CN1742089A

CN1742089A - Method for producing alpha-L-aspartyl-L-phenylalanine-beta-ester and method for producing alpha-L-aspartyl-L-phenylalanine-alpha-methyl ester

Info

Publication number: CN1742089A
Application number: CNA2004800025198A
Authority: CN
Inventors: 横关健三; 大野绫子; 原诚一; 阿部巧
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 2003-01-24
Filing date: 2004-01-23
Publication date: 2006-03-01
Anticipated expiration: 2024-01-23
Also published as: CN100362109C

Abstract

A method for producing an alpha-L-aspartyl-L-phenylalanine-beta-ester (also named alpha-L-(beta-o-substituted aspartyl)-L-phenylalanine), which is an intermediate of an alpha-L-aspartyl-L-phenylalanine-alpha-methyl ester (also named alpha-L-aspartyl-L-phenylalanine methyl ester; product name: aspartame), easily, at high yield and inexpensively without going through a complex synthesis method is provided. Further, an easy, inexpensive and high-yield production method for an alpha-L-aspartyl-L-phenylalanine-alpha-methyl ester is provided. alpha-L-aspartyl-L-phenylalanine-alpha-methyl ester is produced from a L-aspartic acid-alpha,beta-diester and L-phenylalanine using an enzyme or enzyme-containing substance that has an ability to catalyze a reaction in which L-phenylalanine performs no nucleophilic attack on a beta-ester site of L-aspartic acid-alpha,beta-diester but performs a nucleophilic attack on an alpha-ester site thereof.

Description

The production method of the production method of 2-L-aspartyl-L-phenylalanine-β-ester and 2-L-aspartyl-L-phenylalanine-α-methyl esters

Technical field

The present invention relates to 2-L-aspartyl-L-phenylalanine-β-ester and (be also referred to as the production method of α-L-(β-o-replaces aspartoyl)-L-phenylalanine (abbreviation: α-ARP)), and 2-L-aspartyl-L-phenylalanine-α-methyl esters (is also referred to as the production method of 2-L-aspartyl-L-phenylalanine methyl esters (abbreviation: α-APM)).More particularly, the present invention relates to the production method of 2-L-aspartyl-L-phenylalanine-β-ester, and utilizing this production method to produce the method for 2-L-aspartyl-L-phenylalanine-α-methyl esters, described 2-L-aspartyl-L-phenylalanine-β-ester is the huge sweeting agent 2-L-aspartyl-L-phenylalanine-α-methyl esters of a production demand (ProductName: important intermediate aspartame).

Background technology

Common known 2-L-aspartyl-L-phenylalanine-α-methyl esters (following be abbreviated as sometimes " α-APM ") production method comprises chemical synthesis and enzymic synthesis method.The known chemical synthesis method has condensation N-protected L-aspartic anhydride and L-phenylalanine methyl ester to synthesize N-protected APM, sloughs the N-protected group again and obtains APM; The known enzyme synthesis method has condensation N-protected L-aspartic acid and L-phenylalanine methyl ester to synthesize N-protected APM, sloughs the N-protected group again and obtains APM.But these two kinds of methods all must add, the step of deprotection base, and technology bothers very much.On the other hand, the someone has studied the APM production method (referring to the open communique H02-015196 of Japanese Patent) of not using the N-protected base.But this method is owing to the very low suitability for industrialized production that is unsuitable for of output of product.Therefore, in this case, be badly in need of the method for developing low-cost, suitability for industrialized production aspartame.

Disclosure of the Invention

An object of the present invention is to provide easy, cheap, high yield and do not experience the method for the production 2-L-aspartyl-L-phenylalanine-β-ester of complicated synthetic route, 2-L-aspartyl-L-phenylalanine-β-ester is the intermediate of 2-L-aspartyl-L-phenylalanine-α-methyl esters.Another object of the present invention provides, and easy, cheap, the high place of production produces the method for 2-L-aspartyl-L-phenylalanine-α-methyl esters.

Consider above purpose, the present inventor furthers investigate, found that: a kind of newfound enzyme or contain enzyme material can be by L-aspartic acid-α, β-diester and L-phenylalanine are optionally produced 2-L-aspartyl-L-phenylalanine-β-ester, thereby have realized the present invention.

That is, the present invention is as described below.

[1] method of production 2-L-aspartyl-L-phenylalanine-β-ester (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine), this method comprises that use can be connected to L-aspartic acid-α with L-phenylalanine selectivity by peptide bond, the enzyme in the α of β-diester-ester site or contain enzyme material, by L-aspartic acid-α, β-diester and L-phenylalanine generate 2-L-aspartyl-L-phenylalanine-β-ester.

[2] produce the method for 2-L-aspartyl-L-phenylalanine-β-ester (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [1], wherein said enzyme or contain enzyme material and be selected from following one or both or more kinds of: can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the microorganisms cultures in the α of β-diester-ester site, separate from the microorganism cells of described culture and the microorganism cells of described microorganism and handle product.

[3] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [2], wherein said microorganism belongs to and is selected from following genus: Aeromonas (Aeromonas); Azotobacter (Azotobacter); Alcaligenes (Alcaligenes); brevibacterium sp (Brevibacterium); Corynebacterium (Corynebacterium); Escherichia (Escherichia); steady Bacillaceae (Empedobacter); Flavobacterium (Flavobacterium); Microbacterium (Microbacterium); propiono-bacterium (Propionibacterium); bacillus brevis belongs to (Brevibacillus); series bacillus belongs to (Paenibacillus); Rhodopseudomonas (Pseudomonas); serratia (Serratia); oligotrophy zygosaccharomyces (Stenotrophomonas); Sphingobacterium (Sphingobacterium); streptomyces (Streptomyces); xanthomonas (Xanthomonas); intend Weir yeast belong (Williopsis); mycocandida (Candida); Geotrichum (Geotrichum); Pichia (Pichia); yeast belong (Saccharomyces); spore torulopsis (Torulaspora) is arranged; Cytophaga (Cellulophaga); Weeks Bordetella (Weeksella); soil Bacillaceae (Pedobacter); peachiness Bacillaceae (Persicobacter); Flexithrix (Flexithrix); bite chitin Pseudomonas (Chitinophaga); round bar Pseudomonas (Cyclobacterium); ancient character shape Pseudomonas (Runella); hot line Pseudomonas (Thermonema) dwells; cold snake Pseudomonas (Psychroserpens); ice-cold Bacillaceae (Gelidibacter); Dyadobacter; heat color Bacillaceae (Flammeovirga); Spirosoma (Spirosoma); knee Pseudomonas (Flectobacillus); Flexibacter (Tenacibaculum); red thermophilic salt Pseudomonas (Rhodotermus); Zobellia; Muricauda; Salegentibacter; shaping Bacillaceae (Taxeobacter); Cytophage (Cytophaga); the sliding Pseudomonas (Marinilabilia) in sea; rely Wen Pseudomonas (Lewinella); Saprospira (Saprospira) and constraint Bacillaceae (Haliscomenobacter).

[4] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [2], wherein said microorganism is to express (A) or (B) proteinic conversion microorganism:

(A) have the protein of aminoacid sequence of the amino-acid residue ordinal number 23-616 of the described aminoacid sequence of SEQ ID NO:6 in the sequence table,

(B) has the aminoacid sequence of the amino-acid residue ordinal number 23-616 of the described aminoacid sequence of SEQ ID NO:6 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[5] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [2], wherein said microorganism is to express (C) or (D) proteinic conversion microorganism:

(C) have the protein of aminoacid sequence of the amino-acid residue ordinal number 21-619 of the described aminoacid sequence of SEQ ID NO:12 in the sequence table,

(D) has the aminoacid sequence of the amino-acid residue ordinal number 21-619 of the described aminoacid sequence of SEQ ID NO:12 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[6] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [2], wherein said microorganism is to express following (E) or (F) proteinic conversion microorganism:

(E) have the protein of aminoacid sequence of the amino-acid residue ordinal number 23-625 of the described aminoacid sequence of SEQ ID NO:18 in the sequence table,

(F) has the aminoacid sequence of the amino-acid residue ordinal number 23-625 of the described aminoacid sequence of SEQ ID NO:18 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[7] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [2], wherein said microorganism is to express following (G) or (H) proteinic conversion microorganism:

(G) have the protein of aminoacid sequence of the amino-acid residue ordinal number 23-645 of the described aminoacid sequence of SEQ ID NO:23 in the sequence table,

(H) has the aminoacid sequence of the amino-acid residue ordinal number 23-645 of the described aminoacid sequence of SEQ ID NO:23 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[8] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [2], wherein said microorganism is to express following (I) or (J) proteinic conversion microorganism:

(I) have the protein of aminoacid sequence of the amino-acid residue ordinal number 26-620 of the described aminoacid sequence of SEQ ID NO:25 in the sequence table,

(J) has the aminoacid sequence of the amino-acid residue ordinal number 26-620 of the described aminoacid sequence of SEQ ID NO:25 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[9] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [2], wherein said microorganism is to express following (K) or (L) proteinic conversion microorganism:

(K) have the protein of aminoacid sequence of the amino-acid residue ordinal number 18-644 of the described aminoacid sequence of SEQ ID NO:27 in the sequence table,

(L) has the aminoacid sequence of the amino-acid residue ordinal number 18-644 of the described aminoacid sequence of SEQ ID NO:27 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[10] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [2], wherein said microorganism is to express following (M) or (N) proteinic conversion microorganism:

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

(N) has the described aminoacid sequence of SEQ ID NO:6 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site contains the protein in maturation protein district.

[11] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [2], wherein said microorganism is to express following (O) or (P) proteinic conversion microorganism:

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

(P) has the described aminoacid sequence of SEQ ID NO:12 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site contains the protein in maturation protein district.

[12] produce the method for 2-L-aspartyl-L-phenylalanine-β-ester (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to claim [2], wherein said microorganism is to express following (Q) or (R) proteinic conversion microorganism:

(Q) have the described aminoacid sequence of SEQ ID NO:18 in the sequence table, contain the protein in maturation protein district,

(R) has the described aminoacid sequence of SEQ ID NO:18 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond and L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site.

[13] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [3], wherein said microorganism is to express following (S) or (T) proteinic conversion microorganism:

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

(T) has the described aminoacid sequence of SEQ ID NO:23 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site contains the protein in maturation protein district.

[14] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to [2], wherein said microorganism is to express following (U) or (V) proteinic conversion microorganism:

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

(V) has the described aminoacid sequence of SEQ ID NO:25 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site contains the protein in maturation protein district.

[15] produce the method for 2-L-aspartyl-L-phenylalanine-β-esters (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [2], wherein said microorganism is to express following (W) or (X) proteinic conversion microorganism:

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

(X) has the described aminoacid sequence of SEQ ID NO:27 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site contains the protein in maturation protein district.

