifugation disgorging makes it be saturated to 70%.Place the throw outs that generate a night for 4 ℃ again and collect by centrifugation, be dissolved in it in 20mM phosphoric acid buffer (pH7) after, dialyse fully with same damping fluid.

Next, use 20mM phosphoric acid buffer (pH7) that enzyme is adsorbed in DEAE-Fractogel (manufacturing of Merck company) post after the equilibration.After the enzyme stripping of E-test with absorption that contains 0M to 0.5M salt in the described damping fluid, (Amicon company makes, and YM-30) concentrates with the UF film.It is refining in order to the SephacrylS-300 after the described damping fluid equilibration that contains 0.2M salt (manufacturing of Pharmacia company) post concentrated enzyme to be carried out gel-filtration.The maltose phosphorylase active part that collection obtains after dialysing with the above-mentioned damping fluid that contains 1.5M sulphur ammonium, is adsorbed in phenylToyopearl (manufacturing of Tosoh company) post after the equilibration with the above-mentioned damping fluid that contains 1.5M sulphur ammonium with enzyme.After the enzyme stripping of E-test that contains 1.5M to 0M sulphur ammonium in the described damping fluid with absorption, collect the maltose phosphorylase active part that obtains, dialyse with the described damping fluid that contains 0.2M salt.With described UF membrane concentration, carry out gel permeation chromatography once more with the Superdex200 (manufacturing of Pharmacia company) after the described damping fluid equilibration that contains 0.2M salt then, with described method the maltose phosphorylase active part that obtains is concentrated.

Here, active part is to separate the part obtain with methods such as gel permeation chromatographies, is to be that substrate is confirmed activated part when carrying out determination of activity with maltose or trehalose, refers to maltose phosphorylase active part and trehalose phosphorylase active part respectively.That is, when the activity of maltose or trehalose is confirmed,, be the maltose phosphorylase active part near the 0.3M common salt concn then if use the DEAE-Fractogel post, be the trehalose phosphorylase active part near the 0.35M common salt concn.

As the thalline endoenzyme, obtain uniform maltose phosphorylase (activity yield is 30%) with polyacrylamide gel disk electrophoresis method and SDS-polyacrylamide gel electrophoresis method.About the thalline exoenzyme, be initial feed with the nutrient solution supernatant liquor, make with extra care with method same as described above, obtain the refining enzyme that activity yield is about 25% maltose phosphorylase.The refining enzyme that obtains is carried out the mensuration of molecular weight with SDS-polyacrylamide gel electrophoresis method, molecular weight is near 89000 dalton～90000 dalton, and this is very consistent with 87762 dalton that calculated by determined putative amino acid sequence in embodiment 3 described later.With Sephacryl S-200 gel filtration method the molecular weight of refining enzyme is measured, its molecular weight is about 200000 dalton, therefore thinks that this enzyme is made of the homotype dipolymer.

Embodiment 2:

In the thalline and the manufacturing of thalline extracellular trehalose Starch phosphorylase and refining

To containing 1% (w/v) trehalose, 2% yeast extract, 0.15% ammonium phosphate, 0.15% urea, 1% salt, 0.1% Rhodiaphos DKP, 0.02% magnesium sulfate heptahydrate and 0.2% lime carbonate and pH is inoculation series bacillus sp.SH-55 in 7.0 the liquid nutrient medium.Cultivate this liquid nutrient medium and carry out aftertreatment by the method identical, obtain bacterial cell disruption liquid and supernatant liquor with embodiment 1.For the bacterial cell disruption liquid and the supernatant liquor that obtain, use the method identical to make with extra care respectively with embodiment 1.Then, with polyacrylamide gel disk electrophoresis method and SDS-polyacrylamide gel electrophoresis method the described refined liquid that obtains from bacterial cell disruption liquid and supernatant liquor is made with extra care respectively, obtain uniform thalline intracellular trehalose Starch phosphorylase and thalline extracellular trehalose Starch phosphorylase.Activity yield is respectively 30% and 35%.Two kinds of refining enzymes are carried out the mensuration of molecular weight with SDS-polyacrylamide gel electrophoresis method, all near 89000～90000 dalton, this is very consistent with 87151 dalton that calculated by determined putative amino acid sequence in embodiment 3 described later for molecular weight.With Sephacryl S-200 gel filtration method the molecular weight of refining enzyme is measured, its molecular weight is about 190000 dalton, therefore thinks that this enzyme is made of the homotype dipolymer.

Embodiment 3:

The gene clone of maltose phosphorylase and trehalose phosphorylase and order-checking

Carried out the gene clone of maltose phosphorylase as follows.The N-terminal aminoacid sequence of the refining enzyme that embodiment 1 is obtained with ordinary method is measured, and it has the MKQYLKLDEW sequence.With V8 (be a kind of proteolytic enzyme, Sigma makes) refining enzyme is carried out digesting in the gel then.The method of digestion is in the gel, and the refining enzyme of 5 μ g is clicked and entered SDS PAGE gel, adds the V8 protein enzyme solution of 1 μ g and carries out electrophoresis on refining enzyme solution upper strata simultaneously, decomposes the purpose enzyme in gel.With ordinary method two kinds of N-terminal aminoacid sequences of the protein fragments that obtains are measured.The result obtains Ala-Tyr-Ser-Gly-Ser-Ser-Leu-Gln-Gly-Ser-Tyr-Met-Ala-Gly-Val-Tyr-Tyr-Pro-Asp-Lys sequence from segment 1, obtain the Gly-Asp-Val-Ala-Ala-Gln-Gln-Ala-Ile-Arg sequence from segment 2.A kind of amino acid whose dna sequence dna of encoding has 1～6 kind.So, modulated mix primer as follows and antisense mix primer (antisense mix primer) according to the dna sequence dna of the 1st～9 aminoacid sequence in the 1st～9 aminoacid sequence in the segment 1 and the segment 2.

Primer based on segment 1 aminoacid sequence:

5’-CNTARAGRGGNTCNTCNCTNCAYG-3’

Primer based on segment 2 aminoacid sequences:

5’-ATNGCYTGYTGNGCNGCNACYTCG-3’

With the chromosomal DNA is template, uses described mix primer, utilizes Ex Taq (polysaccharase, TaKaRa Bio) to carry out PCR, and dna segment is increased.Reaction conditions is, in 96 ℃ of heating after 2 minutes, 96 ℃ 20 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, repeat this circulation 30 times, be incubated 10 minutes in 72 ℃ at last.After reaction solution carried out agarose gel electrophoresis, detect the dna segment of about 0.8kb base pair.Use TA clone's test kit (Invitrogen) that the dna segment that is obtained by the reaction solution amplification is cloned, modulated the plasmid that is used to measure base sequence.With this plasmid is template, use the terminal cycle sequencing rapid reaction of d rhodamine test kit (dRodamineDye Terminator Cycle Sequencing Ready Reaction Kit, Perkinelmer) carry out the fluorescent mark reaction, (Applied Biosystems) analyzes with dna sequencing instrument 377, determined with the 477th～1304 base sequence in the nucleotide sequence shown in the sequence number 5.

Based on described base sequence, modulated with the primer MF1 shown in 5 '-CAGTTGGTGCTGTTCAACACTTTG-3 ' with the primer MR1 shown in 5 '-ATGGCGATGTAAAGAATAAAG-3 '.On the other hand, behind restriction enzyme XhoI cut-out chromosomal DNA, use Ligation high (ligase enzyme, Japan is spun) in 16 ℃ of connections 1 hour, be template with this DNA, use MF1 and MR1 as primer, utilize LATaq (polysaccharase, TaKaRa Bio) to carry out inverse PCR.After reaction solution carried out agarose gel electrophoresis, detect the dna segment that is about the 7kb base pair.Refining dna segment by the reaction solution amplification, modulation is used to measure the DNA of base sequence.With this dna segment is template, use the terminal cycle sequencing rapid reaction test kit of d rhodamine (Perkinelmer) to carry out the fluorescent mark reaction, (Applied Biosystems) analyzes with dna sequencing instrument 377, obtained having with the aminoacid sequence shown in the sequence number 1 with the gene of the maltose phosphorylase of the nucleotide sequence shown in the sequence number 2.

Then, carried out the gene clone of trehalose phosphorylase as follows.The N-terminal aminoacid sequence of the refining enzyme that embodiment 2 is obtained with ordinary method is measured, and it is the Met-Thr-Lys-Met-Ile-Ser-Asn-Pro-Asp-Leu sequence.Designed mix primer thus with sequence shown in the following primer 1.And, from the aminoacid sequence of known trehalose phosphorylase, retrieve and the higher sequence of its homology, found the aminoacid sequence of Gly-Tyr-Glu-Gly-His-Tyr-Phe-Trp-Asp.Designed the antisense mix primer of sequence shown in the following primer 2 thus.

Primer 1:5 '-ATGACNTGGATGATHAGCAAYC-3 '

Primer 2: 5 '-CCAYAAYTAYTGNCCRTCYTANCC-3 '

With the chromosomal DNA is template, uses described mix primer (primer 1 and primer 2), and utilizes Ex Taq (polysaccharase, TaKaRa Bio) to carry out PCR, and dna segment is increased.Reaction conditions is, in 96 ℃ of heating after 2 minutes, 96 ℃ 20 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, repeat this circulation 30 times, be incubated 10 minutes in 72 ℃ at last.After reaction solution carried out agarose gel electrophoresis, detect the dna segment of about 1.0kb base pair.Use TA clone's test kit (Invitrogen) that the dna segment that is obtained by the reaction solution amplification is cloned, modulated the plasmid that is used to measure base sequence.With this plasmid is template, use the terminal cycle sequencing rapid reaction test kit of d rhodamine (Perkinelmer) to carry out the fluorescent mark reaction, (Applied Biosystems) analyzes with dna sequencing instrument 377, determined with the 1113rd～2168 base sequence in the nucleotide sequence shown in the sequence number 7.

Based on described base sequence, modulated with the primer TF1 shown in 5 '-ACGATGACCAGCTCCAGGAAG-3 ' with the primer TR1 shown in 5 '-TCAGATAGGTACCGCGAATGG-3 '.On the other hand, behind restriction enzyme XhoI cut-out chromosomal DNA, using Ligation high (ligase enzyme, Japan is spun) to connect, is template with this DNA, uses TF1 and TR1 as primer, utilizes LA Taq (polysaccharase, TaKaRa Bio) to carry out inverse PCR.After reaction solution carried out agarose gel electrophoresis, detect the dna segment that is about the 6kb base pair.Refining dna segment by the reaction solution amplification, modulation is used to measure the DNA of base sequence.With this dna segment is template, use the terminal cycle sequencing rapid reaction test kit of d rhodamine (Perkinelmer) to carry out the fluorescent mark reaction, (Applied Biosystems) analyzes with dna sequencing instrument 377, obtained having with the aminoacid sequence shown in the sequence number 3 with the gene of the trehalose phosphorylase of the nucleotide sequence shown in the sequence number 4.

The molecular weight (according to SDS-polyacrylamide gel electrophoresis method) and the iso-electric point of inferring from sequence number 1 and sequence number 3 be respectively, and the molecular weight of maltose phosphorylase is 87762 dalton, and iso-electric point is pH4.98; The molecular weight of trehalose phosphorylase is 87151 dalton, and iso-electric point is pH5.13.

