CN1688698A

CN1688698A - Novel peptide synthase gene

Info

Publication number: CN1688698A
Application number: CNA038224607A
Authority: CN
Inventors: 原诚一; 横关健三; 阿部巧; 外内尚人; 城岛恭子
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 2002-07-26
Filing date: 2003-07-25
Publication date: 2005-10-26
Anticipated expiration: 2023-07-25
Also published as: CN101186904A; CN101186903A; CN101067126A; CN101067125A; CN101067124A; CN101067124B; CN100379868C

Abstract

The present invention relates to a novel enzyme which can easily and cheaply produce peptide in high yield without a complex synthesization method. More specifically, the present invention provides methods of catalyzing the novel enzyme produced by the peptide synthesis reaction of carboxyl ingredient and amine ingredient to produce the microorganism of the enzyme and applying the enzyme or the microorganism to cheaply produce dipeptide. The present invention discloses a novel enzyme capable of effectively synthesizing the peptide form a newly discovered microbe in Empedobacter sp and also discloses a method of simply and cheaply producing the dipeptide.

Description

Novel peptide-forming enzyme gene

Technical field

The present invention relates to needn't be by complexity synthetic method can be easily, high productivity and generate the novel enzyme of peptide at an easy rate.More specifically, the present invention relates to the novel enzyme of catalysis, produce the microorganism of this enzyme by the peptide formation reaction of carboxyl composition and amine component, and the method that adopts this enzyme or microorganisms producing dipeptides.

Background technology

Peptide is used in medicine, field of food and multiple other field.For example, because the stability and the water-soluble L-glutaminate that is higher than of L-alanyl-L-glutamine, so it is widely used as the composition of transfusion and serum free medium.

Chemical synthesis (producing the currently known methods of peptide) is not the method that always is easy to.The known embodiment of described method comprises that the method for the L-glutaminate that uses N-carbobenzoxy-(Cbz) L-Ala (hereinafter referred to as " Z-L-Ala ") and protection is (referring to Bull.Chem.Soc.Jpn.; 34; 739 (1961); Bull.Chem.Soc.Jpn.; 35; 1966 (1962)); the method of the L-L-glutamic acid-γ-methyl esters of use Z-L-Ala and protection is (referring to Bull.Chem.Soc.Jpn.; 37; 200 (1964)); use the method (referring to patent document 1) of Z-alanine ester and unshielded L-glutamic acid, comprise that the propionyl halogenide that replaces with 2-is raw material, synthetic N-(2-replacement)-propionyl glutamine derivative is as the method (referring to patent document 2) of intermediate.

But, because protecting group or use optical activity intermediate need be introduced and slough to all these methods, so these methods can not fully satisfy industrial favourable requirement.

On the other hand; use the well-known example of the typical peptide production method of enzyme to comprise to use the N protection with the unshielded carboxyl composition of C and N is unshielded, the condensation reaction of the amine component of C protection (hereinafter referred to as " reaction 1 ") and use the carboxyl composition N protection and the C protection and N is unshielded, the replacement(metathesis)reaction of the amine component of C protection (hereinafter referred to as " reaction 2 ").The example of reaction 1 is to produce Z-aspartyl-phenylalanine methyl ester (referring to patent document 3) by Z-aspartic acid and alanine methyl ester, and react 2 example is to produce phenyl methyl ketone alanyl leucyl amine (referring to Biochemical J. by acetylphenylalanine ethyl ester and leucyl amine, 163,531 (1977)).Relevant use N is unshielded, the research report example of the method for the carboxyl composition of C protection is considerably less.In international monopoly publication WO 90/01555 (patent document 4), the example of the replacement(metathesis)reaction (hereinafter referred to as " reaction 3 ") of using carboxyl composition that N is unshielded, C protects and the amine component that N is unshielded, C protects has been described.For example, the method for producing the arginyl leucyl amine by arginine ethyl ester and leucyl amine has wherein been described.In European patent publication EP 278787A1 (patent document 5) and the European patent publication EP 359399B1 (patent document 6), described and used that N is unshielded, the carboxyl composition of C protection and N is unshielded, the example of the replacement(metathesis)reaction of the unshielded amine component of C (hereinafter referred to as " reaction 4 ").For example, the method that tyrosine ethyl ester and L-Ala are produced the tyrosyl L-Ala has wherein been described.

Patent document 1; Japanese patent application H1-96194 communique.

Patent document 2; Patent application H6-234715 communique.

Patent document 3; Japanese patent application S53-92729 communique.

Patent document 4; International monopoly publication WO 90/01555 communique

Patent document 5; European patent publication EP 278787A1 communique

Patent document 6; European patent publication EP 359399B1 communique

Non-patent document 1; Biochemical J., 163,531 (1977)

Summary of the invention

In the method for above-mentioned reaction 1-4, the most cheap production method belongs to the minimum reaction of protecting group 4 naturally.

But, there is following subject matter in the example (referring to European patent publication EP 278787A1) of reaction 4 in the prior art: (1) peptide production rate is very low, (2) the peptide productive rate is low, (3) producible peptide is limited to and comprises the high relatively amino acid whose peptide of hydrophobicity, (4) add-on of enzyme very big and (5) need come from the relatively costly carboxypeptidase preparation of mould, yeast or plant.In reaction 4, do not see the method for the enzyme that the bacterium of using beyond the Saccharomycodes or yeast source are arranged, and do not see have the Dipeptiven of production and other to have the method for the peptide of high-hydrophilic.Under this background, need a kind of commercial run of producing the cheapness of these peptides of exploitation.

The purpose of this invention is to provide needn't be by complexity synthetic method can be easily, high productivity and generate the novel enzyme of peptide at an easy rate.More specifically, the purpose of this invention is to provide the novel enzyme of catalysis, produce the microorganism of this enzyme and adopt this enzyme or microorganism to produce the method for dipeptides at an easy rate by the peptide formation reaction of carboxyl composition and amine component.

The inventor has carried out big quantity research according to above-mentioned purpose, found that from the bacterium of finding recently that belongs to steady Bacillaceae etc., can effectively generate the novel enzyme of peptide, and determine the sequence of this enzyme gene, has finished the present invention thus.

That is, the present invention as described below:

[1] coding following (A) or (B) shown in protein DNA:

(A) have the protein of the aminoacid sequence of forming by the 23-616 amino acids residue of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table,

(B) has following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in the aminoacid sequence of forming by the 23-616 amino acids residue of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table displacement, disappearance, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[2] coding following (C) or (D) shown in protein DNA:

(C) have the protein of the aminoacid sequence of forming by the 21-619 amino acids residue of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table,

(D) has following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in the aminoacid sequence of forming by the 21-619 amino acids residue of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table displacement, disappearance, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[3] coding following (E) or (F) shown in protein DNA:

(E) have the protein of the aminoacid sequence of forming by the 23-625 amino acids residue of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table,

(F) has following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in the aminoacid sequence of forming by the 23-625 amino acids residue of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table displacement, disappearance, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[4] coding following (G) or (H) shown in protein DNA:

(G) have the protein of the aminoacid sequence of forming by the 23-645 amino acids residue of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table,

(H) has following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in the aminoacid sequence of forming by the 23-645 amino acids residue of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table displacement, disappearance, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[5] coding following (I) or (J) shown in protein DNA:

(I) have the protein of the aminoacid sequence of forming by the 26-620 amino acids residue of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table,

(J) has following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in the aminoacid sequence of forming by the 26-620 amino acids residue of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table displacement, disappearance, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[6] coding following (K) or (L) shown in protein DNA:

(K) have the protein of the aminoacid sequence of forming by the 18-644 amino acids residue of aminoacid sequence shown in the SEQ ID NO:27 in the sequence table,

(L) has following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in the aminoacid sequence of forming by the 18-644 amino acids residue of aminoacid sequence shown in the SEQ ID NO:27 in the sequence table displacement, disappearance, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[7] coding following (M) or (N) shown in protein DNA:

(M) have the protein of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table,

(N) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in shown in the SEQ ID NO:6 in the sequence table displacement, disappearance in the aminoacid sequence, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[8] coding following (O) or (P) shown in protein DNA:

(O) have the protein of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table,

(P) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in shown in the SEQ ID NO:12 in the sequence table displacement, disappearance in the aminoacid sequence, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[9] coding following (Q) or (R) shown in protein DNA:

(Q) have the protein of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table,

(R) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in shown in the SEQ ID NO:18 in the sequence table displacement, disappearance in the aminoacid sequence, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[10] coding following (S) or (T) shown in protein DNA:

(S) have the protein of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table,

(T) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in shown in the SEQ ID NO:23 in the sequence table displacement, disappearance in the aminoacid sequence, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[11] coding following (U) or (V) shown in protein DNA:

(U) have the protein of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table,

(V) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in shown in the SEQ ID NO:25 in the sequence table displacement, disappearance in the aminoacid sequence, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[12] coding following (W) or (X) shown in protein DNA:

(W) have the protein of aminoacid sequence shown in the SEQ ID NO:27 in the sequence table,

(X) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, shown in aminoacid sequence be included in shown in the SEQ ID NO:27 in the sequence table displacement, disappearance in the aminoacid sequence, insert, add, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

[13] following (a) or (b) shown in DNA:

(a) have the DNA of the base sequence of the 127-1908 bit base of base sequence shown in the SEQ ID NO:5 in the sequence table,

(b) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1908 bit base of base sequence shown in the SEQ IDNO:5 in the sequence table.

[14] following (c) or (d) shown in DNA:

(c) have the DNA of the base sequence of the 121-1917 bit base of base sequence shown in the SEQ ID NO:11 in the sequence table,

(d) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 121-1917 bit base of base sequence shown in the SEQ IDNO:11 in the sequence table.

[15] following (e) or (f) shown in DNA:

(e) have the DNA of the base sequence of the 127-1935 bit base of base sequence shown in the SEQ ID NO:17 in the sequence table,

(f) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1935 bit base of base sequence shown in the SEQ IDNO:17 in the sequence table.

[16] following (g) or (h) shown in DNA:

(g) have the DNA of the base sequence of the 127-1995 bit base of base sequence shown in the SEQ ID NO:22 in the sequence table,

(h) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1995 bit base of base sequence shown in the SEQ IDNO:22 in the sequence table.

[17] following (i) or (j) shown in DNA:

(i) have the DNA of the base sequence of the 104-1888 bit base of base sequence shown in the SEQ ID NO:24 in the sequence table,

(j) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 104-1888 bit base of base sequence shown in the SEQ IDNO:24 in the sequence table.

[18] following (k) or (l) shown in DNA:

(k) have the DNA of the base sequence of the 112-1992 bit base of base sequence shown in the SEQ ID NO:26 in the sequence table,

(l) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 112-1992 bit base of base sequence shown in the SEQ IDNO:26 in the sequence table.

[19] following (m) or (n) shown in DNA:

(m) have the DNA of the base sequence of the 61-1908 bit base of base sequence shown in the SEQ ID NO:5 in the sequence table,

(n) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1908 bit base of base sequence shown in the SEQ ID NO:5 in the sequence table.

[20] following (o) or (p) shown in DNA:

(o) have the DNA of the base sequence of the 61-1917 bit base of base sequence shown in the SEQ ID NO:11 in the sequence table,

(p) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1917 bit base of base sequence shown in the SEQ ID NO:11 in the sequence table.

[21] following (q) or (r) shown in DNA:

(q) have the DNA of the base sequence of the 61-1935 bit base of base sequence shown in the SEQ ID NO:17 in the sequence table,

(r) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1935 bit base of base sequence shown in the SEQ ID NO:17 in the sequence table.

[22] following (s) or (t) shown in DNA:

(s) have the DNA of the 127-1995 bit base sequence of base sequence shown in the SEQ ID NO:22 in the sequence table,

(t) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1995 bit base of base sequence shown in the SEQ ID NO:22 in the sequence table.

[23] following (u) or (DNA v):

(u) have the DNA of the 29-1888 bit base sequence of base sequence shown in the SEQ ID NO:24 in the sequence table,

(v) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 29-1888 bit base of base sequence shown in the SEQ ID NO:24 in the sequence table.

[24] following (w) or (x) shown in DNA:

(w) have the DNA of the 61-1992 bit base sequence of base sequence shown in the SEQ ID NO:26 in the sequence table,

(x) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1992 bit base of base sequence shown in the SEQ ID NO:26 in the sequence table.

[25] according to each DNA of [13]-[24], wherein stringent condition is under the salt concn that is equivalent to 1 * SSC and 0.1%SDS, in 60 ℃ of conditions of washing.

[26] a kind of comprising according to each the recombinant DNA of DNA of [1]-[25].

[27] a kind of transformant that has imported according to the recombinant DNA of [26].

[28] a kind of method of producing peptide-forming enzyme comprises: cultivate the transformant according to [27] in substratum, peptide-forming enzyme is accumulated in substratum and/or transformant.

[29] a kind of method of producing dipeptides comprises: the transformant of cultivating basis [28] in substratum mixes this culture with synthetic dipeptides to obtain culture with carboxyl composition and amine component.

[30] a kind of method of producing dipeptides, comprise: belong to Sphingobacterium and have the culture that generates the active microorganism of dipeptides by carboxyl composition and amine component by use, isolating microorganism cells from this culture, the microorganism cells of this microorganism is handled product or is come from the peptide-forming enzyme of this microorganism, generates dipeptides by carboxyl composition and amine component.

In addition, aminoacid sequence shown in the SEQ ID NO:6 is determined by DNA described in the SEQ ID NO:5 in the sequence table.Aminoacid sequence shown in the SEQ ID NO:12 is determined by DNA described in the SEQID NO:11 in the sequence table.Aminoacid sequence shown in the SEQ ID NO:18 is determined by DNA described in the SEQ ID NO:17 in the sequence table.Aminoacid sequence shown in the SEQ ID NO:23 is determined by DNA described in the SEQ ID NO:22 in the sequence table.Aminoacid sequence shown in the SEQ ID NO:25 is determined by DNA described in the SEQ ID NO:24 in the sequence table.Aminoacid sequence shown in the SEQ ID NO:27 is determined by DNA described in the SEQ ID NO:26 in the sequence table.

The accompanying drawing summary

Fig. 1 is the figure of optimal pH that illustrates the enzyme of steady bacillus of the present invention;

Fig. 2 is the figure of optimum temperuture that illustrates the enzyme of steady bacillus of the present invention;

Fig. 3 illustrates the time-histories figure that is prepared the L-alanyl-L-glutamine by L-alanine methyl ester and L-glutaminate; With

Fig. 4 is the bar graph that illustrates the enzyme amount that exists in kytoplasm part (Cy) and the pericentral siphon part (Pe).

The optimum implementation of invention

It below is the product dipeptides generation enzyme that novel dipeptide of the present invention generates enzyme gene and this gene.

(1) contains the microorganism of DNA of the present invention

Dna encoding of the present invention has the active protein that is generated peptide by carboxyl composition and amine component.In this manual, the carboxyl composition is meant provides the peptide bond (composition in carbonyl site (CO) CONH-), and amine component is meant the composition that amino sites in the peptide bond (NH) is provided.In addition, in this manual, unless refer in particular in addition, term " peptide " is meant the polymer with at least one peptide bond when using separately.In addition, in this manual, " dipeptides " is meant the peptide with a peptide bond.

The example that contains the microorganism of DNA of the present invention comprises and belongs to steady Bacillaceae, Sphingobacterium, soil bacillus (Pedobacter) belongs to, the shaping Bacillaceae, the bacterium that round bar Pseudomonas or Psycloserpens belong to, and its more specifically example comprise short steady bacillus strain ATCC14234 (bacterial strain FERM P-18545, bacterial strain FERM BP-8113), Sphingobacterium sp. bacterial strain FERM BP-8124, separate Vitrum AB soil bacillus (Pedobacter heparinus) bacterial strain IFO 12017, Taxeobacter gelupurpurascens strain DSM Z 11116, sea round bacteria strains A TCC 25205 and Psycloserpens burtonensis strains A TCC700359.Short steady bacillus strain ATCC 14234 (bacterial strain FERM P-18545, bacterial strain FERMBP-8113), Sphingobacterium sp. bacterial strain FERM BP-8124, separate Vitrum AB soil bacillus strain IFO 12017, Taxeobacter gelupurpurascens strain DSM Z 11116, extra large round bacteria strains A TCC 25205 and Psycloserpens burtonens is strains A TCC700359 be the inventor after deliberation and energy the high yield of selecting ground by the microorganism of carboxyl composition and amine component generation peptide.

In the mentioned microorganism bacterial strain, be preserved in Independent Administrative Leged Industrial Technology Complex Inst and speciallyyed permit biological sustenance center (Chuo Dai-6,1-1Higashi 1-Chome with the microorganism of FERM number expression, Tsukuba-shi, Ibaraki-ken Japan), and can obtain with reference to each number.

In the mentioned microorganism bacterial strain, be preserved in American type culture collection (P.O.Box 1549, Manassas, VA 20110, the UnitedStates of America) with the microorganism of ATCC number expression, and can have obtained with reference to each number.

In the mentioned microorganism bacterial strain, with IFO number the expression microorganism be preserved in fermentation research institute, (Osaka-shi Japan), and can obtain with reference to each number Osaka for 2-17-85 Jusanbon-cho, Yodogawa-ku.

In the mentioned microorganism bacterial strain, microorganism with NBRC number expression has been preserved in Japanese state-run technology and evaluation study institute Biological resources center (NITE) (5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, and can obtain Japan), with reference to each number.

In the mentioned microorganism bacterial strain, microorganism with DSMZ number expression has been preserved in DeutcheSammlung von Mikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center) (Mascheroder Weg 1b, 38124Braunschweig, and can obtain Germany), with reference to each number.

Short steady bacillus strain ATCC 14234 (bacterial strain FERM P-18545, bacterial strain FERM BP-8113) is preserved in Independent Administrative Leged Industrial Technology Complex Inst October 1 calendar year 2001 and speciallys permit biological sustenance center (Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, and to specify its preserving number be FERM P-18545 Japan).Subsequently, regulation according to budapest treaty, on July 8th, 2002 Independent Administrative Leged Industrial Technology Complex Inst that is transferred to of this bacterial strain is speciallyyed permit biological sustenance center, and to specify its preserving number be FERM BP-8113 (microorganism performance: short steady bacillus strain AJ 13933).

Sphingobacterium sp. strains A J 110003 is preserved in Independent Administrative Leged Industrial Technology Complex Inst on July 22nd, 2002 and speciallys permit biological sustenance center, and to specify its preserving number be FERM BP-8124.By following qualification test, strains A J 110003 (FERM BP-8124) is accredited as above-mentioned Sphingobacterium sp..Bacterial strain FERM BP-8124 is gram negative bacillus (0.7-0.8 * 1.5-2.0 μ m), does not generate gemma, no mobility.Its bacterium colony is circular, and the edge is smooth fully, comprises low projection, and is glossy, light yellow.This organism is 30 ℃ of growths, the superoxide enzyme positive, and oxidase positive, OF tests (glucose) feminine gender, according to these features it is accredited as the bacterium that belongs to Sphingobacterium.In addition, because following characteristic: nitrate reduction feminine gender, indoles produces negative, derive from the acid product feminine gender of glucose, the arginine dihydrolase feminine gender, the urase positive, the Vitamin C2 hydrolysis positive, the gelatin hydrolysis feminine gender, the beta-galactosidase enzymes positive, the glucose assimilation is positive, the L-arabinose assimilation is negative, the assimilation of D-seminose is positive, the assimilation of D-N.F,USP MANNITOL is negative, N-acetyl-D-glucosamine assimilation is positive, the maltose assimilation is positive, the Potassium Gluconate feminine gender, the n-capric acid feminine gender, the hexanodioic acid assimilation is negative, the assimilation of d1-oxysuccinic acid is negative, the Sodium Citrate assimilation is negative, negative and the Terminal oxidase positive of toluylic acid assimilation can determine that it has the characteristic similar to Sphingobacterium multivorum or Sphingobacterium spiritivorum.In addition, though the homology analysis result of the base sequence of 16S rRNA gene shows that it has the homology (98.8%) with the Sphingobacterium multivorum height, the bacterial isolates that mates fully with this bacterial isolates not.Therefore, this bacterial isolates can be accredited as Sphingobacterium sp.

(2) microorganism culturing

For the microorganism cells of the microorganism that obtains containing DNA of the present invention, this microorganism can be cultivated on the suitable culture base and is grown.The substratum that is used for this purpose there is not special restriction, as long as can make this microorganism growth.This substratum can be the ordinary culture medium that contains common carbon source, nitrogenous source, phosphorus source, sulphur source, mineral ion and organotrophy source as required.

For example, as long as can be utilized, can use any carbon source by mentioned microorganism.The specific examples of spendable carbon source comprises that sugar is such as glucose, fructose, maltose and amylose starch, alcohol is such as Sorbitol Powder, ethanol and glycerine, organic acid such as fumaric acid, citric acid, acetate and propionic acid and salt thereof are such as the carbohydrate of paraffin and their mixture.

The example of spendable nitrogenous source comprises the ammonium salt such as the inorganic salt of ammonium sulfate and ammonium chloride, ammonium salt such as the organic salt of ammonium fumarate and ammonium citrate, such as the nitrate of SODIUMNITRATE and saltpetre, such as the organic nitrogen compound of peptone, yeast extract, meat extract and corn steep liquor and their mixture.

In addition, also the nutrition source such as inorganic salt, trace metal salts and VITAMIN that is used for ordinary culture medium suitably can be mixed use.

Also have no particular limits on culture condition, for example, can make pH and temperature suitably be controlled at the scope that pH is 5-8 under aerobic conditions, temperature be in 15-40 ℃ the scope, to cultivate about 12-about 48 hours.

The purifying of enzyme

Dna encoding peptide-forming enzyme of the present invention.For example, this peptide-forming enzyme can be subordinated to purifying in the bacterium of steady Bacillaceae.The method of separation and purifying peptide-forming enzyme will be described as the example of purifying enzyme from short steady bacillus.

At first, by adopting physical method such as ultrasonication or using the enzyme process smudge cells of molten cell walls enzyme and remove insoluble part by method such as centrifugal, thereby from short steady bacillus, for example (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: preparation microorganism cells extract in the microorganism cells on July 8th, 2002).By associating common method of purifying protein such as anion-exchange chromatography, cation-exchange chromatography or gel permeation chromatography,, the microorganism cells extract solution available from aforesaid method comes the purifying peptide-forming enzyme then through being carried out fractionation.

The example that is used as the carrier of anion-exchange chromatography is Q-Sepharose HP (Amersham production).When the cell extract process that will comprise enzyme has been filled the post of carrier, under the condition of pH8.5, can from non-absorbed portion, reclaim enzyme.

The example that is used as the carrier of cation-exchange chromatography is MonoS HR (Amersham production).Thereby make enzyme be adsorbed on the post by the cell extract that will comprise enzyme through the post of having filled carrier and subsequently after the washing column, with buffered soln wash-out enzyme with high salt concentration.At this moment, salt concn can be increased continuously and maybe concentration gradient can be adopted.For example, under the situation of using MonoS HR, be adsorbed on enzyme on the post with the NaCl wash-out of the about 0.5M of about 0.2-.

Enzyme with the aforesaid method purifying further can be carried out even purifying by gel permeation chromatography etc. then.The example that is used for the carrier of gel permeation chromatography is Sephadex 200pg (Amersham production).

In above-mentioned purification process, can generate activity by the peptide that detects each several part according to the described method of following embodiment, thereby confirm to comprise the part of enzyme.Shown internal amino acid sequence among SEQ ID NO:1 in the sequence table and the SEQ ID NO:2 with the enzyme of aforesaid method purifying.

(4) DNA of the present invention and transformant

(4-1) DNA of the present invention

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center from short steady bacillus strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: on July 8th, 2002), isolate the DNA of the present invention with base sequence of 61-1908 bit base described in the SEQ ID NO:5.The DNA that contains 61-1908 bit base described in the SEQ ID NO:5 is coding region sequence (hereinafter referred to as " CDS ") part.The base sequence of 61-1908 bit base comprises signal sequence district and mature protein district.The signal sequence district is made up of the 61-126 bit base, and ripe protein district is made up of the 127-1908 bit base.That is, the invention provides the peptase protein gene that comprises signal sequence and the peptase protein gene of mature protein form.The signal sequence that comprises in the sequence described in the SEQ ID NO:5 is a kind of leader sequence type, and the major function of inferring the leading peptide that this leader sequence is coded is the secretion in the outside from the cytolemma inboard to cytolemma.By the coded protein of 127-1908 bit base (i.e. site except that leading peptide) is mature protein, but infers that the peptide of its apparent altitude generates active.

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center from Sphingobacterium sp. bacterial strain FERM BP-8124, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002), isolate the DNA with base sequence of 61-1917 bit base described in the SEQ ID NO:11, it also is DNA of the present invention.DNA with base sequence of being made up of the 61-1917 bit base is encoding sequence (CDS) part.The base sequence of being made up of the 61-1917 bit base comprises signal sequence district and mature protein district.The signal sequence district is the zone of 61-120 bit base, and ripe protein district is the zone of 121-1917 bit base.That is, the invention provides the peptase protein gene that comprises signal sequence and the peptase protein gene of mature protein form.The signal sequence that comprises in the sequence described in the SEQ ID NO:5 is the leader sequence type.The major function of inferring the leading peptide that this leader sequence is coded is the secretion in the outside from the cytolemma inboard to cytolemma.By the coded protein of 121-1917 bit base (i.e. part except that leading peptide) is mature protein, but infers that the peptide of its apparent altitude generates active.

From separating Vitrum AB soil bacillus strain IFO 12017 (preservation mechanism: fermentation research institute, Osaka, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) in, isolate the DNA of the present invention of base sequence with the described 61-1935 bit base of SEQ ID NO:17.The DNA that is made up of described 61-1935 bit base among the SEQ ID NO:17 is the CDS part.Comprise signal sequence district and mature protein district in the base sequence of forming by the 61-1935 bit base.The signal sequence district is made up of the 61-126 bit base, and ripe protein district is made up of the 127-1935 bit base.That is, the invention provides the peptase protein gene that comprises signal sequence and the peptase protein gene of mature protein form.The signal sequence that comprises in the sequence described in the SEQ ID NO:17 is the leader sequence type, and the major function of inferring the leading peptide that this leader sequence is coded is the secretion in the outside from the cytolemma inboard to cytolemma.The coded protein of 127-1935 bit base (i.e. site except that leading peptide) is mature protein, but infers that the peptide of its apparent altitude generates active.

From Taxeobacter gelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg1b, 38124 Braunschweig isolate the DNA of the present invention with base sequence of 61-1995 bit base described in the SEQ ID NO:22 in Germany).The DNA that is made up of 61-1995 bit base described in the SEQ ID NO:22 is the CDS part.Comprise signal sequence district and mature protein district in the base sequence by the 61-1995 bit base.The signal sequence district is made up of the 61-126 bit base, and ripe protein district is made up of the 127-1995 bit base.That is, the invention provides the peptase protein gene that comprises signal sequence and the peptase protein gene of mature protein form.The signal sequence that comprises in the sequence described in the SEQ ID NO:22 is the leader sequence type, and the major function of inferring the leading peptide that this leader sequence is coded is the secretion in the outside from the cytolemma inboard to cytolemma.By the coded protein of 127-1995 bit base (i.e. site except that leading peptide) is mature protein, but infers that the peptide of its apparent altitude generates active.

From extra large round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the UnitedStates of America) in, isolates DNA of the present invention with base sequence of 29-1888 bit base described in the SEQ ID NO:24.The DNA that is made up of 29-1888 bit base described in the SEQ ID NO:24 is the CDS part.Contain in the base sequence of 29-1888 bit base and comprise signal sequence district and mature protein district.The signal sequence district is made up of the 29-103 bit base, and ripe protein district is made up of the 104-1888 bit base.That is, the invention provides the peptase protein gene that comprises signal sequence and the peptase protein gene of mature protein form.The signal sequence that is comprised in the described sequence of SEQ ID NO:24 is the leader sequence type, and the major function of inferring the leading peptide that this leader sequence is coded is the secretion in the outside from the cytolemma inboard to cytolemma.By the coded protein of 104-1888 bit base (i.e. site except that leading peptide) is mature protein, but infers that the peptide of its apparent altitude generates active.

From Psycloserpens burtonensis strains A TCC 700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) in, isolates the DNA of the present invention of base sequence with the described 61-1992 bit base of SEQ ID NO:26.The DNA that is made up of 61-1992 bit base described in the SEQ ID NO:26 is the CDS part.Contain in the base sequence of 61-1992 bit base and comprise signal sequence district and mature protein district.The signal sequence district is by the 61-111 bit base, and ripe protein district is made up of the 112-1992 bit base.That is, the invention provides the peptase protein gene that comprises signal sequence and the peptase protein gene of mature protein form.The signal sequence that comprises in the sequence described in the SEQ ID NO:31 is the leader sequence type, and the major function of inferring the leading peptide that this leader sequence is coded is the secretion in the outside from the cytolemma inboard to cytolemma.The coded protein (i.e. site except that leading peptide) of 112-1992 bit base is mature protein, but infers that the peptide of its apparent altitude generates active.

In addition, according to such as Molecular Cloning, 2nd edition, the description in the publication of Cold SpringHarbor Press (1989) can be implemented following several genes recombinant technology.

DNA of the present invention can pass through polymerase chain reaction (hereinafter referred to as " PCR ") (referring to PCR; White T.J. etc., Trends Genet., 5,185 (1989)) or by the hybridization of lacking steady bacillus, Sphingobacterium sp., the chromosomal DNA of separating Vitrum AB soil bacillus, Taxeobactergelupurpurascens, extra large round bacteria or Psycloserpens burtonensis or DNA library obtain.The PCR primer can be according to based on designing by the determined internal amino acid sequence of peptide-forming enzyme of carrying out purifying described in above-mentioned (3) joint.In addition, because the present invention has clearly determined the base sequence (SEQ IDNO:5, SEQ ID NO:11, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26) of peptide-forming enzyme gene, so can be designed for the primer or the probe of hybridization according to these base sequences, also can adopt this gene of probe separates.If will have primer with 5 ' non-translational region and the corresponding sequence of 3 ' non-translational region as PCR primer, the total length of the coding region of the enzyme of the present invention that then can increase.For example, when amplification comprises leader sequence described in the SEQ ID NO:5 and mature protein coding region regional, specifically, the example of 5 ' side primer is the primer with base sequence of the upstream region of 61 bit bases among the SEQ ID NO:5, and the example of 3 ' side primer is the primer that has with the base sequence complementary sequence of 1908 bit base downstream areas.

For example, the 380B type dna synthesizer that can adopt Applied Biosystems to produce according to a conventional method is by phosphoamidite method (referring to Tetrahedron Letters (1981), 22,1859) synthetic primer.For example, can adopt Gene Amp PCR System 9600 (Perkin-Elmer production) and TakaraLA PCR body outer clone test kit (Takara Shuzo production) enforcement PCR to react according to by provider's illustrated method of manufacturers for example.

No matter whether comprise leader sequence, also comprise among the DNA of the present invention with sequence table in SEQID NO:5 described in the same basically DNA of DNA of CDS.That is, the DNA of enzyme of the present invention that can be by having sudden change from coding or contain the cell of this DNA, be separated under the stringent condition with have with sequence table in SEQ ID NO:5 described in the DNA hybridization of CDS complementary base sequence, or with probe hybridization from the preparation of identical base sequence, and coding has peptide and generates active protein DNA and obtain the DNA same basically with DNA of the present invention.

No matter whether comprise leader sequence, also comprise among the DNA of the present invention with sequence table in SEQID NO:11 described in the same basically DNA of DNA of CDS.That is, the DNA of enzyme of the present invention that can be by having sudden change from coding or contain the cell of this DNA, be separated under the stringent condition with have with sequence table in SEQ ID NO:11 described in the DNA hybridization of CDS complementary base sequence, or with probe hybridization from the preparation of identical base sequence, and coding has peptide and generates active protein DNA and obtain the DNA same basically with DNA of the present invention.

No matter whether comprise leader sequence, also comprise among the DNA of the present invention with sequence table in SEQID NO:17 described in the same basically DNA of DNA of CDS.That is, the DNA of enzyme of the present invention that can be by having sudden change from coding or contain the cell of this DNA, be separated under the stringent condition with have with sequence table in SEQ ID NO:17 described in the DNA hybridization of CDS complementary base sequence, or with probe hybridization from the preparation of identical base sequence, and coding has peptide and generates active protein DNA and obtain the DNA same basically with DNA of the present invention

No matter whether comprise leader sequence, also comprise among the DNA of the present invention with sequence table in SEQID NO:22 described in the same basically DNA of DNA of CDS.That is, the DNA of enzyme of the present invention that can be by having sudden change from coding or contain the cell of this DNA, be separated under the stringent condition with have with sequence table in SEQ ID NO:22 described in the DNA hybridization of CDS complementary base sequence, or with probe hybridization from the preparation of identical base sequence, and coding has peptide and generates active protein DNA and obtain the DNA same basically with DNA of the present invention.

No matter whether comprise leader sequence, also comprise among the DNA of the present invention with sequence table in SEQID NO:24 described in the same basically DNA of DNA of CDS.That is, the DNA of enzyme of the present invention that can be by having sudden change from coding or contain the cell of this DNA, be separated under the stringent condition with have with sequence table in SEQ ID NO:24 described in the DNA hybridization of CDS complementary base sequence, or with probe hybridization from the preparation of identical base sequence, and coding has peptide and generates active protein DNA and obtain the DNA same basically with DNA of the present invention.

No matter whether comprise leader sequence, also comprise among the DNA of the present invention with sequence table in SEQID NO:26 described in the same basically DNA of DNA of CDS.That is, the DNA of enzyme of the present invention that can be by having sudden change from coding or contain the cell of this DNA, be separated under the stringent condition with have with sequence table in SEQ ID NO:26 described in the DNA hybridization of CDS complementary base sequence, or with probe hybridization from the preparation of identical base sequence, and coding has peptide and generates active protein DNA and obtain the DNA same basically with DNA of the present invention

For example, can be according to fixed method, prepare probe according to the base sequence described in the SEQ ID NO:5 in the sequence table for example.In addition, also can be according to fixed method, implement by using probe to find DNA with probe hybridization, thus the method that is used to separate target DNA.For example, dna probe can be sheared the base sequence that needs as probe with Restriction Enzyme by the base sequence that increases and clone in plasmid or phage vector, extract required base sequence then and prepare.The part that cuts off can be adjusted according to target DNA.

Term used herein " under stringent condition " is meant the so-called specific hybrid of formation under this condition, and does not form non-specific hybridization.Represent accurately that with numerical value this condition is difficult.For example, these conditions can be, the high DNA of homology (for example, 50% or higher, preferred 80% or higher, more preferably 90% or higher) can hybridize each other and condition that homology can not be hybridized each other than these low DNA, or the usual terms of washing in the Southern hybridization, wherein hybridize with 1 * SSC and 0.1%SDS, under the corresponding salt concn of preferred 0.1 * SSC and 0.1%SDS, carry out in 60 ℃.Though under the described conditions Za Jiao gene comprise occur on the specific position in the sequence terminator or since the active centre in sudden change and those genes of loss of activity, but can be by being connected to commercially available expression vector, thereby the enzymic activity of it being expressed in suitable host and adopting following method to detect expression product is removed it at an easy rate.

But, when base sequence is hybridized under above-mentioned stringent condition, under this base sequence encoded protein condition that is selected in 50 ℃ and pH8 of fine quality, keep have by as the proteinic pact of the coded aminoacid sequence of the original base sequence that keeps half or higher, preferred 80% or higher, more preferably 90% or higher enzymic activity.For example, when being described as follows situation, during the base sequence that for example illustrates under stringent condition and have with the DNA hybridization of the base sequence complementary base sequence of the 127-1908 bit base of base sequence shown in the SEQ ID NO:5, under this base sequence encoded protein condition that is selected in 50 ℃ and pH8 of fine quality, keep the 23-616 amino acids residue with aminoacid sequence shown in the SEQ IDNO:6 aminoacid sequence proteinic pact half or higher, preferred 80% or higher, more preferably 90% or higher enzymic activity.

Shown among the SEQ ID NO:6 in the sequence table by the coded aminoacid sequence of the described CDS of SEQ ID NO:5 in the sequence table.Shown among the SEQ ID NO:12 in the sequence table by the coded aminoacid sequence of the described CDS of SEQ ID NO:11 in the sequence table.Shown among the SEQ ID NO:18 in the sequence table by the coded aminoacid sequence of the described CDS of SEQ ID NO:17 in the sequence table.Shown among the SEQ ID NO:23 in the sequence table by the coded aminoacid sequence of the described CDS of SEQ ID NO:22 in the sequence table.Shown among the SEQ IDNO:25 in the sequence table by the coded aminoacid sequence of the described CDS of SEQ ID NO:24 in the sequence table.Shown among the SEQ ID NO:27 in the sequence table by the coded aminoacid sequence of the described CDS of SEQ ID NO:26 in the sequence table.

Complete amino acid sequence comprises leading peptide and mature protein district described in the SEQ ID NO:6, and wherein 1-22 amino acids residue constitutes leading peptide, and 23-616 amino acids residue constitutes the mature protein district.In addition, complete amino acid sequence comprises leading peptide and mature protein district described in the SEQ ID NO:11, and wherein 1-20 amino acids residue constitutes leading peptide, and 21-619 amino acids residue constitutes the mature protein district.

Complete amino acid sequence comprises leading peptide and mature protein district described in the SEQ ID NO:18, and wherein 1-22 amino acids residue constitutes leading peptide, and 23-625 amino acids residue constitutes the mature protein district.

Complete amino acid sequence described in the SEQ ID NO:23 comprises leading peptide and mature protein district, and wherein 1-22 amino acids residue constitutes leading peptide, and 23-645 amino acids residue constitutes the mature protein district.