[16] produce the method for 2-L-aspartyl-L-phenylalanine-β-ester (being α-L-(β-o-replaces aspartoyl)-L-phenylalanine) according to above [1], wherein said enzyme is selected from least a in following (A)-(X):

(B) has the aminoacid sequence of the amino-acid residue ordinal number 23-616 of the described aminoacid sequence of SEQ ID NO:6 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site

(D) has the aminoacid sequence of the amino-acid residue ordinal number 21-619 of the described aminoacid sequence of SEQ ID NO:12 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site

(F) has the aminoacid sequence of the amino-acid residue ordinal number 23-625 of the described aminoacid sequence of SEQ ID NO:18 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site

(H) has the aminoacid sequence of the amino-acid residue ordinal number 23-645 of the described aminoacid sequence of SEQ ID NO:23 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site

(J) has the aminoacid sequence of the amino-acid residue ordinal number 26-620 of the described aminoacid sequence of SEQ ID NO:25 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site

(L) has the aminoacid sequence of the amino-acid residue ordinal number 18-644 of the described aminoacid sequence of SEQ ID NO:27 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the active protein in the α of β-diester-ester site

(N) has the described aminoacid sequence of SEQ ID NO:6 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site, the protein that contains the maturation protein district

(P) has the described aminoacid sequence of SEQ ID NO:12 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site, the protein that contains the maturation protein district

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

(R) has the described aminoacid sequence of SEQ ID NO:18 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site, the protein that contains the maturation protein district

(T) has the described aminoacid sequence of SEQ ID NO:23 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site, the protein that contains the maturation protein district

(V) has the described aminoacid sequence of SEQ ID NO:25 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site, the protein that contains the maturation protein district

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

(X) have the described aminoacid sequence of SEQ ID NO:27 in the sequence table, and have by peptide bond L-phenylalanine selectivity is connected to L-aspartic acid-α, the activity in the α of β-diester-ester site contains the protein in maturation protein district.

[17] method of production 2-L-aspartyl-L-phenylalanine-α-methyl esters (being the 2-L-aspartyl-L-phenylalanine methyl esters), this method comprises: utilize the production method of the 2-L-aspartyl-L-phenylalanine-β-ester of any one among the claim 1-16, synthetic 2-L-aspartyl-L-phenylalanine-β-methyl esters (is also referred to as the reactions steps of α-L-(β-o-methyl aspartoyl)-L-phenylalanine (abbreviation: α-AMP)); And the reactions steps that 2-L-aspartyl-L-phenylalanine-β-methyl esters (being α-L-(β-o-methyl aspartoyl)-L-phenylalanine) is changed into 2-L-aspartyl-L-phenylalanine-α-methyl esters.

Can easily produce 2-L-aspartyl-L-phenylalanine-β-ester by the present invention.By method of the present invention can be easy, high the place of production produce 2-L-aspartyl-L-phenylalanine-β-ester, reduced and added/use of the complicated synthetic method of deprotection base and so on.

And, by the present invention can be easy, high yield and produce 2-L-aspartyl-L-phenylalanine-α-methyl esters at an easy rate.

When studying carefully together in conjunction with the accompanying drawings, other purpose of the present invention, feature and advantage describe in detail in following the present invention and are specifically stated, or become apparent according to following the present invention's detailed description.

The accompanying drawing summary

Fig. 1 illustrates the enzyme amount that is present in cytoplasm fraction (Cy) and the pericentral siphon component (Pe).

Preferred forms of the present invention

Hereinafter the present invention will be described in the following order:

＜1〉production method of 2-L-aspartyl-L-phenylalanine-β-ester

1. the production method of 2-L-aspartyl-L-phenylalanine-β-ester

2. the present invention microorganism of using

3. the enzyme that uses of the present invention;

＜2〉production method of 2-L-aspartyl-L-phenylalanine-α-methyl esters.

＜1〉production method of 2-L-aspartyl-L-phenylalanine-β-ester

1. the production method of 2-L-aspartyl-L-phenylalanine-β-ester

In 2-L-aspartyl-L-phenylalanine-β of the present invention-ester production method (hereinafter being also referred to as " peptide production method of the present invention "), under situation with the active enzyme existence of described one-tenth peptide, make L-phenylalanine and L-aspartic acid-α, β-diester reaction.Promptly, in peptide production method of the present invention, use can be connected to L-aspartic acid-α with L-phenylalanine selectivity by peptide bond, the enzyme in the α of β-diester-ester site or contain enzyme material, by L-aspartic acid-α, β-diester and L-phenylalanine generate 2-L-aspartyl-L-phenylalanine-β-ester.L-phenylalanine selectivity can be connected to L-aspartic acid-α, the enzyme in the α of β-diester-ester site or contain enzyme material and be meant to have catalytic reaction ability or active enzyme or contain enzyme material, the L-phenylalanine can be substantially to L-aspartic acid-α in this reaction, nucleophillic attack is carried out in the β of β-diester-ester site, and only nucleophillic attack is carried out in its α-ester site.But, shown in reference example hereinafter, can also obtain to have the catalytic reaction ability opposite with aforementioned capabilities, by L-aspartic acid-α, β-diester and L-phenylalanine generate β-L-aspartyl-L-phenylalanine-α-ester and (are also referred to as the enzyme of β-L-(α-o-replaces aspartoyl)-L-phenylalanine (abridge: β-ARP)) or contain enzyme material, the L-phenylalanine can be substantially to L-aspartic acid-α in this reaction, nucleophillic attack is carried out in the α of β-diester-ester site, and only nucleophillic attack is carried out in its β-ester site.

Following formula (I-α) (wherein, " Me " represents methyl) by lifting with L-aspartic acid-α, senecioate is as L-aspartic acid-α, the example of β-diester, listed reaction formula, wherein the L-phenylalanine is to L-aspartic acid-α, and nucleophillic attack is carried out in the α of β-diester-ester site, and generation 2-L-aspartyl-L-phenylalanine-β-ester (α-ARP).Shown in (I-α), in peptide production method of the present invention, the amino of L-phenylalanine and L-aspartic acid-α, the α of senecioate-methyl esters site reaction forms peptide bond.On the other hand, following formula (I-β) has been listed reaction, L-aspartic acid-α wherein, the β of senecioate-methyl esters site experience nucleophillic attack generates β-L-aspartyl-L-phenylalanine-α-methyl esters and (is also referred to as β-L-(α-o-methyl aspartoyl)-L-phenylalanine (abbreviation: β-AMP)).At L-aspartic acid-α, the β of senecioate-methyl esters site forms the peptide bond among β-AMP.The described enzyme that the present invention uses or contain the enzyme material reaction of an accelerating type (I-α) does not basically cause the reaction of formula (I-β) basically.Can produce α-APM by α-AMP by simple reactions steps (formula (II)), but can not be by β-AMP direct production α-APM.In other words, the inventive method is very effective as the production method of α-APM intermediate, can be used for suitability for industrialized production.

Reaction formula I-α

Reaction formula I-β

Reaction formula II

Can be by mixing described enzyme or containing enzyme material and L-aspartic acid-α, β-diester and L-phenylalanine make described enzyme or contain enzyme material to act on L-aspartic acid-α, β-diester and L-phenylalanine.More particularly, can use such method: with described enzyme or contain enzyme material and join realization response in the solution that contains L-aspartic acid-diester and L-phenylalanine.When using the microorganism conduct of producing described enzyme to contain enzyme material, perhaps can react as mentioned above, perhaps can use following or similar method, described method comprises cultivates the microorganism that produces described enzyme, with in microorganism or cultivate and produce in the nutrient solution of described microorganism and accumulate described enzyme, and in nutrient solution, add L-aspartic acid-α, β-diester and L-phenylalanine.Reclaim α-L-aspartic acid-L-phenylalanine-beta-ester of so producing according to ordinary method, can carry out purifying if necessary.

Described " containing enzyme material " can be any material, as long as it contains described enzyme, its specific form comprises the microorganisms cultures that produces described enzyme, the separation microorganism cells product from the microorganism cells of described culture and the described microorganism of handling.Microorganisms cultures is meant the material that obtains by culturing micro-organisms, specifically is meant microorganism cells, is used to cultivate the substratum of described microorganism and the mixture of the material that produced by institute's culturing micro-organisms etc.In addition, described microorganism cells capable of washing is as the microorganism cells that cleans.And the microorganism cells product of handling comprises microorganism cells carried out the product that fragmentation, cracking or freeze-drying gained get, and comprises in addition by handling enzyme crude extract that microorganism cells reclaims and the purifying enzyme that is further purified acquisition.As for the enzyme that purification process is crossed, can use partially purified enzyme by acquisitions such as various purification process.In addition, can use immobilized immobilized enzyme such as, absorption method legal, entrapping method by covalent linkage.And, for some microorganism that will use, can in culturing process, carry out cracking to a part of microorganism cells, in the case, also the supernatant liquor of nutrient solution can be used as the described enzyme material that contains.

In addition, for the microorganism that contains described enzyme, can use wild type strain or express the gene recombination bacterial strain of described enzyme.Such microorganism is not limited to contain the enzyme microorganism cells, can also use the microorganism cells product of handling, for example through the microorganism cells and the freeze-dried microorganisms cell of acetone treatment.In addition, can use immobilizations such as, absorption method legal, entrapping method to handle the fixation of microbial cell that the microorganism cells product obtained or the microorganism cells product of immobilized processing by covalent linkage.

Preferred use can be produced has the wild type strain that 2-L-aspartyl-L-phenylalanine-β-ester generates active one-tenth peptase, because can more easily generate peptide, and need not experience the step for preparing the gene recombination bacterial strain.On the other hand, have the gene recombination bacterial strain that 2-L-aspartyl-L-phenylalanine-β-ester generates active one-tenth peptase, can modify, so that greater amount is produced described one-tenth peptase for transforming to be used for expressing.Therefore, synthetic 2-L-aspartyl-L-phenylalanine-β-ester is possible with bigger output and higher rate.In substratum, cultivate wild-type or gene recombination bacterial strain microorganism, in substratum and/or microorganism, to be accumulated as peptase, and should accumulate product and L-aspartic acid-α, β-diester and L-phenylalanine mix, and can generate 2-L-aspartyl-L-phenylalanine-β-ester.

It is to be noted, during the microorganism cells product of the processing that is obtained at the microorganism cells that uses cultured products, cultured microorganism cell, cleaning with by fragmentation or cracking microorganism cells, in fact there is the enzyme that decomposes formed 2-L-aspartyl-L-phenylalanine-β-ester rather than participate in forming 2-L-aspartyl-L-phenylalanine-β-ester usually.In the case, preferably add inhibitors of metalloproteinase constantly, for example ethylenediamine tetraacetic acid (EDTA) (EDTA) at some.Add-on between 0.1mM-300mM, preferred 1mM-100mM.

If show target effect, enzyme or contain the consumption (significant quantity) of enzyme material may be just much of that.Although persons skilled in the art can be determined this significant quantity easily by simple preliminary experiment, but this consumption for example is about 0.01-100 unit (" U ") under the situation of using enzyme, be about 0.1-500g/L under the situation of using the microorganism cells that cleans.The definition that is noted that 1U is: can be in 1 minute in 25 ℃ by 100mM L-aspartic acid-α, senecioate and 200mM L-phenylalanine are produced the enzyme amount of 1 μ mol L-α-aspartyl-L-phenylalanine-β-methyl esters.

L-aspartic acid-the α that uses in the reaction, β-diester generate any of 2-L-aspartyl-L-phenylalanine-β-ester with the condensation of L-phenylalanine.L-aspartic acid-α, the example of β-diester comprises L-aspartic acid-α, senecioate and L-aspartic acid-α, β-diethyl ester.When making L-aspartic acid-α, when senecioate and the reaction of L-phenylalanine, generation 2-L-aspartyl-L-phenylalanine-β-methyl esters (α-AMP), when making L-aspartic acid-α, during the reaction of β-diethyl ester and L-phenylalanine, generate 2-L-aspartyl-L-phenylalanine-β-ethyl ester and (be also referred to as α-L-(β-o-ethyl aspartoyl)-L-phenylalanine (abbreviation: α-AEP)).