Embodiment 4:

The cultivation of colibacillary conversion, transformant and refining

The initial sequence table of initiator codon the 343rd bit base by the dna sequence dna of the coding maltose phosphorylase of embodiment 3 resulting sequence numbers 5 is shown 5 '-GTGAAACAATATTTAAAGCTTG-3 ', form 5 '-CCGCTCGAGGTGAAACAATATTTAAAGCTTG-3 ' behind 5 ' the terminal place of incision that adds restriction enzyme XhoI of this oligonucleotide, this sequence is primer MF2; Be expressed as 5 '-TTATTTTGAAGCTGCTGTG-3 ' by the initial antisense sequences of terminator codon the 2649th bit base, form 5 '-TTATTTTGAAGCTGCTGTGGGTACCCCG-3 ' after 3 ' of this oligonucleotide is held the place of incision that adds restriction enzyme KpnI, this sequence is primer MR2; With the chromosomal DNA is template, uses above-mentioned two kinds of primers, utilizes Pyrobest (polysaccharase, TaKaRa Bio) to carry out PCR, obtains containing the dna segment that is about the 2.3k base pair of maltose phosphorylase.Reaction conditions is, in 96 ℃ of heating after 2 minutes, 96 ℃ 20 seconds, 55 ℃ 30 seconds, 72 ℃ 2 minutes, repeat this circulation 30 times, be incubated 10 minutes in 72 ℃ at last.Behind the refining dna segment that so obtains, cut off with restriction enzyme XhoI and KpnI.Use Ligation high (ligase enzyme, Japan is spun) this segment and 50ng are connected 2 hour with plasmid vector pRSETA (Invitrogen) after KpnI cuts off in 16 ℃ with restriction enzyme XhoI, make recombinant plasmid pRSMP1, use experience attitude cell method imports e. coli bl21 (DE3) pLysS (F-with this plasmid, ompT hsdSB (rB-mB-) gal dcm (DE3) pLysS (CamR)) bacterial strain, the intestinal bacteria RSMP1 after obtaining transforming.

Similarly, the initial sequence table of initiator codon the 1113rd bit base by the dna sequence dna of the encoding trehalose Starch phosphorylase of embodiment 3 resulting sequence numbers 7 is shown 5 '-ATGACGTGGATGATAAGCAATC-3 ', form 5 '-CGCGGATTCATGACGTGGATGATAAGCAATC-3 ' behind 5 ' the terminal place of incision that adds restriction enzyme BamHI of this oligonucleotide, this sequence is primer TF2; By the initial antisense sequences of terminator codon the 3413rd bit base is 5 '-TTATTTTGAAGCTGCTGTG-3 ', form 5 '-TTATTTTGAAGCTGCTGTGGGTACCCCGGAATTCCGG-3 ' behind 3 ' the terminal place of incision that adds limiting enzyme EcoRI of this oligonucleotide, this sequence is primer TR2; With the chromosomal DNA is template, uses above-mentioned two kinds of primers, utilizes Pyrobest (polysaccharase, TaKaRa Bio) to carry out PCR, obtains containing the dna segment that is about the 2.3k base pair of trehalose phosphorylase.Reaction conditions is, in 96 ℃ of heating after 2 minutes, 96 ℃ 20 seconds, 55 ℃ 30 seconds, 72 ℃ 2 minutes, repeat this circulation 30 times, be incubated 10 minutes in 72 ℃ at last.Behind the refining dna segment that so obtains, cut off with restriction enzyme BamHI and EcoRI.Use Ligationhigh (ligase enzyme, Japan is spun) this segment and 50ng are connected 2 hour with plasmid vector pRSETA (Invitrogen) after EcoRI cuts off in 16 ℃ with restriction enzyme BamHI, make recombinant plasmid pRSTP1, use experience attitude cell method imports e. coli bl21 (DE3) pLysS (F-with this plasmid, ompT hsdSB (rB-mB-) gal dcm (DE3) pLysS (CamR)) bacterial strain, the intestinal bacteria RSTP1 after obtaining transforming.

Cultivate intestinal bacteria RSMP1 and RSTP1 after transforming then respectively, the modulation recombinase.

The penbritin and the ultimate density of at first adding ultimate density and be 50 μ g/mL in LB substratum (1% bacto peptone, 0.5% yeast extract, 1% sodium-chlor) are the paraxin of 34 μ g/mL, to volume is to add the above-mentioned substratum that has added penbritin and paraxin of 30mL in the Erlenmeyer flask of 300mL, single bacterium colony to this inoculation of medium resulting conversion bacterial strain RSMP1 and RSTP1, speed with 180rpm was cultivated the preceding nutrient solution (Seed Pei Juan of modulation RSMP1 and RSTP1 16 hours in 37 ℃

Liquid).

Then, cultivate transformant with following method.Promptly, after volume is to add the LB substratum (1% bacto peptone, 0.5% yeast extract, 1% sodium-chlor) and sterilization of 0.5L in the Erlenmeyer flask of 2L, the interpolation ultimate density is that penbritin and the ultimate density of 50 μ g/mL is the paraxin of 34 μ g/mL, in this substratum, inoculate the preceding nutrient solution of RSMP1 or RSTP1 respectively, the ultimate density of nutrient solution is 1% before making, and being cultured to OD600 (absorbancy under the 600nm) with the speed of 180rpm in 37 ℃ is 0.5.Adding isopropylthiogalactoside (IPTG) afterwards, to make its ultimate density be 1mM, cultivated 3 hours again.After cultivate finishing, with separation and Culture liquid, obtain thalline with centrifugal 10 minutes of the speed of 5000 * g.The intestinal bacteria that obtain with ultrasonic disruption were with the speed centrifugation of 12000 * g 15 minutes.The crude extract that obtains with affinity column (TALON Resin, Clontech company makes) twice of chromatography (cleaning with the 5mM imidazoles, with the stripping of 100mM imidazoles), is obtained maltose phosphorylase (MP) and trehalose phosphorylase (TP) after evenly refining.Use affinity column as shown in table 1 to the refining result of two kinds of enzymes.

Table 1

	Operation	Holoprotein (mg)	Complete active (unit)	Specific activity (unit/mg)	Yield (%)	Purity
							MP	Crude extract	118.5	1380.0	11.7	100.0	1.0
The 1st affine dissolution fluid	21.6	992.5	45.9	71.9	3.9
						The 2nd affine dissolution fluid		19.0	956.7	50.4	69.3	4.3
TP	Crude extract	340.0	2784.0	8.2	100.0								1.0
						The 1st affine dissolution fluid	56.0	2295.7	41.0	82.5	5.0
	The 2nd affine dissolution fluid	53.9	2252.9	41.8	80.9							5.1

As shown in table 1, the maltose phosphorylase (MP) after making with extra care and the specific activity of trehalose phosphorylase (TP) are about 50 units/mg and 42 units/mg respectively.Shown in Fig. 2 A and Fig. 2 B, the molecular weight of reorganization maltose phosphorylase of trying to achieve with SDS-polyacrylamide gel electrophoresis method and reorganization trehalose phosphorylase is about 90000 dalton～92000 dalton (calculated value is 92233 dalton) (Fig. 2 A) and 89000 dalton～91000 dalton (calculated value is 91280 dalton) (Fig. 2 B) respectively.The molecular weight of being tried to achieve respectively is that 92233 dalton and 91280 daltonian numerical value are very consistent with what calculate with putative amino acid sequence.

Embodiment 5:

The conversion of Bacillus subtilus, the cultivation of transformant and refining

Carried out the expression of described enzyme in Bacillus subtilus with following method.Promptly, the maltose phosphorylase that embodiment 3 is obtained and the dna sequence dna of trehalose phosphorylase are connected into Bacillus subtilus respectively with expression vector pHY300PLK (TaKaRa), transform Bacillus subtilus Bacillus subtilisISW1214 (leuA8 metB5 hsrM1) again, obtain transformant BSMP1 and BSTP1.

Then, identical with embodiment 4, with single colony inoculation of the single bacterium colony of the transformant BSMP1 that obtains or BSTP1 to test tube with 5mL PM substratum (4% polyprotein peptone S, 4% maltose, 0.1% yeast extract, 0.2%LAB LEMCO POWDER (Oxoid), 0.1% potassium primary phosphate, 0.02% sal epsom, 0.02% calcium chloride, 15 μ g/mL tsiklomitsins), speed with 120rpm was cultivated the preceding nutrient solution of modulation BSMP1 or BSTP1 24 hours in 30 ℃.

Next, in PM substratum (4% polyprotein peptone S, 4% maltose, 0.1% yeast extract, 0.2%LAB LEMCO POWDER (Oxoid), 0.1% potassium primary phosphate, 0.02% sal epsom, 0.02% calcium chloride, 15 μ g/mL tsiklomitsins), inoculate the preceding nutrient solution of 1% BSMP1 that as above obtains and BSTP1 respectively, cultivated 64 hours in 30 ℃ with the speed of 120rpm.After cultivate finishing, with separation and Culture liquid, obtain nutrient solution supernatant liquor and the thalline of BSMP1 and BSTP1 with centrifugal 10 minutes of the speed of 10000 * g.With the ultrasonic disruption thalline, with the speed centrifugation of 12000 * g 15 minutes, the modulation crude extract.Measure enzymic activity respectively, the result is as follows: for maltose phosphorylase, the nutrient solution supernatant liquor is 0.2 unit/mg, and thalline is 0.1 unit/mg; For trehalose phosphorylase, the nutrient solution supernatant liquor is 0.4 unit/mg, and thalline is 0.2 unit/mg.

Embodiment 6:

The homology of the putative amino acid sequence of maltose phosphorylase and trehalose phosphorylase and the aminoacid sequence of other enzymes

To the maltose phosphorylase gene that obtains among the embodiment 3 and trehalose phosphorylase gene separately amino acid sequence coded carried out FASTA homology search (http://ddbj.nig.ac.jp).The whole amino acid of maltose phosphorylase of the present invention have following homology: with Bacillaceae sp.RK-1 (Bacillus sp.RK-1, AB0084460) has 51.4% homology, with small intestine faecalis (Enterococcus hirae, E21769) has 51.4% homology, with short lactobacillus (Lactobacillus brevis, 1H54A) has 48.3% homology, with newborn Bacterium lacticum (Lactobacillus lactis, E86834) has 48.9% homology, with Lactobacillus sanfrancisco (Lactobacillus sanfranciscensis, LSJA4340) has 45.1% homology, (Neiisseria meningitides F81203) has 48.4% homology with Neisseria meningitidis.In addition, the whole amino acid of trehalose phosphorylase of the present invention have following homology: with bacstearothermophilus (Bacillus stearothermophilus SK-1, AB079610) have 62.7% homology, (Thermoanaerobium brockii ATCC35047 AB073930) has 44.2% homology with the hot anerobe of Bu Shi.

Embodiment 7:

The maltose phosphorylase that series bacillus sp.SH-55 produces and the various zymochemistry character of trehalose phosphorylase

The of the present invention novel maltose phosphorylase that is produced about series bacillus sp.SH-55 and the general zymochemistry characteristic of trehalose phosphorylase are used the method identical with embodiment 1,2,4 and 5 to make with extra care the refining enzyme that obtains and are investigated.In addition, the result of preliminary experiment is, about any one of maltose phosphorylase and trehalose phosphorylase, the enzyme that it is present in the series bacillus sp.SH-55 thalline and thalline is outer and recombinase all have various physico-chemical properties and zymologic property much at one, so the various character of here only representing to obtain among the embodiment 4 with the recombinase of escherichia coli expression.