Complete amino acid sequence described in the SEQ ID NO:25 comprises leading peptide and mature protein district, and wherein 1-25 amino acids residue constitutes leading peptide, and 26-620 amino acids residue constitutes the mature protein district.

Complete amino acid sequence described in the SEQ ID NO:27 comprises leading peptide and mature protein district, and wherein 1-17 amino acids residue constitutes leading peptide, and 18-644 amino acids residue constitutes the mature protein district.

The coded protein of DNA of the present invention is that wherein mature protein has peptide and generates active protein, comprise also among the DNA of the present invention that coding and the protein with aminoacid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:23, SEQ ID NO:25 or the SEQ ID NO:27 in the sequence table goes up same protein DNA substantially, no matter whether it comprises leading peptide (is noted, according to universal code, base sequence is determined by aminoacid sequence).That is, the invention provides protein DNA shown in the coding following (A)-(X):

(B) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 23-616 amino acids residue of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(D) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 21-619 amino acids residue of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(E) have the protein of the aminoacid sequence that the 23-625 amino acids residue of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table forms,

(F) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 23-625 amino acids residue of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(H) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 23-645 amino acids residue of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(J) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 26-620 amino acids residue of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(K) have the protein of the aminoacid sequence of forming by the 18-644 amino acids residue of aminoacid sequence shown in the SEQ ID NO:32 in the sequence table,

(L) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 18-644 amino acids residue of aminoacid sequence shown in the SEQ ID NO:32 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(M) contain the protein of aminoacid sequence shown in the SEQ ID NO:6 in the ordered list,

(N) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in shown in the SEQ ID NO:6 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(P) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in shown in the SEQ ID NO:12 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(R) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in shown in the SEQ ID NO:18 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(T) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in shown in the SEQ ID NO:23 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(V) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in shown in the SEQ ID NO:25 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

(W) have aminoacid sequence shown in the SEQ ID NO:27 in the sequence table protein and

(X) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in shown in the SEQ ID NO:27 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.

Herein, though the implication of term " a plurality of " changes according to the position and the type of amino-acid residue in the proteinic three-dimensional structure, its scope comprises the number that can significantly not damage the amino-acid residue of three-dimensional structure and protein active, is specially 2-50, be preferably 2-30, more preferably 2-10.But, in (B), (D), (F), (H), (J), (L), (N), (P), (R), (T), (V) or proteinic aminoacid sequence (X), comprise displacement, disappearance, insertion, add, and/or under the situation of the aminoacid sequence of the one or more amino acid gained of inversion, protein is preferably under the condition of 50 ℃ and pH8, keep the proteinic pact be under the state that does not comprise sudden change half or higher, preferred 80% or higher, more preferably 90% or higher enzymic activity.For example, the situation to (B) provides explanation; When aminoacid sequence (B) is shown in the SEQ ID NO:6 in sequence table in the aminoacid sequence, comprise displacement, disappearance, insertion, add, and/or under the one or more amino acid whose situations of inversion, this protein is preferably under the condition of 50 ℃ and pH8, keep aminoacid sequence shown in the SEQ ID NO:6 have in the sequence table proteinic pact half or higher, preferred 80% or higher, more preferably 90% or higher enzymic activity.

Amino acid mutation shown in similar above-mentioned (B) etc. can obtain by the following method, for example by the site-directed mutagenesis method, and the base sequence that the amino acid of the privileged site of this enzyme gene is replaced, lack, inserts, adds change.In addition, the DNA of above-mentioned change also can obtain by known mutagenic treatment.Mutagenic treatment is meant, for example, with azanol etc. in the encode method of DNA of this enzyme of extracorporeal treatment, and, handle the method for the Escherichia bacterium of the DNA that comprises this enzyme of encoding such as uviolizing, N-methyl-N '-nitro-N-nitrosoguanidine (NTG) or nitrous acid with the mutagenic compound that are generally used for induced mutations.

In addition, the displacement of above-mentioned base, disappearance, insertion, interpolation and/or inversion comprise that also naturally occurring sudden change is such as because the difference that microbial species or bacterial strain cause.By in suitable cell, expressing DNA with described sudden change and the enzymic activity that detects expression product, can obtain to encode with sequence table in SEQ ID NO:6 or 12 described in protein go up same protein DNA substantially.

(4-2) production of the preparation of transformant and peptide-forming enzyme

Peptide-forming enzyme can by DNA of the present invention is imported suitable host and in this host expressible dna produce.

The example that can be used for expressing by the determined proteinic host of DNA of the present invention comprises that multiple prokaryotic cell prokaryocyte is such as belonging to Escherichia such as intestinal bacteria, steady bacillus, Sphingobacterium, the bacterium of Flavobacterium and Bacillaceae such as Bacillus subtilus, and multiple eucaryon is such as yeast saccharomyces cerevisiae, pichia and aspergillus oryzae.

Be used for the recombinant DNA that DNA imports the host can be inserted with the corresponding carrier of type that will express the host of this DNA by the DNA that will need to import and prepare, can be expressed by the coded protein of this DNA in this mode.Under promotor to the function of peptide-forming enzyme gene in host cell of short steady bacillus etc. was unique situation, this promotor can be used as the promotor of expressing DNA of the present invention.In addition, another promotor that works in host cell can be connected with DNA of the present invention, can under the control of this promotor, express this DNA on demand then.

The example that recombinant DNA is imported the method for transformation of host cell comprises that the method for D.M.Morrison is (referring to Methods in Enzymology, 68,326 (1979)) or by handling the recipient microorganism cell increase the method for DNA permeability (referring to Mandel with calcium chloride, H. and Higa, A., J.Mol.Biol., 53,159 (1970)).

Using under the proteinic situation of recombinant DNA technology mass production, also is to implement preferred implementation of the present invention with the protein bound that produces in the proteinic transformant to form the protein inclusion body.The advantage of this expression and production method comprises the digestion of the proteolytic enzyme that the protection target protein is avoided existing in the microorganism cells, and by the cracking microorganism cells, the centrifugal then simple and easy ground purifying target protein etc. that gets final product.

The protein inclusion body that obtains in this mode dissolves with protein denaturant, by the active regeneration step dissolved protein transduction is turned to suitable fold, physiologically active protein matter, described active regeneration step mainly comprises with protein denaturant crack protein matter, removes denaturing agent then.This process has a lot of examples, comprises Human Inter Leukin-2's active regeneration (opening clear S61-257931 referring to the Japanese Patent spy).

In order from the protein inclusion body, to obtain active protein, need carry out sequence of operations, comprise the dissolving and active regeneration, this method steps than direct preparation active protein complexity many.But, in microorganism cells, microorganism growth is had under the proteinic situation of large volume of deleterious effect producing, can suppress described effect with the protein of the inclusion body form of inactive protein matter by accumulation in microorganism cells.

The example of the method for the large volume target protein of mass production inclusion body form is included in the down independent method of expressing target protein of control of potent promotor, and the method for target protein to express with the form of the fused protein of known large volume expressed protein.

Below, will more specifically explain the present invention as example with preparation intestinal bacteria that transform and the method for using microorganism transformed to produce peptide-forming enzyme.In addition, microorganism such as intestinal bacteria in preparation during peptide-forming enzyme, can mix the DNA of coding precursor protein matter (comprising leader sequence), maybe can mix the DNA that forms by the mature protein district that does not comprise leader sequence, and can be suitable for the DNA of protein coding sequence according to selections such as the working condition of the enzyme that will produce, form, working conditionss.

The promotor that is often used in production heterologous protein in the intestinal bacteria can be used as the promotor of the DNA that expresses the coding peptide-forming enzyme.The example of described promotor comprises T7 promotor, lac promotor, trp promotor, trc promotor, tac promotor, lambda phage PR promotor, PL promotor and other strong promoter.In addition, the example of spendable carrier comprises pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219 and pMW218.In addition, can also use the carrier of phage DNA.And, can use the expression vector that comprises promotor and can express the dna sequence dna (comprising the available promotor) that inserts.

In order to produce the peptide-forming enzyme of fused protein inclusion body form, another protein of will encoding (preferred hydrophilic peptide) thus the gene upstream or the downstream that are connected to peptide-forming enzyme gene obtain the fusion rotein plasmagene.In this mode, another proteinic gene of encoding can be the amount that can increase the cumulative fused protein, and strengthens the deliquescent any gene of fused protein after sex change and regeneration step.Candidate's example of described gene comprises T7 gene 10, beta-galactosidase gene, dihydrofolate reductase gene, gamma-interferon gene, interleukin-2 gene and prochymosin gene.

When these genes are connected with the gene of coding peptide-forming enzyme, full gene is connected so that make the reading frame unanimity of its codon.This gene can be connected to suitable restriction enzyme sites, maybe can use the synthetic DNA that contains suitable sequence.

In addition, in order to increase the output of peptide-forming enzyme, preferably in some cases terminator (transcription termination sequence) is connected to the downstream of fusion rotein plasmagene.This terminator comprises, for example, and T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator and intestinal bacteria trpA gene terminator.

In intestinal bacteria, import the preferred so-called multiple copied type carrier of carrier of the gene of the fused protein between coding peptide-forming enzyme or peptide-forming enzyme and other protein; the example comprise have the replication orgin that comes from Co1E1 plasmid; for example; based on the plasmid of pUC, and based on the plasmid or derivatives thereof of pBR322." derivative " as used herein is meant those plasmids that displacement, disappearance, insertion, interpolation and/or the inversion by base modified.Should be noted that modification as used herein comprises by the modification with the mutagenic treatment of mutagenic compound or UV irradiation, or the modification by spontaneous mutation.

Be the screening transformant, carrier preferably contains the marker such as the Ampicillin Trihydrate resistant gene.Described plasmid comprise can the commercial expression vector that contains effectively start that obtains (based on the carrier (production of Takara Shuzo company) of pUC, based on the carrier (production of ClonetechLaboratories company) of pRROK, pKK233-2 (production of Clonetech Laboratories company) etc.

Obtain recombinant DNA by dna fragmentation being connected to carrier DNA; In described dna fragmentation, connect promotor in the following sequence, coding L-amino acid amide lytic enzyme or comprise the gene (according to circumstances) of L-amino acid amide lytic enzyme and another proteinic fused protein, terminator.

When cultivating the gained intestinal bacteria then with the recombinant DNA transformed into escherichia coli, peptide-forming enzyme or comprise peptide-forming enzyme and another proteinic fused protein and expressed and produce.Can be used as and transform the host though be generally used for the bacterial strain of expression of heterologous genes, preferred, coli strain JM109 for example.The method that the method that implement to transform and being used to filters out transformant is described in Molecular Cloning, and 2nd Edition is in Cold Spring Harbor Press (1989) and other publication.

When the peptide-forming enzyme of expressed fusion protein matter form, can use with peptide-forming enzyme, come the cracking peptide-forming enzyme such as non-existent sequence in factor Xa or the kallikrein as the restricted proteolytic enzyme of recognition sequence.

Can be with being generally used for cultivating colibacillary substratum, such as M9-casamino acids substratum or LB substratum, as producing substratum.In addition, wait according to marker, promotor, the host microorganism type of used carrier and select the suitable culture condition and produce inductive condition.

Following method can be used to reclaim peptide-forming enzyme and comprise peptide-forming enzyme and another proteinic fused protein.If this peptide-forming enzyme or its fused protein are dissolved in microorganism cells, then reclaim this microorganism cells, then with its fragmentation or cracking so that used as crude enzyme liquid.And, can carry out purifying by ordinary skill such as precipitation, filtration or column chromatography to this peptide-forming enzyme or its fused protein as required before using.At this moment, also can use the purifying antibody method that adopts peptide-forming enzyme or its fused protein.

Under the situation that forms the protein inclusion body, dissolve this inclusion body with denaturing agent.Though it can dissolve with microorganism cells protein, considers purification step subsequently, so the preferred inclusion body that takes out dissolves then.Can adopt conventional currently known methods from microorganism cells, to reclaim inclusion body.For example, thus can be by the centrifugal then recovery of cracking microorganism cells inclusion body.The example that can dissolve the denaturing agent of inclusion body comprise Guanidinium hydrochloride (for example, 6M, pH5-8) and urea (for example, 8M) etc.

Having active protein waits by dialysis and removes these denaturing agents and regenerated.The Tris-HCl damping fluid, phosphate buffer solutions etc. can be used as dialyzate used in the dialysis, and its concentration can be, for example, 20mM-0.5M, and its pH can be, and for example, 5-8.

Protein concn during the regeneration step preferably is maintained at about 500 μ g/ml or lower.The dialysis temperature is preferably 5 ℃ or lower so that prevent regenerated peptide-forming enzyme generation self-crosslinking.In addition, except dialysing, the method for removing denaturing agent comprises dilution or ultrafiltration, and which kind of denaturing agent activity no matter expection use all can be reproduced.

(5) characteristic of the coded enzyme of DNA of the present invention

For example, can react in comprising amino acid ester and the amine borate buffer solution as substrate by making enzyme, the peptide that quantitatively generates detects the activity of the coded enzyme of DNA of the present invention then.In embodiment more specifically, adopt the borate buffer solution (pH9.0) that comprises 100mM L-alanine methyl ester and 200mM L-glutaminate, implement the reaction some minutes at 25 ℃.

The definition of the activity unit of used enzyme is among the present invention, 1 unit (U) is the 100mM borate buffer solution (pH9.0) that comprises 100mM L-alanine methyl ester and 200mM L-glutaminate in use, under 25 ℃ the reaction conditions, produce the enzyme amount of 1 micromole (μ mole) peptide in 1 minute.

The coded protein of DNA of the present invention is peptide-forming enzyme protein.Peptide generates the active activity that is generated peptide by carboxyl composition and amine component that is meant.Below, will the characteristic of the preferred implementation of the coded enzyme of DNA of the present invention be described.

A kind of preferred implementation of the enzyme that DNA of the present invention is coded comprises having following active enzyme, and dipeptides productivity is as its indicator.That is, a kind of preferred implementation of enzyme of the present invention comprises having the activity that is generated peptide by carboxyl composition and amine component, and under the condition of following (i)-(iv), in the dipeptides formation reaction, has the 0.03mM/min of being preferably or higher, more preferably 0.3mM/ minute or higher, the enzyme of the productivity of preferred especially 1.0mM/min or higher L-alanyl-L-glutamine.The condition of dipeptides formation reaction is as follows:

(i) the carboxyl composition is a L-alanine methyl ester hydrochloride (100mM);

(ii) amine component is L-glutaminate (200mM);

(iii) pH is 9.0; With,

(iv) according to protein mass, evenly the add-on of the enzyme of purifying is lower than 0.61mg/ml.

Above-mentioned productivity is far above the conventional productivity of using the enzymic synthesis peptide, and enzyme of the present invention has the catalysis peptide with very fast speed synthetic ability.

The above-mentioned add-on indication of enzyme adds the final quantity of the enzyme of reactive system, and by required zymoprotein quality, the enzyme add-on is 0.01mg/ml or higher, preferred 0.02mg/ml or higher.Term " protein mass " is meant by adopting protein determination CBB solution (Nakarai production) and bovine serum albumin as reference material, by the shown value of Xylene Brilliant Cyanine G colorimetry.

In the specific examples of the method for measuring enzymic activity, can in comprising amino acid ester and amine borate buffer solution, react by making enzyme, and the peptide that quantitatively obtains detects enzymic activity as substrate.In example more specifically, method comprises uses the 100mM borate buffer solution (pH9.0) that comprises 100mM L-alanine methyl ester and 200mM L-glutaminate, makes the enzyme reaction some minutes in 25 ℃.

In addition, the preferred implementation of the coded enzyme of DNA of the present invention comprises that have can be with amino acid ester and amino acid amide all as the enzyme of the ability of the substrate of carboxyl composition.Word " with amino acid ester and amino acid amide all as substrate " be meant at least one class or more multiclass amino acid ester and at least one class or more the multiclass amino acid amide can be used as substrate.In addition, a kind of preferred implementation of enzyme of the present invention has the amino acid of all amino acid, C protection and the amine enzyme as the ability of the substrate of amine component.Word " amino acid of amino acid, C protection and amine are as substrate " is meant at least one class or multiclass amino acid more, the amino acid of at least one class or more multiclass C protection and at least one class or more multiclass amine can be used as substrate.Owing to have the substrate specificity widely of carboxyl composition or amine component, enzyme of the present invention is preferred, wherein can select raw material widely, and it is beneficial in the industrial production needs to expense and production unit successively.

The specific examples of carboxyl composition comprises the L-amino acid ester, D-amino acid ester, L-amino acid amide and D-amino acid amide.In addition, amino acid ester not only comprises and the corresponding amino acid ester of naturally occurring amino acid, also comprises the corresponding amino acid ester of amino acid or derivatives thereof that exists with non-natural.In addition, the example of amino acid ester comprise α-An Jisuanzhi and β-, γ-and omega-amino-acid esters etc., it has different amino binding sites.The representative instance of amino acid ester comprises amino acid whose methyl esters, ethyl ester, n-propyl, isopropyl ester, positive butyl ester, isobutyl ester and the tert-butyl ester.

The specific examples of amine component comprises L-amino acid, the L-amino acid of C protection, D-amino acid, the D-amino acid and the amine of C protection.In addition, the example of amine not only comprises naturally occurring amine, also comprises the amine or derivatives thereof that non-natural exists.In addition, amino acid whose example not only comprises naturally occurring amino acid, also comprises the amino acid or derivatives thereof that non-natural exists.It comprises a-amino acid and β-, γ-and omega-amino acid etc., it has different amino binding sites.

In addition, on the other hand, a kind of preferred implementation of the enzyme that DNA of the present invention is coded comprises that the pH scope of catalysis peptide formation reaction is the enzyme of 6.5-10.5.Preferably can be in above-mentioned wide in range pH scope the enzyme of the present invention of catalyzed reaction, it can adapt to the industrial production that multiple restriction may occur neatly.But in the actual generation of peptide, when using enzyme, preferably further be adjusted to the corresponding optimal pH of gained enzyme so that with the katalysis maximization of enzyme.

And on the other hand, a kind of preferred implementation of the enzyme that DNA of the present invention is coded comprises that the temperature range of catalysis peptide formation reaction is 0-60 ℃ a enzyme.Because endonuclease capable of the present invention is catalyzed reaction in above-mentioned wide in range temperature range, so it is preferred, it can adapt to the industrial production that multiple restriction may occur neatly.But in the actual generation of peptide, when using enzyme, preferably further be adjusted to the corresponding optimum temperuture of gained enzyme so that with the katalysis maximization of enzyme.

(6) two peptide production methods

Two peptide production methods of the present invention are included in when having above-mentioned enzyme, the reaction between carboxyl composition and the amine component.Two peptide production methods of the present invention comprise making to have the enzyme that is generated the ability of peptide by carboxyl composition and amine component, thereby or contain the enzyme thing and act on carboxyl composition and the synthetic dipeptides of amine component.

Making the used enzyme of the present invention or contain method that the enzyme thing acts on carboxyl composition and amine component can be to be mixed with each other enzyme or contain the enzyme thing, carboxyl composition and amine component.More specifically, can adopt enzyme or contain the enzyme thing and add the method that the solution that contains carboxyl composition and amine component makes its reaction then.Alternatively, under the situation of the microorganism that use to produce this enzyme, can use to comprise and cultivate the microorganism that produces this enzyme, in microorganism or microbial culture medium, produce and the accumulation enzyme, then carboxyl composition and amine component be added the method for nutrient solution.Collect the dipeptides that produces by fixed method then, and carry out purifying as required.

Term " contains the enzyme thing " and is meant any material that comprises enzyme, the example of its specific form comprises the culture of the microorganism that can produce enzyme, from the isolating microorganism cells of culture with by handling the product (hereinafter referred to as, " microorganism cells processing product ") that microorganism cells obtains.The culture of microorganism is meant by culturing micro-organisms resulting, more specifically, is meant microorganism cells, is used for the substratum of culturing micro-organisms and the mixture of the material that produced by cultured microorganism etc.In addition, can wash microorganism cells, and use with the form of microorganism cells of washing.In addition, microorganism cells is handled product and is comprised cracking, dissolving or freeze dried microorganism cells etc., also comprise by handling thick enzyme that microorganism cells reclaims etc., and the enzyme of the purifying that obtains by the thick enzyme of purifying etc.The partially purified enzyme that obtains by multiple purification process can be used as the enzyme of purifying, maybe can use by method fixed immobilized enzyme such as covalent attachment method, absorption method, entrapping methods.In addition, because certain micro-organisms can generating unit divide cracking (depending on employed microorganism) in the training period, culture supernatant also can be used as and contain the enzyme thing at this moment.

In addition, wild strain can be used as the microorganism that comprises enzyme, or also can use the gene recombination bacterial strain of expressing this enzyme.Microorganism is not limited to complete microorganism cells, can also use the microorganism cells of acetone treatment, cryodesiccated microorganism cells or other treated microorganism cells.Can also use by covalent attachment method, absorption method, entrapping method or other method fixation of microbe cell and microorganism cells and handle fixation of microbial cell and the fixation of microbial cell processing product that product obtains, and the fixation of microbial cell of handling.

In addition, when handling product, wherein can occur not participating in the enzyme that peptide generates usually and decompose the peptide that generates when the microorganism cells that uses culture, cultured microorganism cell, washing or by the microorganism cells that fragmentation or cracking microorganism cells obtain.At this moment, preferred inhibitors of metalloproteinase such as the ethylenediamine tetraacetic acid (EDTA) (EDTA) of adding under some situation.Add-on is 0.1 mmole (mM)-300mM, preferred 1mM-100mM.

The preferred implementation of two peptide production methods of the present invention is following methods, cultivates above-mentioned (4-2) described transformant in substratum, and peptide-forming enzyme is accumulated in substratum and/or transformant.Because by using transformant can easily produce peptide-forming enzyme in a large number, so can be in a large number and produce dipeptides apace.

Reach the target effect if enzyme or the usage quantity that contains the enzyme thing demonstrate, then this amount is enough (significant quantity), and those of ordinary skills can easily determine this significant quantity by simple preliminary experiment.For example, under the situation of using enzyme, usage quantity is the about 100U of about 0.01U-, and under the situation of the microorganism cells that uses washing, usage quantity is the about 500g/L of about 1g/L-.

Any carboxyl composition is spendable, as long as it can generate peptide by the condensation with other material of amine component form.The example of carboxyl composition comprises the L-amino acid ester, D-amino acid ester, L-amino acid amide and D-amino acid amide and the organic acid acetic that does not contain amino.In addition, the example of amino acid ester not only comprises and the corresponding amino acid ester of naturally occurring amino acid, also comprises the corresponding amino acid ester of amino acid or derivatives thereof that exists with non-natural.In addition, the example of amino acid ester comprise α-An Jisuanzhi and β-, γ-and omega-amino-acid esters etc., it has different amino binding sites.The representative instance of amino acid ester comprises amino acid whose methyl esters, ethyl ester, n-propyl, isopropyl ester, positive butyl ester, isobutyl ester and the tert-butyl ester.

Any amine component is spendable, as long as it can generate peptide by the condensation with other material of amine component form.The example of amine component comprises L-amino acid, the L-amino acid of C protection, D-amino acid, the D-amino acid and the amine of C protection.In addition, the example of amine not only comprises naturally occurring amine, also comprises the amine or derivatives thereof that non-natural exists.In addition, amino acid whose example not only comprises naturally occurring amino acid, also comprises the amino acid or derivatives thereof that non-natural exists.It comprises a-amino acid and β-, γ-and omega-amino acid etc., it has different amino binding sites.

Be respectively 1mM to 10M as the carboxyl composition of raw material and the concentration of amine component, be preferably 0.05M to 2M; But, in some cases, preferably to add amine component with carboxyl become to grade mole or the amount of molar excess.In addition, under the situation of the material inhibited reaction of high density, can after being adjusted to the concentration that does not cause suppressing, it during reaction it be added step by step.

Allow that peptide synthetic temperature of reaction is 0-60 ℃, preferred 5-40 ℃.In addition, allow that peptide synthetic reaction pH is 6.5-10.5, preferred 7.0-10.0.

Embodiment

Below, explain the present invention by embodiment.But the present invention is not limited to these embodiment.Except the triketohydrindene hydrate dyeing (qualitative) with membrane chromatographic confirms, implement detection by quantitative so that measure product by following high performance liquid chromatography.Post: Inertsil ODS-2 (production of GLScience company), elutriant: phosphate aqueous solution, wherein comprise 5.0mM 1-perfluorooctane sulfonate (pH2.1): methyl alcohol=100: 15-50, flow velocity: 1.0mL/ minute, detect: 210 nanometers (hereinafter referred to as " nm ").

(embodiment 1) microorganism culturing (short steady bacillus strain FERM BP-8113)

In 500ml slope Kou Shi bottle, add the substratum (pH6.2) that 50mL every liter (L) contains 5 gram (g) glucose, 5g ammonium sulfate, 1g potassium primary phosphate, 3g dipotassium hydrogen phosphate, 0.5g sal epsom, 10g yeast extract and 10g peptone, 115 ℃ of sterilizations 15 minutes.Contained on the same substratum at inoculation 1 platinum loop on this substratum then, cultivated 16 hours the steady bacillus strain FERM of weak point BP-8113 (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst has speciallyyed permit biological sustenance center for 30 ℃, preservation mechanism address: Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: cell on July 8th, 2002), at 30 ℃, 120 reciprocal/minute following concussions were cultivated 16 hours then.

(embodiment 2) produce peptide with microorganism cells

To the medium centrifugal (10,000 rev/mins (rpm), 15 minutes) that obtains among the embodiment 1, collect microorganism cells, then it being suspended to concentration in containing the 100mM borate buffer solution (pH9.0) of 10mM EDTA is 100g/L.Contain in following carboxyl composition of 10mM EDTA, 200mM and the following amino acid whose 100mM borate buffer solution of 400mM (pH9.0) at each 1mL, add this suspension of 1ml respectively, make total amount, 18 ℃ of reactions 2 hours to 2mL.Shown the peptide that this reaction result generated in the table 1.

Table 1

The carboxyl composition	Amine component	The peptide (mM) that generates		The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Ala-OMe	??L-Leu	??L-Ala-L-Leu	??38.2	??Gly-OMe	??L-His	??L-Gly-L-His	??22.1
??L-Met	??L-Ala-L-Met	??68.3	??L-Ser-OMe	??L-Ser	??L-Ser-L-Ser	??29.0			??L-Leu	??L-Ala-L-Leu	??38.2	??Gly-OMe	??L-His	??L-Gly-L-His	??22.1
??L-Met	??L-Ala-L-Met	??68.3	??L-Ser-OMe	??L-Ser	??L-Ser-L-Ser	??29.0	??L-Phe		??L-Ala-L-Phe	??62.4	??L-Val-OMe	??L-Met	??L-Val-L-Met	??10.5
??L-Ser	??L-Ala-L-Ser	??51.3	??L-Met-OMe	??L-Phe	??L-Met-L-Phe	??28.5	??L-Phe		??L-Ala-L-Phe	??62.4	??L-Val-OMe	??L-Met	??L-Val-L-Met	??10.5

??L-His	??L-Ala-L-His	??52.1	??L-Thr-OMe	??L-Leu	??L-Thr-L-Leu	??23.0
??L-His	??L-Ala-L-His	??52.1	??L-Thr-OMe	??L-Leu	??L-Thr-L-Leu	??23.0	??L-Arg	??L-Ala-L-Arg	??72.1	??L-Ile-OMe	??L-Met	??L-Ile-L-Met	??8.3
??L-Gln	??L-Ala-L-Gln	??68.0					??L-Arg	??L-Ala-L-Arg	??72.1	??L-Ile-OMe	??L-Met	??L-Ile-L-Met	??8.3

(all carboxyl compositions are all used hydrochloride.)

The purifying of (embodiment 3) enzyme

Step after centrifugal is on ice or 4 ℃ of enforcements.(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to cultivate short steady bacillus strain FERM BP-8113 according to the method identical with embodiment 1, preservation mechanism address: Chuo Dai-6,1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: on July 8th, 2002), collect microorganism cells by centrifugal (10,000rpm, 15 minutes).After with 50mM Tris-HCl damping fluid (pH8.0) washing 16g microorganism cells, it is suspended in 40 milliliters of (ml or mL) same buffer, carry out ultrasonic degradation with 195 watts then and handled 45 minutes.Centrifugal then this ultrasonic degradation liquid (10,000rpm, 30 minutes) to remove cell debris, obtains ultrasonic degradation liquid supernatant., remove insoluble part by ultracentrifugation (50,000rpm, 30 minutes) then, thereby obtain the soluble part of supernatant liquor form this ultrasonic degradation liquid supernatant dialysed overnight with 50mM Tris-HCl damping fluid (pH8.0).The soluble part adding that obtains is used in Tris-HCl damping fluid (pH8.0) the equilibrated Q-Sepharose HP post (Amersham production) in advance, from non-absorbed portion, collected active part then.With 50mM acetate buffer (pH4.5) to this active part dialysed overnight, then by centrifugal (10,000rpm, 30 minutes) thus remove the part that insoluble part obtains the dialysis of supernatant liquor form.The part that to dialyse then adds to be used in 50mM acetate buffer (pH4.5) the equilibrated Mono S post (Amersham production) in advance, with the linear concentration gradient wash-out enzyme of the same buffer that contains 0-1M NaCl.The part that contains minimum level contaminating protein matter in the active part is added in advance with containing in 50mM acetate buffer (pH4.5) the equilibrated Superdex 200pg post (Amersham product) of 1M NaCl, by making the same buffer (pH4.5) that the contains 1M NaCl post of flowing through implement gel-filtration, obtain active part solution.The result of aforesaid operations confirmed, according to electrophoretic experimental result, used peptide-forming enzyme is by purifying equably among the present invention.The enzyme rate of recovery in the above-mentioned purification step is 12.2%, and purification degrees is 707 times.

(embodiment 4) detect the molecular weight of enzyme

(SDS-gel electrophoresis)

0.3 microgram (μ g) equivalent to the enzyme part of the method gained purifying by embodiment 3 carries out polyacrylamide gel electrophoresis.With 0.3% (w/v) Tris, 1.44% (w/v) glycine and 0.1% (w/v) Sulfuric acid,monododecyl ester, sodium salt are as electrophoretic buffer, gel (the Multigel 10-20 that will have the concentration gradient of 10-20% gel strength, Daiichi Pure Chemicals produces) as polyacrylamide gel, Pharmacia molecular weight marker thing is used as the molecular weight marker thing.After electrophoresis is finished, to gel-colored, find the homogeneous band in the molecular weight position of about 75 kilodaltons (kDa) with Xylene Brilliant Cyanine G R-250.

(gel-filtration)

The enzyme of method gained purifying that will be by embodiment 3 partly adds in advance with containing in 50mM acetate buffer (pH4.5) the equilibrated Superdex 200pg post (Amersham product) of 1M NaCl, by making the same buffer (pH4.5) that contains 1M NaCl thus the post of flowing through is implemented gel-filtration detection molecules amount.The molecular weight marker thing that Pharmacia molecular weight marker thing is used as the known molecular amount is to make working curve.As a result, the molecular weight of this enzyme is about 150kDa.

According to the result of SDS-gel electrophoresis and gel-filtration, pointing out this enzyme is the homodimer with molecular weight of about 75kDa.

The optimal pH of (embodiment 5) enzyme reaction

Generating in the reaction of L-alanyl-L-glutamine, detect the influence of pH by L-alanine methyl ester hydrochloride and L-glutaminate.With acetate buffer (pH3.9-5.4), MES damping fluid (pH5.4-6.4), phosphate buffered saline buffer (pH6.0-7.9), borate buffer solution (pH7.8-9.3), CAPS damping fluid (pH9.3-10.7), and K ₂HPO ₄-NaOH damping fluid (pH10.8-11.6) is as damping fluid.1 microlitre (μ l) the Mono S enzyme part (about 180U/ml) that obtains among the embodiment 3 is added 100 μ l contain 100mM L-alanine methyl ester, in each damping fluid (100mM) of 200mM L-glutaminate and 10mM EDTA, make it 18 ℃ of reactions 5 minutes, detect the influence of pH reaction.Shown among Fig. 1 that the value by will use borate buffer solution (pH9.3) time is decided to be 100% result who represents.As a result, find that optimal pH is 8-9.5.

The optimum temperuture of (embodiment 6) enzyme reaction

Generating in the reaction of L-alanyl-L-glutamine the influence of detected temperatures by L-alanine methyl ester hydrochloride and L-glutaminate.1 μ l is partly added 100 μ l with embodiment 5 used identical enzymes contain 100mM L-alanine methyl ester, in the 100mM borate buffer solution (pH9.0) of 200mM L-glutaminate and 10mM EDTA, it was reacted 5 minutes under each temperature, and detected temperatures is to the influence of reaction.Shown among Fig. 2 by being decided to be 100% result who represents at the activity value under 34 ℃.As a result, optimum temperuture is 30-40 ℃.

(embodiment 7) enzyme inhibitors

Adopt L-alanine methyl ester hydrochloride and L-glutaminate as substrate, detect inhibitor producing the influence of L-alanyl-L-glutamine.2 μ l are partly added 50 μ l with embodiment 5 used identical enzymes contain in the 100mM borate buffer solution (pH9.0) of each enzyme inhibitors shown in the 10mM table 2, make it 25 ℃ of reactions 5 minutes.Notice that before use, with phenanthrolene, phenylmethylsulfonyl fluoride and p-nitrophenyl-p '-guanidine benzoate are dissolved in the methyl alcohol, concentration is 50mM.L-alanyl-L-glutamine generation value when not having inhibitor peptides is decided to be 100, and the enzymic activity under each condition provides with relative reactivity.The result is as shown in table 2.As a result, in the serinase inhibitor that is detected, enzyme is not suppressed by phenylmethyl sulfonylfluoride, but it is suppressed by p-nitrophenyl-p '-guanidine benzoate.

Table 2

Enzyme inhibitors		Produce the relative reactivity (%) of L-Ala-L-Gln
Enzyme inhibitors		Produce the relative reactivity (%) of L-Ala-L-Gln	Do not have		??100
The metalloenzyme inhibitor	??EDTA	??96	Do not have		??100
	??EDTA	??96	Phenanthrolene	??96
	The SH enzyme inhibitors	The N-ethyl maleimide	Phenanthrolene	??96	??110
Single acetoiodide		The N-ethyl maleimide	??101		??110
Single acetoiodide		The serinase inhibitor	??101	Phenylmethyl sulfonylfluoride	??115
4-(2-aminoethyl) benzene sulfonyl is fluoridized thing	??75			Phenylmethyl sulfonylfluoride	??115
4-(2-aminoethyl) benzene sulfonyl is fluoridized thing	??75		P-nitrophenyl-p '-guanidine benzoate	??0.1

(embodiment 8) produce the L-alanyl-L-glutamine by L-alanine methyl ester and L-glutaminate

3 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain 100mM L-alanine methyl ester hydrochloride, 200mM L-glutaminate and 10mM EDTA 100mM borate buffer solution (pH9.0) in, make it 18 ℃ of reactions.The result generates 83mM L-alanyl-L-glutamine (L-Ala-L-Gln) as shown in Figure 3 in adding the enzyme district, the concentration of by product L-Ala-L-Ala-L-Gln is 1.3mM.On the other hand, almost do not observe any generation of L-Ala-L-Gln in not adding the enzyme district, enzyme concn only is about 0.07mM after 120 minutes and react.

(embodiment 9) L-glutaminate concentration is to producing the influence of L-alanyl-L-glutamine

1 μ l is partly added 100 μ l with embodiment 5 used identical enzymes contain 100mM L-alanine methyl ester hydrochloride, in the 100mM borate buffer solution (pH9.0) of the L-glutaminate of concentration shown in the table 3 and 10mM EDTA, make it 18 ℃ of reactions 2 hours.The result is as shown in table 3.

Table 3

L-alanine methyl ester hydrochloride (mM)	L-glutaminate (mM)	??L-Ala-L-Gln(mM)
L-alanine methyl ester hydrochloride (mM)	L-glutaminate (mM)	??L-Ala-L-Gln(mM)	??100	??100	??68.2
??110	??72.1			??100	??68.2
??110	??72.1	??120		??73.3
??130	??75.1	??120		??73.3
??130	??75.1	??150		??75.5
??200	??82.0	??150		??75.5

The substrate specificity (1) of (embodiment 10) enzyme

As under the situation of carboxyl composition, detect the ester specificity at the L-amino acid ester.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the carboxyl composition shown in the 100mM table 4, in the 100mM borate buffer solution (pH9.0) of 200mM L-glutaminate and 10mM EDTA, make it 25 ℃ of reactions 2 hours.The growing amount (HCl represents hydrochloric acid in the table 4) that has shown L-Ala-L-Gln in this reaction in the table 4.

Table 4

The carboxyl composition	The L-Ala-L-Gln (mM) that generates
The carboxyl composition	The L-Ala-L-Gln (mM) that generates	L-alanine methyl ester HCl L-alanine ethyl ester HCl L-L-Ala isopropyl ester HCl L-L-Ala-tert-butyl ester HCl	?84.3 ?91.5 ?78.9 ?7.5

The substrate specificity (2) of (embodiment 11) enzyme

Be used as under the situation of amine component as carboxyl composition and multiple L-amino acid at the L-alanine methyl ester, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain 100mM L-alanine methyl ester hydrochloride, in the 100mM borate buffer solution (pH9.0) of L-amino acid shown in the 150mM table 5 and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 5.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard, and " tr " expression trace).