Although L-aspartic acid-α as raw material, the concentration of β-diester and L-phenylalanine is respectively 1mM-10mM, be preferably 0.05M-2M, but can be in some cases preferably with any substrate adding with respect to mole such as another kind of substrate or above amount, and select as required.In addition, under the situation of high concentration substrate inhibited reaction, it can be adjusted to the concentration of inhibited reaction not and in reaction process stream add.

Allowing the temperature of reaction of production 2-L-aspartyl-L-phenylalanine-β-ester is 0-60 ℃, is preferably 5-40 ℃.In addition, allowing the reaction pH of production 2-L-aspartyl-L-phenylalanine-β-ester is 6.5-10.5, is preferably 7.0-10.0.

2. the present invention microorganism of using

With regard to being used for microorganism of the present invention, can use those can be by L-aspartic acid-α, β-diester and L-phenylalanine are produced the microorganism of 2-L-aspartyl-L-phenylalanine-β-ester, are not specifically limited.Can be by L-aspartic acid-α, the microorganism that β-diester and L-phenylalanine are produced 2-L-aspartyl-L-phenylalanine-β-ester comprises the microorganism that for example belongs to the subordinate: Aeromonas (Aeromonas); Azotobacter (Azotobacter); Alcaligenes (Alcaligenes); brevibacterium sp (Brevibacterium); Corynebacterium (Corynebacterium); Escherichia (Escherichia); steady Bacillaceae (Empedobacter); Flavobacterium (Flavobacterium); Microbacterium (Microbacterium); propiono-bacterium (Propionibacterium); bacillus brevis belongs to (Brevibacillus); series bacillus belongs to (Paenibacillus); Rhodopseudomonas (Pseudomonas); serratia (Serratia); oligotrophy zygosaccharomyces (Stenotrophomonas); Sphingobacterium (Sphingobacterium); streptomyces (Streptomyces); xanthomonas (Xanthomonas); intend Weir yeast belong (Williopsis); mycocandida (Candida); Geotrichum (Geotrichum); Pichia (Pichia); yeast belong (Saccharomyces); spore torulopsis (Torulaspora) is arranged; Cytophaga (Cellulophaga); Weeks Bordetella (Weeksella); soil Bacillaceae (Pedobacter); peachiness Bacillaceae (Persicobacter); Flexithrix (Flexithrix); bite chitin Pseudomonas (Chitinophaga); round bar Pseudomonas (Cyclobacterium); ancient character shape Pseudomonas (Runella); hot line Pseudomonas (Thermonema) dwells; cold snake Pseudomonas (Psychroserpens); ice-cold Bacillaceae (Gelidibacter); Dyadobacter; heat color Bacillaceae (Flammeovirga); Spirosoma (Spirosoma); knee Pseudomonas (Flectobacillus); Flexibacter (Tenacibaculum); red thermophilic salt Pseudomonas (Rhodotermus); Zobellia; Muricauda; Salegentibacter; shaping Bacillaceae (Taxeobacter); Cytophage (Cytophaga); the sliding Pseudomonas (Marinilabilia) in sea; rely Wen Pseudomonas (Lewinella); Saprospira (Saprospira) and constraint Bacillaceae (Haliscomenobacter).Specifically, below can be used as example.

Aeromonas hydrophila (Aeromonas hydrophila) ATCC 13136

Wei Nielande vinelandii (Azotobacter vinelandii) IFO 3741

Alcaligenes faecalis (Alcaligenes faecalis) FERMP-8460

Little bar tyrothricin (Brevibacterium minutiferuna) FERM BP-8277

The excellent bacillus of little Huang (Corynebacterium flavescens) ATCC 10340

Intestinal bacteria (Escherichia coli) FERM BP-8276

Short steady bacillus (Empedobacter brevis) ATCC 14234

Flavobacterium resinovorum (Flavobacterium resinovorum) ATCC 14231

Tree-shaped microbacterium (Microbacterium arborescens) ATCC 4348

Xie Shi propionibacterium (Propionibacterium shermanii) FERM BP-8100

Tyrothricin (Brevibacillus parabrevis) ATCC 8185

Honeycomb series bacillus (Paenibacillus alvei) IFO 14175

Pseudomonas fragi (Pseudomonas fragi) IFO 3458

Ge Shi Serratia (Serratia grimesii) ATCC 14460

Germ oligotrophy unit cell (Stenotrophomonas maltophilia) ATCC 13270

Sphingobacterium kind (Sphingobacterium sp.) FERM BP-8124

Light gray streptomycete (Streptomyces griseolus) NRRL B-1305

Lilac grey streptomycete (Streptomyces lavendulae)

Xanthomonas maltophilia (Xanthomonas maltophilia) FERM BP-5568

Intend Weir yeast (Williopsis saturnus) IFO 0895

Candida magnoliae (Candida magnoliae) IFO 0705

Mould fragrantly (Geotrichum fragrance) CBS 152.25

Geotrichum amycelium IFO 0905

The west is pichia spp (Pichia ciferrii) IFO 0905 not

Saccharomyces unisporus (Saccharomyces unisporus) IFO 0724

There is spore torula (Torulaspora delbrueckii) IFO 0422 in Dell

Phagocyte bacterium (Cellulophaga lytica) NBRC 14961

Poisonous Weeks Salmonella (Weeksella virosa) NBRC 16016

Separate Vitrum AB soil bacillus (Pedobacter heparinus) NBRC 12017

Wandering peachiness bacillus (Persicobacter diffluens) NBRC 15940

Gentle bacterium (Flexithrix dorotheae) NBRC 15987 that sends out of many trailing plants silk

Pine bites chitin bacterium (Chitinophaga pinensis) NBRC 15968

Sea round bacteria (Cyclobacterium marinum) ATCC 25205

Soft ancient character shape bacterium (Runella slithyformis) ATCC 29530 wafts

Death hot line bacterium (Thermonema lapsum) ATCC 43542 of dwelling

The cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359

Cool ice-cold bacillus (Gelidibacter algens) ATCC 700364

Dyadobacter fermentans ATCC 700827

Happiness flare up look bacillus (Flammeovirga aprica) NBRC 15941

Tongue spiral bacterium (Spirosoma linguale) DSMZ 74

Big knee bacterium (Flectobacillus major) DSMZ 103

Bacillus (Tenacibaculum maritimum) ATCC 43398 is subdued in the ocean

The red thermophilic salt bacterium in ocean (Rhodotermus marinus) DSMZ 4252

Zobellia galactanivorans DSMZ 12802

Muricauda ruestringensis DSMZ 13258

Salegentibacter salegens DSMZ 5424

Shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116

Cytophaga hutchinsonii (Cytophaga hutchinsonii) NBRC 15051

Sliding bacterium (Marinilabilia salmonicolor) NBRC 15948 in salmon colour sea

Connect and rely Wen bacterium (Lewinella cohaerens) ATCC 23123

Big corrupt spirobacteria (Saprospira grandis) ATCC 23119

Water constraint bacillus (Haliscomenobacter hydrossis) ATCC 27775

In above-mentioned microorganism strains, specially permit biological sustenance center (Chuo Dai-6 with the microbial preservation that the FERM numbering is described in Independent Administrative Leged Industrial Technology Complex Inst, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), can provide according to numbering separately.

In above-mentioned microorganism strains, the microbial preservation of describing with ATCC numbering is in American type culture collection (P.O.Box 1549, Manassas, VA 20110, theUnited States of America), can provide according to numbering separately.

In above-mentioned microorganism strains, (Osaka-shi Japan), can provide according to numbering separately for 2-17-85 Jusanbon-cho, Yodogawa-ku in Osaka fermentation research institute in the microbial preservation of describing with the IFO numbering.

In above-mentioned microorganism strains, number the microbial preservation of description at the Japanese technological assessment NITE of institute Biological resources center (5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken with NBRC, Japan), can provide according to numbering separately.

In above-mentioned microorganism strains, (Mascheroder Weg 1b, 38124Braunschweig Germany), can provide according to numbering separately at German microorganism and cell culture preservation center in the microbial preservation of describing with DSMZ numbering.

The same with above-mentioned bacterial strain, specially permit biological sustenance center (Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi with the microbial preservation that the FERM numbering is described in Independent Administrative Leged Industrial Technology Complex Inst, Ibaraki-ken, 305-8566 Japan).Alcaligenes faecalis (Alcaligenes faecalis) FERM P-8460 was in preservation on September 30 in 1985, and the preserving number of distribution is FERM P-8460.Xie Shi propionibacterium (Propionibacterium shermanii) FERM P-9737 is at first in preservation on December 4 in 1987, the contrast of this microorganism was transferred to international preservation mechanism on July 1st, 2002 according to being specified in of budapest treaty subsequently, and the preserving number of distribution is FERM BP-8100.Xanthomonas maltophilia (Xanthomonasmaltophilia) FERM BP-5568 is at first in nineteen ninety-five preservation on June 14, and the contrast of this microorganism was transferred to international preservation mechanism on June 14th, 1996 according to being specified in of budapest treaty subsequently.Little bar tyrothricin (Brevibacterium minutiferuna) FERM BP-8277 is according to the international preservation on January 20 in 2002 that is specified in of budapest treaty.Intestinal bacteria (Escherichia coli) FERM BP-8276 was deposited in international preservation mechanism on January 20th, 2002 according to being specified in of budapest treaty.

Short steady bacillus (Empedobacter brevis) strains A TCC 14234 (bacterial strain FERM P-18545, bacterial strain FERM BP-8113) is deposited 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, Japan), the preserving number of distribution is FERM P-18545.The contrast of this microorganism was transferred to Independent Administrative Leged Industrial Technology Complex Inst on July 8th, 2002 according to being specified in of budapest treaty subsequently and speciallys permit biological sustenance center preservation, and the preserving number of distribution is a FERM BP-8113 (microorganism sign: short steady bacillus (Empedobacter brevis) strains A J 13933).

Sphingobacterium kind (Sphingobacterium sp.) strains A J 110003 is deposited in Independent Administrative Leged Industrial Technology Complex Inst on July 22nd, 2002 and speciallys permit (depositary institution address: Chuo Dai-6, biological sustenance center, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), the preserving number of distribution is FERM BP-8124.

It is to be noted that strains A J 110003 (FERM BP-8124) is defined as aforesaid Sphingobacterium kind (Sphingobacterium sp.) by following identification experiment.Bacterial strain FERMBP-8124 is gram negative bacillus (0.7-0.8 * 1.5-2.0 μ m), does not form spore and can not move.Its bacterium colony is the perfectly smooth circle in edge, has low projection, and is glossy, faint yellow.This microorganism is in 30 ℃ of growths, the hydrogen peroxide enzyme positive, and oxidase positive, oxidative fermentation (OF) experiment (glucose) feminine gender judges that based on these characteristics it is to belong to the bacterium that Sphingobacterium (Sphingobacterium) belongs to.And, because it has following characteristic: the nitrate reduction feminine gender, product indoles feminine gender, produce the acid feminine gender by glucose, arginine dihydrolase (dihydrolase) feminine gender, the urase positive, the polychrom 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-glycosamine assimilation is positive, the maltose assimilation is positive, the potassium gluconate assimilation is negative, the n-capric acid assimilation is negative, the hexanodioic acid assimilation is negative, the assimilation of dl-oxysuccinic acid is negative, the Trisodium Citrate assimilation is negative, negative and the Terminal oxidase positive of phenylacetate assimilation determines that its property class is similar to the characteristic of Sphingobacterium multivorum (Sphingobacterium multivorum) or Sphingobacterium spiritivorum (Sphingobacteriumspiritivorum).In addition, although the homology analysis result of 16S rRNA gene base sequence shows and Sphingobacterium multivorum (Sphingobacterium multivorum) height homology (98.8%), there is not the bacterial strain that mates fully with this bacterial isolates.Therefore, this bacterial isolates is differentiated to be Sphingobacterium kind (Sphingobacterium sp.).