(I) effect

Maltose solution and middle maltose phosphorylase and the trehalose phosphorylase (adding the enzyme that the decomposition reaction activity is 5 units respectively) of adding of aqueous trehalose (being dissolved in the 10mM phosphate buffer solution (pH7.0)) to 1% (w/v) with respect to the 1g substrate, after 5 hours, heating made enzyme deactivation in 3 minutes in boiling water bath in 50 ℃ of reactions.With the high speed liquid chromatography method sugar in the saccharification solution that obtains is measured, the result detects glucose and glucose 1-phosphoric acid respectively.With the glucose of 1% (w/v) and β-D-glucose 1-sodium phosphate salt or with the mixing solutions (being dissolved in the Tris-acetate buffer solution (pH7.0) of 10mM) of alpha-D-glucose 1-sodium phosphate salt be substrate, add maltose phosphorylase and trehalose phosphorylase (adding the enzyme of 5 units with respect to the 1g substrate respectively) respectively, in 50 ℃ of reactions 5 hours.The composition of the sugar that above-mentioned processing generates to process is measured, the result detects maltose and trehalose from glucose and β-D-glucose 1-phosphoric acid, confirm the building-up reactions that causes by maltose phosphorylase and trehalose phosphorylase, but but do not detected the building-up reactions of two kinds of sugar that cause by glucose and alpha-D-glucose 1-phosphoric acid.

The sugar that generates is analyzed with following method.At first, the insolubles in the saccharification solution that heated and inactivated is obtained with the filtering membrane in 0.45 μ m aperture filters.With the filtrate that obtains is sample, uses YMC-Pack, ODS-AQ post (AQ-304, YMC makes) to carry out the mensuration of high speed liquid chromatography method.In addition, use water as mobile phase, column temperature is 30 ℃, uses differential refractometer to detect.

(II) substrate specificity (decomposition reaction)

(i) maltose phosphorylase

In front in Ji Zai the enzyme assay method (decomposition reaction), replace maltose as substrate with trehalose, isomaltose, neotrehalose, sucrose, lactose or cellobiose, measured degrading activity, but all do not confirmed enzymic activity in these substrates these substrates.

(ii) trehalose phosphorylase

In same enzyme assay method (decomposition reaction), replace trehalose as substrate with maltose, isomaltose, neotrehalose, sucrose, lactose or cellobiose, measured degrading activity, but all do not confirmed enzymic activity in these substrates these substrates.

(III) action pH scope, optimal pH and stable pH range

The optimal pH of used the refining enzymatic determination that obtains among the embodiment 4 decomposition reaction and building-up reactions.The results are shown in Fig. 3.As shown in Figure 3A, the optimal pH of the decomposition reaction of maltose phosphorylase (white round dot zero) is 7.0～8.0, and the action pH scope is 4.5～9.5; The optimal pH of the building-up reactions of maltose phosphorylase (black round dot ●) is 5.5～6.5, and the action pH scope is 4.5～9.5.

In addition, shown in Fig. 3 B, the optimal pH of the decomposition reaction of trehalose phosphorylase (white round dot zero) is 7.0～8.0, and the optimal pH of its building-up reactions (black round dot ●) is 5.8～7.8; The action pH scope of this enzyme decomposition reaction and building-up reactions is 4.5～9.5.

In addition, in decomposition reaction, used the solution that in 50mM phosphoric acid-citrate buffer solution (pH4.5～8.0), phosphoric acid-borate buffer (pH8.0～9.5) and glycine-NaOH damping fluid (pH9.0～12.0), is added with the 25mM potassiumphosphate.MES (pH5.5～6.5), MOPS (pH6.5～7.0), HEPES (pH7.0～8.0), each damping fluid of Tris-hydrochloric acid (pH7.5～9.0) in building-up reactions, have been used.

Two kinds of enzymes after refining were handled 10 minutes in 50 ℃ in each damping fluid, measured the remaining enzymic activity of these enzymes then with decomposition reaction.As shown in Figure 4, maltose phosphorylase (white round dot zero) is stable in the scope of pH5.5～7.5, and trehalose phosphorylase (black round dot ●) is stable in the scope of pH5.5～9.5.50mM phosphoric acid-citrate buffer solution (pH4.5～8.0), phosphoric acid-borate buffer (pH8.0～9.5) and each damping fluid of glycine-NaOH damping fluid (pH9.0～12.0) have been used when in addition, regulating pH.

(IV) scope of operative temperature and optimum temperuture

Shown in Fig. 5 A, the decomposition reaction of maltose phosphorylase (white round dot zero) near 45 ℃～55 ℃, have optimum temperature (the 50mM phosphoric acid buffer, pH7.0), the operative temperature scope is 20 ℃～60 ℃; Building-up reactions (black round dot ●) has optimum temperature near 50 ℃～55 ℃, the operative temperature scope is 20 ℃～60 ℃.In addition, shown in Fig. 5 B, the decomposition reaction of trehalose phosphorylase (white round dot zero) near 50 ℃～65 ℃, have optimum temperature (the 50mM phosphoric acid buffer, pH7.0), the operative temperature scope is 25 ℃～70 ℃; Building-up reactions (black round dot ●) has optimum temperature near 45 ℃～60 ℃, the operative temperature scope is 25 ℃～70 ℃.

(V) temperature stability

Under the condition of pH6.0 and pH7.0 (50mM phosphoric acid buffer), under all temps, maltose phosphorylase and trehalose phosphorylase were handled 15 minutes respectively, calculate the remaining activity of described enzyme with ordinary method, and then measure the thermotolerance of described maltose phosphorylase and trehalose phosphorylase.The results are shown in Fig. 6.As shown in Figure 6, maltose phosphorylase (white round dot zero) is extremely stable 50 ℃ or 50 ℃ of following temperature, in 70 ℃ of complete deactivations; In addition, trehalose phosphorylase (black round dot ●) is extremely stable 60 ℃ or 60 ℃ of following temperature, in 70 ℃ of complete deactivations.

(VI) inhibitor

In the presence of various mineral ions and inhibitor, measured the degrading activity of maltose phosphorylase and trehalose phosphorylase.The result shows, two kinds of enzymes all are subjected to copper, mercury, N-bromosuccinimide, to the strongly inhibited of chlorine mercury M-nitro benzoic acid (respectively being 1mM).Maltose phosphorylase is subjected to the strongly inhibited of SDS (1%), but trehalose phosphorylase is not suppressed by it.

(VII) iso-electric point

Use the isogel Isogel (manufacturing of FMC BioProducto company) of isoelectric focusing method, measured the iso-electric point of maltose phosphorylase and trehalose phosphorylase, the result is as follows: the iso-electric point of maltose phosphorylase is pH4.8～5.0 (calculated value is pH4.98), and the iso-electric point of trehalose phosphorylase is pH4.8～5.2 (calculated value is pH5.13).

(VIII) with the gel filteration determining molecular weight

Adopt Sephacryl S-200 gel filtration method that the molecular weight of maltose phosphorylase and trehalose phosphorylase is measured.The result is about 190000 dalton with the molecular weight of two kinds of enzymes that gel filtration method records, and the molecular weight of the maltose phosphorylase of measuring with SDS-polyacrylamide gel electrophoresis method is about 89000 dalton～90000 dalton (calculated value is 92233 dalton), the molecular weight of trehalose phosphorylase is about 89000 dalton～90000 dalton (calculated value is 91280 dalton), therefore envisions these enzymes and is made of the homotype dipolymer respectively.The enzyme of the molecular weight ratio wild-type of two recombinases is big slightly, this is to have added histidine label (Histag) because of the N-terminal at recombinase, and added the compartment of 23 aminoacid sequences, so the molecular weight ratio wild-type of two recombinases big about 3500.

Embodiment 8:

In the thalline and the manufacturing of thalline extracellular trehalose Starch phosphorylase

Adopting aseptic technique to contain 1% (w/v) trehalose, 2% yeast extract (manufacturings of Difco company), 0.25% ammonium phosphate, 0.15% urea, 1% salt, 0.1% Rhodiaphos DKP, 0.02% magnesium sulfate heptahydrate and 0.15% lime carbonate and pH to 10 liters (L) is that interpolation 500mL has cultivated the series bacillus sp.SH-55 kind bacterium at a night in advance in 7.5 the liquid nutrient medium with same medium, in speed is that 300rpm, air flow are 1vvm[air flow (L)/substratum (L)/min] condition under, cultivated 24 hours in 35 ℃ of aeration-agitations.Trehalose phosphorylase activity to this nutrient solution is measured, and the activity of the every 1mL nutrient solution of result is 1.8 units.Also use the same method the activity of maltose phosphorylase is measured, but active faint.Next, in this nutrient solution of 4 ℃ of centrifugations 10 minutes, obtain the thalline (weight in wet base) of about 75g and the supernatant liquor of about 10L with the speed of 12000 * g.The UF film (YM-30) that uses Amicon company to make concentrates this supernatant liquor, and the thalline that obtains about 500mL concentrates thick enzyme outward.Enzymic activity in the supernatant liquor is measured, and its activity is about 25% of complete active (being about 14 * 103 units) as a result.With phosphoric acid buffer (pH7) the thorough washing thalline part of 10mM, use ultrasonic wave bacterial cell disruption crusher machine thalline after it is suspended in the same buffer of 240mL.According to the activity of ordinary method mensuration trehalose phosphorylase, the result shows that the activity that thalline contains is about complete active 75%.

Therefore, cultivate series bacillus sp.SH-55 by using trehalose as carbon source, can preferentially produce trehalose phosphorylase, this moment, enzyme comprised wherein according to ratio about 75% in the thalline, thalline outer about 25%.

Embodiment 9:

Contain the interior manufacturing that reaches the thalline exoenzyme of thalline of maltose phosphorylase and trehalose phosphorylase

Except the trehalose of the medium component that will be among the embodiment 2 uses is changed to maltose, 2% yeast extract is changed to 4.5% (w/v) polyprotein peptone FC (Nihon Pharmaceutical Co., Ltd.'s manufacturings), according to method cultivation series bacillus sp.SH-55 same as the previously described embodiments.The activity of maltose phosphorylase and trehalose phosphorylase in the mensuration nutrient solution, the result shows that every 1mL nutrient solution produces the maltose phosphorylase of 0.8 unit and the trehalose phosphorylase of 0.5 unit.Carry out centrifugation according to the method identical, obtain the supernatant liquor of about 50g thalline (weight in wet base) and about 7L with embodiment 8.The thalline that obtains and the maltose phosphorylase in the supernatant liquor are measured, found that accounting for complete active 80% of maltose phosphorylase is contained in the thalline, about 20% is contained in thalline (nutrient solution supernatant liquor) outward.Concentrate the nutrient solution supernatant liquor with the method identical, obtain the concentrated enzyme of about 330mL with embodiment 1.Concentrate in the enzyme and approximately contain the maltose phosphorylase of 550 units and the trehalose phosphorylase of 500 units.

Therefore, cultivate series bacillus sp.SH-55 by using maltose as carbon source, can produce maltose phosphorylase, simultaneously can also produce trehalose phosphorylase, this moment two kinds of enzymes in thalline and the ratio outside the thalline all be that thalline includes and has an appointment 80%, contains outside the thalline and have an appointment 20%.

Embodiment 10:

Make trehalose by maltose

To 10mL 10%, 20%, 30% or 40% (w/v, be dissolved in the phosphoric acid buffer (pH6) of 10mM) each maltose solution in add trehalose phosphorylase of the present invention and maltose phosphorylase (is that the substrate of 1g adds the enzyme that degrading activity is 5 units respectively with respect to weight), in 55 ℃ of reactions 70 hours.After reaction finishes, made enzyme deactivation in 5 minutes, measure the content of trehalose in the saccharification liquid of gained then in 100 ℃ of reacting by heating liquid.The result is as follows: with respect to the weight of substrate, described 4 kinds of maltose solutions generate 58.2%, 58.1%, 58.6% and 57.9% trehalose respectively.