Table 5

Amine component	The peptide (mM) that generates		Amine component	The peptide (mM) that generates
Amine component	The peptide (mM) that generates		Amine component	The peptide (mM) that generates		??Gly ??L-Ala ??L-Val ??L-Leu ??L-Ile ??L-Met ??L-Phe ??L-Trp ??L-Ser ??L-Thr	??L-Ala-Gly ??L-Ala-L-Ala ??L-Ala-L-Val ??L-Ala-L-Leu ??L-Ala-L-Ile ??L-Ala-L-Met ??L-Ala-L-Phe ??L-Ala-L-Trp ??L-Ala-L-Ser ??L-Ala-L-Thr	??13.7 ??25.4 ??20.8 ??45.3 ??33.9 ??83.3 ??74.4 ??53.9 ??62.5 ??53.9	??L-Asn ??L-Gln ??L-Tyr ??L-CySH ??L-Lys ??L-Arg ??L-His ??L-Asp ??L-Glu ??L-Pro	??L-Ala-L-Asn ??L-Ala-L-Gln ??L-Ala-L-Tyr ??L-Ala-L-CySH ??L-Ala-L-Lys ??L-Ala-L-Arg ??L-Ala-L-His ??L-Ala-L-Asp ??L-Ala-L-Glu ??L-Ala-L-Pro	??65.5 ??79.3 ??17.6 ??+ ??71.8 ??88.0 ??66.9 ??2.1 ??42.9 ??tr

The substrate specificity (3) of (embodiment 12) enzyme

Be used as under the situation of amine component as carboxyl composition and L-glutaminate at multiple L-amino acid methyl ester, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the L-amino acid methyl ester hydrochloride (AA-OMeHCl) shown in the 100mM table 6, in the 100mM borate buffer solution (pH9.0) of 150mM L-glutaminate and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 6.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard, and " tr " expression trace).In addition, when using L-Trp-OMe and L-Tyr-Ome, in reactive system, add Tween-80 to final concentration be 0.1%.

Table 6

The carboxyl composition	The peptide (mM) that generates		The carboxyl composition	The peptide (mM) that generates
The carboxyl composition	The peptide (mM) that generates		The carboxyl composition	The peptide (mM) that generates		??Gly-OMe ??L-Ala-OMe ??L-Val-OMe ??L-Leu-OMe ??L-Ile-OMe ??L-Met-OMe ??L-Phe-OMe ??L-Trp-OMe ??L-Ser-OMe ??L-Thr-OMe ??L-Asn-OMe ??L-Gln-OMe	??Gly-L-Gln ??L-Ala-L-Gln ??L-Val-L-Gln ??L-Leu-L-Gln ??L-Ile-L-Gln ??L-Met-L-Gln ??L-Phe-L-Gln ??L-Trp-L-Gln ??L-Ser-L-Gln ??L-Thr-L-Gln ??L-Asn-L-Gln ??L-Gln-L-Gln	??54.7 ??74.6 ??15.4 ??+ ??8.4 ??12.0 ??0.9 ??+ ??24.0 ??81.9 ??+ ??0.3	??L-Tyr-OMe ??CySH-OMe ??L-Lys-OMe ??L-Arg-OMe ??L-His-OMe ??L-Asp-α-OMe ??L-Asp-β-OMe ??L-Glu-α-OMe ??L-Glu-γ-OMe ??L-Pro-OMe	??L-Tyr-L-Gln ??L-CySH-L-Gln ??L-Lys-L-Gln ??L-Arg-L-Gln ??L-His-L-Gln ??α-L-Asp-L-Gln ??β-L-Asp-L-Gln ??α-L-Glu-L-Gln ??γ-L-Glu-L-Gln ??L-Pro-L-Gln	??3.4 ??+ ??+ ??7.1 ??+ ??tr ??tr ??+ ??+ ??2.2

(all carboxyl compositions are all used hydrochloride.)

(embodiment 13) enzyme substrates specificitys (4)

Be used as under the situation of amine component as carboxyl composition and multiple L-amino acid at multiple L-amino acid methyl ester, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the L-amino acid methyl ester hydrochloride (AA-OMeHCl) shown in the 100mM table 7, in the 100mM borate buffer solution (pH9.0) of 150mM L-glutaminate and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown each peptide that is generated in this reaction in the table 7.(" tr " represents trace).In addition, when using L-Trp-OMe, in reactive system, add Tween-80 to final concentration be 0.1%.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard).

Table 7

The carboxyl composition	Amine component	The peptide (mM) that generates		The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		The carboxyl composition	Amine component	The peptide (mM) that generates		??Gly-OMe	??L-CySH	??Gly-L-CySH	??45.6	??L-Met-OMe	??L-Ser	??L-Met-L-	??12.8
??L-Arg	??Gly-L-Arg	??25.5	??L-Met	??L-Met-L-	??25.0				??L-CySH	??Gly-L-CySH	??45.6		??L-Ser	??L-Met-L-	??12.8
??L-Arg	??Gly-L-Arg	??25.5	??L-Met	??L-Met-L-	??25.0	??L-Phe	??Gly-L-Phe		??44.0	??L-Phe	??L-Met-L-		??34.0
??L-His	??Gly-L-His	??31.6	??L-Ile-OMe	??L-Ser	??L-Ile-L-	??L-Phe	??Gly-L-Phe		??44.0	??L-Phe	??L-Met-L-		??34.0	??17.2
??L-His	??Gly-L-His	??31.6		??L-Ser	??L-Ile-L-	??L-Lys	??Gly-L-Lys		??9.8	??L-Met	??L-Ile-L-	??10.0		??17.2
??L-Ser	??Gly-L-Ser	??44.2		??L-Phe	??L-Ile-L-	??L-Lys	??Gly-L-Lys		??9.8	??L-Met	??L-Ile-L-	??10.0	??5.2
??L-Ser	??Gly-L-Ser	??44.2		??L-Phe	??L-Ile-L-	??L-Thr-OMe	??Gly		??L-Thr-Gly	??9.4	??L-Arg-OMe	??L-Ser	??5.2	??L-Arg-L-	??3.6
??L-Ala	??L-Thr-L-Ala	??9.4	??L-Met	??L-Arg-L-	??0.7		??Gly		??L-Thr-Gly	??9.4		??L-Ser		??L-Arg-L-	??3.6
??L-Ala	??L-Thr-L-Ala	??9.4	??L-Met	??L-Arg-L-	??0.7		??L-Val	??L-Thr-L-Val	??0.7	??L-Phe		??L-Arg-L-	??1.9
??L-Leu	??L-Thr-L-Leu	??28.4	??L-Leu-OMe	??L-Met	??L-Leu-L-		??L-Val	??L-Thr-L-Val	??0.7	??L-Phe		??L-Arg-L-	??1.9	??12.2
??L-Leu	??L-Thr-L-Leu	??28.4	??L-Leu-OMe	??L-Met	??L-Leu-L-		??L-Met	??L-Thr-L-Met	??38.6	??L-Trp-OMe	??L-Met	??L-Trp-L-	??4.1	??12.2
??L-Ser	??L-Thr-L-Ser	??58.2	??L-Lys-OMe	??L-Met	??L-Lys-L-		??L-Met	??L-Thr-L-Met	??38.6	??L-Trp-OMe	??L-Met	??L-Trp-L-	??4.1	??6.8
??L-Ser	??L-Thr-L-Ser	??58.2	??L-Lys-OMe	??L-Met	??L-Lys-L-		??L-Ser-OMe	??L-Ser	??L-Ser-L-Ser	??38.0	??L-His-OMe	??L-Met	??L-His-L-	??6.8	??6.5
??L-Met	??L-Ser-L-Met	??12.5	??L-Asn-OMe	??L-Glu	??L-Asn-L-	??10.2		??L-Ser	??L-Ser-L-Ser	??38.0	??L-His-OMe	??L-Met	??L-His-L-		??6.5
??L-Met	??L-Ser-L-Met	??12.5	??L-Asn-OMe	??L-Glu	??L-Asn-L-	??10.2		??L-Phe	??L-Ser-L-Phe	??20.3
??L-Val-OMe	??L-Ser	??L-Val-L-Ser	??30.8					??L-Phe	??L-Ser-L-Phe	??20.3
	??L-Ser	??L-Val-L-Ser	??30.8				??L-Met	??L-Val-L-Met	??10.3
	??L-Phe	??L-Val-L-Phe	??6.1				??L-Met	??L-Val-L-Met	??10.3

(all carboxyl compositions are all used hydrochloride.)

(embodiment 14) enzyme substrates specificitys (5)

Be used as carboxyl composition and L or D type multiple amino acids as under the situation of amine component at L or D type multiple amino acids methyl esters, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the L-amino acid methyl ester hydrochloride (AA-OMeHCl) shown in the 100mM table 8, in the 100mM borate buffer solution (pH9.0) of multiple amino acids shown in the 150mM table 8 and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 8.(" tr " represents trace).

Table 8

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??D-Ala-OMe	??L-Gln	??D-Ala-L-Gln	??69.3
??D-Ala-OMe	??L-Ser	??D-Ala-L-Ser	??20.3	??D-Ala-OMe	??L-Gln	??D-Ala-L-Gln	??69.3
??D-Ala-OMe		??D-Ala-L-Ser	??20.3	??D-Thr-OMe	??D-Thr-L-Ser	??1.0
??D-Ser-OMe		??D-Ser-L-Ser	??3.3	??D-Thr-OMe	??D-Thr-L-Ser	??1.0
??D-Ser-OMe		??D-Ser-L-Ser	??3.3	??D-Val-OMe	??D-Val-L-Ser	??0.6
??D-Met-OMe		??D-Met-L-Ser	??5.1	??D-Val-OMe	??D-Val-L-Ser	??0.6
??D-Met-OMe		??D-Met-L-Ser	??5.1	??L-Ala-OMe	??D-Gln	??L-Ala-D-Gln	??0.3
??L-Ala-OMe	??D-Ser	??L-Ala-D-Ser	??5.4	??L-Ala-OMe	??D-Gln	??L-Ala-D-Gln	??0.3
??L-Ala-OMe		??L-Ala-D-Ser	??5.4	??L-Thr-OMe	??L-Thr-D-Ser	??6.9
??L-Ser-OMe		??L-Ser-D-Ser	??16.2	??L-Thr-OMe	??L-Thr-D-Ser	??6.9
??L-Ser-OMe		??L-Ser-D-Ser	??16.2	??L-Val-OMe	??L-Val-D-Ser	??1.4
??L-Met-OMe		??L-Met-D-Ser	??1.9	??L-Val-OMe	??L-Val-D-Ser	??1.4
??L-Met-OMe		??L-Met-D-Ser	??1.9	??D-Ala-OMe	??D-Gln	??D-Ala-D-Gln	??tr
??D-Ala-OMe	??D-Ser	??D-Ala-D-Ser	??0.2	??D-Ala-OMe	??D-Gln	??D-Ala-D-Gln	??tr
??D-Ala-OMe		??D-Ala-D-Ser	??0.2	??D-Thr-OMe	??D-Thr-D-Ser	??1.1
??D-Ser-OMe		??D-Ser-D-Ser	??2.5	??D-Thr-OMe	??D-Thr-D-Ser	??1.1
??D-Ser-OMe		??D-Ser-D-Ser	??2.5	??D-Val-OMe	??D-Val-D-Ser	??0.5
??D-Met-OMe		??D-Met-D-Ser	??2.7	??D-Val-OMe	??D-Val-D-Ser	??0.5

(all carboxyl compositions are all used hydrochloride.)

The substrate specificity (6) of (embodiment 15) enzyme

Be used as under the situation of amine component as carboxyl composition and multiple L-amino acid at multiple L-amino acid amide, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the L-amino acid amide (AA-NH shown in the 100mM table 9 ₂HCl), in the 100mM borate buffer solution (pH9.0) of L-amino acid shown in the 150mM table 9 and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 9.

Table 9

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Phe-NH ₂	??L-Gln	??L-Phe-L-Gln	??0.2
??L-Phe-NH ₂	??L-Ser	??L-Phe-L-Ser	??0.6	??L-Phe-NH ₂	??L-Gln	??L-Phe-L-Gln	??0.2
??L-Phe-NH ₂	??L-Ser	??L-Phe-L-Ser	??0.6	??L-Ala-NH ₂	??L-Gln	??L-Ala-L-Gln	??7.6
??L-Ala-NH ₂	??L-Met	??L-Ala-L-Met	??3.4	??L-Ala-NH ₂	??L-Gln	??L-Ala-L-Gln	??7.6
??L-Ala-NH ₂	??L-Met	??L-Ala-L-Met	??3.4	??L-Ala-NH ₂	??L-His	??L-Ala-L-His	??3.9
??L-Thr-NH ₂	??L-Gln	??L-Thr-L-Gln	??0.3	??L-Ala-NH ₂	??L-His	??L-Ala-L-His	??3.9

The substrate specificity (7) of (embodiment 16) enzyme

Be used as under the situation of amine component at the L-amino acid of multiple L-alanine methyl ester as carboxyl composition and C protection, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the L-amino acid methyl ester (AA-OMeHCl) shown in the 100mM table 10, in the 100mM borate buffer solution (pH9.0) of L-amino acid amide shown in the 150mM table 10 and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 10.

Table 10

The carboxyl composition	Amine component	The peptide that generates	??(mM)
The carboxyl composition	Amine component	The peptide that generates	??(mM)	??L-Ala-OMe	??Gly-NH ₂	??L-Ala-Gly-NH ₂	??7.4
??L-Ala-NH ₂	??L-Ala-L-Ala-NH ₂	??8.3			??Gly-NH ₂	??L-Ala-Gly-NH ₂	??7.4
??L-Ala-NH ₂	??L-Ala-L-Ala-NH ₂	??8.3	??L-Phe-NH ₂		??L-Ala-L-Phe-NH ₂	??12.2

The substrate specificity (8) of (embodiment 17) enzyme

Be used as under the situation of amine component as carboxyl composition and methylamine at the multiple amino acids methyl esters, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the L-amino acid methyl ester (AA-OMeHCl) shown in the 100mM table 11, in the 100mM borate buffer solution (pH9.0) of methylamine shown in the 150mM table 11 and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 11.

Table 11

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??Gly-OMe	The Gly-methylamine	??1.1
??L-Thr-OMe	The L-Thr-methylamine	??0.2		??Gly-OMe	The Gly-methylamine	??1.1
??L-Thr-OMe	The L-Thr-methylamine	??0.2	??L-Ala-OMe	The L-Ala-methylamine	??0.3

The substrate specificity (9) of (embodiment 18) enzyme

Be used as under the situation of amine component as carboxyl composition and beta-amino acids at beta-amino acid esters, detection of peptides generates.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the carboxyl composition shown in the 100mM table 12, in the 100mM borate buffer solution (pH9.0) of amine component shown in the 150mM table 12 and 10mMEDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 12.(" tr " represents trace).

Table 12

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??Gly-OMe	??β-Ala	??Gly-β-Ala	??2.2
??Gly-OMe	??β-Phe	??Gly-β-Phe	??0.4	??Gly-OMe	??β-Ala	??Gly-β-Ala	??2.2
??Gly-OMe	??β-Phe	??Gly-β-Phe	??0.4	??L-Ala-OMe	??β-Ala	??Ala-β-Ala	??7.7
??L-Ala-OMe	??β-Phe	??Ala-β-Phe	??1.4	??L-Ala-OMe	??β-Ala	??Ala-β-Ala	??7.7
??L-Ala-OMe	??β-Phe	??Ala-β-Phe	??1.4	??L-Thr-OMe	??β-Ala	??Thr-β-Ala	??3.2
??L-Thr-OMe	??β-Phe	??Thr-β-Phe	??1.4	??L-Thr-OMe	??β-Ala	??Thr-β-Ala	??3.2
??L-Thr-OMe	??β-Phe	??Thr-β-Phe	??1.4	??β-Ala-OMe	??L-α-Ala	??β-Ala-L-α-Ala	??tr
??β-Ala-OMe	??β-Ala	??β-Ala-β-Ala	??0.2	??β-Ala-OMe	??L-α-Ala	??β-Ala-L-α-Ala	??tr
??β-Ala-OMe	??β-Ala	??β-Ala-β-Ala	??0.2	??β-Ala-OMe	??L-Gln	??β-Ala-L-Gln	??0.6
??β-Ala-OMe	??L-Ser	??β-Ala-L-Ser	??3.2	??β-Ala-OMe	??L-Gln	??β-Ala-L-Gln	??0.6

The substrate specificity (10) of (embodiment 19) enzyme

Be used as under the situation of amine component as carboxyl composition and peptide at the L-amino acid ester, detect oligopeptides and generate.2 μ l are partly added 100 μ l with embodiment 5 used identical enzymes contain the carboxyl composition shown in the 100mM table 13, in the 100mM borate buffer solution (pH9.0) of amine component shown in the 150mM table 13 and 10mM EDTA, make it 25 ℃ of reactions 3 hours.The growing amount that has shown multiple peptide in this reaction in the table 13.The result clearly shows enzyme of the present invention can not only generate dipeptides, can also be by peptide is generated the long-chain peptide as amine component.

Shown in above-mentioned embodiment 9-20, the enzyme of the present invention that is obtained by short steady bacillus strain FERM BP-18545 is determined has substrate specificity very widely.

Table 13

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Ala-OMe	??L-Ala	??L-Ala-L-Ala	??28.7
??L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala	??57.5			??L-Ala	??L-Ala-L-Ala	??28.7
??L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala	??57.5	??L-Ala-L-Ala-L-Ala		??L-Ala-L-Ala-L-Ala-L-Ala	??44.5
??L-Ala-L-Ala-L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala-L-Ala-L-Ala	??34.8	??L-Ala-L-Ala-L-Ala		??L-Ala-L-Ala-L-Ala-L-Ala	??44.5
??L-Ala-L-Ala-L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala-L-Ala-L-Ala	??34.8	??L-Ala-L-Ala-L-Ala-L-Ala- ??L-Ala		??L-Ala-L-Ala-L-Ala-L-Ala-L- ??Ala-L-Ala	??1.4*
??L-Ala-L-Gln	??L-Ala-L-Ala-L-Gln	??15.2	??L-Ala-L-Ala-L-Ala-L-Ala- ??L-Ala		??L-Ala-L-Ala-L-Ala-L-Ala-L- ??Ala-L-Ala	??1.4*
??L-Ala-L-Gln	??L-Ala-L-Ala-L-Gln	??15.2	??Gly-L-Ala		??L-Ala-Gly-L-Ala	??25.9
??Gly-Gly	??L-Ala-Gly-Gly	??41.7	??Gly-L-Ala		??L-Ala-Gly-L-Ala	??25.9
??Gly-Gly	??L-Ala-Gly-Gly	??41.7	??L-His-L-Ala		??L-Ala-L-His-L-Ala	??55.9
??L-Leu-L-Ala	??L-Ala-L-Leu-L-Ala	??48.3	??L-His-L-Ala		??L-Ala-L-His-L-Ala	??55.9
??L-Leu-L-Ala	??L-Ala-L-Leu-L-Ala	??48.3	??L-Phe-L-Ala		??L-Ala-L-Phe-L-Ala	??49.7
??L-Phe-Gly	??L-Ala-L-Phe-Gly	??43.7	??L-Phe-L-Ala		??L-Ala-L-Phe-L-Ala	??49.7
??L-Phe-Gly	??L-Ala-L-Phe-Gly	??43.7	??Gly-OMe		??L-Ala-L-Tyr	??Gly-L-Ala-L-Tyr	??1.7
??Gly-L-Gln	??Gly-Gly-L-Gln	??7.2			??L-Ala-L-Tyr	??Gly-L-Ala-L-Tyr	??1.7
??Gly-L-Gln	??Gly-Gly-L-Gln	??7.2		??Gly-L-Tyr-L-Ala	??Gly-Gly-L-Tyr-L-Ala	??44.2
??L-Thr-OMe	??Gly-Gly	??L-Thr-Gly-Gly		??Gly-L-Tyr-L-Ala	??Gly-Gly-L-Tyr-L-Ala	??44.2	??83.0

(*: because the solubleness of L-Ala-L-Ala-L-Ala-L-Ala-L-Ala is low, so the concentration of the carboxyl composition that uses in this reactive system is 10mM, the concentration of the amine component that uses is 15mM.Other condition identical with described in the foregoing description.)

(embodiment 20) compare the ability that the catalysis peptide generates with known enzyme

Enzyme of the present invention and known enzyme are compared the ability that the catalysis peptide generates.Sulfydryl endopeptidase described in carboxypeptidase y described in the EP 278787A1 and the EP 359399B1 (ficin, papoid, bromeline and Disken) is used as known enzyme, and its form with the enzyme of purifying is used (Sigma production).The enzyme of even purifying is used as the source of enzyme of the present invention among the embodiment 3.These enzymes are added in the reactive system with the proteinic amount shown in the table 14.Enzyme is added 100 μ l contain in the borate buffer solution (pH9.0) of 100mM L-alanine methyl ester and 200mM L-glutaminate, make gains 25 ℃ of reactions.Note, used carboxypeptidase is dissolved in the 10mM acetate buffer (pH5.0) that contains 1mM EDTA, contain 2mM EDTA and used sulfydryl endopeptidase is dissolved in, in the 10mM acetate buffer (pH5.0) of 0.1M KCl and 5mM dithiothreitol (DTT).The ratio that has shown the L-alanyl-L-glutamine productivity of these enzymes in the table 14.

As a result,, and compare, in the district that adds carboxypeptidase or sulfydryl endopeptidase, observe the slight increase of productivity with the district that does not add enzyme even in the generation of the L-alanyl-L-glutamine that does not have also to observe under the situation of enzyme utmost point trace.On the contrary, observe high relatively L-alanyl-L-glutamine productivity in adding the district of enzyme of the present invention, the productivity of this productivity ratio carboxypeptidase y and sulfydryl endopeptidase is high about 5,000-100,000 times.As mentioned above, enzyme of the present invention is proved and has the high peptide productivity different with known enzyme in the prior art.In addition, enzyme of the present invention is the dimer with molecular weight of about 75,000.On the contrary, it is about 61,000 that the molecular weight of carboxypeptidase y has been reported as, and that the molecular weight of sulfydryl endopeptidase has been reported as is about 23,000-36,000.Therefore, as shown in the Examples, compare with the L-alanyl-L-glutamine productivity of carboxypeptidase y and sulfydryl endopeptidase, even the situation that the L-alanyl-L-glutamine survival rate of enzyme of the present invention also is higher than it when productivity is represented with every molal weight when representing with per unit weight.

Table 14

Enzyme	The add-on of enzyme (protein mg/ml)	L-Ala-L-Gln productivity (mM/min)	The ratio of L-Ala-L-Gln productivity/unit of enzyme weight
Enzyme	The add-on of enzyme (protein mg/ml)	L-Ala-L-Gln productivity (mM/min)	The ratio of L-Ala-L-Gln productivity/unit of enzyme weight	No enzyme	??0	??0.0006
Carboxypeptidase y	??0.61	??0.0257	??0.0191	No enzyme	??0	??0.0006
Carboxypeptidase y	??0.61	??0.0257	??0.0191	Ficin	??2.60	??0.0096	??0.0017
Papoid	??2.30	??0.0106	??0.0021	Ficin	??2.60	??0.0096	??0.0017
Papoid	??2.30	??0.0106	??0.0021	Bromeline	??2.80	??0.0062	??0.0010
Chymopapain	??3.60	??0.0100	??0.0013	Bromeline	??2.80	??0.0062	??0.0010
Chymopapain	??3.60	??0.0100	??0.0013	Enzyme of the present invention	??0.02	??4.4000	??100.0

(embodiment 21) produce the L-alanyl-L-glutamine with the microorganism cells of Sphingobacterium sp.

In 500ml slope Kou Shi bottle, add 50mL every liter (L) and contain 5g glucose, 5g ammonium sulfate, the 1g potassium primary phosphate, the 3g dipotassium hydrogen phosphate, 0.5g sal epsom, the substratum of 10g yeast extract and 10g peptone (pH6.2), (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to be used to cultivate Sphingobacterium sp. bacterial strain FERM BP-8124 in 15 minutes 115 ℃ of sterilizations, preservation mechanism address: Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002).That inoculates 1 platinum loop cell then on this substratum contains 5g glucose at every liter (L), the 10g yeast extract, slant agar substratum (the agar: 20g/L of 10g peptone and 5g NaCl, pH7.0) on, cultivated 24 hours Sphingobacterium sp. bacterial strain FERM BP-8124 (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst has speciallyyed permit biological sustenance center for 30 ℃, preservation mechanism address: Chuo Dai-6,1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002), at 30 ℃, 120 reciprocal/minute following concussions were cultivated 20 hours then.Then this nutrient solution of 1ml is added above-mentioned substratum (50mL/500ml slope Kou Shi bottle), cultivated 18 hours in 30 ℃.After cultivation is finished, by centrifugal from nutrient solution the separate microorganism cell, then it is suspended in the 0.1M borate buffer solution (pH9.0) that contains 10mM EDTA, its concentration is 100g/L, in wet microorganism cells.In this microorganism cells suspension of 0.1ml, add 0.1ml then and contain 10mM EDTA, in the 100mM borate buffer solution (pH9.0) of 200mM L-alanyl methyl ester hydrochloride and 400mM L-glutaminate.0.2mL gained mixture was reacted 120 minutes at 25 ℃.The concentration of the L-alanyl-L-glutamine that produce this moment is 62mM.

(embodiment 22) are from the purifying of the enzyme of Sphingobacterium sp.

Following steps after centrifugal are on ice or 4 ℃ of enforcements.(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to cultivate Sphingobacterium sp. bacterial strain FERM BP-8124 according to the method identical with embodiment 21, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002), collect microorganism cells by centrifugal (10,000rpm, 15 minutes).Behind 20mM Tris-HCl damping fluid (pH7.6) washing 2g microorganism cells, it is suspended in the 8ml same buffer, carry out ultrasonic degradation with 195W then and handled 45 minutes.Centrifugal then this ultrasonic degradation liquid (10,000rpm, 30 minutes) to remove cell debris, obtains ultrasonic degradation liquid supernatant.With 20mM Tris-HCl damping fluid (pH7.6) to this ultrasonic degradation liquid supernatant dialysed overnight, then by ultracentrifugation (50,000rpm, 30 minutes) thus remove the soluble part that insoluble part obtains the supernatant liquor form.The soluble part adding that obtains is used in Tris-HCl damping fluid (pH7.6) the equilibrated Q-Sepharose HP post (Amersham production) in advance, from non-absorbed portion, collected active part.With 20mM acetate buffer (pH5.0) to this active part dialysed overnight, then by centrifugal (10,000rpm, 30 minutes) thus remove the part that insoluble part obtains the dialysis of supernatant liquor form.The part of this dialysis added use in advance in 20mM acetate buffer (pH5.0) the equilibrated SP-Sepharose HP post (Amersham production), with the linear concentration gradient wash-out enzyme of the same buffer that contains 0-1M NaCl.

(embodiment 23) use enzyme part producing L-alanyl-L-glutamine

SP-Sepharose HP part (about 27U/ml) the adding 90 μ l of purifying among the 10 μ l embodiment 22 are contained 111mM L-alanine methyl ester hydrochloride, 222mM L-glutaminate and 11mMEDTA 111mM borate buffer solution (pH9.0) in, make its 25 ℃ the reaction 120 minutes.As a result, in the district that adds enzyme, generate 73mM L-alanyl-L-glutamine.On the other hand, almost do not observe any generation of L-Ala-L-Gln in not adding the district of enzyme, enzyme concn only is about 0.07mM after 120 minutes and react.

The substrate specificity (11) of (embodiment 24) enzyme

Detection comes from Sphingobacterium sp. bacterial strain FERM BP-8124, and (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center, preservation mechanism address: ChuoDai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: the substrate specificity of enzyme on July 22nd, 2002).100 μ l are comprised multiple carboxyl composition shown in the table 15-1 to 15-4 that final concentration is 100mM and final concentration is the various kinds of amine composition of 150mM, the SP-Sepharose HP enzyme of purifying part among the embodiment 22 (adding 0.33 unit in the reaction solution) and the 100mM borate buffer solution (pH9.0) of 10mM EDTA reacted 1.5 hours at 25 ℃.The growing amount that has shown multiple peptide in this reaction in the table 15.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard, and " tr " expression trace).In addition, when using L-Tyr-Ome, in reactive system, add Tween-80 to final concentration be 0.1%.In addition, all use hydrochloride for all carboxyl compositions.

Table 15-1

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Ala-OMe	??Gly	??L-Ala-Gly	??11.1
??L-Ala	??L-Ala-L-Ala	??13.1			??Gly	??L-Ala-Gly	??11.1
??L-Ala	??L-Ala-L-Ala	??13.1	??L-Val		??L-Ala-L-Val	??10.9
??L-Leu	??L-Ala-L-Leu	??33.0	??L-Val		??L-Ala-L-Val	??10.9
??L-Leu	??L-Ala-L-Leu	??33.0	??L-Ile		??L-Ala-L-Ile	??24.7
??L-Met	??L-Ala-L-Met	??86.9	??L-Ile		??L-Ala-L-Ile	??24.7
??L-Met	??L-Ala-L-Met	??86.9	??L-Pro		??L-Ala-L-Pro	??1.5
??L-Phe	??L-Ala-L-Phe	??69.5	??L-Pro		??L-Ala-L-Pro	??1.5
??L-Phe	??L-Ala-L-Phe	??69.5	??L-Trp		??L-Ala-L-Trp	??46.0
??L-Thr	??L-Ala-L-Thr	??47.3	??L-Trp		??L-Ala-L-Trp	??46.0
??L-Thr	??L-Ala-L-Thr	??47.3	??L-Asn		??L-Ala-L-Asn	??52.3
??L-Tyr	??L-Ala-L-Tyr	??11.1	??L-Asn		??L-Ala-L-Asn	??52.3
??L-Tyr	??L-Ala-L-Tyr	??11.1	??L-CySH		??L-Ala-L-CySH	??+
??L-Lys	??L-Ala-L-Lys	??71.2	??L-CySH		??L-Ala-L-CySH	??+
??L-Lys	??L-Ala-L-Lys	??71.2	??L-Arg		??L-Ala-L-Arg	??72.2
??L-His	??L-Ala-L-His	??73.6	??L-Arg		??L-Ala-L-Arg	??72.2
??L-His	??L-Ala-L-His	??73.6	??L-Asp		??L-Ala-L-Asp	??2.3
??L-Glu	??L-Ala-L-Glu	??39.1	??L-Asp		??L-Ala-L-Asp	??2.3
??L-Glu	??L-Ala-L-Glu	??39.1	??L-Ser		??L-Ala-L-Ser	??43.8
??D-Ser	??L-Ala-D-Ser	??3.3	??L-Ser		??L-Ala-L-Ser	??43.8
??D-Ser	??L-Ala-D-Ser	??3.3	??D-Ala-OMe		??L-Ser	??D-Ala-L-Ser	??24.1
??D-Ser	??D-Ala-D-Ser	??5.5			??L-Ser	??D-Ala-L-Ser	??24.1

Table 15-2

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Thr-OMe	??L-Gln	??L-Thr-L-Gln	??36.1
??Gly-OMe	??Gly-L-Gln	??61.1		??L-Thr-OMe		??L-Thr-L-Gln	??36.1
??Gly-OMe	??Gly-L-Gln	??61.1	??L-Ser-OMe	??L-Ser-L-Gln		??12.9
??L-Val-OMe	??L-Val-L-Gln	??8.2	??L-Ser-OMe	??L-Ser-L-Gln		??12.9
??L-Val-OMe	??L-Val-L-Gln	??8.2	??L-Met-OMe	??L-Met-L-Gln		??32.6
??L-Ile-OMe	??L-Ile-L-Gln	??6.4	??L-Met-OMe	??L-Met-L-Gln		??32.6
??L-Ile-OMe	??L-Ile-L-Gln	??6.4	??L-Arg-OMe	??L-Arg-L-Gln		??17.2
??L-Tyr-OMe	??L-Tyr-L-Gln	??0.6	??L-Arg-OMe	??L-Arg-L-Gln		??17.2
??L-Tyr-OMe	??L-Tyr-L-Gln	??0.6	??L-Pro-OMe	??L-Pro-L-Gln		??1.8
??L-Phe-OMe	??L-Phe-L-Gln	??0.8	??L-Pro-OMe	??L-Pro-L-Gln		??1.8
??L-Phe-OMe	??L-Phe-L-Gln	??0.8	??L-Gln-OMe	??L-Gln-L-Gln		??0.1
??Asp-α-OMe	??α-L-Asp-L-G1n	??0.05	??L-Gln-OMe	??L-Gln-L-Gln		??0.1

Table 15-3

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Thr-OMe	??Gly	??L-Thr-Gly	??0.4
??L-Ala	??L-Thr-L-Ala	??5.8			??Gly	??L-Thr-Gly	??0.4
??L-Ala	??L-Thr-L-Ala	??5.8	??L-Val		??L-Thr-L-Val	??1.3
??L-Leu	??L-Thr-L-Leu	??15.3	??L-Val		??L-Thr-L-Val	??1.3
??L-Leu	??L-Thr-L-Leu	??15.3	??L-Met		??L-Thr-L-Met	??28.9
??Gly-OMe	??L-Arg	??Gly-L-Arg	??L-Met		??L-Thr-L-Met	??28.9	??17.9
	??L-Arg	??Gly-L-Arg	??L-Phe	??Gly-L-Phe	??20.0		??17.9
	??L-His	??Gly-L-His	??L-Phe	??Gly-L-Phe	??20.0	??36.2
	??L-His	??Gly-L-His	??L-Lys	??Gly-L-Lys	??48.2	??36.2
	??L-Ser	??Gly-L-Ser	??L-Lys	??Gly-L-Lys	??48.2	??53.8
	??L-Ser	??Gly-L-Ser	??L-Ser-OMe	??L-Ser	??L-Ser-L-Ser	??53.8	??9.9
??L-Met	??L-Ser-L-Met	??7.6		??L-Ser	??L-Ser-L-Ser		??9.9
??L-Met	??L-Ser-L-Met	??7.6		??L-Phe	??L-Ser-L-Phe	??4.3
??L-Val-OMe	??L-Ser	??L-Val-L-Ser		??L-Phe	??L-Ser-L-Phe	??4.3	??31.9
	??L-Ser	??L-Val-L-Ser	??L-Met	??L-Val-L-Met	??6.8		??31.9
	??L-Phe	??L-Val-L-Phe	??L-Met	??L-Val-L-Met	??6.8	??1.0
	??L-Phe	??L-Val-L-Phe	??L-Met-OMe	??L-Ser	??L-Met-L-Ser	??1.0	??25.3
??L-Met	??L-Met-L-Met	??28.4		??L-Ser	??L-Met-L-Ser		??25.3
??L-Met	??L-Met-L-Met	??28.4		??L-Phe	??L-Met-L-Phe	??8.9
??L-Ile-OMe	??L-Ser	??L-Ile-L-Ser		??L-Phe	??L-Met-L-Phe	??8.9	??17.3
	??L-Ser	??L-Ile-L-Ser	??L-Met	??L-Ile-L-Met	??5.1		??17.3
	??L-Phe	??L-Ile-L-Phe	??L-Met	??L-Ile-L-Met	??5.1	??1.5
	??L-Phe	??L-Ile-L-Phe	??L-Arg-OMe	??L-Ser	??L-Arg-L-Ser	??1.5	??2.2
??L-Met	??L-Arg-L-Met	??tr		??L-Ser	??L-Arg-L-Ser		??2.2
??L-Met	??L-Arg-L-Met	??tr		??L-Phe	??L-Arg-L-Phe	??tr

Table 15-4

The carboxyl composition	Amine component	The peptide of producing (mM)
The carboxyl composition	Amine component	The peptide of producing (mM)		??L-Ala-OMe	The Gly acid amides	The L-Ala-Gly acid amides	??15.1
The L-Ala acid amides	The L-Ala-L-Ala acid amides	??9.2			The Gly acid amides	The L-Ala-Gly acid amides	??15.1
The L-Ala acid amides	The L-Ala-L-Ala acid amides	??9.2	The L-Phe acid amides		The L-Ala-Phe acid amides	??27.1
??L-Ala-OMe	Methylamine	The L-Ala-methylamine	The L-Phe acid amides		The L-Ala-Phe acid amides	??27.1	??0.6
??L-Ala-OMe		The L-Ala-methylamine	??L-Thr-OMe	The L-Thr-methylamine	??0.3		??0.6
??Gly-OMe		The Gly-methylamine	??L-Thr-OMe	The L-Thr-methylamine	??0.3	??1.0
??Gly-OMe		The Gly-methylamine	The L-Ala acid amides	??L-Gln	??L-Ala-L-Gln	??1.0	??0.3
??L-Met	??L-Ala-L-Met	??tr		??L-Gln	??L-Ala-L-Gln		??0.3
??L-Met	??L-Ala-L-Met	??tr		??L-His	??L-Ala-L-His	??tr

The substrate specificity (12) of (embodiment 25) enzyme

Detection comes from Sphingobacterium sp. bacterial strain FERM BP-8124, and (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center, preservation mechanism address: ChuoDai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: the substrate specificity about the oligopeptides generation of enzyme on July 22nd, 2002).100 μ l are comprised the various kinds of amine composition shown in the table 16 that multiple carboxyl composition that final concentration is 100mM and final concentration be 150mM, and the SP-Sepharose HP enzyme of purifying part among the embodiment 22 (adding 0.33 unit in the reaction solution) and the 100mM borate buffer solution (pH9.0) of 10mM EDTA reacted 1.5 hours at 25 ℃.The growing amount that has shown each oligopeptides in this reaction in the table 16.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard, and " tr " expression trace).In addition, all use hydrochloride for all carboxyl compositions.