Concerning these microorganisms, or wild type strain or saltant all can use, and perhaps can use by cytogamy or genetic technique (for example genetic manipulation) inductive recombinant bacterial strain.

For obtaining the microorganism cells of described microorganism, can make described microorganism in suitable medium, cultivate and grow.It the substratum that is used for this purposes is not specifically limited, as long as can make microorganism growth.This substratum can be the ordinary culture medium that contains common carbon source, nitrogenous source, phosphorus source, sulphur source, inorganic salt and organotrophy source (adding as required).

For example, can use any carbon source, as long as microorganism can utilize.The specific examples of available carbon source comprises sugar as glucose, fructose, maltose and polysaccharide, and is pure as Sorbitol Powder, ethanol and glycerine, organic acid such as fumaric acid, citric acid, acetate, propionic acid and salt thereof, hydrocarbon polymer such as paraffin, and their mixture.

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

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

Culture condition is had no particular limits can about 12-48 hour as cultivating, and suitably controls pH and temperature simultaneously under aeration condition respectively, and the pH scope is 5-8, and temperature range is 15-40 ℃.

3. the enzyme that uses of the present invention

In the peptide production method of the invention described above, use can be connected to L-aspartic acid-α with L-phenylalanine selectivity by peptide bond, the enzyme in the α of β-diester-ester site.In peptide production method of the present invention, source of restriction enzyme and production method do not need only it and have this activity.Hereinafter will set forth the application of the purifying and the genetic engineering technique of the used enzyme of the present invention.

(3-1) has the microorganism of the enzyme that can be used for production method of the present invention

For the microorganism of production enzyme of the present invention, can use all can be by L-aspartic acid-α, β-diester and L-phenylalanine are produced the microorganism of 2-L-aspartyl-L-phenylalanine-β-ester.Described microorganism comprises and belongs to bacterium that is selected from the subordinate etc.: Aeromonas (Aeromonas); Azotobacter (Azotobacter); Alcaligenes (Alcaligenes); brevibacterium sp (Brevibacterium); Corynebacterium (Corynebacterium); Escherichia (Escherichia); steady Bacillaceae (Empedodacter); Flavobacterium (Flavobacterium); Microbacterium (Microbacterium); propiono-bacterium (Propionibacterium); bacillus brevis belongs to (Brevibacillus); series bacillus belongs to (Paenibacillus); Rhodopseudomonas (Pseudomonas); serratia (Serratia); oligotrophy zygosaccharomyces (Stenotrophomonas); Sphingobacterium (Sphingobacterium); streptomyces (Streptomyces); xanthomonas (Xanthomonas); intend Weir yeast belong (Williopsis); mycocandida (Candida); Geotrichum (Geotrichum); Pichia (Pichia); yeast belong (Saccharomyces); spore torulopsis (Torulaspora) is arranged; Cytophaga (Cellulophaga); Weeks Bordetella (Weeksella); soil Bacillaceae (Pedobacter); peachiness Bacillaceae (Persicobacter); Flexithrix (Flexithrix); bite chitin Pseudomonas (Chitinophaga); round bar Pseudomonas (Cyclobacterium); ancient character shape Pseudomonas (Runella); hot line Pseudomonas (Thermonema) dwells; cold snake Pseudomonas (Psychroserpens); ice-cold Bacillaceae (Gelidibacter); Dyadobacter; heat color Bacillaceae (Flammeovirga); Spirosoma (Spirosoma); knee Pseudomonas (Flectobacillus); Flexibacter (Tenacibaculum); red thermophilic salt Pseudomonas (Rhodotermus); Zobellia; Muricauda; Salegentibacter; shaping Bacillaceae (Taxeobacter); Cytophage (Cytophaga); the sliding Pseudomonas (Marinilabilia) in sea; rely Wen Pseudomonas (Lewinella); Saprospira (Saprospira) and constraint Bacillaceae (Haliscomenobacter).More particularly, described microorganism comprises short steady bacillus (Empedobacter brevis) ATCC 14234 (bacterial strain FERM P-18545, (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 thing shifts day: on July 8th, 2002)), (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to Sphingobacterium kind (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 day: on July 22nd, 2002), separate Vitrum AB soil bacillus (Pedobacterheparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute; 2-17-85Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan), shaping bacillus (Taxeobactergelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany), sea round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) and the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America) etc.The inventor has studied aspartic acid-α by L-, the microbes producing cellulase of β-diester and L-phenylalanine high yield 2-L-aspartyl-L-phenylalanine-β-ester, the result has selected short steady bacillus (Empedodacter brevis) ATCC 14234 (bacterial strain FERM P-18545, (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 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing day: on July 8th, 2002)), (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to Sphingobacterium kind (Sphingobacterium sp.) FERM BP-8124 bacterial strain, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation day: on July 22nd, 2002), separate Vitrum AB soil bacillus (Pedobacterheparinus) IFO 12017 bacterial strains (preservation mechanism: Osaka fermentation research institute; 2-17-85Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan), shaping bacillus (Taxeobactergelupurpurascens) DSMZ 11116 bacterial strains (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany), sea round bacteria (Cyclobacterium marinum) ATCC25205 bacterial strain (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) and the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 bacterial strains (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the United States of America) microorganism such as.

(3-2) purifying of enzyme

As previously mentioned, can go out the one-tenth peptase that the present invention uses by the bacteria purification that for example belongs to steady Bacillaceae (Empedobacter).Set forth as the enzyme purification example to separate with the method that is purified into into peptase by short steady bacillus (Empedobacter brevis).

At first, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center as bacterial strain FERM BP-8113 by short steady bacillus (Empedobacter brevis), preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: microorganism cells on July 8th, 2002) prepares the microorganism cells extract, method is to use physical method such as ultrasonic disruption or uses the enzyme process smudge cells of Lysozyme, and removes indissolvable component by centrifugation etc.Pass through the common method for purifying proteins of combination such as anion-exchange chromatography, cation-exchange chromatography or gel permeation chromatography then, with the cell extract fractional separation that obtains in the above described manner, but the described one-tenth peptase of purifying.

The example that is used for the carrier of anion-exchange chromatography is Q-Sepharose HP (Amersham production).When making this media-filled post of cell extract process that contains described enzyme, do not reclaiming enzyme in the absorbed component under the pH8.5 condition.

The example that is used for the carrier of cation-exchange chromatography is MonoS HR (Amersham production).Make the cell extract that contains described enzyme through this media-filled post, enzyme is adsorbed on the post, washes post then, with high salt concentration buffer solution elution enzyme.At this moment, salt concn can increase gradually, perhaps available employing concentration gradient.For example, when using MonoS HR, be adsorbed on enzyme on the post with the NaCl concentration wash-out of about 0.2-0.5M.

Then can be by the further purifying enzyme of purifying in the above described manner equably such as gel permeation chromatography.The example that is used for the carrier of gel permeation chromatography is Sephadex 200pg (Amersham production).

In aforementioned purification step, can according to hereinafter the method for the embodiment that describes explanation, by measuring the one-tenth peptide activity of each fraction, calibrating contains the fraction of enzyme.The SEQ IDNO:1 of sequence table and SEQ ID NO:2 have listed the internal amino acid sequence of the enzyme of purifying in the above described manner.

(3-3) production of the separation of DNA, transformant and the purifying that becomes peptase

(3-3-1) separation of DNA

At first (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to the present inventor by short steady bacillus (Empedobacter brevis) bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: successfully isolate a kind of DNA that can be used for the one-tenth peptase of peptide production method of the present invention on July 8th, 2002).

It has the base sequence of the base ordinal number 61-1908 of the described base sequence of SEQ ID NO:5 a kind of DNA of the present invention, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to separate certainly short steady bacillus (Empedobacter brevis) bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, 2002).DNA with base sequence of base ordinal number 61-1908 is encoding sequence (CDS) part.The base sequence of base ordinal number 61-1908 comprises signal sequence district and maturation protein district.The signal sequence district is the zone of base ordinal number 61-126, and ripe protein region is the zone of base ordinal number 127-1908.That is, the present invention both provided the one-tenth peptase that contains signal sequence protein gene, and the one-tenth peptase protein gene of maturation protein form is provided again.The signal sequence that the described sequence of SEQ ID NO:5 contains is a kind of leader sequence.By inference, by the major function of the leading peptide of leader sequence coding be by the cytolemma internal secretion outside cytolemma.By base ordinal number 127-1908 encoded protein, promptly do not comprise the ester site of leading peptide, infer that it is a maturation protein, have the one-tenth peptide activity of height.

It has the base sequence of the described base ordinal number of SEQ ID NO:11 61-1917 another kind of DNA of the present invention, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center from Sphingobacterium kind (Sphingobacterium sp.) bacterial strain FERM BP-8124 in separation, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation day: on July 22nd, 2002).DNA with base sequence of base ordinal number 61-1917 is encoding sequence (CDS) part.The base sequence of base ordinal number 61-1917 comprises signal sequence district and maturation protein district.The signal sequence district is the zone that comprises base ordinal number 61-120, and ripe protein region is the zone that comprises base ordinal number 121-1917.That is, the present invention both provided the one-tenth peptase that contains signal sequence protein gene, and the one-tenth peptase protein gene of maturation protein form is provided again.The signal sequence that the described sequence of SEQ ID NO:11 contains is a kind of leader sequence.By inference, by the major function of the leading peptide of leader sequence coding be by the cytolemma internal secretion outside cytolemma.By base ordinal number 121-1917 encoded protein, promptly do not comprise the part of leading peptide, infer that it is a maturation protein, have the one-tenth peptide activity of height.

It has the base sequence of the described base ordinal number of SEQ ID NO:17 61-1935 the another kind of DNA of the present invention, separates and explains Vitrum AB soil bacillus (Pedobacter heparinus) bacterial strain IFO12017 (preservation mechanism: Osaka fermentation research institute by oneself; 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan).DNA with base sequence of the described base ordinal number of SEQ ID NO:17 61-1935 is encoding sequence (CDS) part.The base sequence of base ordinal number 61-1935 comprises signal sequence district and maturation protein district.The signal sequence district is the zone that comprises base ordinal number 61-126, and ripe protein region is the zone that comprises base ordinal number 127-1935.That is, the present invention both provided the one-tenth peptase that contains signal sequence protein gene, and the one-tenth peptase protein gene of maturation protein form is provided again.The signal sequence that the described sequence of SEQ ID NO:17 contains is a kind of leader sequence.By inference, by the major function of the leading peptide of leader sequence coding be by the cytolemma internal secretion outside cytolemma.By base ordinal number 127-1935 encoded protein, promptly do not comprise the part of leading peptide, infer that it is a maturation protein, have the one-tenth peptide activity of height.

It has the base sequence of the described base ordinal number of SEQ ID NO:22 61-1995 another kind of DNA of the present invention, separation is from shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany).DNA with base sequence of the described base ordinal number of SEQ ID NO:22 61-1995 is encoding sequence (CDS) part.The base sequence of base ordinal number 61-1995 comprises signal sequence district and maturation protein district.The signal sequence district is the zone that comprises base ordinal number 61-126, and ripe protein region is the zone that comprises base ordinal number 127-1995.That is, the present invention both provided the one-tenth peptase that contains signal sequence protein gene, and the one-tenth peptase protein gene of maturation protein form is provided again.The signal sequence that the described sequence of SEQID NO:22 contains is a kind of leader sequence.By inference, by the major function of the leading peptide of leader sequence coding be by the cytolemma internal secretion outside cytolemma.By base ordinal number 127-1995 encoded protein, promptly do not comprise the part of leading peptide, infer that it is a maturation protein, have the one-tenth peptide activity of height.