In addition, trehalose quantitatively carries out with following method.Promptly, adding water in the saccharification liquid after heated and inactivated becomes about 1% (w/v) it, in this saccharification liquid of 0.5mL, add glucoamylase (the biochemical industry manufacturing of 0.01 unit then, pure grade 30U/mg), reaction is 1 hour under 50 ℃, the condition of pH5.0, makes unreacted maltose all be decomposed into glucose.Heating made the glucose starch enzyme deactivation in 5 minutes in 100 ℃ boiling water bath then, adopt the membrane filter of 0.45 μ m to remove the insoluble protein of generation, then use YMC-Pack ODS-AQ post (AQ-304, YMC makes) to utilize high speed liquid chromatography method (HPLC method) that the content of trehalose in the gained filtrate is measured.In addition, mensuration makes water as mobile phase, and column temperature is 30 ℃, uses differential refractometer to detect.

Embodiment 11:

Make trehalose by maltose syrups

To 10mL 20% (w/v, be dissolved in the 5mM phosphoric acid buffer (pH6)) the high malt sugar syrup (the japanese food chemical industry is made, trade(brand)name MC-95, sugar composition: 2.5% glucose, 95.2% maltose, 0.8% trisaccharide maltose, 1.5% maltotetrose) adds trehalose phosphorylase of the present invention and maltose phosphorylase (adding the enzyme of 5 units with respect to the 1g substrate respectively) in, carry out the reaction identical with embodiment 10.With the HPLC method trehalose that generates is measured, the result is: the weight with respect to employed substrate has generated 54.3% trehalose.

Industrial utilizability

The invention provides the novel microbial that can produce maltose phosphorylase and trehalose phosphorylase in thalline and outside the thalline, described maltose phosphorylase and trehalose phosphorylase are necessary enzymes when making trehalose with enzyme process. Because microorganism of the present invention is bacterium, compare with basidiomycetes with the known green alga as the trehalose phosphorylase supply source in the past, not only the adquisitiones of enzyme is very simple, can also shorten significantly incubation time, and is favourable economically. And, because necessary two kinds of enzymes when using a kind of microorganism can produce simultaneously with the Production by Enzymes trehalose only, thereby microorganism of the present invention has obvious advantage aspect practicality. And therefore desired all conditions when two kinds of enzymes of the present invention all satisfy with the Production by Enzymes trehalose, are highly susceptible to resulting trehalose is effectively utilized, and also can realize economically significantly improving. That is, maltose phosphorylase of the present invention and trehalose phosphorylase have higher temperature stability, can at high temperature carry out enzyme reaction, so the living contaminants in can avoiding reacting. And because two kinds of enzymes have almost identical optimal pH scope, in reaction, has the advantage that need not numerous and diverse pH control. In addition, as the additive method that obtains enzyme of the present invention, from described bacterial strain, utilize technique for gene engineering to make recombinant microorganism behind the gene of extraction code book invention enzyme, produce maltose phosphorylase and trehalose phosphorylase essential when making trehalose with enzyme process by cultivating this recombinant microorganism, thereby can carry out the improvement of protein engineering aspect to two kinds of enzymes.

Sequence table

＜110〉Independent Administrative Ins

Japan Food Chemical Co., Ltd

＜120〉novel microorganism, maltose phosphorylase and trehalose phosphorylase and manufacture method thereof

<130>04F003

<150>JP 2003-192199

<151>2003-07-04

<160>8

<170>PatentIn version 3.1

<210>1

<211>768

<212>PRT

＜213〉maltose phosphorylase

<220>

<221>MISC_FEATURE

＜223〉contriver: Ri Gao Yu; Qin field brave two; She advances by rattan; Pick is liberal-mindedness and vigorous endurance more;

Jitian is refined great; Middle village believes it; High fields are just protected; In of a specified duration happiness brightness husband

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Val Arg His Glu Ile Gly Ala Ile Arg Tyr Ala Val Thr Pro Leu Asn

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Ala Gln Gly Tyr Lys Ser Leu Cys Asp Arg Leu Gly Val Thr Ala Asp

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Asp Glu Ala Leu Gly Ile Asn Pro Gln Asp Asp Thr Phe Leu Arg Lys

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Ala Val Trp Asp Phe Glu Asn Thr Pro Glu Asp Lys Tyr Pro Leu Leu

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Ala Asp Thr Val Leu Ala His Phe Leu Leu Glu Asp Glu Gln Ser Phe

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Glu Thr Ile Gln Arg Ser Tyr Asp Tyr Tyr Glu Gly Ile Thr Thr His

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Asp Ser Ser Leu Ser Ser Cys Ile Phe Ser Ile Met Ala Ser Lys Ile

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Gly Asn Met Asp Lys Ala Tyr Glu Tyr Phe Ile Glu Thr Ala Arg Leu

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Asp Leu Asp Asn Thr His Gly Asn Thr Lys Asp Gly Leu His Met Ala

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Asn Met Gly Gly Thr Trp Met Ser Ile Val Tyr Gly Phe Ala Gly Met

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Arg Leu Lys Glu Ser Gly Leu Ser Leu Ser Pro Ala Ile Pro Gln Asp

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Trp Glu Lys Tyr Ala Phe Arg Leu Asn Phe Arg Gly Arg Leu Ile Gly

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Val Ser Ile Glu Lys Asp Gly Val Thr Leu Glu Ile Val Glu Gly Asp

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＜213〉trehalose phosphorylase

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gggacaatta cttcgcgtga acgccgcttg catatggaca aggggtattc cgaacgaatc 360

attcagtgga aatcaccgga aggcaaagaa atcaagttga ccttccgtcg cctggtatct 420

ttcacgcgtc gggagctgtt tgctatccat gtacaaatag agccgatcaa tttcaacggc 480

caggtacgea tcgtctccac cgtgaacggc aaggtaaaga actacaccaa tgccaatgat 540

ccgcgggtag gagcaggaca cgcagaacgg atgaccgtca tcgataccgg cgtgaagggc 600

agcgatgcct atgttgtcga cgagacgatg gcttcccagc tgcatgcggc ttgtgtgacc 660

cgtcatcgct tagatgttga tgccgacatt cagctcgaag ccggaaccgg agaagtcacg 720

ttcacggctg cccttccatt gacaggaccg atccagttta cgaaatacaa tctgtatacc 780

gacagtcttc gtcacggtca ggacatgatc gatcgcggta tccagctgca ggaggagtta 840

aaggagcttt ctttcgagga tttgcttgcc gagcaggctg agtatctgaa cgattactgg 900

aaatctgccg atgtggttat tcagaacgat gaccagctcc aggaaggcat ccgcttcaat 960

ctatttcagc tgctgcaatc cgcgggccgt gacaagcaca gcaacatctc tgccaaaggt 1020

ctaagcggcg agggttatga aggccattat ttttgggaca cggagattta catgttccct 1080

gtatttctga tgacgcagcc ggacattgcg cgccagctgc tgctgtatcg ctattccaca 1140

ttggagcaag cgagagacag agcacgagaa atgggacatc gccaaggcgc cctgttcccg 1200

tggcgtacga tctcggggac ggaatgctct tccttcttcc cttcgggaac agcgcaatat 1260

catatcagcg cagatatcgc atacagctat attcaatatt atctcgccga gcaggaccgg 1320

gatttcctct tgtcctatgg ggccgaggta ttgattgaaa ccgcccgcct gtgggctgat 1380

atcggccact attataatgg cgccttccat atcgatgagg tgacggggcc ggatgaatat 1440

acctgctgcg taaacaacaa ctattacaca aacgtcatgg cgaagcataa cctgaagtgg 1500

gctgccaaga gctgctcgat tctggaatcc tacgatgcgc aaggatacaa atcactgtgt 1560

gaccggcttg gcgtaaccgc ggacgagatt tcagcttggg cgaaagccgc ccatgccatg 1620

ctgctgccat acgatgaagc acttggcatt aatccgcagg atgatacgtt cctccgcaaa 1680

gcggtatggg atttcgagaa cacgcctgag gataaatatc cgctgctgct gaactaccat 1740

ccgctcacca tttaccgtta tcaggtatgt aaacaagcgg atacggtgct cgcccacttc 1800

ttactcgaag acgaacaaag ctttgaaacg attcagcgtt cgtatgacta ctatgaaggc 1860

attacaacac atgactcttc cctgtcctcc tgtatcttca gcatcatggc ttcgaagatt 1920

ggcaatatgg acaaagcgta tgagtatttc atcgagacgg cacgccttga cctcgacaat 1980

acacacggca acacgaaaga cggcctgcat atggccaaca tgggcggcac ctggatgtcg 2040

atcgtctacg gctttgctgg tatgcgtctg aaggaaagtg gactttccct ctcccctgcc 2100

attccgcagg actgggagaa gtatgcgttc cgtttgaact tccgcggccg cctcatcggt 2160

gtatccattg agaaggacgg cgtgaccttg gagattgtgg agggcgattc catcgagatc 2220

aagctgtacg acgaagtagt taagcttgag gccgggaagt ccgtgaagca tgcacttcac 2280

aaagcaaata ataaggtt 2298

<210>5

<211>3207

<212>DNA

＜213〉series bacillus sp.SH-55 (maltose phosphorylase)

<220>

<221>CDS

<222>(343)..(2649)