Table 16

The carboxyl composition	Amine component	The peptide (mM) that generates
The carboxyl composition	Amine component	The peptide (mM) that generates		??L-Ala-OMe	??L-Ala	??L-Ala-L-Ala	??25.6
??L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala	??41.1			??L-Ala	??L-Ala-L-Ala	??25.6
??L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala	??41.1	??L-Ala-L-Ala-L-Ala		??L-Ala-L-Ala-L-Ala-L-Ala	??30.1
??L-Ala-L-Ala-L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala-L-Ala-L-Ala	??22.8	??L-Ala-L-Ala-L-Ala		??L-Ala-L-Ala-L-Ala-L-Ala	??30.1
??L-Ala-L-Ala-L-Ala-L-Ala	??L-Ala-L-Ala-L-Ala-L-Ala-L-Ala	??22.8	??Gly-Gly		??L-Ala-Gly-Gly	??33.7
??Gly-Ala	??L-Ala-Gly-L-Ala	??35.1	??Gly-Gly		??L-Ala-Gly-Gly	??33.7
??Gly-Ala	??L-Ala-Gly-L-Ala	??35.1	??L-His-L-Ala		??L-Ala-L-His-L-Ala	??58.0
??L-Phe-Gly	??L-Ala-L-Phe-Gly	??34.0	??L-His-L-Ala		??L-Ala-L-His-L-Ala	??58.0
??L-Phe-Gly	??L-Ala-L-Phe-Gly	??34.0	??L-Leu-L-Ala		??L-Ala-L-Leu-L-Ala	??40.7
??L-Phe-L-Ala	??L-Ala-L-Phe-L-Ala	??24.8	??L-Leu-L-Ala		??L-Ala-L-Leu-L-Ala	??40.7
??L-Phe-L-Ala	??L-Ala-L-Phe-L-Ala	??24.8	??L-Thr-OMe		??Gly-Gly	??L-Thr-Gly-Gly	??8.4
??Gly-OMe	??L-Ala-L-Tyr	??Gly-L-Ala-L-Tyr	??L-Thr-OMe	??0.6	??Gly-Gly	??L-Thr-Gly-Gly	??8.4

The substrate specificity (13) of (embodiment 26) enzyme

Adopt with embodiment 5 employed identical enzyme parts and further estimate substrate specificity.

Table 17

Carboxyl composition (mM)		Amine component (mM)		The peptide (mM) that generates		Reaction times (hr)
Carboxyl composition (mM)		Amine component (mM)		The peptide (mM) that generates		Reaction times (hr)	??H-Ala-OMe ??H-Ala-OMe ??H-Ala-OMe ??H-Ala-OMe	??50mM ??40mM ??40mM ??100mM	??H-p-F-Phe-OH ??H-Cl-F-Phe-OH ??H-p-NO2-Phe-OH ??H-t-Leu-OH	??50mM ??40mM ??40mM ??150mM	??H-Ala-p-F-Phe-OH ??H-Ala-Cl-F-Phe-OH ??H-Ala-p-NO2-Phe-OH ??H-Ala-t-Leu-OH	??21.9mM ??20.8mM ??27.5mM ??0.4mM	??3 ??3 ??3 ??3
??H-p-F-Phe-OMe ??H-Cl-F-Phe-OMe ??H-p-NO ₂-Phe-OMe ??H-t-Leu-OMe ??H-2-Nal-OMe	??100mM ??25mM ??40mM ??100mM ??40mM	??H-Gln-OH ??H-Gln-OH ??H-Gln-OH ??H-Gln-OH ??H-Gln-OH	??150mM ??50mM ??40mM ??150mM ??40mM	??H-p-F-Phe-H-Gln-OH ??H-Cl-F-Phe-H-Gln-OH ??H-p-NO ₂-Phe-H-Gln-OH ??H-t-Leu-H-Gln-OH ??H-2-Nal-H-Gln-OH	??tr ??tr ??1.1mM ??tr ??tr	??3 ??3 ??3 ??3 ??3	??H-Ala-OMe ??H-Ala-OMe ??H-Ala-OMe ??H-Ala-OMe	??50mM ??40mM ??40mM ??100mM		??50mM ??40mM ??40mM ??150mM		??21.9mM ??20.8mM ??27.5mM ??0.4mM	??3 ??3 ??3 ??3
	??100mM ??25mM ??40mM ??100mM ??40mM	??H-Gln-OH ??H-Gln-OH ??H-Gln-OH ??H-Gln-OH ??H-Gln-OH	??150mM ??50mM ??40mM ??150mM ??40mM		??tr ??tr ??1.1mM ??tr ??tr	??3 ??3 ??3 ??3 ??3	??H-Aib-OMe ??H-CHA-OMe	??100mM ??40mM	??H-Phe-OH ??H-Phe-OH	??150mM ??40mM	??H-Aib-Phe-OH ??17.2mM		??3 ??3
??H-Ala-OMe ??H-Ser(tBu)-OMe ??H-Ala-OMe ??H-Asp(OtBu)-OMe	??100mM ??100mM ??100mM ??100mM	??H-Ser(tBu)-OH ??H-Gln-OH ??H-Asp(OtBu)-OH ??H-Gln-OH	??150mM ??150mM ??150mM ??150mM	??H-Ala-Ser(tBu)-OH ??48.8mM ??H-Ser(tBu)-Gln-OH ??H-Ala-Asp(OtBu)-OH	??tr ??62.6mM	??2 ??2 ??2 ??2	??H-Aib-OMe ??H-CHA-OMe	??100mM ??40mM	??H-Phe-OH ??H-Phe-OH	??150mM ??40mM	??H-Aib-Phe-OH ??17.2mM		??3 ??3

100 μ l are comprised reaction soln with the 100mM borate buffer solution (pH9.0) of each the carboxyl composition of final concentration shown in the table 17 and amine component and 10mM EDTA in the reaction times shown in 25 ℃ of reaction tables 17.The growing amount that has shown multiple peptide in this reaction in the table 17.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard, and " tr " expression trace).

(abbreviation)

H-Ala-Ome:L-alanine methyl ester hydrochloride

H-p-F-Phe-Ome:p-fluoro-L-phenylalanine methyl ester hydrochloride

H-Cl-F-Phe-Ome:p-chloro-L-phenylalanine methyl ester hydrochloride

H-p-NO ₂-Phe-Ome:p-nitro-L-phenylalanine methyl ester hydrochloride

H-t-Leu-Ome: uncle-L-leucine methyl ester hydrochloride

H-2-Nal-OMe:3-(2-naphthyl)-L-alanine methyl ester hydrochloride

H-Aib-Ome: α-An Jiyidingsuan methyl ester hydrochloride

H-N-Me-Ala-OMe:N-methyl-L-alanine methyl ester hydrochloride

H-CHA-Ome: β-cyclohexyl-L-alanine methyl ester hydrochloride

H-Ser (the tBu)-Ome:O-tertiary butyl-L-serine methyl ester hydrochloride

H-Asp (OtBu)-Ome:L-Aspartic Acid β-tertiary butyl ester α-methyl ester hydrochloride

H-Lys (Boc)-Ome:N-ε-uncle-butoxy carbonyl-L-lysine methyl ester hydrochloride

H-p-F-Phe-OH:p-fluoro-L-phenylalanine

H-Cl-F-Phe-OH:p-chloro-L-phenylalanine

H-p-NO ₂-Phe-OH:p-nitro-L-phenylalanine

The H-t-Leu-OH:tert-L-leucine

H-2-Nal-OH:3-(2-naphthyl)-L-L-Ala

The H-Gln-OH:L-glutamine

The H-Phe-OH:L-phenylalanine

H-Ser (the tBu)-OH:O-tertiary butyl-L-Serine

H-Asp (OtBu)-OH:L-Aspartic Acid β-tertiary butyl ester

H-Lys (Boc)-OH:N-ε-uncle-butoxy carbonyl-L-Methionin

The substrate specificity (14) of (embodiment 27) enzyme

Adopt the substrate specificity of enzyme part (the come from short steady bacillus) assessment identical about the oligopeptides generation with embodiment 5.To comprise final concentration each carboxyl composition and amine component as shown in Table 18,100 μ l reaction solns of the 100mM borate buffer solution (pH9.0) of enzyme (Board Lot that adds in the reaction soln is shown in table 18) and 10mM EDTA were 25 ℃ of reactions 3 hours.The growing amount that has shown multiple oligopeptides in this reaction in the table 18.(generation of mark "+" expression peptide is confirmed but can not be by quantitatively owing to lack standard, and " tr " expression trace).Should be noted that for all carboxyl compositions and all use hydrochloride.

Table 18

The carboxyl composition	Amine component	Enzyme amount (unit)	The peptide (mM) that generates
The carboxyl composition	Amine component	Enzyme amount (unit)	The peptide (mM) that generates		??Gly-OMe	??L-Phe-L-Met	??1.0	??Gly-Phe-Met	??13.3
??L-Ala-OMe	??L-Phe-L-Met	??0.2	??L-Ala-L-Phe-L-Met	??+	??Gly-OMe	??L-Phe-L-Met	??1.0	??Gly-Phe-Met	??13.3
??L-Ala-OMe	??L-Phe-L-Met	??0.2	??L-Ala-L-Phe-L-Met	??+	??L-Tyr-OMe	??Gly-Gly-L-Phe-L-Met	??1.0	??L-Tyr-Gly-Gly-L-Phe-L-Met	??2.7
??L-Ala-OMe	??Gly-Gly-L-Phe-L-Met	??0.2	??L-Ala-Gly-Gly-L-Phe-L-Met	??+	??L-Tyr-OMe	??Gly-Gly-L-Phe-L-Met	??1.0	??L-Tyr-Gly-Gly-L-Phe-L-Met	??2.7
??L-Ala-OMe	??Gly-Gly-L-Phe-L-Met	??0.2	??L-Ala-Gly-Gly-L-Phe-L-Met	??+	??Gly-OMe	??Gly-L-Phe	??0.1	??Gly-L-Phe	??17.3
??L-Ala-OMe	??Gly-L-Phe	??0.1	??L-Ala-Gly-L-Phe	??+	??Gly-OMe	??Gly-L-Phe	??0.1	??Gly-L-Phe	??17.3
??L-Ala-OMe	??Gly-L-Phe	??0.1	??L-Ala-Gly-L-Phe	??+	??D-Ala-OMe	??Gly-L-Phe	??0.1	??D-Ala-Gly-L-Phe	??Tr

(embodiment 28) come from the separation of the peptide-forming enzyme gene of short steady bacillus

Below, will the separation of peptide-forming enzyme gene have been described.(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to short steady bacillus strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: on July 8th, 2002) be used as microorganism.In the separation of gene, intestinal bacteria JM-109 is used as the host, and pUC118 is used as carrier.

(1) prepares the PCR primer according to the aminoacid sequence of determining

Based on, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to come from short steady bacillus strain FERM BP-8113 according to the EdmanShi decomposition method by the digestion of lysyl endopeptidase, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: the determined aminoacid sequence of lysyl endopeptidase digestion product (SEQ ID NOs:1 and 2) that peptide-forming enzyme on July 8th, 2002) obtains, preparation has the mix primer of base sequence shown in SEQ ID NO:3 and the SEQ ID NO:4 respectively.

(2) preparation of microorganism cells

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center will to lack steady bacillus strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: on July 8th, 2002) (contain 50g/l glucose, 10g/l yeast extract, 110/l peptone, 5g/l sodium-chlor and 20g/l agar at the CM2G nutrient agar, pH7.0) on, cultivated 24 hours in 30 ℃.Inoculation 1 platinum loop gained microorganism cells in the 500ml slope Kou Shi bottle that contains 50ml CM2G liquid nutrient medium (the above-mentioned substratum except that agar) is then 30 ℃ of shaking culture.

(3) from microorganism cells, prepare chromosomal DNA

At first, with 50mL medium centrifugal (12,000rpm, 4 ℃, 15 minutes), collect microorganism cells.Then, adopt QIAGEN Genomic-Tip System (Qiagen),, from microorganism cells, obtain chromosomal DNA according to step described in its specification sheets.

(4) contain the dna fragmentation of the part of peptide-forming enzyme gene by PCR preparation

Adopt LA-Taq (Takara Shuzo production), pass through PCR method, acquisition contains and comes from short steady bacillus strain FERM BP-8113 (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center, preservation mechanism address: Dai-6,1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: the dna fragmentation of the part of peptide-forming enzyme gene on July 8th, 2002).Adopt the primer of the base sequence that contains SEQ ID NOs:3 and 4 then, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to coming from short steady bacillus strain FERMBP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: chromosomal DNA on July 8th, 2002) carries out PCR.

Adopt Takara PCR Thermal Cycler PERSONAL (Takara Shuzo production), implement 30 round-robin PCR reactions by following condition.

94 ℃ 30 seconds

52 ℃ 1 minute

72 ℃ 1 minute

After the reaction, carry out 0.8% agarose electrophoresis with 3 μ l reaction solutions.As a result, confirm that the dna fragmentation that increases is about 1.5 kilobase (kb).

(5) by gene library clone peptide-forming enzyme gene

In order to obtain the total length peptide-forming enzyme gene, adopt the dna fragmentation that in the PCR step, increases to carry out Southern hybridization as probe.Molecular Cloning, 2nd edition has described the step that Southern is hybridized among the ColdSpring Harbor Press (1989).

About 1.5kb dna fragmentation by the amplification of PCR step separates by 0.8% agarose electrophoresis.Downcut the target band then, purifying DNA fragment.Adopt DIG High Prime (Boehringer-Mannheim production), according to step described in the specification sheets of test kit, with probe digoxin (digoxinigen) labeled dna fragment.

Behind the chromosomal DNA of the steady bacillus of weak point that is obtained in the step (3) of complete digestion 5 μ g present embodiments 28 (3), gained DNA was being carried out electrophoresis 37 ℃ of reactions 16 hours with Restriction Enzyme HindIII on 0.8% sepharose.Behind the electrophoresis, electrophoretic chromosomal DNA trace from the sepharose is comprised that then alkaline denaturation, neutralization and fixed handle on the nylon leaching film (Roche Diagnostics production) of positively charged.Adopt EASY HYB (Boehringer-Mannheim production) to implement hybridization.In 50 ℃, the filter prehybridization after 1 hour, is added the probe with digoxigenin labeled by above-mentioned preparation, in 50 ℃ of hybridization 16 hours.Subsequently, with the 2 * SSC that contains 0.1%SDS washing nozzle 20 minutes under room temperature.In addition, again by 0.1 * SSC, 65 ℃, 15 minutes, washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production), detect according to step described in the specification sheets of test kit pair band with probe hybridization.As a result, can detect band with about 3kb of probe hybridization.

Then, the chromosomal DNA for preparing in the step (3) with HindIII complete digestion present embodiment 28 (3).By 0.8% agarose gel electrophoresis, isolate the DNA of about 4kb, adopt Gene Clean II test kit (Funakoshi production) purify DNA then, then DNA is dissolved among the 10 μ l TE.Then 4 these products of μ l are mixed with pUC118 HindIII/BAP (Takara Shuzo production), adopt dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix 5 μ l ligation mixtures and 100 μ l escherichia coli jm109 competent cells (Toyobo production) thereby transformed into escherichia coli.The transformant that will obtain thus places on the suitable solid medium and produces chromosomal dna library then.

In order to obtain the peptide-forming enzyme gene of total length, adopt above-mentioned probe, by colony hybridization screening chromosomal dna library.Molecular Cloning, 2nd edition has described the step of colony hybridization among the Cold SpringHarbor Press (1989).

The clone of chromosomal dna library is gone to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization, (Roche Diagnostics production)), comprise alkaline denaturation, neutralization and fixed processing then.Adopt EASY HYB (Boehringer-Mannheim production) to implement hybridization.In 37 ℃, the filter prehybridization after 1 hour, is added above-mentioned probe with digoxigenin labeled, in 50 ℃ of hybridization 16 hours.Subsequently, with the 2 * SSC that contains 0.1%SDS washing nozzle 20 minutes under room temperature.In addition, again by 0.1 * SSC, 65 ℃, 15 minutes, washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production),, the clone with the probe hybridization of mark is detected according to the explanation described in the specification sheets of test kit.The result confirms to have the probe hybridization of two clones and mark.

(6) come from the base sequence of the peptide-forming enzyme gene of short steady bacillus

The plasmid that e. coli jm109 contained is proved two clones with the probe hybridization of mark by adopting Wizard Plus Minipreps dna purification system (Promega productions) preparation by above-mentioned, determines with probe the part of hybridizing and the base sequence of neighbouring part to take place.Adopt CEQ DTCS-Quick Start Kit (Beckman-Coulter production), implement sequencing reaction according to step described in the specification sheets of test kit.In addition, adopt CEQ 2000-XL (Beckman-Coulter production) to implement electrophoresis.

The result confirms that the proteinic opening code-reading frame that coding comprises the internal amino acid sequence (SEQ ID NOs:1 and 2) of peptide-forming enzyme exists really.Therefore, opening code-reading frame is confirmed to be the gene of coding peptide-forming enzyme.The base sequence and the amino acid sequence corresponding that have shown the total length peptide-forming enzyme gene among the SEQ ID NO:5.With BLASTP the result of the homology analysis of gained opening code-reading frame is shown, have homology between two kinds of enzymes; It demonstrates on amino acid sequence level alpha-Amino acid ester hydrolase with Pasteur's bacillus aceticus (Acetobacter pasteurianus) and has 34% homology (referring to Appl.Environ.Microbiol.; 68 (1); 211-218 (2002)); and on amino acid sequence level, has 26% homology (referring to J.Bacteriol. with glutaryl-7ACA acyltransferase of side spore bacillus pumilus (Brevibacillus laterosporum); 173 (24), 7848-7855 (1991)).

(embodiment 29) come from the expression of peptide-forming enzyme gene in intestinal bacteria of short steady bacillus

With oligonucleotide described in SEQ ID NOs:7 and 8 as primer, by implementing increase target gene district on the promoter region of tryptophan operon on the chromosomal DNA of intestinal bacteria W3110 of PCR, the gained dna fragmentation is connected to pGEM-Teasy carrier (Promega production).Transform e. coli jm109 then in this connection liquid, select to contain the bacterial strain of target plasmid from the resistant strain of Ampicillin Trihydrate, the direction of insertion of the trp promotor of inserting in the described target plasmid is opposite with the direction of lac promotor.Then, will handle product (Takara production) with the EcoO109I/EcoRI of pUC19 and be connected by handle the dna fragmentation that comprises the trp promotor that this plasmid obtains with EcoO109I/EcoRI.Connect liquid transformed into escherichia coli JM109 with this then, from the resistant strain of Ampicillin Trihydrate, select to contain the bacterial strain of target plasmid.Then, will with HindIII/PvuII handle this plasmid and the dna fragmentation that obtains the dna fragmentation that comprises the rrnB terminator that obtains is connected with handle pKK223-3 (Amersham Pharmacia production) with HindIII/HincII.Connect liquid transformed into escherichia coli JM109 with this then, from the resistant strain of Ampicillin Trihydrate, select to contain the bacterial strain of target plasmid, this plasmid called after pTrpT.

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to use short steady bacillus strain FERM BP-8113, preservation mechanism address: Chuo No ChuoDai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: chromosomal DNA on July 8th, 2002) is as template, oligonucleotide shown in the SEQ IDNO:9 and 10 is as primer, by the pcr amplification target gene.Handle this dna fragmentation with NdeI/PstI then, then the gained dna fragmentation is handled product with the NdeI/PstI of pTrpT and be connected.Connect liquid transformed into escherichia coli JM109 with this then, from the resistant strain of Ampicillin Trihydrate, select to contain the bacterial strain of target plasmid, this plasmid called after pTrpT_Gtg2.

The e. coli jm109 that will contain pTrpT_Gtg2 is in containing the LB substratum of 100mg/l Ampicillin Trihydrate, in 30 ℃ of pre-cultivations 24 hours.1ml gained nutrient solution is seeded in contains 50ml substratum (2g/l D glucose, 10g/l yeast extract, 10g/l casamino acids, 5g/l ammonium sulfate, 3g/l potassium primary phosphate, 1g/l dipotassium hydrogen phosphate, 0.5g/l magnesium sulfate heptahydrate, with the 100mg/l Ampicillin Trihydrate) 500ml slope Kou Shi bottle in, cultivated 24 hours at 25 ℃ then.It is the 0.44U/1ml nutrient solution that the L-alanyl-L-glutamine of nutrient solution generates activity, confirms the escherichia coli expression cloned genes.In addition, in the transformant that only imports pTrpT in contrast, find activity.

(prediction signal sequence)

When with the aminoacid sequence of SEQ ID NO:6 described in the Signal P v 1.1 programanalysis sequence tables (referring to Protein Engineering, Vol.12, No.1, pp.3-9,1999), the performance of prediction 1-22 amino acids is secreted semiotic function to the pericentral siphon with peptide, and estimates that mature protein is positioned at the downstream of 23 amino acids.

(excretory checking)

The e. coli jm109 that contains pTrpT_Gtg2 is in containing the LB substratum of 100mg/l Ampicillin Trihydrate, in 30 ℃ of pre-cultivations 24 hours.1ml gained nutrient solution is seeded in contains 50ml substratum (2g/l glucose, 10g/l yeast extract, 10g/l casamino acids, 5g/l ammonium sulfate, 3g/l potassium primary phosphate, 1g/l dipotassium hydrogen phosphate, 0.5g/l magnesium sulfate heptahydrate, with the 100mg/l Ampicillin Trihydrate) 500ml slope Kou Shi bottle in, final cultivate 24 hours at 25 ℃ then to obtain microorganism cells.

With 20 Grams Per Minute liter (g/dl) sucrose solutions,, the cultured microorganism cell is divided into pericentral siphon part and kytoplasm part by the osmotic pressure ballistic method.The microorganism cells that immerses in the 20g/dl sucrose solution is immersed 5mM MgSO ₄In the aqueous solution.With centrifugal supernatant called after pericentral siphon part (" Pe ").In addition, the centrifugal throw out is resuspended, carry out ultrasonic degradation then.With this resultant called after kytoplasm part (" Cy ").The activity of glucose-6-phosphate dehydrogenase (G6PD) (known being present in the kytoplasm) is used as the separated indicator of confirmation kytoplasm.By, comprising the 1mM G-6-P, 0.4mM NADP adds an amount of enzyme in the reaction solution of 10mM MgSO4 and 50mM Tris-Cl (pH8), carry out this detection under 30 ℃, thereby detects the generation that the 340nm absorbancy detects NADPH then.

Fig. 4 has shown when the activity value that will separate the cell-free extract of preparation is decided to be 100%, the amount of enzyme in pericentral siphon part and the kytoplasm part.In pericentral siphon, do not find the glucose-6-phosphate dehydrogenase (G6PD) activity.This prompting pericentral siphon part is not sneaked in the kytoplasm part.Recovered about 60% Ala-Gln generation activity in pericentral siphon, as adopting Signal P v 1.1 programs from what aminoacid sequence doped, Ala-Gln generation enzyme is proved to be secreted to pericentral siphon.

(embodiment 30) produce the L-alanyl-L-glutamine with the microorganism cells of Sphingobacterium sp.

In 500ml slope Kou Shi bottle, add 50mL every liter (L) and contain 5g glucose, 5g ammonium sulfate, the 1g potassium primary phosphate, the 3g dipotassium hydrogen phosphate, 0.5g sal epsom, the substratum of 10g yeast extract and 10g peptone (pH6.2), (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallyyed permit biological sustenance center in order to cultivate Sphingobacterium sp. bacterial strain FERM BP-8124 115 ℃ of sterilizations 15 minutes, preservation mechanism address: Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002).Inoculation 1 platinum loop contains 5g glucose at every liter (L) on this substratum then, the 10g yeast extract, slant agar substratum (the agar: 20g/L of 10g peptone and 5g NaCl, pH7.0) on, cultivated 24 hours Sphingobacterium sp. bacterial strain FERM BP-8124 (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst has speciallyyed permit biological sustenance center for 30 ℃, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: cell on July 22nd, 2002), at 30 ℃, 120 reciprocal/minute following concussions were cultivated 20 hours then.Then this nutrient solution of 1ml is added in the above-mentioned substratum (50mL/500ml slope Kou Shi bottle), cultivated 18 hours in 30 ℃.After cultivation is finished, by centrifugal from nutrient solution the separate microorganism cell, then it is suspended in the 0.1M borate buffer solution (pH9.0) that contains 10mM EDTA, concentration is 100g/L, in wet microorganism cells.In this microorganism cells suspension of 0.1ml, add the 100mM borate buffer solution (pH9.0) that 0.1ml contains 10mM EDTA, 200mM L-alanyl methyl ester hydrochloride and 400mM L-glutaminate then.0.2mL gained mixture was reacted 120 minutes at 25 ℃.The concentration of the L-alanyl-L-glutamine that produce this moment is 62mM.

(embodiment 31) are from the purifying of the enzyme of Sphingobacterium sp.

Following steps after centrifugal are on ice or 4 ℃ of enforcements.(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to cultivate Sphingobacterium sp. bacterial strain FERM BP-8124 according to the method identical with embodiment 21, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002), collect microorganism cells by centrifugal (10,000rpm, 15 minutes).After with 20mM Tris-HCl damping fluid (pH7.6) washing 2g microorganism cells, it is suspended in the 8ml same buffer, carry out ultrasonic degradation with 195W then and handled 45 minutes.Centrifugal then this ultrasonic degradation liquid (10,000rpm, 30 minutes) to remove cell debris, obtains ultrasonic degradation liquid supernatant., remove insoluble part by ultracentrifugation (50,000rpm, 30 minutes) then, thereby obtain the soluble part of supernatant liquor form this ultrasonic degradation liquid supernatant dialysed overnight with 20mM Tris-HCl damping fluid (pH7.6).The soluble part adding that obtains is used in Tris-HCl damping fluid (pH7.6) the equilibrated Q-Sepharose HP post (Amersham production) in advance, from non-absorbed portion, collected active part then., remove insoluble part by centrifugal (10,000rpm, 30 minutes) then, thereby obtain the part of the dialysis of supernatant liquor form this active part dialysed overnight with 20mM acetate buffer (pH5.0).The part adding of this dialysis is used in 20mM acetate buffer (pH5.0) the equilibrated SP-Sepharose HP post (Amersham production) in advance, with the linear concentration gradient wash-out enzyme of the same buffer that contains 0-1M NaCl.

(embodiment 32) produce the L-alanyl-L-glutamine with active part

SP-Sepharose HP part (about 27U/ml) the adding 90 μ l of purifying among the 10 μ l embodiment 31 are contained 111mM L-alanine methyl ester hydrochloride, 222mM L-glutaminate and 11mMEDTA 111mM borate buffer solution (pH9.0) in, make its 25 ℃ the reaction 120 minutes.As a result, in the district that adds enzyme, generate 73mM L-alanyl-L-glutamine.On the other hand, almost do not observe any generation of L-Ala-L-Gln in not adding the district of enzyme, enzyme concn only is about 0.07mM after 120 minutes and react.

(embodiment 33) come from the separation of the peptide-forming enzyme gene of Sphingobacterium sp.

The separation of peptide-forming enzyme gene is described below, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center with Sphingobacterium sp. bacterial strain FERMBP-8124, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002) as microorganism.Adopt bacillus coli DH 5 alpha as the host, pUC118 implements gene isolation as carrier.

(1) preparation of microorganism

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center with Sphingobacterium sp. bacterial strain FERM BP-8124, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: on July 22nd, 2002) (contain 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l sodium-chlor and 20g/l agar, pH7.0) go up and cultivated 24 hours in 25 ℃ at the CM2G nutrient agar.Inoculation 1 platinum loop gained microorganism cells in the 500ml slope Kou Shi bottle that contains 50ml CM2G liquid nutrient medium (the above-mentioned substratum except that agar) is cultivated 25 ℃ of concussions then.

(2) from microorganism cells, prepare chromosomal DNA

The 50ml nutrient solution is carried out centrifugal (12,000rpm, 4 ℃, 15 minutes), collect microorganism cells.Adopt Qiagen Genomic-Tip System (Qiagen) from microorganism cells, to obtain chromosomal DNA then.

(3) prepare probe dna fragment by PCR

Adopt LA-Taq (Takara Shuzo production), obtain comprising by PCR method and come from short steady bacillus strain FERM BP-8113 (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center, preservation mechanism address: Chuo Dai-6,1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation is passed on the date: the dna fragmentation of a peptide-forming enzyme gene part on July 8th, 2002).Adopt the primer of the base sequence that contains SEQ ID NOs:3 and 4 then, to (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center available from short steady bacillus strain FERMBP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki 1-Chome, Japan, international preservation is passed on the date: chromosomal DNA on July 8th, 2002) is implemented the PCR reaction.

94 ℃ 30 seconds

52 ℃ 1 minute

72 ℃ 1 minute

After the reaction, carry out 0.8% agarose electrophoresis with 3 μ l reaction mixtures.The result confirms that the dna fragmentation that increases is about 1.5kb.

(4) by gene library clone peptide-forming enzyme gene

About 1.5kb dna fragmentation by the amplification of PCR step separates by 0.8% agarose electrophoresis.Downcut the target band then, purifying DNA fragment.Adopt DIG High Prime (Boehringer-Mannheim production), according to step described in the specification sheets of test kit, with probe digoxigenin labeled dna fragmentation.

Behind the chromosomal DNA of the Sphingobacterium sp. that is obtained in the step (2) of complete digestion 5 μ g present embodiments 33, it was being carried out 0.8% agarose gel electrophoresis 37 ℃ of reactions 16 hours with Restriction Enzyme SacI.Behind the electrophoresis, electrophoretic chromosomal DNA trace from the sepharose is comprised that then alkaline denaturation, neutralization and fixed handle on the nylon leaching film (Roche Diagnostics production) of positively charged.Adopt EASY HYB (Boehringer-Mannheim production) to hybridize.In 37 ℃, the filter prehybridization after 1 hour, is added the probe with digoxigenin labeled by above-mentioned preparation, in 37 ℃ of hybridization 16 hours.Subsequently, with the 2 * SSC that contains 0.1%SDS washing nozzle 20 minutes under room temperature.In addition, again with the 0.1 * SSC that comprises 0.1%SDS, 60 ℃, washed twice.

Then, the chromosomal DNA for preparing in the step (2) with SacI complete digestion present embodiment 33.Separate the DNA of about 3kb by 0.8% agarose gel electrophoresis, adopt Gene Clean II test kit (Funakoshi production) purify DNA then, then DNA is dissolved among the 10 μ l TE.Then with 4 these products of μ l and SacI under 37 ℃, reacted 16 hours, so that complete digestion, it is mixed with the pUC118 that uses alkaline phosphatase (intestinal bacteria C75) to handle, 37 ℃, 30 minutes and 50 ℃, 30 minutes, adopt dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l bacillus coli DH 5 competent cells (Takara Shuzo production) thereby transformed into escherichia coli.The transformant that will obtain thus places on the suitable solid medium and produces chromosomal dna library then.

The clone of chromosomal dna library is gone to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization, (Roche Diagnostics production)), comprise alkaline denaturation, neutralization and fixed processing then.Adopt EASY HYB (Boehringer-Mannheim production) to implement hybridization.In 37 ℃, the filter prehybridization after 1 hour, is added above-mentioned probe with digoxigenin labeled, in 50 ℃ of hybridization 16 hours.Subsequently, with comprising 0.1 * SSC of 1%SDS in 60 ℃ of washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production),, the clone with the probe hybridization of mark is detected according to the explanation described in the specification sheets of test kit.As a result, confirm to have the probe hybridization of 6 strain clones and mark.

(5) come from the base sequence of the peptide-forming enzyme gene of Sphingobacterium sp.

Adopt Wizard Plus Minipreps dna purification system (Promega productions) preparation plasmid that bacillus coli DH 5 contained from be proved the above-mentioned six strain microorganism cellss with the probe hybridization of mark, thereby determine and the contiguous base sequence of probe hybridization.Adopt CEQDTCS-Quick Start Kit (Beckman-Coulter production), implement sequencing reaction according to step described in the specification sheets of test kit.In addition, adopt CEQ 2000-XL (Beckman-Coulter production) to implement electrophoresis.

As a result, find to exist the opening code-reading frame of coding peptide-forming enzyme.The base sequence and the amino acid sequence corresponding that have shown the total length peptide-forming enzyme gene that comes from Sphingobacterium sp. among the SEQ ID NO:11.The peptide-forming enzyme that comes from Sphingobacterium sp. has 63.5% homology (shown in the BLASTP program detects) with the peptide-forming enzyme that comes from the steady bacillus of above-mentioned weak point on amino acid sequence level.

(embodiment 34) come from the peptide-forming enzyme gene of Sphingobacterium sp. in colibacillary expression

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to adopt Sphingobacterium sp. bacterial strain FERM BP-8124, preservation mechanism address: ChuoDai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation date: chromosomal DNA on July 22nd, 2002) is as template, oligonucleotide is implemented PCR as primer described in the SEQ ID NOs:13 and 14, the amplified target gene.Handle this dna fragmentation with NdeI/XbaI, the dna fragmentation that obtains is handled product with the NdeI/XbaI of pTrpT be connected.Connect liquid transformed into escherichia coli JM109 with this then, from the resistant strain of Ampicillin Trihydrate, select to contain the bacterial strain of target plasmid, this plasmid called after pTrpT_Sm_aet.

Be seeded in by strain cell and contain 3ml substratum (2g/l glucose, 10g/l yeast extract, 10g/l casamino acids, 5g/l ammonium sulfate, 3g/l potassium primary phosphate, 1g/l dipotassium hydrogen phosphate, 0.5g/l magnesium sulfate heptahydrate a platinum loop, with the 100mg/l Ampicillin Trihydrate) common test tube in mode, the e. coli jm109 that will contain pTrpT_Sm_aet was cultivated 20 hours at 25 ℃.The L-alanyl-L-glutamine generates activity and is proved by escherichia coli expression for the cloned genes of 2.1U/1ml nutrient solution.In addition, in the transformant that only imports pTrpT in contrast, find activity.

(prediction signal sequence)

When with the aminoacid sequence of SEQ ID NO:12 described in the Signal P v 1.1 programanalysis sequence tables (referring to Protein Engineering, Vol.12, No.1, pp.3-9,1999), the performance of prediction 1-20 amino acids is secreted semiotic function to the pericentral siphon with peptide, and estimates that mature protein is positioned at the downstream of 21 amino acids.

(confirmation of signal sequence)

Be seeded in by strain cell and contain 50ml substratum (2g/l glucose, 10g/l yeast extract, 10g/l casamino acids, 5g/l ammonium sulfate, 3g/l potassium primary phosphate, 1g/l dipotassium hydrogen phosphate, 0.5g/l magnesium sulfate heptahydrate a platinum loop, with the 100mg/l Ampicillin Trihydrate) common test tube in mode, the e. coli jm109 that will contain pTrpT_Sm_aet was cultivated 20 hours at 25 ℃.

Following steps after centrifugal are on ice or 4 ℃ of enforcements.After cultivation is finished, by centrifugal from nutrient solution the separate microorganism cell, with the 100mM phosphate buffered saline buffer (pH7) washing after, it is suspended in the identical damping fluid.Then this microorganism cells is carried out ultrasonic degradation with 195W and handled 20 minutes, centrifugal then this ultrasonic degradation liquid (12,000rpm, 30 minutes) to remove cell debris, obtains soluble part.The soluble part adding that obtains is used in 100mM phosphate buffered saline buffer (pH7) the equilibrated CHT-II post (Biorad production) in advance, used the 500mM phosphoric acid buffer with linear concentration gradient wash-out enzyme.The solution that the 2M ammonium sulfate of mixed active part and 5 times of volumes and 100mM phosphate buffered saline buffer are obtained, add and use in advance in 2M ammonium sulfate and the 100mM phosphoric acid buffer equilibrated Resource-PHE post (Amersham), with 0-2M ammonium sulfate with linear concentration gradient wash-out enzyme, thereby obtain active part solution.According to the result of these steps, confirmed by electrophoretic mode peptide-forming enzyme purifying equably.

When determining the aminoacid sequence of above-mentioned peptide-forming enzyme, obtain the aminoacid sequence of SEQ ID NO:15, as predicting that by Signal P v 1.1 programs mature protein is proved the downstream that is positioned at 21 amino acids by the EdmanShi decomposition method.

(embodiment 35) come from the separation of the peptide-forming enzyme gene of separating Vitrum AB soil bacillus IFO 12017

Below, will the separation of peptide-forming enzyme gene be described.Microorganism used therefor for separate Vitrum AB soil bacillus strain IFO 12017 (preservation mechanism: fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan).In gene isolation, intestinal bacteria JM-109 is used as the host, and pUC118 is used as carrier.

(1) preparation of microorganism

To separate Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) (contain 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l sodium-chlor and 20g/l agar at the CM2G nutrient agar, pH7.0) on, cultivated 24 hours in 25 ℃.Inoculation 1 platinum loop gained microorganism cells in the 500ml slope Kou Shi bottle that contains 50ml CM2G liquid nutrient medium (the above-mentioned substratum except that agar) is cultivated 25 ℃ of concussions then.

(2) from microorganism cells, prepare chromosomal DNA

With 50mL medium centrifugal (12,000rpm, 4 ℃, 15 minutes), collect microorganism cells.Then, adopt QIAGEN Genomic-Tip System (Qiagen),, from microorganism cells, obtain chromosomal DNA according to step described in its specification sheets.

(3) prepare probe dna fragment by PCR

Adopt LA-Taq (Takara Shuzo production), pass through PCR method, acquisition contains to come from separates Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, the dna fragmentation of the part of peptide-forming enzyme gene Japan).Adopt the primer of the base sequence that contains SEQ ID NOs:15 and 16 then, to available from separating Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, chromosomal DNA Japan) carries out PCR.About 1kb dna fragmentation by pcr amplification separates by 0.8% agarose electrophoresis.Downcut the target band then, the dna fragmentation that purifying obtains.Adopt DIG High Prime (Boehringer-Mannheim production), according to step described in the specification sheets of test kit, with this dna fragmentation of probe digoxigenin labeled.

(4) by gene library clone peptide-forming enzyme gene

In order to obtain the total length peptide-forming enzyme gene, adopt the dna fragmentation that increases in the above-mentioned PCR step to carry out Southern hybridization as probe.Molecular Cloning, 2nd edition has described the step that Southern is hybridized among the Cold Spring Harbor Press (1989).