It has the base sequence of the described base ordinal number of SEQ ID NO:24 29-1888 the another kind of DNA of the present invention, separation is from extra large round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America).DNA with base sequence of the described base ordinal number of SEQ ID NO:24 29-1888 is encoding sequence (CDS) part.The base sequence of base ordinal number 29-1888 comprises signal sequence district and maturation protein district.The signal sequence district is the zone that comprises base ordinal number 29-103, and ripe protein region is the zone that comprises base ordinal number 104-1888.That is, the present invention both provided the one-tenth peptase that contains signal sequence protein gene, and the one-tenth peptase protein gene of maturation protein form is provided again.The signal sequence that the described sequence of SEQ ID NO:24 contains is a kind of leader sequence.By inference, by the major function of the leading peptide of leader sequence coding be by the cytolemma internal secretion outside cytolemma.By base ordinal number 104-1888 encoded protein, promptly do not comprise the part of leading peptide, infer that it is a maturation protein, have the one-tenth peptide activity of height.

It has the base sequence of the described base ordinal number of SEQ ID NO:26 61-1992 another kind of DNA of the present invention, separation is cold snake bacterium (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection from the Christian Breton lake, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) etc.DNA with base sequence of the described base ordinal number of SEQ ID NO:26 61-1992 is encoding sequence (CDS) part.The base sequence of base ordinal number 61-1992 comprises signal sequence district and maturation protein district.The signal sequence district is the zone that comprises base ordinal number 61-111, and ripe protein region is the zone that comprises base ordinal number 112-1992.That is, the present invention both provided the one-tenth peptase that contains signal sequence protein gene, and the one-tenth peptase protein gene of maturation protein form is provided again.The signal sequence that the described sequence of SEQ ID NO:26 contains is a kind of leader sequence.By inference, by the major function of the leading peptide of leader sequence coding be by the cytolemma internal secretion outside cytolemma.By base ordinal number 112-1992 encoded protein, promptly do not comprise the part of leading peptide, infer that it is a maturation protein, have the one-tenth peptide activity of height.

In addition, can be according to molecular cloning, the 2nd edition, the following range gene recombinant technology of narrating is implemented in the description of Cold Spring Harbor Press (1989) and other publication.

Can pass through polymerase chain reaction (PCR, referring to White, T.J. etc., Trends Genet., 5,185 (1989)), perhaps with short steady bacillus (Empedobacter brevis), Sphingobacterium kind (Sphingobacterium sp.), separate Vitrum AB soil bacillus (Pedobacter heparinus), shaping bacillus (Taxeobacter gelupurpurascens), chromosomal DNA of sea round bacteria (Cyclobacteriummarinum) or the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) or the hybridization of DNA library obtain can be used for enzyme coding DNA of the present invention.The primer that PCR uses can design according to the internal amino acid base sequence, and the internal amino acid base sequence is based on the elaboration of chapters and sections (3) as described above, and the one-tenth peptase of purifying is determined.In addition, because the present invention has differentiated the base sequence (SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26) that becomes peptidase genes, so can design primer or hybridization probe according to these base sequences, and use described probe separates gene.If primer sequence has respectively corresponding to 5 '-non-translational region and 3 '-sequence of non-translational region, then it is as PCR primer, the full length coding region of this enzyme that can increase.The zone of containing described leader sequence of SEQ ID NO:5 and mature protein coding region with amplification simultaneously is an example, the specific examples of primer comprise 5 of base ordinal number 61 upstream region base sequences with SEQ ID NO:5 '-the end primer, and have with 3 of the base sequence complementary sequence of base ordinal number 1908 downstream areas '-the end primer.

For example, can use, with phosphoramidite (phosphoamidite) method (referring to Tetrahedron Letters (1981), 22,1859), by the 380B type dna synthesizer that Applied Biosystems produces, synthetic primer according to general method.For example, can use Gene Amp PCRSystem 9600 (Perkin-Elmer) and Takara LA PCR body outer clone test kit (TakaraShuzo),, carry out the PCR reaction according to supplier's method of manufacturer explanation for example.

The coding DNA that can be used for the enzyme of peptide production method of the present invention no matter whether this DNA contains leader sequence, all comprises such DNA: the DNA of the described CDS of SEQ ID NO:5 is roughly the same in itself and the sequence table.Promptly, can be by the mutant enzyme coding DNA or have and isolate following DNA in the cell of this DNA and obtain the DNA roughly the same with DNA of the present invention: the DNA of the complementary base sequence of the described CDS of SEQ ID NO:5 or by the probe hybridize under stringent condition of described base sequence preparation in described DNA and the sequence table, and coding has into the active albumen of peptide.

DNA of the present invention no matter whether it contains leader sequence, comprises such DNA: the DNA of the described CDS of SEQ ID NO:11 is roughly the same in itself and the sequence table.Promptly, can be by the mutant enzyme coding DNA or have and isolate following DNA in the cell of this DNA and obtain the DNA roughly the same with DNA of the present invention: the DNA of the complementary base sequence of the described CDS of SEQ IDNO:11 or by the probe hybridize under stringent condition of described base sequence preparation in described DNA and the sequence table, and coding has into the active albumen of peptide.

DNA of the present invention no matter whether it contains leader sequence, comprises such DNA: the DNA of the described CDS of SEQ ID NO:17 is roughly the same in itself and the sequence table.Promptly, can be by the mutant enzyme coding DNA or have and isolate following DNA in the cell of this DNA and obtain the DNA roughly the same with DNA of the present invention: the DNA of the complementary base sequence of the described CDS of SEQ IDNO:17 or by the probe hybridize under stringent condition of described base sequence preparation in described DNA and the sequence table, and coding has into the active albumen of peptide.

DNA of the present invention no matter whether it contains leader sequence, comprises such DNA: the DNA of the described CDS of SEQ ID NO:22 is roughly the same in itself and the sequence table.Promptly, can be by the mutant enzyme coding DNA or have and isolate following DNA in the cell of this DNA and obtain the DNA roughly the same with DNA of the present invention: the DNA of the complementary base sequence of the described CDS of SEQ IDNO:22 or by the probe hybridize under stringent condition of described base sequence preparation in described DNA and the sequence table, and coding has into the active albumen of peptide.

DNA of the present invention no matter whether it contains leader sequence, comprises such DNA: the DNA of the described CDS of SEQ ID NO:24 is roughly the same in itself and the sequence table.Promptly, can be by the mutant enzyme coding DNA or have and isolate following DNA in the cell of this DNA and obtain the DNA roughly the same with DNA of the present invention: the DNA of the complementary base sequence of the described CDS of SEQ IDNO:24 or by the probe hybridize under stringent condition of described base sequence preparation in described DNA and the sequence table, and coding has into the active albumen of peptide.

DNA of the present invention no matter whether it contains leader sequence, comprises such DNA: the DNA of the described CDS of SEQ ID NO:26 is roughly the same in itself and the sequence table.Promptly, can be by the mutant enzyme coding DNA or have and isolate following DNA in the cell of this DNA and obtain the DNA roughly the same with DNA of the present invention: the DNA of the complementary base sequence of the described CDS of SEQ IDNO:26 or by the probe hybridize under stringent condition of described base sequence preparation in described DNA and the sequence table, and coding has into the active albumen of peptide.

For example can be according to the method for having established, according to the described base sequence of SEQ ID NO:5 in the sequence table for example, produce probe.In addition, also can be according to the method for having established, implement to find DNA with described probe hybridization with probe, thus the method for separating target DNA.For example, can produce dna probe by the following method: amplification is cloned in the base sequence in plasmid vector or the phage vector, cuts out the base sequence of wishing as probe with Restriction Enzyme, extracts the purpose base sequence then.Can adjust the part that will cut out according to the situation of 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.Be difficult to exactly with this condition of numeric representation.For example, can a condition of carrying be: under this condition, DNA with high homology as 50% or more than, preferred 80% or more than, more preferably 90% or more than, mutually between hybridization, the DNA that homology is lower than these values is not hybridized between mutually, or be used for the general condition of DNA hybridization rinsing, under this condition, in 60 ℃ be equivalent to hybridize under the salt concn of 1 * SSC and 0.1%SDS, preferred 0.1 * SSC and 0.1%SDS.Although Za Jiao gene comprises that terminator codon appears at the gene of some position on its sequence or the deactivated gene owing to the active centre sudden change under the described conditions, but they can be removed easily, method is: they are connected with commercially available expression vector, in appropriate host, express, use hereinafter described method to measure the enzymic activity of expression product.

But, for the base sequence of under above-mentioned stringent condition, hybridizing, the enzymic activity that preferred this base sequence proteins encoded keeps under 50 ℃ and pH8 condition, be it by former base sequence as the pact of the proteic enzymic activity of alkali yl coding aminoacid sequence half or more than, preferred 80% or more than, more preferably 90% or more than.As making an explanation with example, for example, under stringent condition, base sequence with the complementary base sequence DNA hybridization of the base sequence of the base ordinal number 127-1908 of the described base sequence of SEQ ID NO:5, the enzymic activity that then preferred this base sequence proteins encoded keeps under 50 ℃ and pH8 condition be the described aminoacid sequence of SEQ ID NO:6 amino-acid residue ordinal number 23-616 aminoacid sequence proteic enzymic activity pact half or more than, preferred 80% or more than, more preferably 90% or more than.

SEQ ID NO:6 has listed the described CDS amino acid sequence coded of SEQ ID NO:5 in the sequence table in the sequence table.SEQ ID NO:12 has listed the described CDS amino acid sequence coded of SEQ IDNO:11 in the sequence table in the sequence table.SEQ ID NO:18 has listed the described CDS amino acid sequence coded of SEQ ID NO:17 in the sequence table in the sequence table.SEQ ID NO:23 has listed the described CDS amino acid sequence coded of SEQ ID NO:22 in the sequence table in the sequence table.SEQ ID NO:25 has listed the described CDS amino acid sequence coded of SEQ IDNO:24 in the sequence table in the sequence table.SEQ ID NO:27 has listed the described CDS amino acid sequence coded of SEQ ID NO:26 in the sequence table in the sequence table.

The described complete amino acid sequence of SEQ ID NO:6 comprises leading peptide and maturation protein district, and leading peptide is made of amino-acid residue ordinal number 1-22, and the maturation protein district is made of amino-acid residue ordinal number 23-616.

The described complete amino acid sequence of SEQ ID NO:11 comprises leading peptide and maturation protein district, and leading peptide is made of amino-acid residue ordinal number 1-20, and the maturation protein district is made of amino-acid residue ordinal number 21-619.

The described complete amino acid sequence of SEQ ID NO:18 comprises leading peptide and maturation protein district, and leading peptide is made of amino-acid residue ordinal number 1-22, and the maturation protein district is made of amino-acid residue ordinal number 23-625.

The described complete amino acid sequence of SEQ ID NO:23 comprises leading peptide and maturation protein district, and leading peptide is made of amino-acid residue ordinal number 1-22, and the maturation protein district is made of amino-acid residue ordinal number 23-645.

The described complete amino acid sequence of SEQ ID NO:25 comprises leading peptide and maturation protein district, and leading peptide is made of amino-acid residue ordinal number 1-25, and the maturation protein district is made of amino-acid residue ordinal number 26-620.

The described complete amino acid sequence of SEQ ID NO:27 comprises leading peptide and maturation protein district, and leading peptide is made of amino-acid residue ordinal number 1-17, and the maturation protein district is made of amino-acid residue ordinal number 18-644.