<223>

<400>5

gacgccatca tcgatggtac gaagaccagt caggcgaagc cggatccaga agtattcacg 60

ctgggagctc atgagcttgg cgctcagcca gaagcttgcg ttgtattcga agatgccgaa 120

gccggaatcg aagctgctat ccgtgcgggg atgcgcagcg ttggcatcgg ttcgcctgaa 180

actttgggtc aggccaacat cgttcttccc tccctggaag gattcacggt ggaccgtctg 240

ctggaattgt aggcgaatac gcacatatgc ttacagcatc cagcagtttc atcctatata 300

gatctgcaac catttttcct aaaaacaaag gagccgatga tt gtg aaa caa tat 354

Val Lys Gln Tyr

1

tta aag ctt gat gaa tgg tca att att gaa gag gga ttt gat cct cac 402

Leu Lys Leu Asp Glu Trp Ser Ile Ile Glu Glu Gly Phe Asp Pro His

5 10 15 20

acc cat gaa atc tcg gag agt gta ttc agt atc ggg aac ggg ttt atg 450

Thr His Glu Ile Ser Glu Ser Val Phe Ser Ile Gly Asn Gly Phe Met

25 30 35

ggc cag cgc gcc aac ttc gaa gaa gcc tac agc ggc tct tct ctg caa 498

Gly Gln Arg Ala Asn Phe Glu Glu Ala Tyr Ser Gly Ser Ser Leu Gln

40 45 50

ggc agt tat atg gct ggc gta tat tat ccg gat aaa acc cgt gtt ggc 546

Gly Ser Tyr Met Ala Gly Val Tyr Tyr Pro Asp Lys Thr Arg Val Gly

55 60 65

tgg tgg aaa aac ggc tac cct gag tac ttc gec aaa gtg ttg aac agc 594

Trp Trp Lys Asn Gly Tyr Pro Glu Tyr Phe Ala Lys Val Leu Asn Ser

70 75 80

acc aac tgg atc ggc att gac att caa atc gac ggc aca ccg ctg gat 642

Thr Asn Trp Ile Gly Ile Asp Ile Gln Ile Asp Gly Thr Pro Leu Asp

85 90 95 100

ctc gcc aaa tgt acg gtg aag gat ttt gta cgt gag ctg aat atg aag 690

Leu Ala Lys Cys Thr Val Lys Asp Phe Val Arg Glu Leu Asn Met Lys

105 110 115

gaa ggc ttc ctc tcc cgc cgt ttc act gcc gtt acg gaa gac ggc aag 738

Glu Gly Phe Leu Ser Arg Arg Phe Thr Ala Val Thr Glu Asp Gly Lys

120 125 130

gag ctc aaa gtc gaa gcc atc cgt ttt gtc agc att gtt cgt cac gag 786

Glu Leu Lys Val Glu Ala Ile Arg Phe Val Ser Ile Val Arg His Glu

135 140 145

atc ggt gcc atc cgc tat gcc gta act ccg ctt aat ttc aag ggt gag 834

Ile Gly Ala Ile Arg Tyr Ala Val Thr Pro Leu Asn Phe Lys Gly Glu

150 155 160

ctt acc att act cct tat ctt gat ggc gat gta aag aat aaa gat tcg 882

Leu Thr Ile Thr Pro Tyr Leu Asp Gly Asp Val Lys Asn Lys Asp Ser

165 170 175 180

aat tat gac gag aaa ttc tgg ctt gaa gtg ttt aag gaa gcg acg caa 930

Asn Tyr Asp Glu Lys Phe Trp Leu Glu Val Phe Lys Glu Ala Thr Gln

185 190 195

ggc tcg gcc gca gta acg gtg aaa acg aag aaa ctc gat ttc cat gta 978

Gly Ser Ala Ala Val Thr Val Lys Thr Lys Lys Leu Asp Phe His Val

200 205 210

acc tcc gtt atg tct tat acg atc tta aag aac ggt gag aag ctg gag 1026

Thr Ser Val Met Ser Tyr Thr Ile Leu Lys Asn Gly Glu Lys Leu Glu

215 220 225

ctc caa gcc gag ctg gtc gaa aaa gag aaa tat gcg ggg aac cgc gtc 1074

Leu Gln Ala Glu Leu Val Glu Lys Glu Lys Tyr Ala Gly Asn Arg Val

230 235 240

agt atg ccg gta tct gag ggg gaa acc gtc acc gtc tac aaa tat gta 1122

Ser Met Pro Val Ser Glu Gly Glu Thr Val Thr Val Tyr Lys Tyr Val

245 250 255 260

gcg aac gtt act tcc cgc aat cat gga ttc ggc gaa ttg gtg gaa gct 1170

Ala Asn Val Thr Ser Arg Asn His Gly Phe Gly Glu Leu Val Glu Ala

265 270 275

gca cgc gcc gtg ctg gaa ccg gca gtg gag aca ggt ttt gaa cag ctg 1218

Ala Arg Ala Val Leu Glu Pro Ala Val Glu Thr Gly Phe Glu Gln Leu

280 285 290

ctg aag gag caa gcc gat gct tgg ggc gac aaa tgg aag gaa agc gat 1266

Leu Lys Glu Gln Ala Asp Ala Trp Gly Asp Lys Trp Lys Glu Ser Asp

295 300 305

att gtc att gaa ggc gat gta gca gcc cag cag gcg atc cgc ttt aac 1314

Ile Val Ile Glu Gly Asp Val Ala Ala Gln Gln Ala Ile Arg Phe Asn

310 315 320

atc ttc cag ctg aat caa acc tac agc ggt gaa gac gac cgt ctg aac 1362

Ile Phe Gln Leu Asn Gln Thr Tyr Ser Gly Glu Asp Asp Arg Leu Asn

325 330 335 340

atc ggg ccg aag ggc ttc acc ggg gag aaa tac ggc ggc agc acc tac 1410

Ile Gly Pro Lys Gly Phe Thr Gly Glu Lys Tyr Gly Gly Ser Thr Tyr

345 350 355

tgg gat acc gag gct tac tgc ctt ccg ttc tac ctg agc acg gcg gat 1458

Trp Asp Thr Glu Ala Tyr Cys Leu Pro Phe Tyr Leu Ser Thr Ala Asp

360 365 370

gcc agc atc tcg cgc aat ctg ctg att tat cgt tac aag cac ttg gaa 1506

Ala Ser Ile Ser Arg Asn Leu Leu Ile Tyr Arg Tyr Lys His Leu Glu

375 380 385

aag gcg aag gaa aac gcc aaa aag ctc ggc ttt acc aaa ggc gcg ctc 1554

Lys Ala Lys Glu Asn Ala Lys Lys Leu Gly Phe Thr Lys Gly Ala Leu

390 395 400

tac ccg atg gtg acg atg aac ggt gag gag tgc cat aac gag tgg gag 1602

Tyr Pro Met Val Thr Met Asn Gly Glu Glu Cys His Asn Glu Trp Glu

405 410 415 420

att acg ttt gaa gag att cac cgt aac ggc gcg att gcc tac gct atc 1650

Ile Thr Phe Glu Glu Ile His Arg Asn Gly Ala Ile Ala Tyr Ala Ile

425 430 435

tat aac tat gta aat tat acc ggt gac ttc tct tat ctc ggt cag tat 1698

Tyr Asn Tyr Val Asn Tyr Thr Gly Asp Phe Ser Tyr Leu Gly Gln Tyr

440 445 450

ggt ctg gaa gtg ctt gtg gaa atc tcc cgc ttc tgg gag gag cgc gtg 1746

Gly Leu Glu Val Leu Val Glu Ile Ser Arg Phe Trp Glu Glu Arg Val

455 460 465

aat tac gta gcg gcg aag gat cag tat atg atg ctg ggc gtt acc ggt 1794

Asn Tyr Val Ala Ala Lys Asp Gln Tyr Met Met Leu Gly Val Thr Gly

470 475 480

cca aac gag tac gag aac aac gta aac aac aac tgg tac aca aac cgc 1842

Pro Asn Glu Tyr Glu Asn Asn Val Asn Asn Asn Trp Tyr Thr Asn Arg

485 490 495 500

att gcc agc tgg acg atg gaa tac acg ctc gac gtg ctt gaa tat ttg 1890

Ile Ala Ser Trp Thr Met Glu Tyr Thr Leu Asp Val Leu Glu Tyr Leu

505 510 515

aaa gag aac gag aat gcc cgc tat gac gag ctt aca gcg aag ctt gcc 1938

Lys Glu Asn Glu Asn Ala Arg Tyr Asp Glu Leu Thr Ala Lys Leu Ala

520 525 530

ctg cag gaa gcc gaa acg acc aaa tgg cag gat atc atc caa aaa atg 1986

Leu Gln Glu Ala Glu Thr Thr Lys Trp Gln Asp Ile Ile Gln Lys Met

535 540 545

tac tat ccg gtc gac gag gag ctt ggc gta ttc ctg cag cag gac ggc 2034

Tyr Tyr Pro Val Asp Glu Glu Leu Gly Val Phe Leu Gln Gln Asp Gly

550 555 560

ttc ctt gac aag gag ctt gtt ccg gtg aag gaa ttg gac cct gtg cac 2082

Phe Leu Asp Lys Glu Leu Val Pro Val Lys Glu Leu Asp Pro Val His

565 570 575 580

ctg cca ctg aac cag aac tgg tct tgg gac cgc att ctg cgt tca gtt 2130

Leu Pro Leu Asn Gln Asn Trp Ser Trp Asp ArgIle Leu Arg Ser Val

585 590 595

tac atc aag cag gca gat gtg ctg caa ggc ttg ttc ttc ctg ggc gac 2178

Tyr Ile Lys Gln Ala Asp Val Leu Gln Gly Leu Phe Phe Leu Gly Asp

600 605 610

agg tac gat ctg gct aca aag aaa cgc aac ttc gac ttc tat gaa ccg 2226

Arg Tyr Asp Leu Ala Thr Lys Lys Arg Asn Phe Asp Phe Tyr Glu Pro

615 620 625

ttc acg gta cac gag tcc tcc ctc tca cct tgc gtg cac tcc att ctc 2274

Phe Thr Val His Glu Ser Ser Leu Ser Pro Cys Val His Ser Ile Leu

630 635 640

gca tgt gag ctt ggc tat cag gag aag gca tac gag atg tac ctc cgt 2322

Ala Cys Glu Leu Gly Tyr Gln Glu Lys Ala Tyr Glu Met Tyr Leu Arg

645 650 655 660

acc gcc cgc ctg gac ctg gac aac tat aat aac gat acc gag gac ggc 2370

Thr Ala Arg Leu Asp Leu Asp Asn Tyr Asn Asn Asp Thr Glu Asp Gly

665 670 675

tgc cac act acc agt atg gca ggt aca tgg atg tcc gtt gtg cat ggc 2418

Cys His Thr Thr Ser Met Ala Gly Thr Trp Met Ser Val Val His Gly

680 685 690

ttt ggc ggt ttg cgt gtg aag gac ggc gta ctg cat ctg aac cca ttc 2466

Phe Gly Gly Leu Arg Val Lys Asp Gly Val Leu His Leu Asn Pro Phe

695 700 705

att ccg ggt cat tgg tct tcc ttc tcc ttc aaa gtg atg ttc cgc ggg 2514

Ile Pro Gly His Trp Ser Ser Phe Ser Phe Lys Val Met Phe Arg Gly

710 715 720

tct cgc ctg aag gtt agc gtc aaa ggg aat gaa act att atc gtg aat 2562

Ser Arg Leu Lys Val Ser Val Lys Gly Asn Glu Thr Ile Ile Val Asn

725 730 735 740

gaa aca gaa aca cct gcc ctt ctg aat gta agc ggg aag gaa ttc agt 2610

Glu Thr Glu Thr Pro Ala Leu Leu Asn Val Ser Gly Lys Glu Phe Ser

745 750 755

atc gat gga ttt ggg gaa atc aca gca gct tca aaa taa gaaaaacatc 2659

Ile Asp Gly Phe Gly Glu Ile Thr Ala Ala Ser Lys

760 765

gctttatatc aaagaattgc ataaaaaaca agagcccgat gggagaatgc cagcgggccc 2719

ttgctctata ggaaaacgga cccgcacaag aaccattctt aatccttgac gggtccattt 2779

tcatgtttcg taccaggcat ctacaacgtc acactttaac cggacgcacg gaatttttta 2839

gctgccgctc ctcacacttt catgggttcg agtatgtatg ctatgaatag gttattcgag 2899

agtgcggttg aagcggaggt ctttcagctg gctttccgaa taaataccct tgtgagagct 2959

caattccaat atctcggcaa aaatgaaact cctctcgtcg ctcaatgcct tcagctaata 3019

ccatggctcc aaaatcatgg gccatattcg tgatgatctc aagctgtttc tgctgggcgt 3079

gactccgatc acaatggtcg ataaggcttc gatcaatctt tacataatcc ggcttaagcc 3139

tcaccatctg ctccaatgtg gaataacctg ctcccacgtc gtccatggca acaggatatt 3199

ccgtggct 3207

<210>6

<211>768

<212>PRT

＜213〉series bacillus sp.SH-55 (maltose phosphorylase)