By with Restriction Enzyme HindIII in 37 ℃ of reactions 16 hours and Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute is separated in complete digestion, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) behind the chromosomal DNA, it is carried out 0.8% agarose gel electrophoresis.Behind the electrophoresis, electrophoretic chromosomal DNA trace from the sepharose is comprised then that at the nylon leaching film (Roche Diagnostics production) of positively charged alkaline denaturation, neutralization and fixed handle.Adopt EASY HYB (Boehringer-Mannheim production) to hybridize.In 50 ℃, the filter prehybridization after 1 hour, is added the probe with digoxigenin labeled by above-mentioned preparation, in 50 ℃ of hybridization 16 hours.Subsequently, with the 0.1 * SSC that comprises 0.1%SDS, 60 ℃, washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production), detect according to step described in the specification sheets of test kit pair band with probe hybridization.As a result, can detect band with about 5kb of probe hybridization.

Then, separate Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, chromosomal DNA Japan) with HindIII complete digestion 5 μ g.Separate the DNA of about 5kb by 0.8% agarose gel electrophoresis, adopt Gene Clean II test kit (Funakoshi production) purify DNA then, then it is dissolved among the 10 μ l TE.Then 4 these products of μ l are mixed with pUC118 HindIII/BAP (Takara Shuzo production), adopt dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l e. coli jm109s (Takara Shuzo production), thus transformed into escherichia coli.The transformant that will obtain thus places on the suitable solid medium and produces chromosomal dna library then.

The clone of chromosomal dna library is gone to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization, (Roche Diagnostics production)), comprise alkaline denaturation, neutralization and fixed processing then.Adopt EASY HYB (Boehringer-Mannheim production) to implement hybridization.In 37 ℃, the filter prehybridization after 1 hour, is added above-mentioned probe with digoxigenin labeled, in 37 ℃ of hybridization 16 hours.Subsequently, with comprising 0.1 * SSC of 1%SDS in 60 ℃ of washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production), the clone with the probe hybridization of mark is detected according to the explanation described in the specification sheets of test kit.As a result, confirm to have the probe hybridization of 1 strain clone and mark.

(5) come from the base sequence of the peptide-forming enzyme gene of separating Vitrum AB soil bacillus strain IFO-12017

The plasmid that e. coli jm109 contained determines to take place with probe the contiguous base sequence of hybridization by the above-mentioned microorganism cells bacterial strain preparation that is proved with the probe hybridization of mark.Adopt CEQDTCS-Quick Start Kit (Beckman-Coulter production), implement sequencing reaction according to step described in the specification sheets of test kit.In addition, adopt CEQ 2000-XL (Beckman-Coulter production) to implement electrophoresis.

As a result, find to exist the opening code-reading frame of coding peptide-forming enzyme.Shown among the SEQ IDNO:17 in the sequence table to come from and separated Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, the base sequence of total length peptide-forming enzyme gene Japan), and amino acid sequence corresponding.

(embodiment 36) come from the expression of peptide-forming enzyme gene in intestinal bacteria of separating Vitrum AB soil bacillus strain IFO-12017

Vitrum AB soil bacillus strain IFO-12017 (preservation mechanism: fermentation research institute is separated in use, Osaka, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) chromosomal DNA is as template, and the oligonucleotide shown in the SEQ ID NO:19 and 20 is as primer, by the pcr amplification target gene.Handle this dna fragmentation with NdeI/HindIII then, then the gained dna fragmentation is handled product with the NdeI/HindIII of pTrpT and be connected.Connect liquid transformed into escherichia coli JM109 with this then, from the resistant strain of Ampicillin Trihydrate, select to contain the bacterial strain of target plasmid, this plasmid called after pTrpT_Ph_aet.

Be seeded in by strain cell and contain 3ml substratum (2g/l glucose, 10g/l yeast extract, 10g/l casamino acids, 5g/l ammonium sulfate, 3g/l potassium primary phosphate, 1g/l dipotassium hydrogen phosphate, 0.5g/l magnesium sulfate heptahydrate a platinum loop, with the 100mg/l Ampicillin Trihydrate) common test tube in mode, the e. coli jm109 that will contain pTrpT_Ph_aet was cultivated 20 hours at 25 ℃.The L-alanyl-L-glutamine generates activity and is proved by escherichia coli expression for the cloned genes of 0.3U/1ml nutrient solution.In addition, in the transformant that only imports pTrpT in contrast, find activity.

(embodiment 37) are from the separation of the peptide-forming enzyme gene of Taxeobacter gelupurpurascens strain DSM Z11116

Below, will the separation of peptide-forming enzyme gene be described.Employed microorganism is Taxeobacter gelupurpurascens strain DSM Z a 11116 (preservation mechanism: DeutcheSammlung von Mikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany).In gene isolation, intestinal bacteria JM-109 is used as the host, and pUC118 is used as carrier.

(1) preparation of microorganism

With Taxeobacter gelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg1b, 38124 Braunschweig, Germany) (contain 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l sodium-chlor and 20g/l agar, pH7.0) go up and cultivated 24 hours at the CM2G nutrient agar in 25 ℃.Inoculation 1 platinum loop gained microorganism cells in the 500ml slope Kou Shi bottle that contains 50ml CM2G liquid nutrient medium (the above-mentioned substratum except that agar) is cultivated 25 ℃ of concussions then.

(2) from microorganism cells, prepare chromosomal DNA

The 50mL nutrient solution is carried out centrifugal (12,000rpm, 4 ℃, 15 minutes), collect microorganism cells.Then, adopt QIAGEN Genomic-Tip System (Qiagen),, from microorganism cells, obtain chromosomal DNA according to step described in its specification sheets.

(3) prepare probe dna fragment by PCR

Adopt LA-Taq (Takara Shuzo production), pass through PCR method, acquisition contains and comes from Taxeobacter gelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg1b, 38124 Braunschweig, the dna fragmentation of the part of peptide-forming enzyme gene Germany).Adopt the primer of the base sequence that contains SEQ ID NOs:21 and 16 then, to available from Taxeobacter gelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg1b, 38124 Braunschweig, chromosomal DNA Germany) carries out PCR.To separate by 0.8% agarose electrophoresis by about 1kb dna fragmentation of pcr amplification.Downcut the target band then, the dna fragmentation that purifying obtains.Adopt DIG High Prime (Boehringer-Mannheim production), according to step described in the specification sheets of test kit, with this dna fragmentation of probe digoxigenin labeled.

(4) from gene library clone peptide-forming enzyme gene

Reacting 16 hours and complete digestion Taxeobactergelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung vonMikroorganismen und Zellkulturen GmbH (German microorganism and cell culture preservation center) at 37 ℃ with Restriction Enzyme PstI, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany) behind the chromosomal DNA, it is carried out 0.8% agarose gel electrophoresis.Behind the electrophoresis, electrophoretic chromosomal DNA trace from the sepharose is comprised then that at the nylon leaching film (Roche Diagnostics production) of positively charged alkaline denaturation, neutralization and fixed handle.Adopt EASY HYB (Boehringer-Mannheim production) to hybridize.In 50 ℃, the filter prehybridization after 1 hour, is added the probe with digoxigenin labeled by above-mentioned preparation, in 50 ℃ of hybridization 16 hours.Subsequently, with the 0.1 * SSC that comprises 0.1%SDS, 60 ℃, washed twice.

With HindIII complete digestion 5 μ g Taxeobacter gelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, chromosomal DNA Germany).Separate the DNA of about 5kb by 0.8% agarose gel electrophoresis, adopt Gene Clean II test kit (Funakoshi production) purify DNA then, it is dissolved among the 10 μ l TE.Then 4 these products of μ l are mixed with pUC118 PstI/BAP (Takara Shuzo production), adopt dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l e. coli jm109s (Takara Shuzo production) and transformed into escherichia coli.The transformant that will obtain thus places on the suitable solid medium and produces chromosomal dna library then.

The clone of chromosomal dna library is gone to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization, (Roche Diagnostics production)), comprise alkaline denaturation, neutralization and fixed processing then.Adopt EASY HYB (Boehringer-Mannheim production) to implement hybridization.In 37 ℃, the filter prehybridization after 1 hour, is added above-mentioned probe with digoxigenin labeled, in 37 ℃ of hybridization 16 hours.Subsequently, with the 0.1 * SSC that comprises 1%SDS in twice of 60 ℃ of washing nozzle.

(5) come from the base sequence of the peptide-forming enzyme gene of Taxeobacter gelupurpurascens strain DSM Z 11116

As a result, find to exist the opening code-reading frame of coding peptide-forming enzyme.Shown among the SEQ IDNO:22 in the sequence table and come from Taxeobacter gelupurpurascens strain DSM Z 11116 (preservation mechanism: Deutche Sammlung von Mikroorganismen undZellkulturen GmbH (German microorganism and cell culture preservation center), preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, the base sequence of total length peptide-forming enzyme gene Germany), and amino acid sequence corresponding.

(embodiment 38) come from the separation of the peptide-forming enzyme gene of extra large round bacteria strains A TCC 25205

Below, will the separation of peptide-forming enzyme gene be described.Used microorganism is extra large round bacteria strains A TCC 25205 (a preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America).In gene isolation, intestinal bacteria JM-109 is used as the host, and pUC118 is used as carrier.

(1) preparation of microorganism cells

With extra large round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the UnitedStates of America) (contains 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l sodium-chlor and 20g/l agar at the CM2G nutrient agar, pH7.0) on, cultivated 24 hours in 25 ℃.Inoculation 1 platinum loop gained microorganism cells in the 500ml slope Kou Shi bottle that contains 50ml CM2G liquid nutrient medium (the above-mentioned substratum except that agar) is cultivated 25 ℃ of concussions then.

(2) from microorganism cells, prepare chromosomal DNA

(3) prepare probe dna fragment by PCR

Adopt LA-Taq (Takara Shuzo production), pass through PCR method, acquisition contains and comes from extra large round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United Statesof America) the dna fragmentation of a part of peptide-forming enzyme gene.Adopt the primer of the base sequence that contains SEQID NOs:15 and 16 then, to available from extra large round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box1549, Manassas, VA 20110, the United States of America) chromosomal DNA carry out PCR.About 1kb dna fragmentation by pcr amplification separates by 0.8% agarose electrophoresis.Downcut the target band then, the dna fragmentation that purifying obtains.Adopt DIG HighPrime (Boehringer-Mannheim production), according to step described in the specification sheets of test kit, with this dna fragmentation of probe digoxigenin labeled.

(4) by gene library clone peptide-forming enzyme gene

Reacting 16 hours and complete digestion sea round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection at 37 ℃ with Restriction Enzyme HincII, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) behind the chromosomal DNA, it is implemented 0.8% agarose gel electrophoresis respectively.Behind the electrophoresis, electrophoretic chromosomal DNA trace from the sepharose is comprised then that at the nylon leaching film (RocheDiagnostics production) of positively charged alkaline denaturation, neutralization and fixed handle.Adopt EASY HYB (Boehringer-Mannheim production) to hybridize.In 50 ℃, the filter prehybridization after 1 hour, is added the probe with digoxigenin labeled by above-mentioned preparation, in 50 ℃ of hybridization 16 hours.Subsequently, with the 0.1 * SSC that comprises 0.1%SDS, 60 ℃, washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production), detect according to step described in the specification sheets of test kit pair band with probe hybridization.As a result, in the PstI digestion product, detect the band with about 7k of probe hybridization, and in the HincII digestion product, detect the band with about 2k of probe hybridization.

With PstI or HincII complete digestion 5 μ g sea round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) chromosomal DNA.Isolate the DNA of about 7kb or 2kb by 0.8% agarose gel electrophoresis, adopt Gene Clean II test kit (Funakoshi production) purify DNA then, it is dissolved among the 10 μ l TE.Then 4 these products of μ l are mixed with pUC118 PstI/BAP (Takara Shuzo production) or pUC118 HincII/BAP (Takara Shuzo production), adopt dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l e. coli jm109s (Takara Shuzo production) respectively and transformed into escherichia coli.The transformant that will obtain thus places on the suitable solid medium and produces chromosomal dna library then.

(5) come from the base sequence of the peptide-forming enzyme gene of extra large round bacteria strains A TCC 25205

As a result, find to exist the opening code-reading frame of coding peptide-forming enzyme.Shown among the SEQ IDNO:24 in the sequence table and come from extra large round bacteria strains A TCC 25205 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the base sequence of total length peptide-forming enzyme gene the United States of America), and amino acid sequence corresponding.

(embodiment 39) come from the separation of the peptide-forming enzyme gene of Psycloserpens burtonensis strains A TCC 700359

Below, will the separation of peptide-forming enzyme gene be described.Microorganism used therefor is Psycloserpensburtonensis strains A TCC 700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the UnitedStates of America).In isolated genes, intestinal bacteria JM-109 is used as the host, and pUC118 is as carrier.

(1) preparation of microorganism

With Psycloserpens burtonensis strains A TCC 700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) (contains 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l sodium-chlor and 20g/l agar, pH7.0) go up and cultivated 24 hours at the CM2G nutrient agar in 10 ℃.Inoculation 1 platinum loop gained microorganism cells in the 500ml slope Kou Shi bottle that contains 50ml CM2G liquid nutrient medium (the above-mentioned substratum except that agar) is cultivated 10 ℃ of concussions then.

(2) from microorganism cells, prepare chromosomal DNA

(3) prepare probe dna fragment by PCR

Adopt LA-Taq (Takara Shuzo production), pass through PCR method, acquisition contains and comes from Psycloserpens burtonensis strains A TCC 700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) the dna fragmentation of a part of peptide-forming enzyme gene.Adopt the primer of the base sequence that contains SEQ ID NOs:15 and 16 then, to available from Psycloserpens burtonensis strains A TCC 700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) chromosomal DNA carry out PCR.About 1kb dna fragmentation by pcr amplification separates by 0.8% agarose electrophoresis.Downcut the target band then, the dna fragmentation that purifying obtains.Adopt DIG High Prime (Boehringer-Mannheim production), according to step described in the specification sheets of test kit, with this dna fragmentation of probe digoxigenin labeled.

(4) by gene library clone peptide-forming enzyme gene

In order to obtain the total length peptide-forming enzyme gene, adopt the dna fragmentation that in above-mentioned PCR step, increases to carry out Southern hybridization as probe.Molecular Cloning, 2nd edition has described the step that Southern is hybridized among the Cold Spring Harbor Press (1989).

Reacting 16 hours and complete digestion Psycloserpens burtonensis strains A TCC 700359 (preservation mechanism: American type culture collection at 37 ℃ with Restriction Enzyme EcoRI, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the United States of America) behind the chromosomal DNA, it is carried out 0.8% agarose gel electrophoresis.Behind the electrophoresis, electrophoretic chromosomal DNA trace from the sepharose is comprised then that at the nylon leaching film (Roche Diagnostics production) of positively charged alkaline denaturation, neutralization and fixed handle.Adopt EASY HYB (Boehringer-Mannheim production) to hybridize.In 50 ℃, the filter prehybridization after 1 hour, is added the probe with mark by above-mentioned preparation, in 50 ℃ of hybridization 16 hours.Subsequently, with the 0.1 * SSC that comprises 0.1%SDS, 60 ℃, washed twice.

Adopt DIG kit for detecting nucleic acid (Boehringer-Mannheim production), detect according to step described in the specification sheets of test kit pair band with probe hybridization.As a result, can detect band with about 7kb of probe hybridization.

With EcoRI complete digestion 5 μ g Psycloserpens burtonensis strains A TCC700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box1549, Manassas, VA 20110, the United States of America) chromosomal DNA.Separate the DNA of about 7kb by 0.8% agarose gel electrophoresis, adopt Gene Clean II test kit (Funakoshi production) purify DNA then, it is dissolved among the 10 μ l TE.Then 4 these products of μ l are mixed with pUC118 EcoRI/BAP (Takara Shuzo production), adopt dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l e. coli jm109s (Takara Shuzo production) and transformed into escherichia coli.The transformant that will obtain thus places on the suitable solid medium and produces chromosomal dna library then.

Adopt DIG Nucleotide Detection Kit (Boehringer-Mannheim production), according to the clone of specification sheets detection with the probe hybridization of mark.As a result, confirm the probe hybridization of 1 strain clone and mark.

(5) come from the base sequence of the peptide-forming enzyme gene of Psycloserpens burtonensis strains A TCC 700359

The plasmid that e. coli jm109 contained determines to take place with probe the contiguous base sequence of hybridization by the above-mentioned microorganism cells bacterial strain preparation that is proved with the probe hybridization of mark.According to the described step of specification sheets, adopt CEQ DTCS-Quick Start Kit (Beckman-Coulter production) to carry out sequencing reaction.In addition, adopt CEQ 2000-XL (Beckman-Coulter production) to implement electrophoresis.

As a result, find to exist the opening code-reading frame of coding peptide-forming enzyme.Shown among the SEQ IDNO:31 in the sequence table and come from Psycloserpens burtonensis strains A TCC 700359 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box1549, Manassas, VA 20110, the base sequence of total length peptide-forming enzyme gene the United States of America), and amino acid sequence corresponding.

Industrial applicability

According to the present invention, provide by reducing such as the complicated synthetic method of introducing and eliminate protecting group, and can be easily, high productivity and produce at an easy rate the novel enzyme of peptide. Use endonuclease capable of the present invention to realize the efficient industrial production of peptide.

Sequence table

SEQ ID NO:3: synthetic primer 1

SEQ ID NO:4: synthetic primer 2

SEQ ID NO:5: the gene of coding peptide-forming enzyme

SEQ ID NO:7: the synthetic primer that is used to prepare pTrpT

SEQ ID NO:8: the synthetic primer that is used to prepare pTrpT

SEQ ID NO:9: the synthetic primer that is used to prepare pTrpT_Gtg2

SEQ ID NO:10: the synthetic primer that is used to prepare pTrpT_Gtg2

SEQ ID NO:11: the gene of coding peptide-forming enzyme

SEQ ID NO:13: the synthetic primer that is used to prepare pTrpT_Sm_aet

SEQ ID NO:14: the synthetic primer that is used to prepare pTrpT_Sm_aet

SEQ ID NO:15: the mix primer 1 that is used for Aet

SEQ ID NO:16: the mix primer 2 that is used for Aet

SEQ ID NO:19: the primer 1 that is used to make up the aet expression vector that comes from Pedobacter.

SEQ ID NO:20: the primer 2 that is used to make up the aet expression vector that comes from Pedobacter.

SEQ ID NO:21: the mix primer 3 that is used for Aet.

Sequence table

<110>AJINOMOTO?CO.，LTD.

＜120〉novel peptide-forming enzyme gene

<130>PAMA-15377，PAMA-03030

<150>JP2002-218957

<151>2002-07-26

<150>JP2003-016765

<151>2003-01-24

<160>27

<170>PatentIn?Ver.2.1

<210>1

<211>9

<212>PRT

＜213〉lack steady stalk bacterium

<220>

<223>Inventor：HARA，Seiichi

Inventor：YOKOZEKI，Kenzo

Inventor：ABE，Isao

Inventor：TONOUCHI，Naoto

Inventor：JOJIMA，Yasuko

<400>1

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

1?????????????5

<210>2

<211>9

<212>PRT

＜213〉lack steady stalk bacterium

<400>2

Thr?Asn?Val?Thr?Tyr?Thr?Met?Pro?Asp

1?????????????5

<210>3

<211>20

<212>DNA

＜213〉composition sequence

<220>

＜223〉artificial sequence description: synthetic primer 1

<400>3

ttyacngcna?thtaycarcc??????????????????????????????????????????????????20

<210>4

<211>23

<212>DNA

＜213〉composition sequence

<220>

＜223〉artificial sequence description: synthetic primer 2

<400>4

tcnggcatng?trtangtnac?rtt??????????????????????????????????????????????23

<210>5

<211>2024

<212>DNA

＜213〉lack steady stalk bacterium

<220>

<221>CDS

<222>(61)..(1908)

＜223〉gene of encoded peptide synthetic enzyme

<400>5

atttcttaat?aaaaactgaa?atcttaatac?atttatacta?tcgtaaaatt?tattgaacac?60

gtg?aaa?aaa?tta?aca?tta?aaa?gta?act?cta?ctt?aca?ctt?ttg?ttg?gga???108

Val?Lys?Lys?Leu?Thr?Leu?Lys?Val?Thr?Leu?Leu?Thr?Leu?Leu?Leu?Gly

1???????????????5???????????????????10??????????????????15

agt?aca?gtt?gga?ttt?gcg?caa?gat?gca?aaa?gca?gat?tct?gct?tat?gtg???156

Ser?Thr?Val?Gly?Phe?Ala?Gln?Asp?Ala?Lys?Ala?Asp?Ser?Ala?Tyr?Val

20??????????????????25??????????????????30

cgc?gac?aat?tac?gaa?aaa?ata?gaa?caa?gta?att?ccg?atg?cgc?gat?ggt???204

Arg?Asp?Asn?Tyr?Glu?Lys?Ile?Glu?Gln?Val?Ile?Pro?Met?Arg?Asp?Gly

35??????????????????40??????????????????45

aca?aag?tta?ttt?aca?gct?att?tat?cag?cca?aaa?gat?aaa?aca?aaa?caa???252

Thr?Lys?Leu?Phe?Thr?Ala?Ile?Tyr?Gln?Pro?Lys?Asp?Lys?Thr?Lys?Gln

50??????????????????55??????????????????60

tat?ccc?gtt?ttg?tta?aat?cgt?acg?cct?tat?aca?gtt?gcg?cct?tat?ggt???300

Tyr?Pro?Val?Leu?Leu?Asn?Arg?Thr?Pro?Tyr?Thr?Val?Ala?Pro?Tyr?Gly

65??????????????????70??????????????????75??????????????????80

gta?aat?gaa?tac?aag?aaa?tcg?tta?gga?aat?ttt?cct?aca?gaa?atg?cgc???348

Val?Asn?Glu?Tyr?Lys?Lys?Ser?Leu?Gly?Asn?Phe?Pro?Thr?Glu?Met?Arg

85??????????????????90??????????????????95

gaa?ggt?ttt?att?ttt?gtt?tac?caa?gat?gtg?aga?gga?aaa?tgg?atg?agc???396

Glu?Gly?Phe?Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Lys?Trp?Met?Ser

100?????????????????105?????????????????110

gaa?ggc?gaa?ttt?gaa?gat?gtt?cga?cct?ata?aat?cct?tca?aaa?agt?aaa???444

Glu?Gly?Glu?Phe?Glu?Asp?Val?Arg?Pro?Ile?Asn?Pro?Ser?Lys?Ser?Lys

115?????????????????120?????????????????125

aag?gca?att?gac?gaa?agc?aca?gat?aca?ttt?gat?acg?cta?gaa?tgg?ctt???492

Lys?Ala?Ile?Asp?Glu?Ser?Thr?Asp?Thr?Phe?Asp?Thr?Leu?Glu?Trp?Leu

130?????????????????135?????????????????140

gct?aaa?aac?ttg?aag?aat?tac?acg?aaa?aaa?gct?gga?att?tat?gga?att???540

Ala?Lys?Asn?Leu?Lys?Asn?Tyr?Thr?Lys?Lys?Ala?Gly?Ile?Tyr?Gly?Ile

145?????????????????150?????????????????155?????????????????160

tcg?tat?cct?ggt?ttt?tat?tcg?aca?atg?agt?ttg?gtt?aat?tcg?cat?cca???588

Ser?Tyr?Pro?Gly?Phe?Tyr?Ser?Thr?Met?Ser?Leu?Val?Asn?Ser?His?Pro

165?????????????????170?????????????????175

act?cta?aaa?gcc?gtt?tcg?cca?caa?gcg?ccc?gtt?acc?aat?tgg?ttt?tta???636

Thr?Leu?Lys?Ala?Val?Ser?Pro?Gln?Ala?Pro?Val?Thr?Asn?Trp?Phe?Leu

180?????????????????185?????????????????190

ggt?gac?gat?ttt?cat?cat?aat?gga?gtt?tta?ttc?ttg?aat?gat?tct?ttc???684

Gly?Asp?Asp?Phe?His?His?Asn?Gly?Val?Leu?Phe?Leu?Asn?Asp?Ser?Phe

195?????????????????200?????????????????205

tca?ttt?atg?act?ttt?ttt?ggt?gta?aaa?cgt?ccg?caa?cca?att?acg?cca???732

Ser?Phe?Met?Thr?Phe?Phe?Gly?Val?Lys?Arg?Pro?Gln?Pro?Ile?Thr?Pro

210?????????????????215?????????????????220

gat?aaa?ggt?ccg?aaa?cgt?ttt?gaa?tat?cca?ata?aaa?gat?aat?tat?aga???780

Asp?Lys?Gly?Pro?Lys?Arg?Phe?Glu?Tyr?Pro?Ile?Lys?Asp?Asn?Tyr?Arg

225?????????????????230?????????????????235?????????????????240

ttt?tat?gca?agt?ggc?tct?gta?aaa?gag?ttg?aaa?gat?aaa?tat?ttg?caa???828

Phe?Tyr?Ala?Ser?Gly?Ser?Val?Lys?Glu?Leu?Lys?Asp?Lys?Tyr?Leu?Gln

245?????????????????250?????????????????255

gat?aat?atc?aag?ttt?tac?aat?gat?tta?ttt?gcg?cat?cca?gat?tac?gat???876

Asp?Asn?Ile?Lys?Phe?Tyr?Asn?Asp?Leu?Phe?Ala?His?Pro?Asp?Tyr?Asp

260?????????????????265?????????????????270

caa?ttt?tgg?caa?gat?cgt?aat?gtt?tta?cca?cat?tta?act?aac?gtg?caa???924

Gln?Phe?Trp?Gln?Asp?Arg?Asn?Val?Leu?Pro?His?Leu?Thr?Asn?Val?Gln

275?????????????????280?????????????????285

cct?gct?gta?atg?acg?gtt?gga?ggt?ttt?ttt?gat?gca?gaa?gat?gtc?tac???972

Pro?Ala?Val?Met?Thr?Val?Gly?Gly?Phe?Phe?Asp?Ala?Glu?Asp?Val?Tyr

290?????????????????295?????????????????300

ggc?gct?ttc?gaa?acg?tat?aaa?gca?att?gag?aaa?caa?aat?ccg?aaa?gca???1020

Gly?Ala?Phe?Glu?Thr?Tyr?Lys?Ala?Ile?Glu?Lys?Gln?Asn?Pro?Lys?Ala

305?????????????????310?????????????????315?????????????????320

aca?aat?att?atg?gtt?gcc?gga?cct?tgg?ttt?cat?ggt?ggt?tgg?gtt?cgt???1068

Thr?Asn?Ile?Met?Val?Ala?Gly?Pro?Trp?Phe?His?Gly?Gly?Trp?Val?Arg

325?????????????????330?????????????????335

agc?aac?gga?agt?act?ttt?gga?gat?atg?caa?ttt?gca?tcg?aat?aca?agt???1116

Ser?Asn?Gly?Ser?Thr?Phe?Gly?Asp?Met?Gln?Phe?Ala?Ser?Asn?Thr?Ser

340?????????????????345?????????????????350

gag?cat?tat?cag?caa?gaa?ata?gaa?ttg?cct?ttt?ttt?aat?tat?tac?tta???1164

Glu?His?Tyr?Gln?Gln?Glu?Ile?Glu?Leu?Pro?Phe?Phe?Asn?Tyr?Tyr?Leu

355?????????????????360?????????????????365

aaa?gat?aaa?ggt?aat?ttt?aaa?cca?acc?gaa?gct?aca?att?ttt?att?acg???1212

Lys?Asp?Lys?Gly?Asn?Phe?Lys?Pro?Thr?Glu?Ala?Thr?Ile?Phe?Ile?Thr

370?????????????????375?????????????????380

gga?tct?aac?gaa?tgg?aaa?caa?ttt?gat?gct?tgg?cca?cca?aaa?aat?gta???1260

Gly?Ser?Asn?Glu?Trp?Lys?Gln?Phe?Asp?Ala?Trp?Pro?Pro?Lys?Asn?Val

385?????????????????390?????????????????395?????????????????400

aca?aca?caa?aaa?att?tat?ttg?caa?caa?aat?ggt?aaa?ata?gct?ttt?aat???1308

Thr?Thr?Gln?Lys?Ile?Tyr?Leu?Gln?Gln?Asn?Gly?Lys?Ile?Ala?Phe?Asn

405?????????????????410?????????????????415

aaa?acc?aat?aca?aca?act?act?ttt?gac?gaa?tat?gtt?gca?gat?cca?aat???1356

Lys?Thr?Asn?Thr?Thr?Thr?Thr?Phe?Asp?Glu?Tyr?Val?Ala?Asp?Pro?Asn

420?????????????????425?????????????????430

tct?cca?gtt?cct?tat?tca?gga?gga?gtt?tta?gaa?act?cgt?tca?aga?gaa???1404

Ser?Pro?Val?Pro?Tyr?Ser?Gly?Gly?Val?Leu?Glu?Thr?Arg?Ser?Arg?Glu

435?????????????????440?????????????????445

tat?atg?gtc?gat?gat?caa?cgc?ttt?gct?tct?act?cgt?cct?gat?gtt?atg???1452

Tyr?Met?Val?Asp?Asp?Gln?Arg?Phe?Ala?Ser?Thr?Arg?Pro?Asp?Val?Met

450?????????????????455?????????????????460

gtg?tat?caa?tct?gat?att?ttg?aca?gaa?gat?att?acg?ctt?gct?ggt?cct???1500

Val?Tyr?Gln?Ser?Asp?Ile?Leu?Thr?Glu?Asp?Ile?Thr?Leu?Ala?Gly?Pro

465?????????????????470?????????????????475?????????????????480

gtt?atc?aat?cat?tta?gtg?gtt?tct?act?acg?gga?aca?gac?gct?gat?tat???1548

Val?Ile?Asn?His?Leu?Val?Val?Ser?Thr?Thr?Gly?Thr?Asp?Ala?Asp?Tyr

485?????????????????490?????????????????495

gtt?gta?aaa?ttg?att?gat?gtt?tat?cct?gaa?aac?acg?cca?aaa?ttt?aat???1596

Val?Val?Lys?Leu?Ile?Asp?Val?Tyr?Pro?Glu?Asn?Thr?Pro?Lys?Phe?Asn

500?????????????????505?????????????????510

aac?aaa?tta?atg?gct?gga?tat?caa?aat?ttg?att?cgt?gca?gaa?att?atg???1644

Asn?Lys?Leu?Met?Ala?Gly?Tyr?Gln?Asn?Leu?Ile?Arg?Ala?Glu?Ile?Met

515?????????????????520?????????????????525

cgc?gga?aaa?tat?aga?aat?agt?ttc?tct?aac?ccc?gaa?gct?atg?gtt?ccg???1692

Arg?Gly?Lys?Tyr?Arg?Asn?Ser?Phe?Ser?Asn?Pro?Glu?Ala?Met?Val?Pro

530?????????????????535?????????????????540

aat?aaa?gaa?aca?aat?gta?acg?tac?acg?atg?cca?gat?gtt?gga?cat?aca???1740

Asn?Lys?Glu?Thr?Asn?Val?Thr?Tyr?Thr?Met?Pro?Asp?Val?Gly?His?Thr

545?????????????????550?????????????????555?????????????????560

ttt?aag?aaa?gga?cat?cgc?att?atg?att?caa?gtt?cag?aac?agt?tgg?ttt???1788

Phe?Lys?Lys?Gly?His?Arg?Ile?Met?Ile?Gln?Val?Gln?Asn?Ser?Trp?Phe

565?????????????????570?????????????????575

cct?tta?gca?gat?cgc?aat?ccg?caa?caa?ttt?atg?aat?gtt?tac?gaa?gca???1836

Pro?Leu?Ala?Asp?Arg?Asn?Pro?Gln?Gln?Phe?Met?Asn?Val?Tyr?Glu?Ala

580?????????????????585?????????????????590

act?tct?aaa?gat?tat?tta?aaa?caa?acg?caa?cga?att?tat?cat?act?tct???1884

Thr?Ser?Lys?Asp?Tyr?Leu?Lys?Gln?Thr?Gln?Arg?Ile?Tyr?His?Thr?Ser

595?????????????????600?????????????????605

tat?atc?gaa?att?ccg?gta?ttg?aaa?taacaaaaaa?atccagctaa?ttagctggat??1938

Tyr?Ile?Glu?Ile?Pro?Val?Leu?Lys

610?????????????????615

tttttttata?atgttacttt?tcctattttt?cctttatttc?caactaaaat?tacatatttt?1998

ttatcgggcg?aaaccgtaca?agtatg??????????????????????????????????????2024

<210>6

<211>616

<212>PRT

＜213〉lack steady stalk bacterium

<400>6

Val?Lys?Lys?Leu?Thr?Leu?Lys?Val?Thr?Leu?Leu?Thr?Leu?Leu?Leu?Gly

1???????????????5???????????????????10??????????????????15

Ser?Thr?Val?Gly?Phe?Ala?Gln?Asp?Ala?Lys?Ala?Asp?Ser?Ala?Tyr?Val

20??????????????????25??????????????????30

Arg?Asp?Asn?Tyr?Glu?Lys?Ile?Glu?Gln?Val?Ile?Pro?Met?Arg?Asp?Gly

35??????????????????40??????????????????45

Thr?Lys?Leu?Phe?Thr?Ala?Ile?Tyr?Gln?Pro?Lys?Asp?Lys?Thr?Lys?Gln

50??????????????????55??????????????????60

Tyr?Pro?Val?Leu?Leu?Asn?Arg?Thr?Pro?Tyr?Thr?Val?Ala?Pro?Tyr?Gly

65??????????????????70??????????????????75??????????????????80

Val?Asn?Glu?Tyr?Lys?Lys?Ser?Leu?Gly?Asn?Phe?Pro?Thr?Glu?Met?Arg

85??????????????????90??????????????????95

Glu?Gly?Phe?Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Lys?Trp?Met?Ser

100?????????????????105?????????????????110

Glu?Gly?Glu?Phe?Glu?Asp?Val?Arg?Pro?Ile?Asn?Pro?Ser?Lys?Ser?Lys

115?????????????????120?????????????????125

Lys?Ala?Ile?Asp?Glu?Ser?Thr?Asp?Thr?Phe?Asp?Thr?Leu?Glu?Trp?Leu

130?????????????????135?????????????????140

Ala?Lys?Asn?Leu?Lys?Asn?Tyr?Thr?Lys?Lys?Ala?Gly?Ile?Tyr?Gly?Ile

145?????????????????150?????????????????155?????????????????160

Ser?Tyr?Pro?Gly?Phe?Tyr?Ser?Thr?Met?Ser?Leu?Val?Asn?Ser?His?Pro

165?????????????????170?????????????????175

Thr?Leu?Lys?Ala?Val?Ser?Pro?Gln?Ala?Pro?Val?Thr?Asn?Trp?Phe?Leu

180?????????????????185?????????????????190

Gly?Asp?Asp?Phe?His?His?Asn?Gly?Val?Leu?Phe?Leu?Asn?Asp?Ser?Phe

195?????????????????200?????????????????205

Ser?Phe?Met?Thr?Phe?Phe?Gly?Val?Lys?Arg?Pro?Gln?Pro?Ile?Thr?Pro

210?????????????????215?????????????????220

Asp?Lys?Gly?Pro?Lys?Arg?Phe?Glu?Tyr?Pro?Ile?Lys?Asp?Asn?Tyr?Arg

225?????????????????230?????????????????235?????????????????240

Phe?Tyr?Ala?Ser?Gly?Ser?Val?Lys?Glu?Leu?Lys?Asp?Lys?Tyr?Leu?Gln

245?????????????????250?????????????????255

Asp?Asn?Ile?Lys?Phe?Tyr?Asn?Asp?Leu?Phe?Ala?His?Pro?Asp?Tyr?Asp

260?????????????????265?????????????????270

Gln?Phe?Trp?Gln?Asp?Arg?Asn?Val?Leu?Pro?His?Leu?Thr?Asn?Val?Gln

275?????????????????280?????????????????285

Pro?Ala?Val?Met?Thr?Val?Gly?Gly?Phe?Phe?Asp?Ala?Glu?Asp?Val?Tyr

290?????????????????295?????????????????300

Gly?Ala?Phe?Glu?Thr?Tyr?Lys?Ala?Ile?Glu?Lys?Gln?Asn?Pro?Lys?Ala

305?????????????????310?????????????????315?????????????????320

Thr?Asn?Ile?Met?Val?Ala?Gly?Pro?Trp?Phe?His?Gly?Gly?Trp?Val?Arg

325?????????????????330?????????????????335

Ser?Asn?Gly?Ser?Thr?Phe?Gly?Asp?Met?Gln?Phe?Ala?Ser?Asn?Thr?Ser

340?????????????????345?????????????????350

Glu?His?Tyr?Gln?Gln?Glu?Ile?Glu?Leu?Pro?Phe?Phe?Asn?Tyr?Tyr?Leu

355?????????????????360?????????????????365

Lys?Asp?Lys?Gly?Asn?Phe?Lys?Pro?Thr?Glu?Ala?Thr?Ile?Phe?Ile?Thr

370?????????????????375?????????????????380

Gly?Ser?Asn?Glu?Trp?Lys?Gln?Phe?Asp?Ala?Trp?Pro?Pro?Lys?Asn?Val

385?????????????????390?????????????????395?????????????????400

Thr?Thr?Gln?Lys?Ile?Tyr?Leu?Gln?Gln?Asn?Gly?Lys?Ile?Ala?Phe?Asn

405?????????????????410?????????????????415

Lys?Thr?Asn?Thr?Thr?Thr?Thr?Phe?Asp?Glu?Tyr?Val?Ala?Asp?Pro?Asn

420?????????????????425?????????????????430

Ser?Pro?Val?Pro?Tyr?Ser?Gly?Gly?Val?Leu?Glu?Thr?Arg?Ser?Arg?Glu

435?????????????????440?????????????????445

Tyr?Met?Val?Asp?Asp?Gln?Arg?Phe?Ala?Ser?Thr?Arg?Pro?Asp?Val?Met

450?????????????????455?????????????????460

Val?Tyr?Gln?Ser?Asp?Ile?Leu?Thr?Glu?Asp?Ile?Thr?Leu?Ala?Gly?Pro

465?????????????????470?????????????????475?????????????????480

Val?Ile?Asn?His?Leu?Val?Val?Ser?Thr?Thr?Gly?Thr?Asp?Ala?Asp?Tyr

485?????????????????490?????????????????495

Val?Val?Lys?Leu?Ile?Asp?Val?Tyr?Pro?Glu?Asn?Thr?Pro?Lys?Phe?Asn

500?????????????????505?????????????????510

Asn?Lys?Leu?Met?Ala?Gly?Tyr?Gln?Asn?Leu?Ile?Arg?Ala?Glu?Ile?Met

515?????????????????520?????????????????525

Arg?Gly?Lys?Tyr?Arg?Asn?Ser?Phe?Ser?Asn?Pro?Glu?Ala?Met?Val?Pro

530?????????????????535?????????????????540

Asn?Lys?Glu?Thr?Asn?Val?Thr?Tyr?Thr?Met?Pro?Asp?Val?Gly?His?Thr

545?????????????????550?????????????????555?????????????????560

Phe?Lys?Lys?Gly?His?Arg?Ile?Met?Ile?Gln?Val?Gln?Asn?Ser?Trp?Phe

565?????????????????570?????????????????575

Pro?Leu?Ala?Asp?Arg?Asn?Pro?Gln?Gln?Phe?Met?Asn?Val?Tyr?Glu?Ala

580?????????????????585?????????????????590

Thr?Ser?Lys?Asp?Tyr?Leu?Lys?Gln?Thr?Gln?Arg?Ile?Tyr?His?Thr?Ser

595?????????????????600?????????????????605

Tyr?Ile?Glu?Ile?Pro?Val?Leu?Lys

610?????????????????615

<210>7

<211>40

<212>DNA

＜213〉composition sequence

<220>

＜223〉artificial sequence description: be the synthetic primer of preparation pTrpT

<400>7

gtatcacgag?gccctagctg?tggtgtcatg?gtcggtgatc????????????????????????????40

<210>8

<211>40

<212>DNA

＜213〉composition sequence

<220>

<400>8

ttcggggatt?ccatatgata?ccctttttac?gtgaacttgc????????????????????????????40

<210>9

<211>38

<212>DNA

＜213〉composition sequence

<220>

＜223〉artificial sequence description: be the synthetic primer of preparation pTrpT_Gtg2

<400>9

gggaattcca?tatgaaaaaa?ttaacattaa?aagtaact??????????????????????????????38

<210>10

<211>36

<212>DNA

＜213〉composition sequence

<220>

<400>10

gggggctgca?gtacttgtac?ggtttcgccc?gataaa????????????????????????????????36

<210>11

<211>1935

<212>DNA

＜213〉Sphingobacterium sp.