The albumen of dna encoding of the present invention is that wherein maturation protein has into the active protein of peptide, and SEQ ID NO:6, SEQ ID NO:12, SEQID NO:18, SEQ ID NO:23, SEQ ID NO:25 or the roughly the same DNA of the described aminoacid sequence albumen of SEQ ID NO:27 in its proteins encoded and the sequence table, no matter whether it contains leading peptide, also be included among the DNA of the present invention.(it is to be noted that base sequence is specified by aminoacid sequence according to universal code coding.) promptly, the invention provides proteic DNA shown in the coding following (A)-(X):

(A) have the protein of the amino-acid residue ordinal number 23-616 of the described aminoacid sequence of SEQ ID NO:6 in the sequence table,

(B) has the amino-acid residue 23-616 of the described aminoacid sequence of SEQ ID NO:6 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the protein in the α of β-diester-ester site

(C) have the protein of the amino-acid residue ordinal number 21-619 of the described aminoacid sequence of SEQ ID NO:12 in the sequence table,

(D) has the amino-acid residue 21-619 of the described aminoacid sequence of SEQ ID NO:12 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the protein in the α of β-diester-ester site

(E) have the protein of the amino-acid residue ordinal number 23-625 of the described aminoacid sequence of SEQ ID NO:18 in the sequence table,

(F) has the amino-acid residue 23-625 of the described aminoacid sequence of SEQ ID NO:18 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the protein in the α of β-diester-ester site

(G) have the protein of the amino-acid residue ordinal number 23-645 of the described aminoacid sequence of SEQ ID NO:23 in the sequence table,

(H) has the amino-acid residue 23-645 of the described aminoacid sequence of SEQ ID NO:23 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the protein in the α of β-diester-ester site

(I) have the protein of the amino-acid residue ordinal number 26-620 of the described aminoacid sequence of SEQ ID NO:25 in the sequence table,

(J) has the amino-acid residue 26-620 of the described aminoacid sequence of SEQ ID NO:25 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the protein in the α of β-diester-ester site

(K) have the protein of the amino-acid residue ordinal number 18-644 of the described aminoacid sequence of SEQ ID NO:27 in the sequence table,

(L) has the amino-acid residue 18-644 of the described aminoacid sequence of SEQ ID NO:27 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the protein in the α of β-diester-ester site

(N) has the described aminoacid sequence of SEQ ID NO:6 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the α of β-diester-ester site, the protein that contains the maturation protein district

(P) has the described aminoacid sequence of SEQ ID NO:12 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the α of β-diester-ester site, the protein that contains the maturation protein district

(R) has the described aminoacid sequence of SEQ ID NO:18 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the α of β-diester-ester site, the protein that contains the maturation protein district

(T) has the described aminoacid sequence of SEQ ID NO:23 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the α of β-diester-ester site, the protein that contains the maturation protein district

(V) has the described aminoacid sequence of SEQ ID NO:25 in the sequence table, have wherein that one or more amino acid are replaced, lack, inserted, increase and/or inversion, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the α of β-diester-ester site, the protein that contains the maturation protein district

(X) have the described aminoacid sequence of SEQ ID NO:27 in the sequence table, and can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the α of β-diester-ester site contains the protein in maturation protein district.

At this, although the implication of term " a plurality of " changes to some extent according to the position of amino-acid residue in the protein three-dimensional structure and type, its in the scope of the three-dimensional structure of significantly not damaging amino-acid residue and protein-active, 2-50 specifically, preferred 2-30, more preferably 2-10.But, for the aminoacid sequence that one or more amino acid replacements, disappearance, insertion, increase and/or inversion are arranged in (B), (D), (F), (H), (J), (L), (N), (P), (R), (T), (V) or the Argine Monohydrochloride sequence (X), the enzymic activity that preferred its proteins encoded keeps under 50 ℃ and pH8 condition, be no mutation status protease activity pact half or more than, more preferably 80% or more than, more more preferably 90% or more than.For example, with (B) is that example is set forth: for the aminoacid sequence (B) that one or more amino acid replacements, disappearance, insertion, increase and/or inversion are arranged in the described aminoacid sequence of SEQ ID NO:6 in the sequence table, the enzymic activity that preferred its proteins encoded keeps under 50 ℃ and pH8 condition, be the described amino acid sequence encode protease activity of SEQ ID NO:6 in the sequence table pact half or more than, more preferably 80% or more than, more more preferably 90% or more than.

Can pass through the modified base sequence,, obtain at the amino acid mutation described in aforementioned (B) etc. for example by the site-directed mutagenesis of ester, replacement, disappearance, insertion or increase the amino acid of specifying the ester site in the enzyme gene of the present invention.In addition, also can obtain above-mentioned modifying DNA by mutagenic treatment known in the art.Mutagenic treatment for example is meant the method with the DNA of extracorporeal treatment code book invention enzymes such as azanol, and the method for handling Escherichia (Escherichia) bacterium that contains enzyme coding DNA of the present invention with the mutagenic compound that are generally used for induced mutations (for example uv-radiation, N-methyl-N '-nitro-N-nitrosoguanidine (NTG) or nitrous acid).

In addition, naturally occurring sudden change for example is attributable to the difference of microbial species or bacterial strain, is also included within above-mentioned base replacement, disappearance, insertion, increase and/or the inversion.By the DNA of the described sudden change of expression tool in suitable cell, and the enzymic activity of research expression product, SEQ ID NO:6 in its encoded protein and the sequence table, the roughly the same DNA of 12,18,23,25 or 27 described albumen can be obtained.

(3-3-2) preparation of transformant and the production that becomes peptase

Above (3-3-1) described DNA is imported among the suitable host, and in this host, express described DNA, can produce the one-tenth peptase that can be used for peptide production method of the present invention.

About the proteic expressive host of determining by described DNA, spendable host's example comprises various prokaryotic cell prokaryocytes, comprise Escherichia (Escherichia) bacterium such as intestinal bacteria (Escherichia coli), steady Bacillaceae (Empedobacter) bacterium, Sphingobacterium (Sphingobacterium) bacterium, Flavobacterium (Flavobacterium) bacterium and Bacillus subtilus (Bacillus subtilis), and various eukaryotic cells, comprise yeast saccharomyces cerevisiae (Saccharomycescerevisiae), pichia stipitis (Pichia stipitis) and aspergillus oryzae (Aspergillusoryzae).

Be inserted in the pairing carrier of the host type of wanting expressible dna by the DNA that will import, can prepare the recombinant DNA that is used for DNA is imported the host, can express the albumen of described dna encoding with this form.If the exclusive promotor of one-tenth peptidase genes of short steady bacillus (Empedobacter brevis) etc. works in host cell, then this promotor can be used as the promotor of expressing DNA of the present invention.In addition, the promotor that another can be worked in host cell is connected to DNA of the present invention, can express described DNA as required under this promotor control.

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

Just use recombinant DNA technology mass production albumen, in producing this proteic transformant, described protein binding forms the albumen inclusion body, also is to implement an optimal way of the present invention.This expresses and the advantage of production method comprises the proteasome degradation of protecting target protein to avoid existing in the microorganism cells, and by purification of target albumen simply easily such as disruption of microorganisms cell and centrifugation subsequently.

The albumen inclusion body of Huo Deing is dissolved in protein denaturant in this way, and by mainly comprising the active regeneration step of removing denaturing agent, albumen changes correct folding physiologically active protein into.There are numerous such examples, comprise the active regeneration (referring to Japanese Patent Application Publication communique S61-257931) of human interleukin-2.

In order to obtain activated protein by inclusion body, need series of steps, comprise dissolving and active regeneration, these steps are more complicated than direct production activated protein.But, for mass production in microorganism cells microorganism growth is had the proteic situation of evil effect, then the inactivating protein of accumulation inclusion body form can suppress this effect in microorganism cells.

The example of the large scale production method of inclusion body form target protein is included in the independent down method of expressing target protein of strong promoter control, and with the albumen of known great expression with the fusion protein form expression target protein.

Hereinafter will more specifically set forth the present invention producing method that transformed into escherichia coli and this conversion microorganisms producing of use become peptase as an example.And, for in the microorganism of intestinal bacteria and so on, producing peptase, the coding DNA that can be integrated into the precursor protein that contains leader sequence maybe can be integrated into the DNA that only is made of the maturation protein district that does not contain leader sequence, and can suitably select albumen coded sequence DNA according to the working condition of production enzyme, form, working conditions etc.

Usually be used in the promotor of producing foreign protein in the intestinal bacteria and can be used as the promotor that is expressed as peptidase-encoding DNA.The example of such promotor comprises T7 promotor, lac promotor, trp promotor, trc promotor, tac promotor, lambda particles phage PR promotor, PL promotor and other strong promoter.In addition, available carrier example comprises pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219 and pMW218.In addition, can also use the phage DNA carrier.And, can use the expression vector that contains promotor and can express the dna sequence dna that inserts.

For producing the one-tenth peptase of fusion rotein inclusion body form, the gene of the another kind of albumen of will encoding (preferred hydrophilic peptide) is connected to into the upstream or the downstream of peptidase genes, to obtain antigen-4 fusion protein gene.In this method, the another kind of proteic gene of encoding can be any gene, as long as it increases the semi-invariant of fusion rotein, and strengthens the solvability of sex change and regeneration step rear fusion protein.Candidate's example of this gene comprises T7 gene 10, beta-galactosidase gene, Tetrahydrofolate dehydrogenase (dehydrofolate reductase) gene, gamma-interferon gene, interleukin-2 gene and prochymosin gene.

When these genes when becoming the peptidase-encoding gene to join, connect gene and make the codon frame link up.Recommend to use and be connected the gene in suitable restriction enzyme-ester site or synthetic DNA with proper sequence.

In addition, for increasing into the peptide production of enzyme, preferably will be connected the antigen-4 fusion protein gene downstream sometimes for the terminator of transcription termination sequence.Terminator comprises for example T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator and intestinal bacteria trpA gene terminator.

Import carrier for the gene that in intestinal bacteria, will be encoded into peptase or fusion rotein (becoming peptase and another kind of albumen to merge); preferred so-called multiple copied type carrier; the example comprises the plasmid with ColE1 source replicon, for example based on the plasmid of pUC, based on the plasmid or derivatives thereof of pBR322.Term used herein " derivative " is meant by base replacement, disappearance, insertion, increase and/or inversion and has carried out the plasmid of modifying.It is to be noted that modification used herein comprises the modification that suddenlys change and handled with mutagenic compound or uv-radiation, perhaps spontaneous mutation is modified.

Be the screening transformant, described carrier preferably has mark, for example ampicillin resistance gene.Such plasmid is commercially available expression vector, has effective promotor, as based on pUC carrier (Takara Shuzo, Co., Ltd. production), based on pRROK carrier (ClonetechLaboratories, Inc. produce), pKK233-2 (Clonetech Laboratories, Inc. produces) etc.

Obtain recombinant DNA by dna fragmentation being connected to carrier DNA.In the case, the encoding gene of the fusion rotein of forming according to promotor, L-amino acid amide lytic enzyme or by L-amino acid amide lytic enzyme and another kind of albumen, and being linked in sequence of the terminator of according to circumstances selecting for use.

When using the recombinant DNA transformed into escherichia coli and cultivating the intestinal bacteria that obtained, express and produce peptase or by the fusion rotein that becomes peptase and another kind of albumen to form.Although being generally used for the bacterial strain of expression alien gene can be used as and want host transformed, preference such as coli strain JM109.Molecular cloning, the 2nd edition, Cold Spring Harbor Press (1989) and other publication have been described the method for implementing to transform and filter out transformant.