<400>6

Val Lys Gln Tyr Leu Lys Leu Asp Glu Trp Ser Ile Ile Glu Glu Gly

1 5 10 15

Phe Asp Pro His Thr His Glu Ile Ser Glu Ser Val Phe Ser Ile Gly

20 25 30

Asn Gly Phe Met Gly Gln Arg Ala Asn Phe Glu Glu Ala Tyr Ser Gly

35 40 45

Ser Ser Leu Gln Gly Ser Tyr Met Ala Gly Val Tyr Tyr Pro Asp Lys

50 55 60

Thr Arg Val Gly Trp Trp Lys Asn Gly Tyr Pro Glu Tyr Phe Ala Lys

65 70 75 80

Val Leu Asn Ser Thr Asn Trp Ile Gly Ile Asp Ile Gln Ile Asp Gly

85 90 95

Thr Pro Leu Asp Leu Ala Lys Cys Thr Val Lys Asp Phe Val Arg Glu

100 105 110

Leu Asn Met Lys Glu Gly Phe Leu Ser Arg Arg Phe Thr Ala Val Thr

115 120 125

Glu Asp Gly Lys Glu Leu Lys Val Glu Ala Ile Arg Phe Val Ser Ile

130 135 140

Val Arg His Glu Ile Gly Ala Ile Arg Tyr Ala Val Thr Pro Leu Asn

145 150 155 160

Phe Lys Gly Glu Leu Thr Ile Thr Pro Tyr Leu Asp Gly Asp Val Lys

165 170 175

Asn Lys Asp Ser Asn Tyr Asp Glu Lys Phe Trp Leu Glu Val Phe Lys

180 185 190

Glu Ala Thr Gln Gly Ser Ala Ala Val Thr Val Lys Thr Lys Lys Leu

195 200 205

Asp Phe His Val Thr Ser Val Met Ser Tyr Thr Ile Leu Lys Asn Gly

210 215 220

Glu Lys Leu Glu Leu Gln Ala Glu Leu Val Glu Lys Glu Lys Tyr Ala

225 230 235 240

Gly Asn Arg Val Ser Met Pro Val Ser Glu Gly Glu Thr Val Thr Val

245 250 255

Tyr Lys Tyr Val Ala Asn Val Thr Ser Arg Asn His Gly Phe Gly Glu

260 265 270

Leu Val Glu Ala Ala Arg Ala Val Leu Glu Pro Ala Val Glu Thr Gly

275 280 285

Phe Glu Gln Leu Leu Lys Glu Gln Ala Asp Ala Trp Gly Asp Lys Trp

290 295 300

Lys Glu Ser Asp Ile Val Ile Glu Gly Asp Val Ala Ala Gln Gln Ala

305 310 315 320

Ile Arg Phe Asn Ile Phe Gln Leu Asn Gln Thr Tyr Ser Gly Glu Asp

325 330 335

Asp Arg Leu Asn Ile Gly Pro Lys Gly Phe Thr Gly Glu Lys Tyr Gly

340 345 350

Gly Ser Thr Tyr Trp Asp Thr Glu Ala Tyr Cys Leu Pro Phe Tyr Leu

355 360 365

Ser Thr Ala Asp Ala Ser Ile Ser Arg Asn Leu Leu Ile Tyr Arg Tyr

370 375 380

Lys His Leu Glu Lys Ala Lys Glu Asn Ala Lys Lys Leu Gly Phe Thr

385 390 395 400

Lys Gly Ala Leu Tyr Pro Met Val Thr Met Asn Gly Glu Glu Cys His

405 410 415

Asn Glu Trp Glu Ile Thr Phe Glu Glu Ile His Arg Asn Gly Ala Ile

420 425 430

Ala Tyr Ala Ile Tyr Asn Tyr Val Asn Tyr Thr Gly Asp Phe Ser Tyr

435 440 445

Leu Gly Gln Tyr Gly Leu Glu Val Leu Val Glu Ile Ser Arg Phe Trp

450 455 460

Glu Glu Arg Val Asn Tyr Val Ala Ala Lys Asp Gln Tyr Met Met Leu

465 470 475 480

Gly Val Thr Gly Pro Asn Glu Tyr Glu Asn Asn Val Asn Asn Asn Trp

485 490 495

Tyr Thr Asn Arg Ile Ala Ser Trp Thr Met Glu Tyr Thr Leu Asp Val

500 505 510

Leu Glu Tyr Leu Lys Glu Asn Glu Asn Ala Arg Tyr Asp Glu Leu Thr

515 520 525

Ala Lys Leu Ala Leu Gln Glu Ala Glu Thr Thr Lys Trp Gln Asp Ile

530 535 540

Ile Gln Lys Met Tyr Tyr Pro Val Asp Glu Glu Leu Gly Val Phe Leu

545 550 555 560

Gln Gln Asp Gly Phe Leu Asp Lys Glu Leu Val Pro Val Lys Glu Leu

565 570 575

Asp Pro Val His Leu Pro Leu Asn Gln Asn Trp Ser Trp Asp Arg Ile

580 585 590

Leu Arg Ser Val Tyr Ile Lys Gln Ala Asp Val Leu Gln Gly Leu Phe

595 600 605

Phe Leu Gly Asp Arg Tyr Asp Leu Ala Thr Lys Lys Arg Asn Phe Asp

610 615 620

Phe Tyr Glu Pro Phe Thr Val His Glu Ser Ser Leu Ser Pro Cys Val

625 630 635 640

His Ser Ile Leu Ala Cys Glu Leu Gly Tyr Gln Glu Lys Ala Tyr Glu

645 650 655

Met Tyr Leu Arg Thr Ala Arg Leu Asp Leu Asp Asn Tyr Asn Asn Asp

660 665 670

Thr Glu Asp Gly Cys His Thr Thr Ser Met Ala Gly Thr Trp Met Ser

675 680 685

Val Val His Gly Phe Gly Gly Leu Arg Val Lys Asp Gly Val Leu His

690 695 700

Leu Asn Pro Phe Ile Pro Gly His Trp Ser Ser Phe Ser Phe Lys Val

705 710 715 720

Met Phe Arg Gly Ser Arg Leu Lys Val Ser Val Lys Gly Asn Glu Thr

725 730 735

Ile Ile Val Asn Glu Thr Glu Thr Pro Ala Leu Leu Asn Val Ser Gly

740 745 750

Lys Glu Phe Ser Ile Asp Gly Phe Gly Glu Ile Thr Ala Ala Ser Lys

755 760 765

<210>7

<211>3868

<212>DNA

＜213〉series bacillus sp.SH-55 (trehalose phosphorylase)

<220>

<221>CDS

<222>(1113)..(3413)