<220>

<221>CDS

<222>(61)..(1917)

＜223〉peptide synthetase gene

<400>11

gaaaccaagt?gtaaaattat?aatttacacc?aaagaatgta?ctgaacaaat?aattatctga?60

atg?aaa?aat?aca?att?tcg?tgc?cta?act?tta?gcg?ctt?tta?agc?gca?agc???108

Met?Lys?Asn?Thr?Ile?Ser?Cys?Leu?Thr?Leu?Ala?Leu?Leu?Ser?Ala?Ser

1???????????????5???????????????????10??????????????????15

cag?tta?cat?gct?caa?aca?gct?gcc?gac?tcg?gct?tat?gtt?aga?gat?cat???156

Gln?Leu?His?Ala?Gln?Thr?Ala?Ala?Asp?Ser?Ala?Tyr?Val?Arg?Asp?His

20??????????????????25??????????????????30

tat?gaa?aag?acc?gaa?gta?gca?att?ccc?atg?cga?gat?ggg?aaa?aaa?tta???204

Tyr?Glu?Lys?Thr?Glu?Val?Ala?Ile?Pro?Met?Arg?Asp?Gly?Lys?Lys?Leu

35??????????????????40??????????????????45

ttt?act?gcg?atc?tac?agt?cca?aaa?gac?aaa?tcc?aag?aaa?tat?cca?gtt???252

Phe?Thr?Ala?Ile?Tyr?Ser?Pro?Lys?Asp?Lys?Ser?Lys?Lys?Tyr?Pro?Val

50??????????????????55??????????????????60

ttg?ctc?aat?aga?acg?ccc?tac?acg?gtt?tca?cct?tat?ggg?cag?aac?gaa???300

Leu?Leu?Asn?Arg?Thr?Pro?Tyr?Thr?Val?Ser?Pro?Tyr?Gly?Gln?Asn?Glu

65??????????????????70??????????????????75??????????????????80

tat?aaa?aaa?agc?ttg?gga?aac?ttt?ccc?caa?atg?atg?cgt?gaa?ggc?tat???348

Tyr?Lys?Lys?Ser?Leu?Gly?Asn?Phe?Pro?Gln?Met?Met?Arg?Glu?Gly?Tyr

85??????????????????90??????????????????95

att?ttc?gtt?tac?cag?gat?gtc?cgt?ggc?aag?tgg?atg?agc?gaa?ggt?gat???396

Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Lys?Trp?Met?Ser?Glu?Gly?Asp

100?????????????????105?????????????????110

ttt?gaa?gat?ata?cgt?ccg?acc?acg?tac?agc?aaa?gat?aaa?aaa?gca?atc???444

Phe?Glu?Asp?Ile?Arg?Pro?Thr?Thr?Tyr?Ser?Lys?Asp?Lys?Lys?Ala?Ile

115?????????????????120?????????????????125

gat?gaa?agt?acg?gat?acc?tat?gat?gcg?ctt?gaa?tgg?tta?cag?aaa?aat???492

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

130?????????????????135?????????????????140

ctc?aaa?aac?tat?aat?ggc?aaa?gcc?ggg?ctc?tat?ggg?att?tcc?tat?cca???540

Leu?Lys?Asn?Tyr?Asn?Gly?Lys?Ala?Gly?Leu?Tyr?Gly?Ile?Ser?Tyr?Pro

145?????????????????150?????????????????155?????????????????160

ggc?ttc?tat?tct?acc?gtc?gga?ttg?gtc?aaa?aca?cac?ccg?agc?ttg?aag???588

Gly?Phe?Tyr?Ser?Thr?Val?Gly?Leu?Val?Lys?Thr?His?Pro?Ser?Leu?Lys

165?????????????????170?????????????????175

gca?gtc?tcc?cca?cag?gct?ccc?gta?aca?gac?tgg?tat?atc?ggc?gac?gac???636

Ala?Val?Ser?Pro?Gln?Ala?Pro?Val?Thr?Asp?Trp?Tyr?Ile?Gly?Asp?Asp

180?????????????????185?????????????????190

ttc?cac?cat?aat?ggc?gta?ttg?ttt?ctt?cag?gat?gca?ttt?aca?ttc?atg???684

Phe?His?His?Asn?Gly?Val?Leu?Phe?Leu?Gln?Asp?Ala?Phe?Thr?Phe?Met

195?????????????????200?????????????????205

tca?acc?ttt?ggt?gtc?cct?cgt?cca?aaa?ccc?att?aca?ccg?gat?caa?ttt???732

Ser?Thr?Phe?Gly?Val?Pro?Arg?Pro?Lys?Pro?Ile?Thr?Pro?Asp?Gln?Phe

210?????????????????215?????????????????220

aag?ggc?aaa?att?cag?atc?aaa?gaa?gcc?gat?aaa?tat?aac?ttt?ttt?gca???780

Lys?Gly?Lys?Ile?Gln?Ile?Lys?Glu?Ala?Asp?Lys?Tyr?Asn?Phe?Phe?Ala

225?????????????????230?????????????????235?????????????????240

gaa?gca?gga?aca?gcg?cgg?gaa?ctc?aaa?gaa?aag?tat?ttt?ggt?gac?tcc???828

Glu?Ala?Gly?Thr?Ala?Arg?Glu?Leu?Lys?Glu?Lys?Tyr?Phe?Gly?Asp?Ser

245?????????????????250?????????????????255

gta?caa?ttt?tgg?aat?gac?ctg?ttt?aag?cat?ccc?gac?tat?gat?gat?ttt???876

Val?Gln?Phe?Trp?Asn?Asp?Leu?Phe?Lys?His?Pro?Asp?Tyr?Asp?Asp?Phe

260?????????????????265?????????????????270

tgg?aaa?tcg?cgt?gtg?atc?acg?aat?tct?tta?cag?gag?gta?aaa?cca?gct???924

Trp?Lys?Ser?Arg?Val?Ile?Thr?Asn?Ser?Leu?Gln?Glu?Val?Lys?Pro?Ala

275?????????????????280?????????????????285

gtg?atg?gtg?gtt?ggt?ggt?ttc?ttt?gac?gcg?gaa?gat?gct?tat?gga?aca???972

Val?Met?Val?Val?Gly?Gly?Phe?Phe?Asp?Ala?Glu?Asp?Ala?Tyr?Gly?Thr

290?????????????????295?????????????????300

ttt?aag?acc?tac?caa?tcg?att?gag?gat?aaa?agc?aaa?aaa?aac?aac?tcg???1020

Phe?Lys?Thr?Tyr?Gln?Ser?Ile?Glu?Asp?Lys?Ser?Lys?Lys?Asn?Asn?Ser

305?????????????????310?????????????????315?????????????????320

att?tta?gtc?gcg?gga?cct?tgg?tat?cat?ggc?ggt?tgg?gtt?cgt?gca?gaa???1068

Ile?Leu?Val?Ala?Gly?Pro?Trp?Tyr?His?Gly?Gly?Trp?Val?Arg?Ala?Glu

325?????????????????330?????????????????335

gga?aac?tat?tta?ggt?gat?atc?caa?ttt?gag?aaa?aaa?acc?agt?att?act???1116

Gly?Asn?Tyr?Leu?Gly?Asp?Ile?Gln?Phe?Glu?Lys?Lys?Thr?Ser?Ile?Thr

340?????????????????345?????????????????350

tat?cag?gaa?caa?ttt?gaa?caa?cca?ttt?ttc?aaa?tat?tac?cta?aaa?gat???1164

Tyr?Gln?Glu?Gln?Phe?Glu?Gln?Pro?Phe?Phe?Lys?Tyr?Tyr?Leu?Lys?Asp

355?????????????????360?????????????????365

gaa?gga?aac?ttc?gcc?cct?tcc?gaa?gct?aac?att?ttt?gtt?tca?ggc?agc???1212

Glu?Gly?Asn?Phe?Ala?Pro?Ser?Glu?Ala?Asn?Ile?Phe?Val?Ser?Gly?Ser

370?????????????????375?????????????????380

aac?gaa?tgg?aaa?cat?ttc?gaa?cag?tgg?cca?cca?aaa?aat?gta?gag?aca???1260

Asn?Glu?Trp?Lys?His?Phe?Glu?Gln?Trp?Pro?Pro?Lys?Asn?Val?Glu?Thr

385?????????????????390?????????????????395?????????????????400

aaa?aaa?cta?tac?ttc?caa?cct?cag?ggg?aaa?ctt?gga?ttt?gac?aaa?gtt???1308

Lys?Lys?Leu?Tyr?Phe?Gln?Pro?Gln?Gly?Lys?Leu?Gly?Phe?Asp?Lys?Val

405?????????????????410?????????????????415

caa?cgt?aca?gat?tcc?tgg?gat?gaa?tat?gta?aca?gac?cct?aat?aaa?cct???1356

Gln?Arg?Thr?Asp?Ser?Trp?Asp?Glu?Tyr?Val?Thr?Asp?Pro?Asn?Lys?Pro

420?????????????????425?????????????????430

gtt?ccg?cat?caa?ggt?ggg?gta?att?caa?aac?cga?aca?cgg?gag?tat?atg???1404

Val?Pro?His?Gln?Gly?Gly?Val?Ile?Gln?Asn?Arg?Thr?Arg?Glu?Tyr?Met

435?????????????????440?????????????????445

gta?gat?gat?caa?cgt?ttc?gcg?gct?agt?cgc?cct?gat?gtc?atg?gtt?tat???1452

Val?Asp?Asp?Gln?Arg?Phe?Ala?Ala?Ser?Arg?Pro?Asp?Val?Met?Val?Tyr

450?????????????????455?????????????????460

caa?acg?gaa?ccg?ttg?acg?gag?gac?ctg?acg?ata?gta?ggc?cca?atc?aaa???1500

Gln?Thr?Glu?Pro?Leu?Thr?Glu?Asp?Leu?Thr?Ile?Val?Gly?Pro?Ile?Lys

465?????????????????470?????????????????475?????????????????480

aac?ttt?ctc?aaa?gtt?tct?tca?aca?gga?aca?gac?gcg?gac?tat?gtt?gtc???1548

Asn?Phe?Leu?Lys?Val?Ser?Ser?Thr?Gly?Thr?Asp?Ala?Asp?Tyr?Val?Val

485?????????????????490?????????????????495

aaa?ctg?att?gac?gtt?tat?ccg?aat?gat?gca?gca?agt?tat?caa?gga?aaa???1596

Lys?Leu?Ile?Asp?Val?Tyr?Pro?Asn?Asp?Ala?Ala?Ser?Tyr?Gln?Gly?Lys

500?????????????????505?????????????????510

aca?atg?gct?gga?tat?caa?atg?atg?gta?cgt?ggt?gag?atc?atg?gcg?ggg???1644

Thr?Met?Ala?Gly?Tyr?Gln?Met?Met?Val?Arg?Gly?Glu?Ile?Met?Ala?Gly

515?????????????????520?????????????????525

aaa?tac?cga?aat?ggt?ttc?gat?aaa?gcg?cag?gcc?ttg?act?cca?ggt?atg???1692

Lys?Tyr?Arg?Asn?Gly?Phe?Asp?Lys?Ala?Gln?Ala?Leu?Thr?Pro?Gly?Met

530?????????????????535?????????????????540

gtc?gaa?aag?gtg?aat?ttt?gaa?atg?cca?gac?gtt?gcg?cat?acc?ttc?aaa????1740

Val?Glu?Lys?Val?Asn?Phe?Glu?Met?Pro?Asp?Val?Ala?His?Thr?Phe?Lys

545?????????????????550?????????????????555?????????????????560

aaa?gga?cat?cgc?att?atg?gtt?cag?gta?caa?aac?tca?tgg?ttt?ccg?ctg????1788

Lys?Gly?His?Arg?Ile?Met?Val?Gln?Val?Gln?Asn?Ser?Trp?Phe?Pro?Leu

565?????????????????570?????????????????575

gca?gaa?cga?aat?cca?cag?gtg?ttt?tta?gca?cct?tat?aca?gct?acc?aaa????1836

Ala?Glu?Arg?Asn?Pro?Gln?Val?Phe?Leu?Ala?Pro?Tyr?Thr?Ala?Thr?Lys

580?????????????????585?????????????????590

gct?gat?ttc?cgc?aaa?gct?acc?caa?cgt?att?ttt?cac?gat?gtg?aac?aat????1884

Ala?Asp?Phe?Arg?Lys?Ala?Thr?Gln?Arg?Ile?Phe?His?Asp?Val?Asn?Asn

595?????????????????600?????????????????605

gcc?aca?tac?atc?gaa?ttt?tct?gtc?ctc?aaa?gat?tagcaggtaa?attcgaaa????1935

Ala?Thr?Tyr?Ile?Glu?Phe?Ser?Val?Leu?Lys?Asp

610?????????????????615

<210>12

<211>619

<212>PRT

＜213〉Sphingobacterium sp.

<400>12

Met?Lys?Asn?Thr?Ile?Ser?Cys?Leu?Thr?Leu?Ala?Leu?Leu?Ser?Ala?Ser

1???????????????5???????????????????10??????????????????15

Gln?Leu?His?Ala?Gln?Thr?Ala?Ala?Asp?Ser?Ala?Tyr?Val?Arg?Asp?His

20??????????????????25??????????????????30

Tyr?Glu?Lys?Thr?Glu?Val?Ala?Ile?Pro?Met?Arg?Asp?Gly?Lys?Lys?Leu

35??????????????????40??????????????????45

Phe?Thr?Ala?Ile?Tyr?Ser?Pro?Lys?Asp?Lys?Ser?Lys?Lys?Tyr?Pro?Val

50??????????????????55??????????????????60

Leu?Leu?Asn?Arg?Thr?Pro?Tyr?Thr?Val?Ser?Pro?Tyr?Gly?Gln?Asn?Glu

65??????????????????70??????????????????75??????????????????80

Tyr?Lys?Lys?Ser?Leu?Gly?Asn?Phe?Pro?Gln?Met?Met?Arg?Glu?Gly?Tyr

85??????????????????90??????????????????95

Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Lys?Trp?Met?Ser?Glu?Gly?Asp

100?????????????????105?????????????????110

Phe?Glu?Asp?Ile?Arg?Pro?Thr?Thr?Tyr?Ser?Lys?Asp?Lys?Lys?Ala?Ile

115?????????????????120?????????????????125

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

130?????????????????135?????????????????140

Leu?Lys?Asn?Tyr?Asn?Gly?Lys?Ala?Gly?Leu?Tyr?Gly?Ile?Ser?Tyr?Pro

145?????????????????150?????????????????155?????????????????160

Gly?Phe?Tyr?Ser?Thr?Val?Gly?Leu?Val?Lys?Thr?His?Pro?Ser?Leu?Lys

165?????????????????170?????????????????175

Ala?Val?Ser?Pro?Gln?Ala?Pro?Val?Thr?Asp?Trp?Tyr?Ile?Gly?Asp?Asp

180?????????????????185?????????????????190

Phe?His?His?Asn?Gly?Val?Leu?Phe?Leu?Gln?Asp?Ala?Phe?Thr?Phe?Met

195?????????????????200?????????????????205

Ser?Thr?Phe?Gly?Val?Pro?Arg?Pro?Lys?Pro?Ile?Thr?Pro?Asp?Gln?Phe

210?????????????????215?????????????????220

Lys?Gly?Lys?Ile?Gln?Ile?Lys?Glu?Ala?Asp?Lys?Tyr?Asn?Phe?Phe?Ala

225?????????????????230?????????????????235?????????????????240

Glu?Ala?Gly?Thr?Ala?Arg?Glu?Leu?Lys?Glu?Lys?Tyr?Phe?Gly?Asp?Ser

245?????????????????250?????????????????255

Val?Gln?Phe?Trp?Asn?Asp?Leu?Phe?Lys?His?Pro?Asp?Tyr?Asp?Asp?Phe

260?????????????????265?????????????????270

Trp?Lys?Ser?Arg?Val?Ile?Thr?Asn?Ser?Leu?Gln?Glu?Val?Lys?Pro?Ala

275?????????????????280?????????????????285

Val?Met?Val?Val?Gly?Gly?Phe?Phe?Asp?Ala?Glu?Asp?Ala?Tyr?Gly?Thr

290?????????????????295?????????????????300

Phe?Lys?Thr?Tyr?Gln?Ser?Ile?Glu?Asp?Lys?Ser?Lys?Lys?Asn?Asn?Ser

305?????????????????310?????????????????315?????????????????320

Ile?Leu?Val?Ala?Gly?Pro?Trp?Tyr?His?Gly?Gly?Trp?Val?Arg?Ala?Glu

325?????????????????330?????????????????335

Gly?Asn?Tyr?Leu?Gly?Asp?Ile?Gln?Phe?Glu?Lys?Lys?Thr?Ser?Ile?Thr

340?????????????????345?????????????????350

Tyr?Gln?Glu?Gln?Phe?Glu?Gln?Pro?Phe?Phe?Lys?Tyr?Tyr?Leu?Lys?Asp

355?????????????????360?????????????????365

Glu?Gly?Asn?Phe?Ala?Pro?Ser?Glu?Ala?Asn?Ile?Phe?Val?Ser?Gly?Ser

370?????????????????375?????????????????380

Asn?Glu?Trp?Lys?His?Phe?Glu?Gln?Trp?Pro?Pro?Lys?Asn?Val?Glu?Thr

385?????????????????390?????????????????395?????????????????400

Lys?Lys?Leu?Tyr?Phe?Gln?Pro?Gln?Gly?Lys?Leu?Gly?Phe?Asp?Lys?Val

405?????????????????410?????????????????415

Gln?Arg?Thr?Asp?Ser?Trp?Asp?Glu?Tyr?Val?Thr?Asp?Pro?Asn?Lys?Pro

420?????????????????425?????????????????430

Val?Pro?His?Gln?Gly?Gly?Val?Ile?Gln?Asn?Arg?Thr?Arg?Glu?Tyr?Met

435?????????????????440?????????????????445

Val?Asp?Asp?Gln?Arg?Phe?Ala?Ala?Ser?Arg?Pro?Asp?Val?Met?Val?Tyr

450?????????????????455?????????????????460

Gln?Thr?Glu?Pro?Leu?Thr?Glu?Asp?Leu?Thr?Ile?Val?Gly?Pro?Ile?Lys

465?????????????????470?????????????????475?????????????????480

Asn?Phe?Leu?Lys?Val?Ser?Ser?Thr?Gly?Thr?Asp?Ala?Asp?Tyr?Val?Val

485?????????????????490?????????????????495

Lys?Leu?Ile?Asp?Val?Tyr?Pro?Asn?Asp?Ala?Ala?Ser?Tyr?Gln?Gly?Lys

500?????????????????505?????????????????510

Thr?Met?Ala?Gly?Tyr?Gln?Met?Met?Val?Arg?Gly?Glu?Ile?Met?Ala?Gly

515?????????????????520?????????????????525

Lys?Tyr?Arg?Asn?Gly?Phe?Asp?Lys?Ala?Gln?Ala?Leu?Thr?Pro?Gly?Met

530?????????????????535?????????????????540

Val?Glu?Lys?Val?Asn?Phe?Glu?Met?Pro?Asp?Val?Ala?His?Thr?Phe?Lys

545?????????????????550?????????????????555?????????????????560

Lys?Gly?His?Arg?Ile?Met?Val?Gln?Val?Gln?Asn?Ser?Trp?Phe?Pro?Leu

565?????????????????570?????????????????575

Ala?Glu?Arg?Asn?Pro?Gln?Val?Phe?Leu?Ala?Pro?Tyr?Thr?Ala?Thr?Lys

580?????????????????585?????????????????590

Ala?Asp?Phe?Arg?Lys?Ala?Thr?Gln?Arg?Ile?Phe?His?Asp?Val?Asn?Asn

595?????????????????600?????????????????605

Ala?Thr?Tyr?Ile?Glu?Phe?Ser?Val?Leu?Lys?Asp

610?????????????????615

<210>13

<211>30

<212>DNA

＜213〉composition sequence

<220>

＜223〉artificial sequence description: be the synthetic primer of preparation pTrpT_Sm_aet

<400>13

gggaattcca?tatgaaaaat?acaatttcgt???????????????????????????????????????30

<210>14

<211>29

<212>DNA

＜213〉composition sequence

<220>

<400>14

gctctagact?aatctttgag?gacagaaaa????????????????????????????????????????29

<210>15

<211>17

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the mix primer 1 of Aet

<400>15

gaygayttyc?aycayaa?????????????????????????????????????????????????????17

<210>16

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the mix primer 2 of Aet

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉any base

<400>16

tgrtcrtcna?ccatrtaytc??????????????????????????????????????????????????20

<210>17

<211>1974

<212>DNA

＜213〉separate Vitrum AB soil bacillus

<220>

<221>CDS

<222>(61)..(1935)