When the one-tenth peptase of expressed fusion protein form, can use restricted proteolytic enzyme to cut out into peptase, described restricted proteolytic enzyme uses into non-existent sequence in the peptase, as blood coagulation factor Xa or kallikrein, as recognition sequence.

Be generally used for cultivating colibacillary substratum, for example M9-casamino acids substratum or LB substratum can be used as the production substratum.In addition, according to the mark of use carrier, promotor, host microorganism type etc., select culture condition rightly and produce inductive condition.

The fusion rotein that can use following method to be recycled into peptase or to constitute by one-tenth peptase and another kind of albumen.If become peptase or its fusion rotein to be dissolved in the microorganism cells, then after reclaiming microorganism cells, fragmentation or cracking microorganism cells can be used as it and slightly carry enzyme liquid.And, can become peptase or its fusion rotein by ordinary skill such as precipitation, filtration or column chromatography purification as required before use.In the case, can also use peptase or its fusion rotein purifying antibody method utilized into.

When forming the albumen inclusion body, dissolve inclusion body with denaturing agent.They can dissolve with microorganism cells albumen.But, consider the subsequent purification step, preferably take out inclusion body, then dissolving.Can use common known method to reclaim inclusion body by microorganism cells.For example, can pass through the disruption of microorganisms cell, inclusion body is reclaimed in centrifugation then.The denaturing agent example that can dissolve inclusion body comprise Guanidinium hydrochloride (6M for example, pH5-8) and urea (for example 8M).

Remove these denaturing agents activated albumen of regenerating by dialysis.Tris-HCl damping fluid or phosphoric acid buffer etc. can be used as the dialysis solution that uses in the dialysis, and concentration can be 20mM to 0.5M for example, and pH can be 5-8 for example.

Protein concentration in the regeneration step is preferably maintained in the range of from about 500 μ g/ml or lower.The dialysis temperature is preferably 5 ℃ or lower, becomes peptase self-crosslinking to occur to suppress regenerated.And except dialysis, dilution or ultrafiltration can be used for removing denaturing agent, and no matter using which kind of denaturing agent to expect can regeneration activity.

＜2〉production method of 2-L-aspartyl-L-phenylalanine-α-methyl esters

α of the present invention-APM production method comprises: the first step, according to " production method of＜1〉2-L-aspartyl-L-phenylalanine-β-ester " synthetic 2-L-aspartyl-L-phenylalanine-β-methyl esters; In second step, change 2-L-aspartyl-L-phenylalanine-β-methyl esters into 2-L-aspartyl-L-phenylalanine-α-methyl esters.

Optimum condition in the first step is described like " production method of＜1〉2-L-aspartyl-L-phenylalanine-β-ester ".In addition, can implement for second step according to currently known methods, but method and optimum condition that the open H4-41155 of reference example such as Japanese Patent etc. describe.Utilize α of the present invention-APM production method, can produce important sweeting agent α-APM at an easy rate by high yield.

Embodiment

Hereinafter will explain the present invention by embodiment.But, the invention is not restricted to these embodiment.In order to measure product, except confirming, can carry out quantitative assay by following high performance liquid chromatography by membrane chromatographic triketohydrindene hydrate dyeing (qualitative).Post: Inertsil ODS-2 (GLScience, Inc. produces), wash-out: the phosphate aqueous solution (pH2.1) that contains the hot sodium sulfonate of 5.0mM 1-: methyl alcohol=100: 15-50, flow velocity: 1.0ml/ minute, detect: 210 nanometers (nm).

Embodiment 1

Produce the microorganism of 2-L-aspartyl-L-phenylalanine-β-methyl esters

50ml is contained 20g glycerine, 5g ammonium sulfate, 1g potassium primary phosphate, 3g dipotassium hydrogen phosphate, 0.5g sal epsom, 10g yeast extract and 10g peptone in 1L substratum (pH7.0) is transferred to 500ml Sakaguchi and shakes in the bottle, in 15 minutes (substratum 1) of 115 ℃ of sterilizations, be used to cultivate the bacterium and the actinomycetes that are shown in table 1-1.Preparation contains the slant agar substratum (pH7.0) of 5g/L glucose, 10g/L yeast extract, 10g/L peptone, 5g/L NaCl and 20g/L agar in substratum 1, and cultivates the microorganism shown in the table 1 24 hours in 30 ℃ on this slant agar substratum.Then, the described microorganism of a loopful was cultivated 24 hours in 30 ℃ in substratum 1, then in 30 ℃ with 120 times/minute shaking culture 17 hours.After finishing cultivation, microorganism cells is separated with nutrient solution, and in containing the 0.1M borate buffer solution (pH9.0) of 10mMEDTA, be suspended into 100g/L (in the microorganism cells weight in wet base) by centrifugal.

50ml is contained 10g glucose, 10g glycerine, 5g ammonium sulfate, 1g potassium primary phosphate, 3g dipotassium hydrogen phosphate, 0.5g sal epsom, 5g yeast extract, 5g malt extract and 10g peptone in 1L substratum (pH6.0) is transferred to 500ml Sakaguchi and shakes in the bottle, and, use this culture medium culturing to be shown in the yeast of table 1-1 in 15 minutes (substratum 2) of 115 ℃ of sterilizations.Preparation contains the slant agar substratum (pH6.0) of 5g/L glucose, 5g/L yeast extract, 5g/L malt extract, 10g/L peptone, 5g/L NaCl and 20g/L agar in substratum 2, and cultivates the yeast shown in the table 1 24 hours in 30 ℃ on this slant medium.Then, with the described yeast of a loopful in substratum 2 in 30 ℃ of shaking culture 24 hours, then in 25 ℃ with 120 times/minute shaking culture 17 hours.After finishing cultivation, microorganism cells is separated with these nutrient solutions, and be suspended into 100g/L (in the microorganism cells weight in wet base) with the 0.1M borate buffer solution (pH9.0) that contains 10mM EDTA by centrifugal.