<223>

<400>7

gctgtaggga ggtatcgata ttcttaatga tttgcttgtt attctccacc gcagcctcaa 60

tcatggtatt ccggttattc tccacgtaca cccaggatac gatgccgccg atcagaagga 120

gaggaacgat gataaaggcg gagaacgccg caatcagttt tttctccaga cttaagtgaa 180

gcattttctg ctttagtttc ttcatgggcc gtgtctctct tcttgttgtt cgttatggga 240

tcttgtcttt accgcgctgt accgctgatt cgaaattgcc ttggcgtcat tcccgtaaaa 300

ttcttgaata cttgcgtata ataactctgg ctgcagaatt tgaacgtaaa cgcaatttcg 360

gatattgatt gattcgtatt ggccagcaaa tgcttggatt cctcaatctt ccgccgattg 420

ataaaagtcg taatcgcctc acctgtctct cgcttaaagc gatcggacag atagctgggg 480

tgaaccttca aatattgcgc aaccccctgc aaggtcagct cctcaaaaat atgctccctg 540

atgtaataga tcgccagatt cacgatatga gagaaagaaa tgtcttcctt ctcggagcga 600

atcatcgaca caaactgtac cagcatttca tattcaaaat ggaccagccg ctctacatcg 660

gtcattttct caatctcaag gattaacgta tcacttaaat gaaaagctat ctccggatcc 720

acgccgcccc tgatgatcgc ccgggtgaat aaagtgcaag aggcaatcaa ggcgttcttc 780

ttcgaccgca gtggatacgt tgcaagcaca gccgcttcca gctcgttaat cttgcgcagc 840

atgtccatcg ctcgattctc atcgccgaaa cggattgctt ccagcagttc gtgctcatag 900

tcaaacgggg gatggataaa attctcgcgc aaataatgca tgcgggaaga caggtagaag 960

ggttggttac catgacttcc tgagcgtgaa ggctctttca ttatttacac accacctaaa 1020

aatattgata aaaaccttgt tattttagta taacataacc ccgattggtt ctaaaattca 1080

agcataacca tgctcggaag gacgtgtaga cc atg acg tgg atg ata agc aat 1133

Met Thr Trp Met Ile Ser Asn

1 5

cct gat ttg tcg cag caa gcc ttg ctg aat atg gaa agt att ttt gca 1181

Pro Asp Leu Ser Gln Gln Ala Leu Leu Asn Met Glu Ser Ile Phe Ala

10 15 20

ctc gga aac ggt tac ctg ggt gtg cgc ggc aac ttc gaa gaa ggt tac 1229

Leu Gly Asn Gly Tyr Leu Gly Val Arg Gly Asn Phe Glu Glu Gly Tyr

25 30 35

gga gag agc atg tcc acc att cgc ggt acc tat ctg aac gct ttc cac 1277

Gly Glu Ser Met Ser Thr Ile Arg Gly Thr Tyr Leu Asn Ala Phe His

40 45 50 55

gac gtt att gaa att cct tac ggc gag aag ctc ttc gcc ttt ccc gat 1325

Asp Val Ile Glu Ile Pro Tyr Gly Glu Lys Leu Phe Ala Phe Pro Asp

60 65 70

aca cag caa aaa ctc gtt aac aat att gat gcc caa acg gtc ctg atc 1373

Thr Gln Gln Lys Leu Val Asn Asn Ile Asp Ala G1n Thr Val Leu Ile

75 80 85

tat tta ggc gat gag aag gag cct ttc cgc ctc gat cac ggg aca att 1421

Tyr Leu Gly Asp Glu Lys Glu Pro Phe Arg Leu Asp His Gly Thr Ile

90 95 100

act tcg cgt gaa cgc cgc ttg cat atg gac aag ggg tat tcc gaa cga 1469

Thr Ser Arg Glu Arg Arg Leu His Met Asp Lys Gly Tyr Ser Glu Arg

105 110 115

atc att cag tgg aaa tca ccg gaa ggc aaa gaa atc aag ttg acc ttc 1517

Ile Ile Gln Trp Lys Ser Pro Glu Gly Lys Glu Ile Lys Leu Thr Phe

120 125 130 135

cgt cgc ctg gta tct ttc acg cgt cgg gag ctg ttt gct atc cat gta 1565

Arg Arg Leu Val Ser Phe Thr Arg Arg Glu Leu Phe Ala Ile His Val

140 145 150

caa ata gag ccg atc aat ttc aac ggc cag gta cgc atc gtc tcc acc 1613

Gln Ile Glu Pro Ile Asn Phe Asn Gly Gln Val Arg Ile Val Ser Thr

155 160 165

gtg aac ggc aag gta aag aac tac acc aat gcc aat gat ccg cgg gta 1661

Val Asn Gly Lys Val Lys Asn Tyr Thr Asn Ala Asn Asp Pro Arg Val

170 175 180

gga gca gga cac gca gaa cgg atg acc gtc atc gat acc ggc gtg aag 1709

Gly Ala Gly His Ala Glu Arg Met Thr Val Ile Asp Thr Gly Val Lys

185 190 195

ggc agc gat gcc tat gtt gtc gac gag acg atg gct tcc cag ctg cat 1757

Gly Ser Asp Ala Tyr Val Val Asp Glu Thr Met Ala Ser Gln Leu His

200 205 210 215

gcg gct tgt gtg acc cgt cat cgc tta gat gtt gat gcc gac att cag 1805

Ala Ala Cys Val Thr Arg His Arg Leu Asp Val Asp Ala Asp Ile Gln

220 225 230

ctc gaa gcc gga acc gga gaa gtc acg ttc acg gct gcc ctt cca ttg 1853

Leu Glu Ala Gly Thr Gly Glu Val Thr Phe Thr Ala Ala Leu Pro Leu

235 240 245

aca gga ccg atc cag ttt acg aaa tac aat ctg tat acc gac agt ctt 1901

Thr Gly Pro Ile Gln Phe Thr Lys Tyr Asn Leu Tyr Thr Asp Ser Leu

250 255 260

cgt cac ggt cag gac atg atc gat cgc ggt atc cag ctg cag gag gag 1949

Arg His Gly Gln Asp Met Ile Asp Arg Gly Ile Gln Leu Gln Glu Glu

265 270 275

tta aag gag ctt tct ttc gag gat ttg ctt gcc gag cag gct gag tat 1997

Leu Lys Glu Leu Ser Phe Glu Asp Leu Leu Ala Glu Gln Ala Glu Tyr

280 285 290 295

ctg aac gat tac tgg aaa tct gcc gat gtg gtt att cag aac gat gac 2045

Leu Asn Asp Tyr Trp Lys Ser Ala Asp Val Val Ile Gln Asn Asp Asp

300 305 310

cag ctc cag gaa ggc atc cgc ttc aat cta ttt cag ctg ctg caa tcc 2093

Gln Leu Gln Glu Gly Ile Arg Phe Asn Leu Phe Gln Leu Leu Gln Ser

315 320 325

gcg ggc cgt gac aag cac agc aac atc tct gcc aaa ggt cta agc ggc 2141

Ala Gly Arg Asp Lys His Ser Asn Ile Ser Ala Lys Gly Leu Ser Gly

330 335 340

gag ggt tat gaa ggc cat tat ttt tgg gac acg gag att tac atg ttc 2189

Glu Gly Tyr Glu Gly His Tyr Phe Trp Asp Thr Glu Ile Tyr Met Phe

345 350 355

cct gta ttt ctg atg acg cag ccg gac att gcg cgc cag ctg ctg ctg 2237

Pro Val Phe Leu Met Thr Gln Pro Asp Ile Ala Arg Gln Leu Leu Leu

360 365 370 375

tat cgc tat tcc aca ttg gag caa gcg aga gac aga gca cga gaa atg 2285

Tyr Arg Tyr Ser Thr Leu Glu Gln Ala Arg Asp Arg Ala Arg Glu Met

380 385 390

gga cat cgc caa ggc gcc ctg ttc ccg tgg cgt acg atc tcg ggg acg 2333

Gly His Arg Gln Gly Ala Leu Phe Pro Trp Arg Thr Ile Ser Gly Thr

395 400 405

gaa tgc tct tcc ttc ttc cct tcg gga aca gcg caa tat cat atc agc 2381

Glu Cys Ser Ser Phe Phe Pro Ser Gly Thr Ala Gln Tyr His Ile Ser

410 415 420

gca gat atc gca tac agc tat att caa tat tat ctc gcc gag cag gac 2429

Ala Asp Ile Ala Tyr Ser Tyr Ile Gln Tyr Tyr Leu Ala Glu Gln Asp

425 430 435

cgg gat ttc ctc ttg tcc tat ggg gcc gag gta ttg att gaa acc gcc 2477

Arg Asp Phe Leu Leu Ser Tyr Gly Ala Glu Val Leu Ile Glu Thr Ala

440 445 450 455

cgc ctg tgg gct gat atc ggc cac tat tat aat ggc gcc ttc cat atc 2525

Arg Leu Trp Ala Asp Ile Gly His Tyr Tyr Asn Gly Ala Phe His Ile

460 465 470

gat gag gtg acg ggg ccg gat gaa tat acc tgc tgc gta aac aac aac 2573

Asp Glu Val Thr Gly Pro Asp Glu Tyr Thr Cys Cys Val Asn Asn Asn

475 480 485

tat tac aca aac gtc atg gcg aag cat aac ctg aag tgg gct gcc aag 2621

Tyr Tyr Thr Asn Val Met Ala Lys His Asn Leu Lys Trp Ala Ala Lys

490 495 500

agc tgc tcg att ctg gaa tcc tac gat gcg caa gga tac aaa tca ctg 2669

Ser Cys Ser Ile Leu Glu Ser Tyr Asp Ala Gln Gly Tyr Lys Ser Leu

505 510 515

tgt gac cgg ctt ggc gta acc gcg gac gag att tca gct tgg gcg aaa 2717

Cys Asp Arg Leu Gly Val Thr Ala Asp Glu Ile Ser Ala Trp Ala Lys

520 525 530 535

gcc gcc cat gcc atg ctg ctg cca tac gat gaa gca ctt ggc att aat 2765

Ala Ala His Ala Met Leu Leu Pro Tyr Asp Glu Ala Leu Gly Ile Asn

540 545 550

ccg cag gat gat acg ttc ctc cgc aaa gcg gta tgg gat ttc gag aac 2813

Pro Gln Asp Asp Thr Phe Leu Arg Lys Ala Val Trp Asp Phe Glu Asn

555 560 565

acg cct gag gat aaa tat ccg ctg ctg ctg aac tac cat ccg ctc acc 2861

Thr Pro Glu Asp Lys Tyr Pro Leu Leu Leu Asn Tyr His Pro Leu Thr

570 575 580

att tac cgt tat cag gta tgt aaa caa gcg gat acg gtg ctc gcc cac 2909

Ile Tyr Arg Tyr Gln Val Cys Lys Gln Ala Asp Thr Val Leu Ala His

585 590 595

ttc tta ctc gaa gac gaa caa agc ttt gaa acg att cag cgt tcg tat 2957

Phe Leu Leu Glu Asp Glu Gln Ser Phe Glu Thr Ile Gln Arg Ser Tyr

600 605 610 615

gac tac tat gaa ggc att aca aca cat gac tct tcc ctg tcc tcc tgt 3005

Asp Tyr Tyr Glu Gly Ile Thr Thr His Asp Ser Ser Leu Ser Ser Cys

620 625 630

atc ttc agc atc atg gct tcg aag att ggc aat atg gac aaa gcg tat 3053

Ile Phe Ser Ile Met Ala Ser Lys Ile Gly Asn Met Asp Lys Ala Tyr

635 640 645

gag tat ttc atc gag acg gca cgc ctt gac ctc gac aat aca cac ggc 3101

Glu Tyr Phe Ile Glu Thr Ala Arg Leu Asp Leu Asp Asn Thr His Gly

650 655 660

aac acg aaa gac ggc ctg cat atg gcc aac atg ggc ggc acc tgg atg 3149

Asn Thr Lys Asp Gly Leu His Met Ala Asn Met Gly Gly Thr Trp Met

665 670 675

tcg atc gtc tac ggc ttt gct ggt atg cgt ctg aag gaa agt gga ctt 3197

Ser Ile Val Tyr Gly Phe Ala Gly Met Arg Leu Lys Glu Ser Gly Leu

680 685 690 695

tcc ctc tcc cct gcc att ccg cag gac tgg gag aag tat gcg ttc cgt 3245

Ser Leu Ser Pro Ala Ile Pro Gln Asp Trp Glu Lys Tyr Ala Phe Arg

700 705 710

ttg aac ttc cgc ggc cgc ctc atc ggt gta tcc att gag aag gac ggc 3293

Leu Asn Phe Arg Gly Arg Leu Ile Gly Val Ser Ile Glu Lys Asp Gly

715 720 725

gtg acc ttg gag att gtg gag ggc gat tcc atc gag atc aag ctg tac 3341

Val Thr Leu Glu Ile Val Glu Gly Asp Ser Ile Glu Ile Lys Leu Tyr

730 735 740

gac gaa gta gtt aag ctt gag gcc ggg aag tcc gtg aag cat gca ctt 3389

Asp Glu Val Val Lys Leu Glu Ala Gly Lys Ser Val Lys His Ala Leu

745 750 755

cac aaa gca aat aat aag gtt tag gctgtctgat attgcaaaaa ggagccataa 3443

His Lys Ala Asn Asn Lys Val

760 765

gaacatgaac gaaacacgca agctgaaggc cgttattttt gacctggacg gcgtcattac 3503

cgatacggcg gagtatcatt atcaggcatg gaaagccaca gcaaccgaac ttggcattcc 3563

cttcacccgt gaattcgact tcttcggcaa tggtcacgaa gtcgccttcc ttcttcaccg 3623

taaacttgat aaccgaagag aaggcctgca gcgcttcttc aatgcccgtt gccccctgaa 3683

tcttggccat ccacttgatc acattgagtg tgttatatat aaaatgcgga cgaatctgcg 3743

actggagcgc attcatctcg agggttcgtt tctcctcttc ttccacatag atacgtttaa 3803

ttaaatcacg aatttcgtgc agcatctgat ccaccgtatt gctgatcacg ccgatttcgc 3863

cctga 3868

<210>8

<211>766

<212>PRT

＜213〉series bacillus sp.SH-55 (trehalose phosphorylase)