<223>

<400>17

aaacctatcc?cgtattcagc?aatcaattcc?atatatttat?ccttaaaaaa?accttcctct????60

atg?act?cct?ttc?aaa?tcg?ttc?tcc?ttc?att?ttt?ctc?ttt?att?ttt?acc?????108

Met?Thr?Pro?Phe?Lys?Ser?Phe?Ser?Phe?Ile?Phe?Leu?Phe?Ile?Phe?Thr

1???????????????5???????????????????10??????????????????15

agt?ctt?tct?gct?tct?gca?caa?cag?tcc?gac?tct?gct?tat?ata?cgt?cag?????156

Ser?Leu?Ser?Ala?Ser?Ala?Gln?Gln?Ser?Asp?Ser?Ala?Tyr?Ile?Arg?Gln

20??????????????????25??????????????????30

aac?tat?acc?aaa?ata?gaa?agg?ctg?atc?cct?atg?cgg?gat?ggc?att?aag?????204

Asn?Tyr?Thr?Lys?Ile?Glu?Arg?Leu?Ile?Pro?Met?Arg?Asp?Gly?Ile?Lys

35??????????????????40??????????????????45

cta?ttt?aca?gcc?att?tac?atc?ccc?aaa?gac?aaa?agc?aag?aag?tat?cct?????252

Leu?Phe?Thr?Ala?Ile?Tyr?Ile?Pro?Lys?Asp?Lys?Ser?Lys?Lys?Tyr?Pro

50??????????????????55??????????????????60

ttt?atg?ctc?aac?cgt?act?cct?tat?acc?gtt?tcg?cct?tat?ggc?gaa?aac?????300

Phe?Met?Leu?Asn?Arg?Thr?Pro?Tyr?Thr?Val?Ser?Pro?Tyr?Gly?Glu?Asn

65??????????????????70??????????????????75??????????????????80

aat?tat?aaa?aca?agc?ctt?ggc?ccc?tct?ccg?ctc?ttt?ata?aaa?gaa?ggc?????348

Asn?Tyr?Lys?Thr?Ser?Leu?Gly?Pro?Ser?Pro?Leu?Phe?Ile?Lys?Glu?Gly

85??????????????????90??????????????????95

ttt?atc?ttt?gtt?tat?cag?gat?gta?agg?ggc?aaa?tgg?atg?agt?gag?gga?????396

Phe?Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Lys?Trp?Met?Ser?Glu?Gly

100?????????????????105?????????????????110

aaa?ttt?gaa?gac?gta?agg?ccg?caa?ata?gcc?agc?aag?aaa?cgc?aaa?acg?????444

Lys?Phe?Glu?Asp?Val?Arg?Pro?Gln?Ile?Ala?Ser?Lys?Lys?Arg?Lys?Thr

115?????????????????120?????????????????125

gat?att?gat?gaa?agc?tcc?gat?act?tat?gat?acg?atc?gac?tgg?ctg?atc?????492

Asp?Ile?Asp?Glu?Ser?Ser?Asp?Thr?Tyr?Asp?Thr?Ile?Asp?Trp?Leu?Ile

130?????????????????135?????????????????140

agg?aac?att?cct?gga?aac?aac?cgt?aaa?acc?ggt?att?tac?ggt?atc?tca?????540

Arg?Asn?Ile?Pro?Gly?Asn?Asn?Arg?Lys?Thr?Gly?Ile?Tyr?Gly?Ile?Ser

145?????????????????150?????????????????155?????????????????160

tac?cca?ggc?ttt?tat?gct?act?gct?gcc?cta?cca?gat?gcg?cat?cca?tct?????588

Tyr?Pro?Gly?Phe?Tyr?Ala?Thr?Ala?Ala?Leu?Pro?Asp?Ala?His?Pro?Ser

165?????????????????170?????????????????175

tta?aag?gca?gta?tcg?ccc?cag?gct?ccg?gtt?acc?gac?tgg?ttt?ata?ggc?????636

Leu?Lys?Ala?Val?Ser?Pro?Gln?Ala?Pro?Val?Thr?Asp?Trp?Phe?Ile?Gly

180?????????????????185?????????????????190

gat?gat?ttt?cat?cac?aat?ggc?acc?ttg?ttc?ctt?gca?gat?atc?ttt?agc?????684

Asp?Asp?Phe?His?His?Asn?Gly?Thr?Leu?Phe?Leu?Ala?Asp?Ile?Phe?Ser

195?????????????????200?????????????????205

ttc?tat?tat?acc?ttc?ggg?gta?ccg?cga?cct?caa?cca?att?acg?ccc?gac?????732

Phe?Tyr?Tyr?Thr?Phe?Gly?Val?Pro?Arg?Pro?Gln?Pro?Ile?Thr?Pro?Asp

210?????????????????215?????????????????220

aaa?cgt?cca?aaa?ccc?ttt?gat?ttc?ccg?gtt?aaa?gac?aac?tac?cgt?ttt?????780

Lys?Arg?Pro?Lys?Pro?Phe?Asp?Phe?Pro?Val?Lys?Asp?Asn?Tyr?Arg?Phe

225?????????????????230?????????????????235?????????????????240

ttt?ctt?gaa?ctg?ggc?ccc?tta?aaa?aac?atc?acc?aaa?aaa?tat?tat?ggc?????828

Phe?Leu?Glu?Leu?Gly?Pro?Leu?Lys?Asn?Ile?Thr?Lys?Lys?Tyr?Tyr?Gly

245?????????????????250?????????????????255

gat?acc?ata?cga?ttc?tgg?aat?gat?atc?aat?gcg?cat?acc?aat?tat?gat?????876

Asp?Thr?Ile?Arg?Phe?Trp?Asn?Asp?Ile?Asn?Ala?His?Thr?Asn?Tyr?Asp

260?????????????????265?????????????????270

gcc?ttc?tgg?aaa?gcc?cgt?aac?att?acg?ccg?cat?tta?att?ggt?gta?aaa?????924

Ala?Phe?Trp?Lys?Ala?Arg?Asn?Ile?Thr?Pro?His?Leu?Ile?Gly?Val?Lys

275?????????????????280?????????????????285

cct?gca?gtt?ttg?gta?gtt?ggc?ggc?ttc?ttt?gat?gca?gaa?gac?ctt?tac?????972

Pro?Ala?Val?Leu?Val?Val?Gly?Gly?Phe?Phe?Asp?Ala?Glu?Asp?Leu?Tyr

290?????????????????295?????????????????300

ggt?acg?ctt?aaa?acc?tat?cag?gcc?atc?gaa?aaa?caa?aat?cca?tcc?tca?????1020

Gly?Thr?Leu?Lys?Thr?Tyr?Gln?Ala?Ile?Glu?Lys?Gln?Asn?Pro?Ser?Ser

305?????????????????310?????????????????315?????????????????320

aaa?aac?aac?ctc?gtt?atg?ggc?ccc?tgg?tac?cat?ggt?ggc?tgg?gca?aga?????1068

Lys?Asn?Asn?Leu?Val?Met?Gly?Pro?Trp?Tyr?His?Gly?Gly?Trp?Ala?Arg

325?????????????????330?????????????????335

agt?acg?gga?agc?agt?ttc?ggg?gat?att?aat?ttc?gga?cag?cca?acc?agt?????1116

Ser?Thr?Gly?Ser?Ser?Phe?Gly?Asp?Ile?Asn?Phe?Gly?Gln?Pro?Thr?Ser

340?????????????????345?????????????????350

act?tca?tac?cag?caa?aat?gtt?gag?ttc?cct?ttc?ttt?atg?caa?tac?ctc?????1164

Thr?Ser?Tyr?Gln?Gln?Asn?Val?Glu?Phe?Pro?Phe?Phe?Met?Gln?Tyr?Leu

355?????????????????360?????????????????365

aaa?gag?gca?ccg?gat?gca?aaa?att?gca?gag?gca?acc?att?ttt?atc?act????1212

Lys?Glu?Ala?Pro?Asp?Ala?Lys?Ile?Ala?Glu?Ala?Thr?Ile?Phe?Ile?Thr

370?????????????????375?????????????????380

ggc?agc?aat?gaa?tgg?aag?aaa?ttt?agc?tcc?tgg?cca?cct?cag?gat?aca????1260

Gly?Ser?Asn?Glu?Trp?Lys?Lys?Phe?Ser?Ser?Trp?Pro?Pro?Gln?Asp?Thr

385?????????????????390?????????????????395?????????????????400

gaa?gaa?aga?aca?tta?tac?ctg?cag?ccc?aat?ggc?aaa?ctg?agc?ttt?gag????1308

Glu?Glu?Arg?Thr?Leu?Tyr?Leu?Gln?Pro?Asn?Gly?Lys?Leu?Ser?Phe?Glu

405?????????????????410?????????????????415

aag?gta?cag?cgg?acc?gac?agc?tgg?gat?gaa?tat?gta?agt?gat?ccc?aat????1356

Lys?Val?Gln?Arg?Thr?Asp?Ser?Trp?Asp?Glu?Tyr?Val?Ser?Asp?Pro?Asn

420?????????????????425?????????????????430

tca?cct?gtc?cct?tat?cag?gat?ggc?ata?caa?acc?agc?aga?acc?cgg?gaa????1404

Ser?Pro?Val?Pro?Tyr?Gln?Asp?Gly?Ile?Gln?Thr?Ser?Arg?Thr?Arg?Glu

435?????????????????440?????????????????445

tat?atg?atc?gat?gac?cag?cgt?ttt?gcc?tcg?cgc?aga?ccg?gat?gta?agg????1452

Tyr?Met?Ile?Asp?Asp?Gln?Arg?Phe?Ala?Ser?Arg?Arg?Pro?Asp?Val?Arg

450?????????????????455?????????????????460

gta?ttc?caa?aca?gag?ccc?ctc?agt?tcc?gac?ctt?aca?ctt?acc?ggc?ccg????1500

Val?Phe?Gln?Thr?Glu?Pro?Leu?Ser?Ser?Asp?Leu?Thr?Leu?Thr?Gly?Pro

465?????????????????470?????????????????475?????????????????480

gta?ttg?gcc?aaa?ctg?gtg?gta?tca?acc?aca?ggt?acg?gat?gca?gat?tat????1548

Val?Leu?Ala?Lys?Leu?Val?Val?Ser?Thr?Thr?Gly?Thr?Asp?Ala?Asp?Tyr

485?????????????????490?????????????????495

gtg?gta?aaa?ctg?ata?gat?gta?tat?ccg?gaa?gat?aca?cca?aat?cct?gta????1596

Val?Val?Lys?Leu?Ile?Asp?Val?Tyr?Pro?Glu?Asp?Thr?Pro?Asn?Pro?Val

500?????????????????505?????????????????510

cct?aac?cct?aaa?aac?ctg?atc?atg?ggt?ggt?tac?cag?atg?ctg?gta?cgc????1644

Pro?Asn?Pro?Lys?Asn?Leu?Ile?Met?Gly?Gly?Tyr?Gln?Met?Leu?Val?Arg

515?????????????????520?????????????????525

ggc?gag?atc?atg?cgt?gga?aaa?tac?cgt?aat?agc?ttt?gaa?aaa?ccc?gag????1692

Gly?Glu?Ile?Met?Arg?Gly?Lys?Tyr?Arg?Asn?Ser?Phe?Glu?Lys?Pro?Glu

530?????????????????535?????????????????540

cct?ttt?gtt?cct?gga?aca?att?aca?aaa?gta?aac?tat?gcc?ctt?ccg?gat????1740

Pro?Phe?Val?Pro?Gly?Thr?Ile?Thr?Lys?Val?Asn?Tyr?Ala?Leu?Pro?Asp

545?????????????????550?????????????????555?????????????????560

gta?gcc?cat?acc?ttt?aaa?aaa?ggc?cac?cgc?atc?atg?atc?cag?gtc?cag????1788

Val?Ala?His?Thr?Phe?Lys?Lys?Gly?His?Arg?Ile?Met?Ile?Gln?Val?Gln

565?????????????????570?????????????????575

aat?tca?tgg?ttt?ccc?ctg?gcc?gac?cgg?aat?cca?cag?cag?ttt?atg?gac????1836

Asn?Ser?Trp?Phe?Pro?Leu?Ala?Asp?Arg?Asn?Pro?Gln?Gln?Phe?Met?Asp

580?????????????????585?????????????????590

att?tac?cag?gcc?gaa?cct?ggc?gat?ttc?aga?aaa?gct?acg?cat?agg?atc????1884

Ile?Tyr?Gln?Ala?Glu?Pro?Gly?Asp?Phe?Arg?Lys?Ala?Thr?His?Arg?Ile

595?????????????????600?????????????????605

ttc?cac?gat?gta?cac?aat?gca?tct?gca?att?acg?gta?aac?gta?ctg?aaa????1932

Phe?His?Asp?Val?His?Asn?Ala?Ser?Ala?Ile?Thr?Val?Asn?Val?Leu?Lys

610?????????????????615?????????????????620

cct?taaaacggat?gaaaccagta?tattgtgcca?tccttactt?????????????????????1974

Pro

625

<210>18

<211>625

<212>PRT

＜213〉separate Vitrum AB soil bacillus

<400>18

Met?Thr?Pro?Phe?Lys?Ser?Phe?Ser?Phe?Ile?Phe?Leu?Phe?Ile?Phe?Thr

1???????????????5???????????????????10??????????????????15

Ser?Leu?Ser?Ala?Ser?Ala?Gln?Gln?Ser?Asp?Ser?Ala?Tyr?Ile?Arg?Gln

20??????????????????25??????????????????30

Asn?Tyr?Thr?Lys?Ile?Glu?Arg?Leu?Ile?Pro?Met?Arg?Asp?Gly?Ile?Lys

35??????????????????40??????????????????45

Leu?Phe?Thr?Ala?Ile?Tyr?Ile?Pro?Lys?Asp?Lys?Ser?Lys?Lys?Tyr?Pro

50??????????????????55??????????????????60

Phe?Met?Leu?Asn?Arg?Thr?Pro?Tyr?Thr?Val?Ser?Pro?Tyr?Gly?Glu?Asn

65??????????????????70??????????????????75??????????????????80

Asn?Tyr?Lys?Thr?Ser?Leu?Gly?Pro?Ser?Pro?Leu?Phe?Ile?Lys?Glu?Gly

85??????????????????90??????????????????95

Phe?Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Lys?Trp?Met?Ser?Glu?Gly

100?????????????????105?????????????????110

Lys?Phe?Glu?Asp?Val?Arg?Pro?Gln?Ile?Ala?Ser?Lys?Lys?Arg?Lys?Thr

115?????????????????120?????????????????125

Asp?Ile?Asp?Glu?Ser?Ser?Asp?Thr?Tyr?Asp?Thr?Ile?Asp?Trp?Leu?Ile

130?????????????????135?????????????????140

Arg?Asn?Ile?Pro?Gly?Asn?Asn?Arg?Lys?Thr?Gly?Ile?Tyr?Gly?Ile?Ser

145?????????????????150?????????????????155?????????????????160

Tyr?Pro?Gly?Phe?Tyr?Ala?Thr?Ala?Ala?Leu?Pro?Asp?Ala?His?Pro?Ser

165?????????????????170?????????????????175

Leu?Lys?Ala?Val?Ser?Pro?Gln?Ala?Pro?Val?Thr?Asp?Trp?Phe?Ile?Gly

180?????????????????185?????????????????190

Asp?Asp?Phe?His?His?Asn?Gly?Thr?Leu?Phe?Leu?Ala?Asp?Ile?Phe?Ser

195?????????????????200?????????????????205

Phe?Tyr?Tyr?Thr?Phe?Gly?Val?Pro?Arg?Pro?Gln?Pro?Ile?Thr?Pro?Asp

210?????????????????215?????????????????220

Lys?Arg?Pro?Lys?Pro?Phe?Asp?Phe?Pro?Val?Lys?Asp?Asn?Tyr?Arg?Phe

225?????????????????230?????????????????235?????????????????240

Phe?Leu?Glu?Leu?Gly?Pro?Leu?Lys?Asn?Ile?Thr?Lys?Lys?Tyr?Tyr?Gly

245?????????????????250?????????????????255

Asp?Thr?Ile?Arg?Phe?Trp?Asn?Asp?Ile?Asn?Ala?His?Thr?Asn?Tyr?Asp

260?????????????????265?????????????????270

Ala?Phe?Trp?Lys?Ala?Arg?Asn?Ile?Thr?Pro?His?Leu?Ile?Gly?Val?Lys

275?????????????????280?????????????????285

Pro?Ala?Val?Leu?Val?Val?Gly?Gly?Phe?Phe?Asp?Ala?Glu?Asp?Leu?Tyr

290?????????????????295?????????????????300

Gly?Thr?Leu?Lys?Thr?Tyr?Gln?Ala?Ile?Glu?Lys?Gln?Asn?Pro?Ser?Ser

305?????????????????310?????????????????315?????????????????320

Lys?Asn?Asn?Leu?Val?Met?Gly?Pro?Trp?Tyr?His?Gly?Gly?Trp?Ala?Arg

325?????????????????330?????????????????335

Ser?Thr?Gly?Ser?Ser?Phe?Gly?Asp?Ile?Asn?Phe?Gly?Gln?Pro?Thr?Ser

340?????????????????345?????????????????350

Thr?Ser?Tyr?Gln?Gln?Asn?Val?Glu?Phe?Pro?Phe?Phe?Met?Gln?Tyr?Leu

355?????????????????360?????????????????365

Lys?Glu?Ala?Pro?Asp?Ala?Lys?Ile?Ala?Glu?Ala?Thr?Ile?Phe?Ile?Thr

370?????????????????375?????????????????380

Gly?Ser?Asn?Glu?Trp?Lys?Lys?Phe?Ser?Ser?Trp?Pro?Pro?Gln?Asp?Thr

385?????????????????390?????????????????395?????????????????400

Glu?Glu?Arg?Thr?Leu?Tyr?Leu?Gln?Pro?Asn?Gly?Lys?Leu?Ser?Phe?Glu

405?????????????????410?????????????????415

Lys?Val?Gln?Arg?Thr?Asp?Ser?Trp?Asp?Glu?Tyr?Val?Ser?Asp?Pro?Asn

420?????????????????425?????????????????430

Ser?Pro?Val?Pro?Tyr?Gln?Asp?Gly?Ile?Gln?Thr?Ser?Arg?Thr?Arg?Glu

435?????????????????440?????????????????445

Tyr?Met?Ile?Asp?Asp?Gln?Arg?Phe?Ala?Ser?Arg?Arg?Pro?Asp?Val?Arg

450?????????????????455?????????????????460

Val?Phe?Gln?Thr?Glu?Pro?Leu?Ser?Ser?Asp?Leu?Thr?Leu?Thr?Gly?Pro

465?????????????????470?????????????????475?????????????????480

Val?Leu?Ala?Lys?Leu?Val?Val?Ser?Thr?Thr?Gly?Thr?Asp?Ala?Asp?Tyr

485?????????????????490?????????????????495

Val?Val?Lys?Leu?Ile?Asp?Val?Tyr?Pro?Glu?Asp?Thr?Pro?Asn?Pro?Val

500?????????????????505?????????????????510

Pro?Asn?Pro?Lys?Asn?Leu?Ile?Met?Gly?Gly?Tyr?Gln?Met?Leu?Val?Arg

515?????????????????520?????????????????525

Gly?Glu?Ile?Met?Arg?Gly?Lys?Tyr?Arg?Asn?Ser?Phe?Glu?Lys?Pro?Glu

530?????????????????535?????????????????540

Pro?Phe?Val?Pro?Gly?Thr?Ile?Thr?Lys?Val?Asn?Tyr?Ala?Leu?Pro?Asp

545?????????????????550?????????????????555?????????????????560

Val?Ala?His?Thr?Phe?Lys?Lys?Gly?His?Arg?Ile?Met?Ile?Gln?Val?Gln

565?????????????????570?????????????????575

Asn?Ser?Trp?Phe?Pro?Leu?Ala?Asp?Arg?Asn?Pro?Gln?Gln?Phe?Met?Asp

580?????????????????585?????????????????590

Ile?Tyr?Gln?Ala?Glu?Pro?Gly?Asp?Phe?Arg?Lys?Ala?Thr?His?Arg?Ile

595?????????????????600?????????????????605

Phe?His?Asp?Val?His?Asn?Ala?Ser?Ala?Ile?Thr?Val?Asn?Val?Leu?Lys

610?????????????????615?????????????????620

Pro

625

<210>19

<211>38

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer 1 of the aet expression vector that comes from Pedobacter

<400>19

gggaattcca?tatgactcct?ttcaaatcgt?tctccttc??????????????????????????????38

<210>20

<211>30

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer 2 of the aet expression vector that comes from Pedobacter

<400>20

cccaagcttt?taaggtttca?gtacgtttac???????????????????????????????????????30

<210>21

<211>17

<212>DNA

＜213〉artificial

<220>

＜223〉be used for the mix primer 3 of Aet

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉any base

<400>21

athttygtnt?aycarga?????????????????????????????????????????????????????17

<210>22

<211>2018

<212>DNA

<213>Taxeobacter?gelupurpurascens

<220>

<221>CDS

<222>(61)..(1995)

<223>

<400>22

ctgaatgtct?gctgacgaat?tggaactaca?ttaggctcgt?tcttcaccta?cccttccact????60

atg?ccc?tac?tct?ttc?ccg?aaa?gtt?gcc?gcc?ctg?agt?ggc?cta?ctg?gtg?????108

Met?Pro?Tyr?Ser?Phe?Pro?Lys?Val?Ala?Ala?Leu?Ser?Gly?Leu?Leu?Val

1???????????????5???????????????????10??????????????????15

gcc?ggt?tta?tcc?ggt?gcc?cac?gcc?caa?act?cct?gtt?acc?tat?ccg?ctg?????156

Ala?Gly?Leu?Ser?Gly?Ala?His?Ala?Gln?Thr?Pro?Val?Thr?Tyr?Pro?Leu

20??????????????????25??????????????????30

gct?tct?gag?gct?gaa?aaa?gcg?cag?ctg?gcg?gtg?gta?cta?gcc?gat?acg?????204

Ala?Ser?Glu?Ala?Glu?Lys?Ala?Gln?Leu?Ala?Val?Val?Leu?Ala?Asp?Thr

35??????????????????40??????????????????45

gct?tac?atc?aag?gag?cgc?tat?acc?aaa?aca?gaa?tat?cag?att?ccg?atg?????252

Ala?Tyr?Ile?Lys?Glu?Arg?Tyr?Thr?Lys?Thr?Glu?Tyr?Gln?Ile?Pro?Met

50??????????????????55??????????????????60

cgc?gat?ggg?gtg?aag?ttg?tac?acc?att?gtg?tac?gcg?ccc?aac?gat?gcc?????300

Arg?Asp?Gly?Val?Lys?Leu?Tyr?Thr?Ile?Val?Tyr?Ala?Pro?Asn?Asp?Ala

65??????????????????70??????????????????75??????????????????80

aac?aag?gta?aag?tac?cct?att?ctg?ctc?aac?cgt?acc?cct?tac?gct?att?????348

Asn?Lys?Val?Lys?Tyr?Pro?Ile?Leu?Leu?Asn?Arg?Thr?Pro?Tyr?Ala?Ile

85??????????????????90??????????????????95

ggc?ccc?tac?ggc?ccc?ggc?aaa?tac?aag?ctc?aac?ctg?ggc?ccc?agc?agc?????396

Gly?Pro?Tyr?Gly?Pro?Gly?Lys?Tyr?Lys?Leu?Asn?Leu?Gly?Pro?Ser?Ser

100?????????????????105?????????????????110

acg?atg?atg?cat?gag?gga?tac?atc?ttc?gcc?tac?cag?gat?gtg?cgt?ggg?????444

Thr?Met?Met?His?Glu?Gly?Tyr?Ile?Phe?Ala?Tyr?Gln?Asp?Val?Arg?Gly

115?????????????????120?????????????????125

cga?tat?atg?tcg?gaa?gga?gag?ttt?gtg?gat?gtg?cgc?ccc?gaa?aag?gac?????492

Arg?Tyr?Met?Ser?Glu?Gly?Glu?Phe?Val?Asp?Val?Arg?Pro?Glu?Lys?Asp

130?????????????????135?????????????????140

atg?cac?aaa?ggc?aag?aac?gac?atc?gat?gaa?ggc?acc?gac?acc?tac?gat?????540

Met?His?Lys?Gly?Lys?Asn?Asp?Ile?Asp?Glu?Gly?Thr?Asp?Thr?Tyr?Asp

145?????????????????150?????????????????155?????????????????160

acc?att?gag?tgg?ctt?ctg?aag?cac?ggg?ccc?aag?aat?aac?ggc?cgc?gta?????588

Thr?Ile?Glu?Trp?Leu?Leu?Lys?His?Gly?Pro?Lys?Asn?Asn?Gly?Arg?Val

165?????????????????170?????????????????175

ggc?cag?tgg?ggc?atc?tcc?tac?ccc?ggc?tac?tat?acc?gct?act?ggc?cta?????636

Gly?Gln?Trp?Gly?Ile?Ser?Tyr?Pro?Gly?Tyr?Tyr?Thr?Ala?Thr?Gly?Leu

180?????????????????185?????????????????190

ctg?agc?cgc?cac?aag?gcc?cta?aag?gca?tcc?tca?ccg?cag?gcc?cct?att?????684

Leu?Ser?Arg?His?Lys?Ala?Leu?Lys?Ala?Ser?Ser?Pro?Gln?Ala?Pro?Ile

195?????????????????200?????????????????205

gcc?gac?tgg?ttc?tgg?gac?gat?ttt?cac?cac?aac?ggc?gcg?ttc?ttc?ctg?????732

Ala?Asp?Trp?Phe?Trp?Asp?Asp?Phe?His?His?Asn?Gly?Ala?Phe?Phe?Leu

210?????????????????215?????????????????220

ccg?cac?gct?ttc?aac?ttc?ctg?gcc?tcc?ttt?ggg?ctg?gcc?cgc?ccc?cag?????780

Pro?His?Ala?Phe?Asn?Phe?Leu?Ala?Ser?Phe?Gly?Leu?Ala?Arg?Pro?Gln

225?????????????????230?????????????????235?????????????????240

ccc?acg?cct?acc?ggc?aac?ccc?ggc?ttc?aag?cac?ggc?acc?ccc?gat?ggc?????828

Pro?Thr?Pro?Thr?Gly?Asn?Pro?Gly?Phe?Lys?His?Gly?Thr?Pro?Asp?Gly

245?????????????????250?????????????????255

tac?gat?ttt?ttc?ctg?aag?atg?ggt?ccg?ctg?aaa?aac?gct?gat?gcc?aac?????876

Tyr?Asp?Phe?Phe?Leu?Lys?Met?Gly?Pro?Leu?Lys?Asn?Ala?Asp?Ala?Asn

260?????????????????265?????????????????270

tac?tac?aaa?ggc?aaa?gtg?gcc?ttc?tgg?aac?gaa?atg?gcc?agc?cac?ccc?????924

Tyr?Tyr?Lys?Gly?Lys?Val?Ala?Phe?Trp?Asn?Glu?Met?Ala?Ser?His?Pro

275?????????????????280?????????????????285

aac?tac?gac?gaa?ttc?tgg?cag?gcc?cgt?aac?cta?cgc?ccc?cac?ctc?aag?????972

Asn?Tyr?Asp?Glu?Phe?Trp?Gln?Ala?Arg?Asn?Leu?Arg?Pro?His?Leu?Lys

290?????????????????295?????????????????300

aac?ctc?aac?aaa?ggc?acc?gcg?gtg?ctc?acg?gtt?ggt?ggc?ttc?aat?gat????1020

Asn?Leu?Asn?Lys?Gly?Thr?Ala?Val?Leu?Thr?Val?Gly?Gly?Phe?Asn?Asp

305?????????????????310?????????????????315?????????????????320

gcc?gag?gac?ctg?ttt?ggc?gcc?ctg?aaa?acc?tac?gaa?agc?atc?gag?aag????1068

Ala?Glu?Asp?Leu?Phe?Gly?Ala?Leu?Lys?Thr?Tyr?Glu?Ser?Ile?Glu?Lys

325?????????????????330?????????????????335

caa?aac?ccc?ggc?atg?cgc?aac?ggc?ctc?gtg?atg?ggg?ccg?tgg?gta?cac????1116

Gln?Asn?Pro?Gly?Met?Arg?Asn?Gly?Leu?Val?Met?Gly?Pro?Trp?Val?His

340?????????????????345?????????????????350

ggt?ggc?tgg?gcc?cgc?ggc?act?ggc?gaa?atg?gta?ggc?aat?gtg?gcc?tac????1164

Gly?Gly?Trp?Ala?Arg?Gly?Thr?Gly?Glu?Met?Val?Gly?Asn?Val?Ala?Tyr

355?????????????????360?????????????????365

ggc?gag?tcg?ccg?tcg?ttg?tat?tac?cag?aag?cag?att?gaa?gcg?ccg?ttc????1212

Gly?Glu?Ser?Pro?Ser?Leu?Tyr?Tyr?Gln?Lys?Gln?Ile?Glu?Ala?Pro?Phe

370?????????????????375?????????????????380

ttc?aaa?tca?tat?ctg?aag?gat?ggc?aaa?cct?gcc?gct?acc?ccc?gag?gct????1260

Phe?Lys?Ser?Tyr?Leu?Lys?Asp?Gly?Lys?Pro?Ala?Ala?Thr?Pro?Glu?Ala

385?????????????????390?????????????????395????????????????400

acc?atc?ttt?gaa?agc?ggc?acc?aac?cgc?tgg?cgc?agc?ttc?gaa?acc?tgg????1308

Thr?Ile?Phe?Glu?Ser?Gly?Thr?Asn?Arg?Trp?Arg?Ser?Phe?Glu?Thr?Trp

405?????????????????410?????????????????415

ccg?ccc?aaa?gaa?gcc?aaa?gag?cgc?act?ttg?tac?ttt?cag?tcg?gcc?ggg????1356

Pro?Pro?Lys?Glu?Ala?Lys?Glu?Arg?Thr?Leu?Tyr?Phe?Gln?Ser?Ala?Gly

420?????????????????425?????????????????430

aaa?atc?ggc?ttc?gag?aag?cct?gcc?agt?ggc?cta?gag?tac?gac?cag?ttc????1404

Lys?Ile?Gly?Phe?Glu?Lys?Pro?Ala?Ser?Gly?Leu?Glu?Tyr?Asp?Gln?Phe

435?????????????????440?????????????????445

ctc?agc?gac?ccg?gct?cac?cca?gtg?cct?ttc?acc?gaa?gct?acg?gct?acg????1452

Leu?Ser?Asp?Pro?Ala?His?Pro?Val?Pro?Phe?Thr?Glu?Ala?Thr?Ala?Thr

450?????????????????455?????????????????460

ggc?atg?acc?cgc?gag?tac?atg?acc?gac?gac?cag?cgc?ttc?gcc?agc?cgc????1500

Gly?Met?Thr?Arg?Glu?Tyr?Met?Thr?Asp?Asp?Gln?Arg?Phe?Ala?Ser?Arg

465?????????????????470?????????????????475?????????????????480

cgc?ccc?gac?gtg?ctg?acc?tac?cag?acc?gaa?gcg?ctt?acc?gag?gac?atg????1548

Arg?Pro?Asp?Val?Leu?Thr?Tyr?Gln?Thr?Glu?Ala?Leu?Thr?Glu?Asp?Met

485?????????????????490?????????????????495

acg?ctg?gct?ggc?cct?atc?gag?gcg?ctg?ttg?cag?gta?gcc?acc?acc?ggc????1596

Thr?Leu?Ala?Gly?Pro?Ile?Glu?Ala?Leu?Leu?Gln?Val?Ala?Thr?Thr?Gly

500?????????????????505?????????????????510

acc?gat?gcc?gac?tgg?gta?gtg?aag?att?att?gat?gtg?tac?ccc?gac?gat????1644

Thr?Asp?Ala?Asp?Trp?Val?Val?Lys?Ile?Ile?Asp?Val?Tyr?Pro?Asp?Asp

515?????????????????520?????????????????525

acg?ccc?aac?aac?ccc?agc?acg?aac?ccc?gcc?gtg?aaa?ctg?ggc?ggc?tac????1692

Thr?Pro?Asn?Asn?Pro?Ser?Thr?Asn?Pro?Ala?Val?Lys?Leu?Gly?Gly?Tyr

530?????????????????535?????????????????540

cag?cag?atg?gtt?cgc?tcc?gag?gtg?atg?cgc?ggt?cgt?ttc?cgc?aac?agc????1740

Gln?Gln?Met?Val?Arg?Ser?Glu?Val?Met?Arg?Gly?Arg?Phe?Arg?Asn?Ser

545?????????????????550?????????????????555?????????????????560

ttc?tcc?aag?ccc?gaa?gcc?ttt?gta?ccg?gaa?cag?gta?acg?gcc?gtg?ccc????1788

Phe?Ser?Lys?Pro?Glu?Ala?Phe?Val?Pro?Glu?Gln?Val?Thr?Ala?Val?Pro

565?????????????????570?????????????????575

ttc?acg?gtg?cag?gac?ctg?tgc?cac?acc?ttc?cgg?aaa?gga?cac?cgc?ctg????1836

Phe?Thr?Val?Gln?Asp?Leu?Cys?His?Thr?Phe?Arg?Lys?Gly?His?Arg?Leu

580?????????????????585?????????????????590

atg?gtg?cag?gtg?caa?agc?agc?tgg?ttc?ccg?att?gtt?gac?cgc?aac?ccg????1884

Met?Val?Gln?Val?Gln?Ser?Ser?Trp?Phe?Pro?Ile?Val?Asp?Arg?Asn?Pro

595?????????????????600?????????????????605

cag?acc?ttc?gta?ccc?aat?att?ttc?gag?gcc?gat?gag?aag?gat?ttc?cag????1932

Gln?Thr?Phe?Val?Pro?Asn?Ile?Phe?Glu?Ala?Asp?Glu?Lys?Asp?Phe?Gln

610?????????????????615?????????????????620

gcc?gcc?acg?cat?cgg?ctg?tac?cat?tcg?ccg?gcg?cat?agc?tcg?cag?ctc????1980

Ala?Ala?Thr?His?Arg?Leu?Tyr?His?Ser?Pro?Ala?His?Ser?Ser?Gln?Leu

625?????????????????630?????????????????635?????????????????640

acg?ttg?cgc?gtt?ctg?taggccactc?taaacaggct?cgg??????????????????????2018

Thr?Leu?Arg?Val?Leu

645

<210>23

<211>645

<212>PRT

<213>Taxeobacter?gelupurpurascens

<400>23

Met?Pro?Tyr?Ser?Phe?Pro?Lys?Val?Ala?Ala?Leu?Ser?Gly?Leu?Leu?Val

1???????????????5???????????????????10??????????????????15

Ala?Gly?Leu?Ser?Gly?Ala?His?Ala?Gln?Thr?Pro?Val?Thr?Tyr?Pro?Leu

20??????????????????25??????????????????30

Ala?Ser?Glu?Ala?Glu?Lys?Ala?Gln?Leu?Ala?Val?Val?Leu?Ala?Asp?Thr

35??????????????????40??????????????????45

Ala?Tyr?Ile?Lys?Glu?Arg?Tyr?Thr?Lys?Thr?Glu?Tyr?Gln?Ile?Pro?Met

50??????????????????55??????????????????60

Arg?Asp?Gly?Val?Lys?Leu?Tyr?Thr?Ile?Val?Tyr?Ala?Pro?Asn?Asp?Ala

65??????????????????70??????????????????75??????????????????80

Asn?Lys?Val?Lys?Tyr?Pro?Ile?Leu?Leu?Asn?Arg?Thr?Pro?Tyr?Ala?Ile

85??????????????????90??????????????????95

Gly?Pro?Tyr?Gly?Pro?Gly?Lys?Tyr?Lys?Leu?Asn?Leu?Gly?Pro?Ser?Ser

100?????????????????105?????????????????110

Thr?Met?Met?His?Glu?Gly?Tyr?Ile?Phe?Ala?Tyr?Gln?Asp?Val?Arg?Gly

115?????????????????120?????????????????125

Arg?Tyr?Met?Ser?Glu?Gly?Glu?Phe?Val?Asp?Val?Arg?Pro?Glu?Lys?Asp

130?????????????????135?????????????????140

Met?His?Lys?Gly?Lys?Asn?Asp?Ile?Asp?Glu?Gly?Thr?Asp?Thr?Tyr?Asp

145?????????????????150?????????????????155?????????????????160

Thr?Ile?Glu?Trp?Leu?Leu?Lys?His?Gly?Pro?Lys?Asn?Asn?Gly?Arg?Val

165?????????????????170?????????????????175

Gly?Gln?Trp?Gly?Ile?Ser?Tyr?Pro?Gly?Tyr?Tyr?Thr?Ala?Thr?Gly?Leu

180?????????????????185?????????????????190

Leu?Ser?Arg?His?Lys?Ala?Leu?Lys?Ala?Ser?Ser?Pro?Gln?Ala?Pro?Ile

195?????????????????200?????????????????205

Ala?Asp?Trp?Phe?Trp?Asp?Asp?Phe?His?His?Asn?Gly?Ala?Phe?Phe?Leu

210?????????????????215?????????????????220

Pro?His?Ala?Phe?Asn?Phe?Leu?Ala?Ser?Phe?Gly?Leu?Ala?Arg?Pro?Gln

225?????????????????230?????????????????235?????????????????240

Pro?Thr?Pro?Thr?Gly?Asn?Pro?Gly?Phe?Lys?His?Gly?Thr?Pro?Asp?Gly

245?????????????????250?????????????????255

Tyr?Asp?Phe?Phe?Leu?Lys?Met?Gly?Pro?Leu?Lys?Asn?Ala?Asp?Ala?Asn

260?????????????????265?????????????????270

Tyr?Tyr?Lys?Gly?Lys?Val?Ala?Phe?Trp?Asn?Glu?Met?Ala?Ser?His?Pro

275?????????????????280?????????????????285

Asn?Tyr?Asp?Glu?Phe?Trp?Gln?Ala?Arg?Asn?Leu?Arg?Pro?His?Leu?Lys

290?????????????????295?????????????????300

Asn?Leu?Asn?Lys?Gly?Thr?Ala?Val?Leu?Thr?Val?Gly?Gly?Phe?Asn?Asp

305?????????????????310?????????????????315?????????????????320

Ala?Glu?Asp?Leu?Phe?Gly?Ala?Leu?Lys?Thr?Tyr?Glu?Ser?Ile?Glu?Lys

325?????????????????330?????????????????335

Gln?Asn?Pro?Gly?Met?Arg?Asn?Gly?Leu?Val?Met?Gly?Pro?Trp?Val?His

340?????????????????345?????????????????350

Gly?Gly?Trp?Ala?Arg?Gly?Thr?Gly?Glu?Met?Val?Gly?Asn?Val?Ala?Tyr

355?????????????????360?????????????????365

Gly?Glu?Ser?Pro?Ser?Leu?Tyr?Tyr?Gln?Lys?Gln?Ile?Glu?Ala?Pro?Phe

370?????????????????375?????????????????380

Phe?Lys?Ser?Tyr?Leu?Lys?Asp?Gly?Lys?Pro?Ala?Ala?Thr?Pro?Glu?Ala

385?????????????????390?????????????????395????????????????400

Thr?Ile?Phe?Glu?Ser?Gly?Thr?Asn?Arg?Trp?Arg?Ser?Phe?Glu?Thr?Trp

405?????????????????410?????????????????415

Pro?Pro?Lys?Glu?Ala?Lys?Glu?Arg?Thr?Leu?Tyr?Phe?Gln?Ser?Ala?Gly

420?????????????????425?????????????????430

Lys?Ile?Gly?Phe?Glu?Lys?Pro?Ala?Ser?Gly?Leu?Glu?Tyr?Asp?Gln?Phe

435?????????????????440?????????????????445

Leu?Ser?Asp?Pro?Ala?His?Pro?Val?Pro?Phe?Thr?Glu?Ala?Thr?Ala?Thr

450?????????????????455?????????????????460

Gly?Met?Thr?Arg?Glu?Tyr?Met?Thr?Asp?Asp?Gln?Arg?Phe?Ala?Ser?Arg

465?????????????????470?????????????????475?????????????????480

Arg?Pro?Asp?Val?Leu?Thr?Tyr?Gln?Thr?Glu?Ala?Leu?Thr?Glu?Asp?Met

485?????????????????490?????????????????495

Thr?Leu?Ala?Gly?Pro?Ile?Glu?Ala?Leu?Leu?Gln?Val?Ala?Thr?Thr?Gly

500?????????????????505?????????????????510

Thr?Asp?Ala?Asp?Trp?Val?Val?Lys?Ile?Ile?Asp?Val?Tyr?Pro?Asp?Asp

515?????????????????520?????????????????525

Thr?Pro?Asn?Asn?Pro?Ser?Thr?Asn?Pro?Ala?Val?Lys?Leu?Gly?Gly?Tyr

530?????????????????535?????????????????540

Gln?Gln?Met?Val?Arg?Ser?Glu?Val?Met?Arg?Gly?Arg?Phe?Arg?Asn?Ser

545?????????????????550?????????????????555?????????????????560

Phe?Ser?Lys?Pro?Glu?Ala?Phe?Val?Pro?Glu?Gln?Val?Thr?Ala?Val?Pro

565?????????????????570?????????????????575

Phe?Thr?Val?Gln?Asp?Leu?Cys?His?Thr?Phe?Arg?Lys?Gly?His?Arg?Leu

580?????????????????585?????????????????590

Met?Val?Gln?Val?Gln?Ser?Ser?Trp?Phe?Pro?Ile?Val?Asp?Arg?Asn?Pro

595?????????????????600?????????????????605

Gln?Thr?Phe?Val?Pro?Asn?Ile?Phe?Glu?Ala?Asp?Glu?Lys?Asp?Phe?Gln

610?????????????????615?????????????????620

Ala?Ala?Thr?His?Arg?Leu?Tyr?His?Ser?Pro?Ala?His?Ser?Ser?Gln?Leu

625?????????????????630?????????????????635?????????????????640

Thr?Leu?Arg?Val?Leu

645

<210>24

<211>1931

<212>DNA

＜213〉extra large round bacteria

<220>

<221>CDS

<222>(29)..(1888)

<223>

<400>24

cccaaagcat?taacaaaata?atttagtc?atg?aaa?cac?tgt?tac?aaa?ctt?ctg???????52

Met?Lys?His?Cys?Tyr?Lys?Leu?Leu

1???????????????5

gtc?ttt?tac?aca?tta?ttt?ttg?atg?acc?aca?aac?tgg?gct?tta?tca?caa?????100

Val?Phe?Tyr?Thr?Leu?Phe?Leu?Met?Thr?Thr?Asn?Trp?Ala?Leu?Ser?Gln

10??????????????????15??????????????????20

gcc?att?aat?gga?tat?gat?aag?gca?gcc?tat?gac?att?cct?atg?cga?gat?????148

Ala?Ile?Asn?Gly?Tyr?Asp?Lys?Ala?Ala?Tyr?Asp?Ile?Pro?Met?Arg?Asp

25??????????????????30??????????????????35??????????????????40

gga?gtt?cac?ctt?cac?acc?atc?gtc?tat?agc?ccc?aaa?gat?tta?tcg?cag?????196

Gly?Val?His?Leu?His?Thr?Ile?Val?Tyr?Ser?Pro?Lys?Asp?Leu?Ser?Gln

45??????????????????50??????????????????55

ccc?tat?cct?ata?ttg?atg?caa?agg?aca?cct?tac?agc?gcc?ggc?cct?tat?????244

Pro?Tyr?Pro?Ile?Leu?Met?Gln?Arg?Thr?Pro?Tyr?Ser?Ala?Gly?Pro?Tyr

60??????????????????65??????????????????70

ggt?cct?gga?aat?atg?aaa?aat?aag?ctt?ggc?cct?tct?cag?ttt?tta?atg?????292

Gly?Pro?Gly?Asn?Met?Lys?Asn?Lys?Leu?Gly?Pro?Ser?Gln?Phe?Leu?Met

75??????????????????80??????????????????85

aac?gat?ggc?tat?ata?ttt?gtt?tac?cag?gat?gta?aga?ggg?cgg?tgg?atg?????340

Asn?Asp?Gly?Tyr?Ile?Phe?Val?Tyr?Gln?Asp?Val?Arg?Gly?Arg?Trp?Met

90??????????????????95??????????????????100

tcg?gaa?gga?tcc?tat?gac?aac?atg?cgc?cct?acc?cta?tcc?aaa?tca?gaa?????388

Ser?Glu?Gly?Ser?Tyr?Asp?Asn?Met?Arg?Pro?Thr?Leu?Ser?Lys?Ser?Glu

105?????????????????110?????????????????115?????????????????120

aga?aat?tcc?aac?caa?ata?gac?gaa?agc?aca?gac?acc?tat?gat?acc?ata?????436

Arg?Asn?Ser?Asn?Gln?Ile?Asp?Glu?Ser?Thr?Asp?Thr?Tyr?Asp?Thr?Ile

125?????????????????130?????????????????135

gaa?tgg?ttg?ctc?gcc?aat?atc?aaa?aat?cac?aat?gaa?aaa?gta?ggc?cta?????484

Glu?Trp?Leu?Leu?Ala?Asn?Ile?Lys?Asn?His?Asn?Glu?Lys?Val?Gly?Leu

140?????????????????145?????????????????150

tgg?gga?atc?agc?tat?ccc?gga?ttt?tat?agt?gct?gca?gcc?ctt?cct?ttt?????532

Trp?Gly?Ile?Ser?Tyr?Pro?Gly?Phe?Tyr?Ser?Ala?Ala?Ala?Leu?Pro?Phe

155?????????????????160?????????????????165

gcc?cat?cca?aac?ctg?aaa?gcc?gtt?tcc?cct?caa?gca?ccc?ata?ggg?gat?????580

Ala?His?Pro?Asn?Leu?Lys?Ala?Val?Ser?Pro?Gln?Ala?Pro?Ile?Gly?Asp

170?????????????????175?????????????????180

ttt?tac?ttt?gat?gat?ttt?cat?cat?aac?ggt?gct?tac?tta?tta?agt?tat?????628

Phe?Tyr?Phe?Asp?Asp?Phe?His?His?Asn?Gly?Ala?Tyr?Leu?Leu?Ser?Tyr

185?????????????????190?????????????????195?????????????????200

tgg?ttg?gcc?act?tct?gtt?ttc?ggc?tac?caa?aaa?gac?ggc?cct?aca?cag?????676

Trp?Leu?Ala?Thr?Ser?Val?Phe?Gly?Tyr?Gln?Lys?Asp?Gly?Pro?Thr?Gln

205?????????????????210?????????????????215

gaa?gca?tgg?tat?ggc?atg?gtg?aat?ccg?gaa?aca?aat?gac?ggc?tat?cag?????724

Glu?Ala?Trp?Tyr?Gly?Met?Val?Asn?Pro?Glu?Thr?Asn?Asp?Gly?Tyr?Gln

220?????????????????225?????????????????230

ttt?ttt?atg?gat?atg?ggg?cca?tta?aaa?aat?gcc?gat?aaa?tgg?tat?ggt?????772

Phe?Phe?Met?Asp?Met?Gly?Pro?Leu?Lys?Asn?Ala?Asp?Lys?Trp?Tyr?Gly

235?????????????????240?????????????????245

gaa?gac?aat?ttt?ttc?tgg?caa?caa?ctt?aaa?aac?aat?cct?gat?tac?aac?????820

Glu?Asp?Asn?Phe?Phe?Trp?Gln?Gln?Leu?Lys?Asn?Asn?Pro?Asp?Tyr?Asn

250?????????????????255?????????????????260

gct?ttc?tgg?caa?aag?aga?agt?att?att?cct?cac?tta?aaa?gaa?gtg?aag?????868

Ala?Phe?Trp?Gln?Lys?Arg?Ser?Ile?Ile?Pro?His?Leu?Lys?Glu?Val?Lys

265?????????????????270?????????????????275?????????????????280

cct?gca?gtt?tta?acc?gtt?ggg?ggc?tgg?ttt?gat?gca?gaa?gat?ctc?tat?????916

Pro?Ala?Val?Leu?Thr?Val?Gly?Gly?Trp?Phe?Asp?Ala?Glu?Asp?Leu?Tyr

285?????????????????290?????????????????295

gga?cca?ctt?aca?att?tat?aaa?acc?att?gaa?aaa?aat?aat?cct?gag?acc?????964

Gly?Pro?Leu?Thr?Ile?Tyr?Lys?Thr?Ile?Glu?Lys?Asn?Asn?Pro?Glu?Thr

300?????????????????305?????????????????310

tac?aat?acc?att?gtc?atg?ggc?cct?tgg?tcc?cac?gga?gat?tgg?tca?agg????1012

Tyr?Asn?Thr?Ile?Val?Met?Gly?Pro?Trp?Ser?His?Gly?Asp?Trp?Ser?Arg

315?????????????????320?????????????????325

gaa?cct?gga?tca?cag?gtc?att?tca?aat?att?tat?ttt?ggt?gat?tct?atc????1060

Glu?Pro?Gly?Ser?Gln?Val?Ile?Ser?Asn?Ile?Tyr?Phe?Gly?Asp?Ser?Ile

330?????????????????335?????????????????340

tcc?aca?tgg?tat?caa?aaa?aat?ata?gaa?cgt?gtt?ttt?ttc?aat?cat?ttt????1108

Ser?Thr?Trp?Tyr?Gln?Lys?Asn?Ile?Glu?Arg?Val?Phe?Phe?Asn?His?Phe

345?????????????????350?????????????????355?????????????????360

cta?aaa?gaa?tcc?gaa?aat?agc?aat?cct?gcc?ctt?cct?gaa?gcc?tac?atg????1156

Leu?Lys?Glu?Ser?Glu?Asn?Ser?Asn?Pro?Ala?Leu?Pro?Glu?Ala?Tyr?Met

365?????????????????370?????????????????375

ttt?gat?acc?gga?aaa?cat?aaa?tgg?gaa?aaa?ttt?gac?gat?tgg?cct?cct????1204

Phe?Asp?Thr?Gly?Lys?His?Lys?Trp?Glu?Lys?Phe?Asp?Asp?Trp?Pro?Pro

380?????????????????385?????????????????390

aaa?gaa?agc?caa?tgg?aaa?agc?ttt?tac?ttt?caa?gag?aaa?gga?gag?tta????1252

Lys?Glu?Ser?Gln?Trp?Lys?Ser?Phe?Tyr?Phe?Gln?Glu?Lys?Gly?Glu?Leu

395?????????????????400?????????????????405

act?gag?gta?aca?cct?gag?gga?aat?agg?ttt?act?acc?tat?gtc?tca?gac????1300

Thr?Glu?Val?Thr?Pro?Glu?Gly?Asn?Arg?Phe?Thr?Thr?Tyr?Val?Ser?Asp

410?????????????????415?????????????????420

ccc?tct?aat?cct?gtc?ccc?tat?agt?caa?gat?att?aaa?cta?aac?ttc?act????1348

Pro?Ser?Asn?Pro?Val?Pro?Tyr?Ser?Gln?Asp?Ile?Lys?Leu?Asn?Phe?Thr

425?????????????????430?????????????????435?????????????????440

ccg?aga?aaa?tac?atg?gcc?gat?gac?cag?cga?ttt?gca?gcc?aga?aga?ccg????1396

Pro?Arg?Lys?Tyr?Met?Ala?Asp?Asp?Gln?Arg?Phe?Ala?Ala?Arg?Arg?Pro

445?????????????????450?????????????????455

gac?gta?ctg?acc?ttt?acg?agc?gaa?gta?tta?agt?caa?gac?atg?acg?ctt????1444

Asp?Val?Leu?Thr?Phe?Thr?Ser?Glu?Val?Leu?Ser?Gln?Asp?Met?Thr?Leu

460?????????????????465?????????????????470

gcg?ggg?gaa?gtc?atg?gca?aac?tta?aaa?gtt?gcc?act?tca?caa?act?gat????1492

Ala?Gly?Glu?Val?Met?Ala?Asn?Leu?Lys?Val?Ala?Thr?Ser?Gln?Thr?Asp

475?????????????????480?????????????????485

gct?gat?tgg?gta?gtt?aaa?atc?atc?gat?ata?ttt?ccc?gga?gat?cag?cca????1540

Ala?Asp?Trp?Val?Val?Lys?Ile?Ile?Asp?Ile?Phe?Pro?Gly?Asp?Gln?Pro

490?????????????????495?????????????????500

aat?cat?gcc?tat?gtt?tta?gat?ggg?gtg?gac?atg?ggc?aat?tac?cac?cta????1588

Asn?His?Ala?Tyr?Val?Leu?Asp?Gly?Val?Asp?Met?Gly?Asn?Tyr?His?Leu

505?????????????????510?????????????????515?????????????????520

atg?gtt?cgt?tca?gag?gta?att?aga?ggg?agg?tat?aga?gaa?agt?ttt?gag????1636

Met?Val?Arg?Ser?Glu?Val?Ile?Arg?Gly?Arg?Tyr?Arg?Glu?Ser?Phe?Glu

525?????????????????530?????????????????535

ttt?cct?aaa?ccc?ttt?gtt?cct?gat?caa?atc?act?gct?gtt?gat?ttc?agg????1684

Phe?Pro?Lys?Pro?Phe?Val?Pro?Asp?Gln?Ile?Thr?Ala?Val?Asp?Phe?Arg

540?????????????????545?????????????????550

tta?caa?gat?ctt?ttc?cat?act?ttc?aaa?aag?ggg?cat?aaa?att?caa?ata????1732

Leu?Gln?Asp?Leu?Phe?His?Thr?Phe?Lys?Lys?Gly?His?Lys?Ile?Gln?Ile

555?????????????????560?????????????????565

caa?ata?caa?agt?act?tgg?ttt?ccc?cta?att?gat?cga?aat?ccc?caa?aaa????1780

Gln?Ile?Gln?Ser?Thr?Trp?Phe?Pro?Leu?Ile?Asp?Arg?Asn?Pro?Gln?Lys

570?????????????????575?????????????????580

tat?gta?caa?aac?ata?ttt?gaa?gct?gag?gaa?gcc?gat?ttt?gtc?aaa?gcc????1828

Tyr?Val?Gln?Asn?Ile?Phe?Glu?Ala?Glu?Glu?Ala?Asp?Phe?Val?Lys?Ala

585?????????????????590?????????????????595?????????????????600

acc?cat?agg?gtt?ttt?cat?aca?gaa?aag?ttt?gcc?agc?aaa?att?gaa?gta????1876

Thr?His?Arg?Val?Phe?His?Thr?Glu?Lys?Phe?Ala?Ser?Lys?Ile?Glu?Val

605?????????????????610?????????????????615

atg?gtt?ctt?cct?tagaattaga?atggtttaaa?attactattt?gtagcagaag?ata????1931

Met?Val?Leu?Pro

620

<210>25

<211>620

<212>PRT

＜213〉extra large round bacteria

<400>25

Met?Lys?His?Cys?Tyr?Lys?Leu?Leu?Val?Phe?Tyr?Thr?Leu?Phe?Leu?Met

1???????????????5???????????????????10??????????????????15

Thr?Thr?Asn?Trp?Ala?Leu?Ser?Gln?Ala?Ile?Asn?Gly?Tyr?Asp?Lys?Ala

20??????????????????25??????????????????30

Ala?Tyr?Asp?Ile?Pro?Met?Arg?Asp?Gly?Val?His?Leu?His?Thr?Ile?Val

35??????????????????40??????????????????45

Tyr?Ser?Pro?Lys?Asp?Leu?Ser?Gln?Pro?Tyr?Pro?Ile?Leu?Met?Gln?Arg

50??????????????????55??????????????????60

Thr?Pro?Tyr?Ser?Ala?Gly?Pro?Tyr?Gly?Pro?Gly?Asn?Met?Lys?Asn?Lys

65??????????????????70??????????????????75??????????????????80

Leu?Gly?Pro?Ser?Gln?Phe?Leu?Met?Asn?Asp?Gly?Tyr?Ile?Phe?Val?Tyr

85??????????????????90??????????????????95

Gln?Asp?Val?Arg?Gly?Arg?Trp?Met?Ser?Glu?Gly?Ser?Tyr?Asp?Asn?Met

100?????????????????105?????????????????110

Arg?Pro?Thr?Leu?Ser?Lys?Ser?Glu?Arg?Asn?Ser?Asn?Gln?Ile?Asp?Glu

115?????????????????120?????????????????125

Ser?Thr?Asp?Thr?Tyr?Asp?Thr?Ile?Glu?Trp?Leu?Leu?Ala?Asn?Ile?Lys

130?????????????????135?????????????????140

Asn?His?Asn?Glu?Lys?Val?Gly?Leu?Trp?Gly?Ile?Ser?Tyr?Pro?Gly?Phe

145?????????????????150?????????????????155?????????????????160

Tyr?Ser?Ala?Ala?Ala?Leu?Pro?Phe?Ala?His?Pro?Asn?Leu?Lys?Ala?Val

165?????????????????170?????????????????175

Ser?Pro?Gln?Ala?Pro?Ile?Gly?Asp?Phe?Tyr?Phe?Asp?Asp?Phe?His?His

180?????????????????185?????????????????190

Asn?Gly?Ala?Tyr?Leu?Leu?Ser?Tyr?Trp?Leu?Ala?Thr?Ser?Val?Phe?Gly

195?????????????????200?????????????????205

Tyr?Gln?Lys?Asp?Gly?Pro?Thr?Gln?Glu?Ala?Trp?Tyr?Gly?Met?Val?Asn

210?????????????????215?????????????????220

Pro?Glu?Thr?Asn?Asp?Gly?Tyr?Gln?Phe?Phe?Met?Asp?Met?Gly?Pro?Leu

225?????????????????230?????????????????235?????????????????240

Lys?Asn?Ala?Asp?Lys?Trp?Tyr?Gly?Glu?Asp?Asn?Phe?Phe?Trp?Gln?Gln

245?????????????????250?????????????????255

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

260?????????????????265?????????????????270

Ile?Pro?His?Leu?Lys?Glu?Val?Lys?Pro?Ala?Val?Leu?Thr?Val?Gly?Gly

275?????????????????280?????????????????285

Trp?Phe?Asp?Ala?Glu?Asp?Leu?Tyr?Gly?Pro?Leu?Thr?Ile?Tyr?Lys?Thr

290?????????????????295?????????????????300

Ile?Glu?Lys?Asn?Asn?Pro?Glu?Thr?Tyr?Asn?Thr?Ile?Val?Met?Gly?Pro

305?????????????????310?????????????????315?????????????????320

Trp?Ser?His?Gly?Asp?Trp?Ser?Arg?Glu?Pro?Gly?Ser?Gln?Val?Ile?Ser

325?????????????????330?????????????????335

Asn?Ile?Tyr?Phe?Gly?Asp?Ser?Ile?Ser?Thr?Trp?Tyr?Gln?Lys?Asn?Ile

340?????????????????345?????????????????350

Glu?Arg?Val?Phe?Phe?Asn?His?Phe?Leu?Lys?Glu?Ser?Glu?Asn?Ser?Asn

355?????????????????360?????????????????365

Pro?Ala?Leu?Pro?Glu?Ala?Tyr?Met?Phe?Asp?Thr?Gly?Lys?His?Lys?Trp

370?????????????????375?????????????????380

Glu?Lys?Phe?Asp?Asp?Trp?Pro?Pro?Lys?Glu?Ser?Gln?Trp?Lys?Ser?Phe

385?????????????????390?????????????????395?????????????????400

Tyr?Phe?Gln?Glu?Lys?Gly?Glu?Leu?Thr?Glu?Val?Thr?Pro?Glu?Gly?Asn

405?????????????????410?????????????????415

Arg?Phe?Thr?Thr?Tyr?Val?Ser?Asp?Pro?Ser?Asn?Pro?Val?Pro?Tyr?Ser

420?????????????????425?????????????????430

Gln?Asp?Ile?Lys?Leu?Asn?Phe?Thr?Pro?Arg?Lys?Tyr?Met?Ala?Asp?Asp

435?????????????????440?????????????????445

Gln?Arg?Phe?Ala?Ala?Arg?Arg?Pro?Asp?Val?Leu?Thr?Phe?Thr?Ser?Glu

450?????????????????455?????????????????460

Val?Leu?Ser?Gln?Asp?Met?Thr?Leu?Ala?Gly?Glu?Val?Met?Ala?Asn?Leu

465?????????????????470?????????????????475?????????????????480

Lys?Val?Ala?Thr?Ser?Gln?Thr?Asp?Ala?Asp?Trp?Val?Val?Lys?Ile?Ile

485?????????????????490?????????????????495

Asp?Ile?Phe?Pro?Gly?Asp?Gln?Pro?Asn?His?Ala?Tyr?Val?Leu?Asp?Gly

500?????????????????505?????????????????510

Val?Asp?Met?Gly?Asn?Tyr?His?Leu?Met?Val?Arg?Ser?Glu?Val?Ile?Arg

515?????????????????520?????????????????525

Gly?Arg?Tyr?Arg?Glu?Ser?Phe?Glu?Phe?Pro?Lys?Pro?Phe?Val?Pro?Asp

530?????????????????535?????????????????540

Gln?Ile?Thr?Ala?Val?Asp?Phe?Arg?Leu?Gln?Asp?Leu?Phe?His?Thr?Phe

545?????????????????550?????????????????555?????????????????560

Lys?Lys?Gly?His?Lys?Ile?Gln?Ile?Gln?Ile?Gln?Ser?Thr?Trp?Phe?Pro

565?????????????????570?????????????????575

Leu?Ile?Asp?Arg?Asn?Pro?Gln?Lys?Tyr?Val?Gln?Asn?Ile?Phe?Glu?Ala

580?????????????????585?????????????????590

Glu?Glu?Ala?Asp?Phe?Val?Lys?Ala?Thr?His?Arg?Val?Phe?His?Thr?Glu

595?????????????????600?????????????????605

Lys?Phe?Ala?Ser?Lys?Ile?Glu?Val?Met?Val?Leu?Pro

610?????????????????615?????????????????620

<210>26

<211>2036

<212>DNA

<213>Psycloserpens?burtonensis

<220>

<221>CDS

<222>(61)..(1992)

<223>

<400>26

catattcgta?aaatagctat?aagtttttgt?aaatttagtc?aatcaaaatt?ttaaatgtaa????60

atg?aag?act?ctt?ttt?aaa?ttg?ttg?ctc?cta?ttt?gta?ttt?gtt?cta?acg?????108

Met?Lys?Thr?Leu?Phe?Lys?Leu?Leu?Leu?Leu?Phe?Val?Phe?Val?Leu?Thr

1???????????????5???????????????????10??????????????????15

tct?tgt?aat?aag?gcc?aac?aaa?gac?gct?act?gaa?att?gtg?aaa?acc?gaa?????156

Ser?Cys?Asn?Lys?Ala?Asn?Lys?Asp?Ala?Thr?Glu?Ile?Val?Lys?Thr?Glu

20??????????????????25??????????????????30

gta?gaa?gat?act?tac?gtt?aaa?gat?aat?tat?aac?aaa?caa?gag?gtg?act?????204

Val?Glu?Asp?Thr?Tyr?Val?Lys?Asp?Asn?Tyr?Asn?Lys?Gln?Glu?Val?Thr

35??????????????????40??????????????????45

att?gaa?atg?cgc?gat?ggt?ata?aaa?ctt?cac?acg?acc?att?tat?tca?cca?????252

Ile?Glu?Met?Arg?Asp?Gly?Ile?Lys?Leu?His?Thr?Thr?Ile?Tyr?Ser?Pro

50??????????????????55??????????????????60

aaa?gat?gaa?agt?cag?acc?tat?cct?att?tta?atg?atg?aga?aca?cca?tat?????300

Lys?Asp?Glu?Ser?Gln?Thr?Tyr?Pro?Ile?Leu?Met?Met?Arg?Thr?Pro?Tyr

65??????????????????70??????????????????75??????????????????80

agt?tct?caa?cct?tat?ggt?gac?aat?gag?ttt?aag?acg?aaa?att?ggt?cct?????348

Ser?Ser?Gln?Pro?Tyr?Gly?Asp?Asn?Glu?Phe?Lys?Thr?Lys?Ile?Gly?Pro

85??????????????????90??????????????????95

aat?gtt?cat?tta?atg?aaa?gaa?ggg?aat?att?gtt?gtg?tat?caa?gat?gta?????396

Asn?Val?His?Leu?Met?Lys?Glu?Gly?Asn?Ile?Val?Val?Tyr?Gln?Asp?Val

100?????????????????105?????????????????110

cga?ggt?cgt?tgg?atg?agt?gaa?ggt?gtc?tat?gat?aat?atg?cgt?gct?tat?????444

Arg?Gly?Arg?Trp?Met?Ser?Glu?Gly?Val?Tyr?Asp?Asn?Met?Arg?Ala?Tyr

115?????????????????120?????????????????125

atc?cca?aat?aaa?aca?gag?gat?tct?caa?att?gat?gag?gca?tca?gac?act?????492

Ile?Pro?Asn?Lys?Thr?Glu?Asp?Ser?Gln?Ile?Asp?Glu?Ala?Ser?Asp?Thr

130?????????????????135?????????????????140

tat?gac?acg?att?gac?tgg?ctg?gta?aat?aac?gta?gaa?aat?aat?aac?ggg?????540

Tyr?Asp?Thr?Ile?Asp?Trp?Leu?Val?Asn?Asn?Val?Glu?Asn?Asn?Asn?Gly

145?????????????????150?????????????????155?????????????????160

aat?gtt?ggt?act?tgg?gga?att?tca?tat?cct?ggt?ttt?tat?gct?aca?tat?????588

Asn?Val?Gly?Thr?Trp?Gly?Ile?Ser?Tyr?Pro?Gly?Phe?Tyr?Ala?Thr?Tyr

165?????????????????170?????????????????175

tct?act?ata?gac?gca?cac?cca?gct?tta?aaa?gca?gca?tcg?cct?caa?gcg?????636

Ser?Thr?Ile?Asp?Ala?His?Pro?Ala?Leu?Lys?Ala?Ala?Ser?Pro?Gln?Ala

180?????????????????185?????????????????190

tgt?att?gga?gat?ttc?ttt?ttt?gac?gat?ttt?cat?cat?aat?ggt?gct?ttt?????684

Cys?Ile?Gly?Asp?Phe?Phe?Phe?Asp?Asp?Phe?His?His?Asn?Gly?Ala?Phe

195?????????????????200?????????????????205

tta?tta?agt?tat?ttt?aga?gca?gtg?tct?tta?ttt?ggt?acg?aca?aaa?gat?????732

Leu?Leu?Ser?Tyr?Phe?Arg?Ala?Val?Ser?Leu?Phe?Gly?Thr?Thr?Lys?Asp

210?????????????????215?????????????????220

aaa?cct?aca?gat?tct?gct?tgg?tat?aag?ttt?cca?gaa?atg?aaa?aca?caa?????780

Lys?Pro?Thr?Asp?Ser?Ala?Trp?Tyr?Lys?Phe?Pro?Glu?Met?Lys?Thr?Gln

225?????????????????230?????????????????235?????????????????240

gat?caa?tat?caa?ttt?ttt?ctt?gat?gct?gga?cct?tta?agt?aat?ttg?aac?????828

Asp?Gln?Tyr?Gln?Phe?Phe?Leu?Asp?Ala?Gly?Pro?Leu?Ser?Asn?Leu?Asn

245?????????????????250?????????????????255

aag?tat?ttc?caa?tat?gac?aca?cca?gac?gac?aca?tct?gta?tcc?aag?tct?????876

Lys?Tyr?Phe?Gln?Tyr?Asp?Thr?Pro?Asp?Asp?Thr?Ser?Val?Ser?Lys?Ser

260?????????????????265?????????????????270

gat?agg?ata?gat?gat?gtg?ttt?tgg?aaa?gaa?att?gta?gag?cat?cca?aac?????924

Asp?Arg?Ile?Asp?Asp?Val?Phe?Trp?Lys?Glu?Ile?Val?Glu?His?Pro?Asn

275?????????????????280?????????????????285

tac?gat?acg?ata?tgg?aaa?tct?aaa?ggt?tta?att?caa?aac?cta?aaa?gat?????972

Tyr?Asp?Thr?Ile?Trp?Lys?Ser?Lys?Gly?Leu?Ile?Gln?Asn?Leu?Lys?Asp

290?????????????????295?????????????????300

att?aag?cca?agt?gta?gcg?aca?atg?att?gtg?gga?ggg?tta?ttt?gat?gcc????1020

Ile?Lys?Pro?Ser?Val?Ala?Thr?Met?Ile?Val?Gly?Gly?Leu?Phe?Asp?Ala

305?????????????????310?????????????????315?????????????????320

gaa?gat?tta?tat?ggg?cca?ttt?gaa?act?tat?aaa?acg?ata?gaa?aaa?cat????1068

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

325?????????????????330?????????????????335

aat?cct?gat?aat?tat?aat?att?atg?gtt?ttt?ggg?cct?tgg?gat?cat?ggt????1116

Asn?Pro?Asp?Asn?Tyr?Asn?Ile?Met?Val?Phe?Gly?Pro?Trp?Asp?His?Gly

340?????????????????345?????????????????350

cgt?tgg?gct?agg?agt?gac?gtt?aaa?aat?tat?gtt?gga?aat?tat?ttc?ttc????1164

Arg?Trp?Ala?Arg?Ser?Asp?Val?Lys?Asn?Tyr?Val?Gly?Asn?Tyr?Phe?Phe

355?????????????????360?????????????????365

gga?gat?tct?ata?tct?cta?aaa?ttt?caa?cgt?gat?gtt?gaa?acg?aag?ttt????1212

Gly?Asp?Ser?Ile?Ser?Leu?Lys?Phe?Gln?Arg?Asp?Val?Glu?Thr?Lys?Phe

370?????????????????375?????????????????380

ttt?aat?cat?ttt?tta?aaa?gga?aaa?ggc?gac?aag?aac?tca?ggg?tta?cca????1260

Phe?Asn?His?Phe?Leu?Lys?Gly?Lys?Gly?Asp?Lys?Asn?Ser?Gly?Leu?Pro

385?????????????????390?????????????????395?????????????????400

gaa?gca?tat?gta?ttt?gat?tct?ggt?aaa?aag?gaa?tgg?agt?agc?ttt?gac????1308

Glu?Ala?Tyr?Val?Phe?Asp?Ser?Gly?Lys?Lys?Glu?Trp?Ser?Ser?Phe?Asp

405?????????????????410?????????????????415

agc?tgg?cct?cca?aag?caa?gca?gaa?aaa?caa?gcc?atg?tat?ctt?aat?gcc????1356

Ser?Trp?Pro?Pro?Lys?Gln?Ala?Glu?Lys?Gln?Ala?Met?Tyr?Leu?Asn?Ala

420?????????????????425?????????????????430

aac?caa?gag?cta?tca?gat?tca?aaa?aaa?gga?aat?act?agt?gag?aca?ttt????1404

Asn?Gln?Glu?Leu?Ser?Asp?Ser?Lys?Lys?Gly?Asn?Thr?Ser?Glu?Thr?Phe

435?????????????????440?????????????????445

gtt?agt?gat?tta?aaa?cgc?cct?gta?cct?tat?tcc?gaa?gat?att?aaa?aca????1452

Val?Ser?Asp?Leu?Lys?Arg?Pro?Val?Pro?Tyr?Ser?Glu?Asp?Ile?Lys?Thr

450?????????????????455?????????????????460

gtt?ttc?aca?cca?cga?aaa?tac?atg?aca?gac?gat?cag?cgt?ttt?gca?gca????1500

Val?Phe?Thr?Pro?Arg?Lys?Tyr?Met?Thr?Asp?Asp?Gln?Arg?Phe?Ala?Ala

465?????????????????470?????????????????475?????????????????480

cga?cgt?cct?gat?gtt?ctt?ata?ttt?gag?acc?gat?att?ctt?gag?gaa?gat????1548

Arg?Arg?Pro?Asp?Val?Leu?Ile?Phe?Glu?Thr?Asp?Ile?Leu?Glu?Glu?Asp

485?????????????????490?????????????????495

ata?acc?tta?gct?ggt?gat?att?tta?gcg?cag?ctt?aat?gtg?tca?act?aca????1596

Ile?Thr?Leu?Ala?Gly?Asp?Ile?Leu?Ala?Gln?Leu?Asn?Val?Ser?Thr?Thr

500?????????????????505?????????????????510

ggg?aca?gat?gca?gat?tgg?att?gtc?aaa?ata?gta?gat?gtt?cat?cca?gca????1644

Gly?Thr?Asp?Ala?Asp?Trp?Ile?Val?Lys?Ile?Val?Asp?Val?His?Pro?Ala

515?????????????????520?????????????????525

gat?gct?gag?gag?caa?aaa?gaa?ggt?atg?caa?gac?cat?tta?tca?atg?agt????1692

Asp?Ala?Glu?Glu?Gln?Lys?Glu?Gly?Met?Gln?Asp?His?Leu?Ser?Met?Ser

530?????????????????535?????????????????540

aat?tat?cat?ttg?atg?gtg?agg?agt?gaa?gtg?atg?cgc?ggt?cgt?ttt?aga????1740

Asn?Tyr?His?Leu?Met?Val?Arg?Ser?Glu?Val?Met?Arg?Gly?Arg?Phe?Arg

545?????????????????550?????????????????555?????????????????560

aat?agt?ttt?gaa?aac?cca?gag?cca?ttt?gtg?cca?aac?caa?cca?aca?gat????1788

Asn?Ser?Phe?Glu?Asn?Pro?Glu?Pro?Phe?Val?Pro?Asn?Gln?Pro?Thr?Asp

565?????????????????570?????????????????575

gtc?aat?atc?aag?tta?caa?gat?gta?cat?cat?aca?ttt?aaa?aaa?ggt?cac????1836

Val?Asn?Ile?Lys?Leu?Gln?Asp?Val?His?His?Thr?Phe?Lys?Lys?Gly?His

580?????????????????585?????????????????590

aaa?tta?caa?gtg?caa?gtt?cag?agt?acg?tgg?ttt?cca?ctt?att?gat?ttg????1884

Lys?Leu?Gln?Val?Gln?Val?Gln?Ser?Thr?Trp?Phe?Pro?Leu?Ile?Asp?Leu

595?????????????????600?????????????????605

aac?ccg?caa?aca?ttt?gtg?cct?aat?att?tat?aaa?gca?aaa?gaa?agc?gat????1932

Asn?Pro?Gln?Thr?Phe?Val?Pro?Asn?Ile?Tyr?Lys?Ala?Lys?Glu?Ser?Asp

610?????????????????615?????????????????620

ttt?aaa?acc?caa?aca?cat?tcg?gtt?ttt?aac?gat?tct?aaa?att?gag?ttt????1980

Phe?Lys?Thr?Gln?Thr?His?Ser?Val?Phe?Asn?Asp?Ser?Lys?Ile?Glu?Phe

625?????????????????630?????????????????635?????????????????640

acg?gtt?ttg?aaa?taagagtaga?tgactaaatt?tgccaaggta?gatttagtct?tttt???2036

Thr?Val?Leu?Lys

<210>27

<211>644

<212>PRT

<213>Psycloserpens?burtonensis

<400>27

Met?Lys?Thr?Leu?Phe?Lys?Leu?Leu?Leu?Leu?Phe?Val?Phe?Val?Leu?Thr

1???????????????5???????????????????10??????????????????15

Ser?Cys?Asn?Lys?Ala?Asn?Lys?Asp?Ala?Thr?Glu?Ile?Val?Lys?Thr?Glu

20??????????????????25??????????????????30

Val?Glu?Asp?Thr?Tyr?Val?Lys?Asp?Asn?Tyr?Asn?Lys?Gin?Glu?Val?Thr

35??????????????????40??????????????????45

Ile?Glu?Met?Arg?Asp?Gly?Ile?Lys?Leu?His?Thr?Thr?Ile?Tyr?Ser?Pro

50??????????????????55??????????????????60

Lys?Asp?Glu?Ser?Gln?Thr?Tyr?Pro?Ile?Leu?Met?Met?Arg?Thr?Pro?Tyr

65??????????????????70??????????????????75??????????????????80

Ser?Ser?Gln?Pro?Tyr?Gly?Asp?Asn?Glu?Phe?Lys?Thr?Lys?Ile?Gly?Pro

85??????????????????90??????????????????95

Asn?Val?His?Leu?Met?Lys?Glu?Gly?Asn?Ile?Val?Val?Tyr?Gln?Asp?Val

100?????????????????105?????????????????110

Arg?Gly?Arg?Trp?Met?Ser?Glu?Gly?Val?Tyr?Asp?Asn?Met?Arg?Ala?Tyr

115?????????????????120?????????????????125

Ile?Pro?Asn?Lys?Thr?Glu?Asp?Ser?Gln?Ile?Asp?Glu?Ala?Ser?Asp?Thr

130?????????????????135?????????????????140

Tyr?Asp?Thr?Ile?Asp?Trp?Leu?Val?Asn?Asn?Val?Glu?Asn?Asn?Asn?Gly

145?????????????????150?????????????????155?????????????????160

Asn?Val?Gly?Thr?Trp?Gly?Ile?Ser?Tyr?Pro?Gly?Phe?Tyr?Ala?Thr?Tyr

165?????????????????170?????????????????175

Ser?Thr?Ile?Asp?Ala?His?Pro?Ala?Leu?Lys?Ala?Ala?Ser?Pro?Gln?Ala

180?????????????????185?????????????????190

Cys?Ile?Gly?Asp?Phe?Phe?Phe?Asp?Asp?Phe?His?His?Asn?Gly?Ala?Phe

195?????????????????200?????????????????205

Leu?Leu?Ser?Tyr?Phe?Arg?Ala?Val?Ser?Leu?Phe?Gly?Thr?Thr?Lys?Asp

210?????????????????215?????????????????220

Lys?Pro?Thr?Asp?Ser?Ala?Trp?Tyr?Lys?Phe?Pro?Glu?Met?Lys?Thr?Gln

225?????????????????230?????????????????235?????????????????240

Asp?Gln?Tyr?Gln?Phe?Phe?Leu?Asp?Ala?Gly?Pro?Leu?Ser?Asn?Leu?Asn

245?????????????????250?????????????????255

Lys?Tyr?Phe?Gln?Tyr?Asp?Thr?Pro?Asp?Asp?Thr?Ser?Val?Ser?Lys?Ser

260?????????????????265?????????????????270

Asp?Arg?Ile?Asp?Asp?Val?Phe?Trp?Lys?Glu?Ile?Val?Glu?His?Pro?Asn

275?????????????????280?????????????????285

Tyr?Asp?Thr?Ile?Trp?Lys?Ser?Lys?Gly?Leu?Ile?Gln?Asn?Leu?Lys?Asp

290?????????????????295?????????????????300

Ile?Lys?Pro?Ser?Val?Ala?Thr?Met?Ile?Val?Gly?Gly?Leu?Phe?Asp?Ala

305?????????????????310?????????????????315?????????????????320

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

325?????????????????330?????????????????335

Asn?Pro?Asp?Asn?Tyr?Asn?Ile?Met?Val?Phe?Gly?Pro?Trp?Asp?His?Gly

340?????????????????345?????????????????350

Arg?Trp?Ala?Arg?Ser?Asp?Val?Lys?Asn?Tyr?Val?Gly?Asn?Tyr?Phe?Phe

355?????????????????360?????????????????365

Gly?Asp?Ser?Ile?Ser?Leu?Lys?Phe?Gln?Arg?Asp?Val?Glu?Thr?Lys?Phe

370?????????????????375?????????????????380

Phe?Asn?His?Phe?Leu?Lys?Gly?Lys?Gly?Asp?Lys?Asn?Ser?Gly?Leu?Pro

385?????????????????390?????????????????395?????????????????400

Glu?Ala?Tyr?Val?Phe?Asp?Ser?Gly?Lys?Lys?Glu?Trp?Ser?Ser?Phe?Asp

405?????????????????410?????????????????415

Ser?Trp?Pro?Pro?Lys?Gln?Ala?Glu?Lys?Gln?Ala?Met?Tyr?Leu?Asn?Ala

420?????????????????425?????????????????430

Asn?Gln?Glu?Leu?Ser?Asp?Ser?Lys?Lys?Gly?Asn?Thr?Ser?Glu?Thr?Phe

435?????????????????440?????????????????445

Val?Ser?Asp?Leu?Lys?Arg?Pro?Val?Pro?Tyr?Ser?Glu?Asp?Ile?Lys?Thr

450?????????????????455?????????????????460

Val?Phe?Thr?Pro?Arg?Lys?Tyr?Met?Thr?Asp?Asp?Gln?Arg?Phe?Ala?Ala

465?????????????????470?????????????????475?????????????????480

Arg?Arg?Pro?Asp?Val?Leu?Ile?Phe?Glu?Thr?Asp?Ile?Leu?Glu?Glu?Asp

485?????????????????490?????????????????495

Ile?Thr?Leu?Ala?Gly?Asp?Ile?Leu?Ala?Gln?Leu?Asn?Val?Ser?Thr?Thr

500?????????????????505?????????????????510

Gly?Thr?Asp?Ala?Asp?Trp?Ile?Val?Lys?Ile?Val?Asp?Val?His?Pro?Ala

515?????????????????520?????????????????525

Asp?Ala?Glu?Glu?Gln?Lys?Glu?Gly?Met?Gln?Asp?His?Leu?Ser?Met?Ser

530?????????????????535?????????????????540

Asn?Tyr?His?Leu?Met?Val?Arg?Ser?Glu?Val?Met?Arg?Gly?Arg?Phe?Arg

545?????????????????550?????????????????555?????????????????560

Asn?Ser?Phe?Glu?Asn?Pro?Glu?Pro?Phe?Val?Pro?Asn?Gln?Pro?Thr?Asp

565?????????????????570?????????????????575

Val?Asn?Ile?Lys?Leu?Gln?Asp?Val?His?His?Thr?Phe?Lys?Lys?Gly?His

580?????????????????585?????????????????590

Lys?Leu?Gln?Val?Gln?Val?Gln?Ser?Thr?Trp?Phe?Pro?Leu?Ile?Asp?Leu

595?????????????????600?????????????????605

Asn?Pro?Gln?Thr?Phe?Val?Pro?Asn?Ile?Tyr?Lys?Ala?Lys?Glu?Ser?Asp

610?????????????????615?????????????????620

Phe?Lys?Thr?Gln?Thr?His?Ser?Val?Phe?Asn?Asp?Ser?Lys?Ile?Glu?Phe

625?????????????????630?????????????????635?????????????????640

Thr?Val?Leu?Lys

Claims

Coding following (A) or (B) shown in protein DNA:

(A) have the protein of the aminoacid sequence of forming by the 23-616 amino acids residue of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table,

(B) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 23-616 amino acids residue of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (C) or (D) shown in protein DNA:

(C) have the protein of the aminoacid sequence of forming by the 21-619 amino acids residue of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table,

(D) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 21-619 amino acids residue of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (E) or (F) shown in protein DNA:

(E) have the protein of the aminoacid sequence of forming by the 23-625 amino acids residue of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table,

(F) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 23-625 amino acids residue of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (G) or (H) shown in protein DNA:

(G) have the protein of the aminoacid sequence of forming by the 23-645 amino acids residue of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table,

(H) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 23-645 amino acids residue of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (I) or (J) shown in protein DNA:

(I) have the protein of the aminoacid sequence of forming by the 26-620 amino acids residue of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table,

(J) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 26-620 amino acids residue of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (K) or (L) shown in protein DNA:

(K) have the protein of the aminoacid sequence of forming by the 18-644 amino acids residue of aminoacid sequence shown in the SEQ ID NO:27 in the sequence table,

(L) has following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence is included in displacement in the aminoacid sequence of being made up of the 18-644 amino acids residue of aminoacid sequence shown in the SEQ ID NO:27 in the sequence table, disappearance, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (M) or (N) shown in protein DNA:

(M) have the protein of aminoacid sequence shown in the SEQ ID NO:6 in the sequence table,

(N) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence comprises shown in the SEQ ID NO:6 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (O) or (P) shown in protein DNA:

(O) have the protein of aminoacid sequence shown in the SEQ ID NO:12 in the sequence table,

(P) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence comprises shown in the SEQ ID NO:12 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (Q) or (R) shown in protein DNA:

(Q) have the protein of aminoacid sequence shown in the SEQ ID NO:18 in the sequence table,

(R) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence comprises shown in the SEQ ID NO:18 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Coding following (S) or (T) shown in protein DNA:

(S) have the protein of aminoacid sequence shown in the SEQ ID NO:23 in the sequence table,

(T) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence comprises shown in the SEQ ID NO:23 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
11. coding following (U) or (V) shown in protein DNA:

(U) have the protein of aminoacid sequence shown in the SEQ ID NO:25 in the sequence table,

(V) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence comprises shown in the SEQ ID NO:25 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
12. coding following (W) or (X) shown in protein DNA:

(W) have the protein of aminoacid sequence shown in the SEQ ID NO:27 in the sequence table,

(X) contain the mature protein district, have following aminoacid sequence and have peptide and generate active protein, described aminoacid sequence comprises shown in the SEQ ID NO:27 in the sequence table displacement, disappearance in the aminoacid sequence, inserts, adds, and/or the aminoacid sequence of the one or more amino acid gained of inversion.
Following 13. (a) or (b) shown in DNA:

(a) have the DNA of the base sequence of the 127-1908 bit base of base sequence shown in the SEQ ID NO:5 in the sequence table,

(b) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1908 bit base of base sequence shown in the SEQ IDNO:5 in the sequence table.
Following 14. (c) or (d) shown in DNA:

(c) have the DNA of the base sequence of the 121-1917 bit base of base sequence shown in the SEQ ID NO:11 in the sequence table,

(d) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 121-1917 bit base of base sequence shown in the SEQ IDNO:11 in the sequence table.
Following 15. (e) or (f) shown in DNA:

(e) have the DNA of the base sequence of the 127-1935 bit base of base sequence shown in the SEQ ID NO:17 in the sequence table,

(f) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1935 bit base of base sequence shown in the SEQ IDNO:17 in the sequence table.
Following 16. (g) or (h) shown in DNA:

(g) have the DNA of the base sequence of the 127-1995 bit base of base sequence shown in the SEQ ID NO:22 in the sequence table,

(h) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1995 bit base of base sequence shown in the SEQ IDNO:22 in the sequence table.
Following 17. (i) or (j) shown in DNA:

(i) have the DNA of the base sequence of the 104-1888 bit base of base sequence shown in the SEQ ID NO:24 in the sequence table,

(j) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 104-1888 bit base of base sequence shown in the SEQ IDNO:24 in the sequence table.
Following 18. (k) or (l) shown in DNA:

(k) have the DNA of the base sequence of the 112-1992 bit base of base sequence shown in the SEQ ID NO:26 in the sequence table,

(l) under stringent condition, have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 112-1992 bit base of base sequence shown in the SEQ IDNO:26 in the sequence table.
Following 19. (m) or (n) shown in DNA:

(m) have the DNA of the base sequence of the 61-1908 bit base of base sequence shown in the SEQ ID NO:5 in the sequence table,

(n) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1908 bit base of base sequence shown in the SEQ ID NO:5 in the sequence table.
Following 20. (o) or (p) shown in DNA:

(o) have the DNA of the base sequence of the 61-1917 bit base of base sequence shown in the SEQ ID NO:11 in the sequence table,

(p) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1917 bit base of base sequence shown in the SEQ ID NO:11 in the sequence table.
Following 21. (q) or (r) shown in DNA:

(q) have the DNA of the base sequence of the 61-1935 bit base of base sequence shown in the SEQ ID NO:17 in the sequence table,

(r) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1935 bit base of base sequence shown in the SEQ ID NO:17 in the sequence table.
Following 22. (s) or (t) shown in DNA:

(s) have the DNA of the 127-1995 bit base sequence of base sequence shown in the SEQ ID NO:22 in the sequence table,

(t) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 127-1995 bit base of base sequence shown in the SEQ ID NO:22 in the sequence table.
Following 23. (u) or (DNA v):

(u) have the DNA of the 29-1888 bit base sequence of base sequence shown in the SEQ ID NO:24 in the sequence table,

(v) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 29-1888 bit base of base sequence shown in the SEQ ID NO:24 in the sequence table.
Following 24. (w) or (x) shown in DNA:

(w) have the DNA of the 61-1992 bit base sequence of base sequence shown in the SEQ ID NO:26 in the sequence table,

(x) under stringent condition, comprise the mature protein district and have peptide with DNA hybridization with following base sequence and coding and generate active protein DNA, described base sequence and the base sequence complementation of forming by the 61-1992 bit base of base sequence shown in the SEQ ID NO:26 in the sequence table.
25. according to DNA any among the claim 13-24, wherein stringent condition is under the salt concn that is equivalent to 1 * SSC and 0.1% SDS, in 60 ℃ of conditions of washing.
26. recombinant DNA that comprises according to DNA any among the claim 1-25.
27. transformant that has imported according to the recombinant DNA of claim 26.
28. a method of producing peptide-forming enzyme comprises: in substratum, cultivate transformant, peptide-forming enzyme is accumulated in substratum and/or transformant according to claim 27.
29. a method of producing dipeptides comprises: cultivation, mixes this culture with synthetic dipeptides to obtain culture according to the transformant of claim 28 with carboxyl composition and amine component in substratum.
30. method of producing dipeptides, comprise: belong to Sphingobacterium and have the culture that generates the active microorganism of dipeptides by carboxyl composition and amine component by use, isolating microorganism cells from this culture, the microorganism cells of this microorganism is handled product or is come from the peptide-forming enzyme of this microorganism, generates dipeptides by carboxyl composition and amine component.