<400>8

Met Thr Trp Met Ile Ser Asn Pro Asp Leu Ser Gln Gln Ala Leu Leu

l 5 10 15

Asn Met Glu Ser Ile Phe Ala Leu Gly Asn Gly Tyr Leu Gly Val Arg

20 25 30

Gly Asn Phe Glu Glu Gly Tyr Gly Glu Ser Met Ser Thr Ile Arg Gly

35 40 45

Thr Tyr Leu Asn Ala Phe His Asp Val Ile Glu Ile Pro Tyr Gly Glu

50 55 60

Lys Leu Phe Ala Phe Pro Asp Thr Gln Gln Lys Leu Val Asn Asn Ile

65 70 75 80

Asp Ala Gln Thr Val Leu Ile Tyr Leu Gly Asp Glu Lys Glu Pro Phe

85 90 95

Arg Leu Asp His Gly Thr Ile Thr Ser Arg Glu Arg Arg Leu His Met

100 105 110

Asp Lys Gly Tyr Ser Glu Arg Ile Ile Gln Trp Lys Ser Pro Glu Gly

115 120 125

Lys Glu Ile Lys Leu Thr Phe Arg Arg Leu Val Ser Phe Thr Arg Arg

130 135 140

Glu Leu Phe Ala Ile His Val Gln Ile Glu Pro Ile Asn Phe Asn Gly

145 150 155 160

Gln Val Arg Ile Val Ser Thr Val Asn Gly Lys Val Lys Asn Tyr Thr

165 170 175

Asn Ala Asn Asp Pro Arg Val Gly Ala Gly His Ala Glu Arg Met Thr

180 185 190

Val Ile Asp Thr Gly Val Lys Gly Ser Asp Ala Tyr Val Val Asp Glu

195 200 205

Thr Met Ala Ser Gln Leu His Ala Ala Cys Val Thr Arg His Arg Leu

210 215 220

Asp Val Asp Ala Asp Ile Gln Leu Glu Ala Gly Thr Gly Glu Val Thr

225 230 235 240

Phe Thr Ala Ala Leu Pro Leu Thr Gly Pro Ile Gln Phe Thr Lys Tyr

245 250 255

Asn Leu Tyr Thr Asp Ser Leu Arg His Gly Gln Asp Met Ile Asp Arg

260 265 270

Gly Ile Gln Leu Gln Glu Glu Leu Lys Glu Leu Ser Phe Glu Asp Leu

275 280 285

Leu Ala Glu Gln Ala Glu Tyr Leu Asn Asp Tyr Trp Lys Ser Ala Asp

290 295 300

Val Val Ile Gln Asn Asp Asp Gln Leu Gln Glu Gly Ile Arg Phe Asn

305 310 315 320

Leu Phe Gln Leu Leu Gln Ser Ala Gly Arg Asp Lys His Ser Asn Ile

325 330 335

Ser Ala Lys Gly Leu Ser Gly Glu Gly Tyr Glu Gly His Tyr Phe Trp

340 345 350

Asp Thr Glu Ile Tyr Met Phe Pro Val Phe Leu Met Thr Gln Pro Asp

355 360 365

Ile Ala Arg Gln Leu Leu Leu Tyr Arg Tyr Ser Thr Leu Glu Gln Ala

370 375 380

Arg Asp Arg Ala Arg Glu Met Gly His Arg Gln Gly Ala Leu Phe Pro

385 390 395 400

Trp Arg Thr Ile Ser Gly Thr Glu Cys Ser Ser Phe Phe Pro Ser Gly

405 410 415

Thr Ala Gln Tyr His Ile Ser Ala Asp Ile Ala Tyr Ser Tyr Ile Gln

420 425 430

Tyr Tyr Leu Ala Glu Gln Asp Arg Asp Phe Leu Leu Ser Tyr Gly Ala

435 440 445

Glu Val Leu Ile Glu Thr Ala Arg Leu Trp Ala Asp Ile Gly His Tyr

450 455 460

Tyr Asn Gly Ala Phe His Ile Asp Glu Val Thr Gly Pro Asp Glu Tyr

465 470 475 480

Thr Cys Cys Val Asn Asn Asn Tyr Tyr Thr Asn Val Met Ala Lys His

485 490 495

Asn Leu Lys Trp Ala Ala Lys Ser Cys Ser Ile Leu Glu Ser Tyr Asp

500 505 510

Ala Gln Gly Tyr Lys Ser Leu Cys Asp Arg Leu Gly Val Thr Ala Asp

515 520 525

Glu Ile Ser Ala Trp Ala Lys Ala Ala His Ala Met Leu Leu Pro Tyr

530 535 540

Asp Glu Ala Leu Gly Ile Asn Pro Gln Asp Asp Thr Phe Leu Arg Lys

545 550 555 560

Ala Val Trp Asp Phe Glu Asn Thr Pro Glu Asp Lys Tyr Pro Leu Leu

565 570 575

Leu Asn Tyr His Pro Leu Thr Ile Tyr Arg Tyr Gln Val Cys Lys Gln

580 585 590

Ala Asp Thr Val Leu Ala His Phe Leu Leu Glu Asp Glu Gln Ser Phe

595 600 605

Glu Thr Ile Gln Arg Ser Tyr Asp Tyr Tyr Glu Gly Ile Thr Thr His

610 615 620

Asp Ser Ser Leu Ser Ser Cys Ile Phe Ser Ile Met Ala Ser Lys Ile

625 630 635 640

Gly Asn Met Asp Lys Ala Tyr Glu Tyr Phe Ile Glu Thr Ala Arg Leu

645 650 655

Asp Leu Asp Asn Thr His Gly Asn Thr Lys Asp Gly Leu His Met Ala

660 665 670

Asn Met Gly Gly Thr Trp Met Ser Ile Val Tyr Gly Phe Ala Gly Met

675 680 685

Arg Leu Lys Glu Ser Gly Leu Ser Leu Ser Pro Ala Ile Pro Gln Asp

690 695 700

Trp Glu Lys Tyr Ala Phe Arg Leu Asn Phe Arg Gly Arg Leu Ile Gly

705 710 715 720

Val Ser Ile Glu Lys Asp Gly Val Thr Leu Glu Ile Val Glu Gly Asp

725 730 735

Ser Ile Glu Ile Lys Leu Tyr Asp Glu Val Val Lys Leu Glu Ala Gly

740 745 750

Lys Ser Val Lys His Ala Leu His Lys Ala Asn Asn Lys Val

755 760 765

Claims (19)

1. series bacillus microorganism belonging to genus, it has the ability of producing maltose phosphorylase and trehalose phosphorylase.

2. the microorganism with the ability of producing maltose phosphorylase and trehalose phosphorylase as claimed in claim 1, this microorganism is that preserving number is the series bacillus sp.SH-55 of FERM BP-8420.

3. maltose phosphorylase, it has following physico-chemical property:

(I) effect: to the α in the maltose-1,4-Glucopyranose glycosidic bond carries out reversibility and adds the phosphoric acid decomposition, generates glucose and β-D-glucose 1-phosphoric acid;

(II) substrate specificity of decomposition reaction: act on maltose, but do not act on trehalose, sucrose, lactose and cellobiose etc.;

(III) optimal pH and stable pH range: the optimal pH of decomposition reaction is 7.0～8.0, and the optimal pH of building-up reactions is 5.5～6.5; Under 10 minutes heating condition of 50 ℃ of heating, stable in the scope of pH 5.5～7.5;

(IV) scope of operative temperature and optimum temperuture: the operative temperature scope is 20 ℃～60 ℃; The optimum temperuture of decomposition reaction is 45 ℃～55 ℃, and the optimum temperuture of building-up reactions is 50 ℃～55 ℃;

(V) temperature stability: under 15 minutes heating condition of pH 6.0 heating, in extremely stable below 50 ℃ or 50 ℃; In 70 ℃ of complete deactivations;

(VI) molecular weight of measuring with SDS-polyacrylamide gel electrophoresis method is about 89000 dalton～90000 dalton, is about 190000 dalton with the molecular weight of gel filteration determining; Described enzyme is made of the homotype dipolymer.

4. maltose phosphorylase as claimed in claim 3, it has following aminoacid sequence: so that the sequence of one or more aminoacid deletion, replacement, inversion, increase or insertion to be arranged in the represented aminoacid sequence of the represented aminoacid sequence of sequence number 1 or sequence number 1.

5. maltose phosphorylase as claimed in claim 3, aminoacid sequence that it had and the homology that has with the represented aminoacid sequence of sequence number 1 more than or equal to 52%.

6. polynucleotide, the aminoacid sequence of its maltose phosphorylase as claimed in claim 3 of encoding, described polynucleotide are selected from the group that following (a)～(c) forms:

(a) coding has the polynucleotide of the polypeptide of the aminoacid sequence shown in the sequence number 1 of sequence table;

(b) coding has the polynucleotide of polypeptide of following aminoacid sequence, and described aminoacid sequence is the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in the sequence number 1 of sequence table;

(c) have the polynucleotide of the nucleotide sequence shown in the sequence number 2 of sequence table.

7. recombinant vectors, it has polynucleotide as claimed in claim 6.

8. microorganism, it transforms with the described recombinant vectors of claim 7.

9. trehalose phosphorylase, it has following physico-chemical property;

(I) effect: to the α in the trehalose-1,1-Glucopyranose glycosidic bond carries out reversibility and adds the phosphoric acid decomposition, generates glucose and β-D-glucose 1-phosphoric acid;

(II) substrate specificity of decomposition reaction: act on trehalose, but do not act on maltose, sucrose, lactose and cellobiose etc.;

(III) optimal pH and stable pH range: the optimal pH of decomposition reaction is 7.0～8.0, and the optimal pH of building-up reactions is 5.8～7.8; Under 10 minutes heating condition of 50 ℃ of heating, stable in the scope of pH 5.5～9.5;

(IV) scope of operative temperature and optimum temperuture: the operative temperature scope is 25 ℃～70 ℃; The optimum temperuture of decomposition reaction is 50 ℃～65 ℃, and the optimum temperuture of building-up reactions is 45 ℃～60 ℃;

(V) temperature stability: under 15 minutes heating condition of pH 7.0 heating, in extremely stable below 60 ℃ or 60 ℃; In 70 ℃ of complete deactivations;

(VI) molecular weight of measuring with SDS-polyacrylamide gel electrophoresis method is about 89000 dalton～90000 dalton, is about 190000 dalton with the molecular weight of gel filteration determining; Described enzyme is made of the homotype dipolymer.

10. trehalose phosphorylase as claimed in claim 9, it has following aminoacid sequence: so that the sequence of one or more aminoacid deletion, replacement, inversion, increase or insertion to be arranged in the represented aminoacid sequence of the represented aminoacid sequence of sequence number 3 or sequence number 3.

11. trehalose phosphorylase as claimed in claim 9, aminoacid sequence that it had and the homology that has with the represented aminoacid sequence of sequence number 3 more than or equal to 63%.

12. polynucleotide, the aminoacid sequence of its trehalose phosphorylase as claimed in claim 9 of encoding, described polynucleotide are selected from the group that following (d)～(f) forms:

(d) coding has the polynucleotide of the polypeptide of the aminoacid sequence shown in the sequence number 3 of sequence table;

(e) coding has the polynucleotide of polypeptide of following aminoacid sequence, and described aminoacid sequence is the aminoacid sequence that one or more aminoacid deletion, replacement, inversion, increase or insertion are arranged in the aminoacid sequence shown in the sequence number 3 of sequence table;

(f) have the polynucleotide of the nucleotide sequence shown in the sequence number 4 of sequence table.

13. recombinant vectors, it has polynucleotide as claimed in claim 12.

14. microorganism, it transforms with the described recombinant vectors of claim 13.

15. the manufacture method of the mixture of maltose phosphorylase or trehalose phosphorylase or above-mentioned two kinds of enzymes, it is characterized in that, cultivation has the series bacillus microorganism belonging to genus of producing maltose phosphorylase and trehalose phosphorylase ability, generate and accumulate as claim 3 or 4 described maltose phosphorylases and/or as at least a in claim 9 or the 10 described trehalose phosphorylases, and the enzyme that generated of extraction.

16. manufacture method as claimed in claim 15, it is the manufacture method of maltose phosphorylase or maltose phosphorylase and trehalose phosphorylase mixture, it is characterized in that, carry out described cultivation containing in the presence of the carbon source of maltose, generate and accumulate maltose phosphorylase and trehalose phosphorylase.

17. manufacture method as claimed in claim 15, it is the manufacture method of trehalose phosphorylase or maltose phosphorylase and trehalose phosphorylase mixture, it is characterized in that, carry out described cultivation containing in the presence of the carbon source of trehalose, preferentially generate and accumulate trehalose phosphorylase.

18. the manufacture method of the thick enzyme of maltose phosphorylase and/or trehalose phosphorylase, described method are selected from following (i) any one method in (iii):

(i) cultivate series bacillus microorganism belonging to genus, directly extract isolating thalline from the nutrient solution that obtains with production maltose phosphorylase and trehalose phosphorylase ability;

(ii) cultivate and have the series bacillus microorganism belonging to genus of producing maltose phosphorylase and trehalose phosphorylase ability, separating thallus from the nutrient solution that obtains, the thick enzyme of extraction maltose phosphorylase and/or trehalose phosphorylase from isolating thalline;

(iii) cultivate and have the series bacillus microorganism belonging to genus of producing maltose phosphorylase and trehalose phosphorylase ability, separating thallus from resulting nutrient solution, the supernatant liquor of extraction nutrient solution.

19. the manufacture method of trehalose is characterized in that, in the presence of phosphoric acid, will act on maltose as claim 3 or 4 described maltose phosphorylases and as claim 9 or 10 described trehalose phosphorylases, makes trehalose with this.

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