Microorganism shown in the following cultivation table 1-2.Use contains the 1g Tryptones in 1L Daigo artificial seawater SP, agar solid medium (the pH7.2 of 1g yeast extract and 15g agar, in 120 ℃ of sterilizations 15 minutes) cultivation phagocyte bacterium (Cellulophaga lytica) NBRC14961 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) or gentle bacterium (Flexithrix dorotheae) NBRC 15987 (the preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center of sending out of many trailing plants silk, preservation mechanism address: 5-8Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan).Will be in 48 hours phagocyte bacterium of 30 ℃ of inoculation culture (Cellulophaga lytica) NBRC 14961 (preservation mechanism: NITE Japan technological assessment institute Biological resources center on this substratum, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) or gentle bacterium (Flexithrix dorotheae) NBRC 15987 (preservation mechanism: the NITE Japan technological assessment institute Biological resources center of sending out of many trailing plants silk, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use sheep blood agar culture-medium (Nissui Plate, Nissui Pharmaceutical) cultivates poisonous Weeks Salmonella (Weeksella virosa) NBRC 16016 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center, preservation mechanism address: 5-8 Kazusa-Kamaashi2-Chome, Kisarazu-shi, Chiba-ken, Japan).Will be in 48 hours poisonous Weeks Salmonella (Weeksella virosa) NBRC 16016 of 30 ℃ of inoculation culture (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center on this substratum, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 10g peptone, 2g yeast extract and 1gMgSO in 1L distilled water ₄7H ₂Agar solid medium (the pH7.0 of O and 15g agar, in 120 ℃ of sterilizations 15 minutes) cultivate and separate Vitrum AB soil bacillus (Pedobacter heparinus) NBRC 12017 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan).Will be on this substratum separate Vitrum AB soil bacillus (Pedobacterheparinus) NBRC 12017 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center in 30 ℃ of inoculation culture 48 hours, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 0.5g KNO in 1L Daigo artificial seawater SP ₃, 0.1g Sodium Glycerophosphate, 1g Tutofusin tris, 5g Tryptones, 5g yeast extract, 15g agar and 1ml trace element solution agar solid medium (pH7.0, in 120 ℃ of sterilizations 15 minutes) wandering peachiness bacillus (Persicobacter diffluens) NBRC 15940 ((the preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center of cultivation, preservation mechanism address: 5-8Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan).Attention: trace element solution comprises 2.85g H ₃BO ₄, 1.8g MnCl ₂4H ₂O, 1.36g FeSO ₄7H ₂O, 26.9mg CuCl ₂2H ₂O, 20.8mg ZnCl ₂, 40.4mg CoCl ₂6H ₂O, 25.2mgNa ₂MoO ₄2H ₂O and 1.77g sodium tartrate).Will be in 48 hours wandering peachiness bacillus (Persicobacter diffluens) NBRC 15940 of 25 ℃ of inoculation culture (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center on this substratum, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains the 3g bacterium and uses junket peptone (bactocasitone), 1g yeast extract, 1.36g CaCl in 1L distilled water ₂2H ₂Agar solid medium (the pH7.0 of O and 15g agar, in 120 ℃ of sterilizations 15 minutes) cultivate pine and bite chitin bacterium (Chitinophaga pinensis) NBRC15968 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan).To on this substratum, bite chitin bacterium (Chitinophaga pinensis) NBRC 15968 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center in 48 hours pine of 25 ℃ of inoculation culture, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains 5g peptone, 1g yeast extract, 0.2g FeSO in 1L Daigo artificial seawater SP ₄7H ₂Agar solid medium (the pH7.0 of O and 15g agar, in 120 ℃ of sterilizations 15 minutes) cultivate extra large round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America).Will be in 48 hours extra large round bacteria of 25 ℃ of inoculation culture (Cyclobacterium marinum) ATCC25205 (preservation mechanism: American type culture collection on this substratum, preservation mechanism address: P.O.Box1549, Manassas, VA 20110, the United States of America) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains the agar solid medium (pH7.0 of 1g peptone, 1g yeast extract, 1g glucose and 15g agar in 1L distilled water, in 120 ℃ of sterilizations 15 minutes) cultivate soft ancient character shape bacterium (Runella slithyformis) ATCC 29530 (the preservation mechanism: American type culture collection of wafing, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the United States of America).Will be in 48 hours soft ancient character shape bacterium (Runella slithyformis) ATCC 29530 (the preservation mechanism: American type culture collection of wafing of 25 ℃ of inoculation culture on this substratum, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains 0.2g nitrilotriacetic acid(NTA), 2ml 0.03%FeCl in 1L distilled water ₃Solution, 0.12g CaSO ₄2H ₂O, 0.2g MgSO ₄7H ₂O, 0.016g NaCl, 0.21gKNO ₃, 1.4g NaNO ₃, 0.22g Na ₂HPO ₄, 2ml trace element solution and 15g agar agar solid medium (pH8.2, in 120 ℃ of sterilizations 15 minutes) cultivate death hot line bacterium (Thermonema lapsum) ATCC 43542 ((the preservation mechanism: American type culture collection of dwelling, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the UnitedStates of America).Should be noted that: trace element solution comprises 0.5ml H ₂SO ₄, 2.2g MnSO ₄, 0.5g ZnSO ₄, 0.5g H ₃BO ₃, 0.016g CuSO ₄, 0.025gNa ₂MoO ₄With 0.046g CoCl ₂).Will be on this substratum in 48 hours death of 60 ℃ of inoculation culture hot line bacterium (Thermonema lapsum) ATCC 43542 (the preservation mechanism: American type culture collection of dwelling, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the United States of America) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use ocean agar (Marine Agar) 2216 (producing) to cultivate cool ice-cold bacillus (Gelidibacter algens) ATCC 700364 (preservation mechanism: American type culture collection by Difco, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the UnitedStates of America), connect and rely Wen bacterium (Lewinella cohaerens) ATCC 23123 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America), the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America) or Salegentibacter salegens DSMZ 5424 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: MascheroderWeg 1b, 38124 Braunschweig, Germany).For the ice-cold bacillus of cold (Gelidibacteralgens) ATCC 700364 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States ofAmerica) or the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America), will be in the cold snake bacterium of 72 hours cool ice-cold bacillus of 10 ℃ of inoculation culture (Gelidibacter algens) ATCC700364 or Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection on this substratum, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) microorganism cells coating is then in 10 ℃ of main cultivations 72 hours.Rely Wen bacterium (Lewinella cohaerens) ATCC 23123 (preservation mechanism: American type culture collection for connecting, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America), to on this substratum, rely Wen bacterium (Lewinellacohaerens) ATCC 23123 (preservation mechanisms: American type culture collection in 48 hours connection of 30 ℃ of inoculation culture, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States ofAmerica) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.For Salegentibacter salegens DSMZ 5424 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany), will be in 48 hours Salegentibacter salegens DSMZ 5424 of 25 ℃ of inoculation culture (preservation mechanism: German microorganism and cell culture preservation center on this substratum, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains 0.8g NH in 1L distilled water ₄Cl, 0.25g KH ₂PO ₄, 0.4gK ₂HPO ₄, 0.505g KNO ₃, 15mg CaCl ₂2H ₂O, 20mg MgCl ₂6H ₂O, 7mgFeSO ₄7H ₂O, 5mg Na ₂SO ₄, 5mg MnCl ₂4H ₂O, 0.5mg H ₃BO ₃, 0.5mgZnCl ₂, 0.5mg CoCl ₂6H ₂O, 0.5mg NiSO ₄6H ₂O, 0.3mg CuCl ₂2H ₂O, 10mg Na ₂MoO ₄2H ₂Agar solid medium (the pH7.0 of O, 0.5g yeast extract, 0.5g peptone, 0.5g casamino acids, 0.5g glucose, 0.5g Zulkovsky starch, 0.5g Sodium.alpha.-ketopropionate and 15g agar, sterilized 15 minutes for 120 ℃) cultivation Dyadobacterfermentans ATCC 700827 (preservation mechanisms: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States ofAmerica).Will be in 48 hours Dyadobacterfermentans ATCC 700827 (preservation mechanism: American type culture collection of 25 ℃ of inoculation culture on this substratum, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States ofAmerica) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains the agar solid medium (pH7.2 of 2g Tryptones, 0.5g meat extract, 0.5g yeast extract, 0.2g sodium acetate and 15g agar in 1L Daigo artificial seawater SP, in 120 ℃ of sterilizations 15 minutes) cultivate and like flare up look bacillus (Flammeovirga aprica) NBRC15941 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan).Will be in 48 hours happiness flare up look bacillus (Flammeovirga aprica) NBRC 15941 of 25 ℃ of inoculation culture (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center on this substratum, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains 1g glucose in 1L distilled water, the 1g peptone, agar solid medium (the pH7.0 of 1g yeast extract and 15g agar, sterilized 15 minutes for 120 ℃) cultivation tongue spiral bacterium (Spirosoma linguale) DSMZ 74 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany) or big knee bacterium (Flectobacillus major) DSMZ 103 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany).Will be in 48 hours tongue spiral bacterium (Spirosoma linguale) DSMZ 74 (preservation mechanism: German microorganism and cell culture preservation center of 25 ℃ of inoculation culture on this substratum, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) or big knee bacterium (Flectobacillus major) DSMZ 103 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains the agar solid medium (pH7.0 of 0.5g Tryptones, 0.5g yeast extract, 0.2g meat extract, 0.2g sodium acetate and 15g agar in 300ml distilled water and 700ml Daigo artificial seawater SP, in 120 ℃ of sterilizations 15 minutes) cultivate the ocean and subdue bacillus (Tenacibaculum maritimum) ATCC 43398 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America).Will be on this substratum subdue bacillus (Tenacibaculum maritimum) ATCC 43398 (preservation mechanism: American type culture collection in 48 hours ocean of 25 ℃ of inoculation culture, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Use contains 2.5g yeast extract, 2.5g Tryptones, 100mg nitrilotriacetic acid(NTA), 40mg CaSO in 1L ₄2H ₂O, 200mg MgCl ₂6H ₂Agar solid medium (the pH7.2 of the 0.01M ironic citrate of O, 0.5ml, 0.5ml trace element solution, 100ml phosphoric acid buffer, 900ml distilled water and 28g agar, sterilized 15 minutes for 120 ℃) the cultivation red thermophilic salt bacterium in ocean (Rhodotermus marinus) DSMZ 4252 ((preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany).Attention: described trace element solution comprises 12.8g nitrilotriacetic acid(NTA), 1g FeCl ₂4H ₂O, 0.5g MnCl ₂4H ₂O, 0.3g CoCl ₂4H ₂O, 50mg CuCl ₂2H ₂O, 50mg Na ₂MoO ₄2H ₂O, 20mg H ₃BO ₃And 20mgNiCl ₂6H ₂O).Will be in the red thermophilic salt bacterium in 48 hours ocean of 60 ℃ of inoculation culture (Rhodotermus marinus) DSMZ 4252 (preservation mechanism: German microorganism and cell culture preservation center on this substratum, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany) microorganism cells is coated on the same medium, then in 60 ℃ of main cultivations 48 hours.

Use contains agar solid medium (1.5% agar of BACTO MARINE BROTH (DIFCO 2216), pH7.6, sterilized 15 minutes for 120 ℃) cultivation Zobellia galactanivoransDSMZ 12802 (preservation mechanisms: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany).Will be in 48 hours Zobellia galactanivoransDSMZ 12802 of 30 ℃ of inoculation culture (preservation mechanism: German microorganism and cell culture preservation center on this substratum, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 1.5g yeast extract, 2.5g peptone, 2g n-Hexadecane, 17.7g NaCl, 0.48g KCl, 3.4g MgCl in 1L distilled water ₂6H ₂O, 4.46g MgSO ₄7H ₂O, 0.98g CaCl ₂Agar solid medium (pH7.2 with 15g agar, sterilized 15 minutes for 120 ℃) cultivation Muricauda ruestringensis DSMZ 13258 (preservation mechanisms: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany).Will be in 48 hours Muricauda ruestringensis DSMZ 13258 of 30 ℃ of inoculation culture (preservation mechanism: German microorganism and cell culture preservation center preservation mechanism address: Mascheroder Weg 1b on this substratum, 38124Braunschweig, Germany) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 3g junket peptone, 1g yeast extract, 1.36gCaCl in 1L distilled water ₂2H ₂Agar solid medium (the pH7.2 of O and 15g agar, sterilized 15 minutes for 120 ℃) cultivation shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: MascheroderWeg 1b, 38124 Braunschweig, Germany).Will be in 48 hours shaping bacillus of 30 ℃ of inoculation culture (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center on this substratum, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 3g junket peptone, 1g yeast extract, 1.36gCaCl in 1L distilled water ₂2H ₂Agar solid medium (the pH7.2 of O, 5g cellobiose and 15g agar, in 120 ℃ of sterilizations 15 minutes) cultivation Cytophaga hutchinsonii (Cytophaga hutchinsonii) NBRC15051 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan).Will be in 48 hours Cytophaga hutchinsonii of 30 ℃ of inoculation culture (Cytophaga hutchinsonii) NBRC 15051 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center on this substratum, preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 10g peptone, 2g yeast extract, 0.5g MgSO in 250ml distilled water and 750ml Daigo artificial seawater SP ₄7H ₂Agar solid medium (the pH7.2 of O and 15g agar; in 120 ℃ of sterilizations 15 minutes) cultivation salmon colour sea sliding bacterium (Marinilabiliasalmonicolor) NBRC 15948 (preservation mechanisms: the Japanese technological assessment NITE of institute Biological resources center; preservation mechanism address: 5-8 Kazusa-Kamaashi 2-Chome; Kisarazu-shi; Chiba-ken, Japan).Will be in 48 hours salmon colour of 30 ℃ of inoculation culture sea sliding bacterium (Marinilabilia salmonicolor) NBRC 15948 (preservation mechanism: the Japanese technological assessment NITE of institute Biological resources center on this substratum; preservation mechanism address: 5-8Kazusa-Kamaashi 2-Chome; Kisarazu-shi; Chiba-ken; Japan) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 0.5g KNO in 1L Daigo artificial seawater SP ₃, 0.1g Sodium Glycerophosphate, 1g Tutofusin tris, 2g Tryptones, 2g yeast extract, 15g agar and 1ml trace element solution agar solid medium (pH7.0, in 120 ℃ of sterilizations 15 minutes) cultivation big corrupt spirobacteria (Saprospira grandis) ATCC 23119 ((preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America).Attention: described trace element solution comprises 2.85g H ₃BO ₄, 1.8g MnCl ₂4H ₂O, 1.36g FeSO ₄7H ₂O, 26.9mgCuCl ₂2H ₂O, 20.8mg ZnCl ₂, 40.4mg CoCl ₂6H ₂O, 25.2mgNa ₂MoO ₄2H ₂O and 1.77g sodium tartrate).Will be in 48 hours big corrupt spirobacteria of 30 ℃ of inoculation culture (Saprospira grandis) ATCC 23119 (preservation mechanism: American type culture collection on this substratum, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) microorganism cells is coated on the same medium, then in 30 ℃ of main cultivations 48 hours.

Use contains 27mg KH in 1L distilled water ₂PO ₄, 40mg K ₂HPO ₄, 40mgNa ₂HPO ₄2H ₂O, 50mg CaCl ₂2H ₂O, 75mg MgSO ₄7H ₂O, 5mgFeCl ₃6H ₂O, 3mg MnSO ₄H ₂O, 1.31g L-glutamic acid, 2.5mg do not have the agar solid medium (pH7.5 of trypticase soy broth, 0.4mg VitB1,0.01mg vitamins B 12,2g glucose and the 1ml trace element solution of glucose, in 120 ℃ of sterilizations 15 minutes) cultivation water constraint bacillus (Haliscomenobacter hydrossis) ATCC 27775 ((preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America).Attention: described trace element solution comprises 0.1g ZnSO ₄7H ₂O, 0.03g MnCl ₂4H ₂O, 0.3g H ₃BO ₃, 0.2gCoCl ₂6H ₂O, 0.01g CuCl ₂2H ₂O, 0.02g NiCl ₂6H ₂O and 0.03gNa ₂MoO ₄H ₂O).Will be in 48 hours water of 25 ℃ of inoculation culture constraint bacillus (Haliscomenobacter hydrossis) ATCC 27775 (preservation mechanism: American type culture collection on this substratum, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America) microorganism cells is coated on the same medium, then in 25 ℃ of main cultivations 48 hours.

Therefore the microorganism cells that obtains each collect since described nutrient agar, and in containing the 0.1M borate buffer solution (pH9.0) of 10mM EDTA, be suspended into 100g/L (in the microorganism cells weight in wet base).

In the various microorganism cells suspension of these microorganisms of 0.1ml, add 0.1ml and contain 10mM EDTA, 100mM hydrochloric acid L-aspartic acid-α, the 100mM borate buffer solution (pH9.0) of senecioate and 200mM L-phenylalanine, making total amount is 0.2ml.Then, when using microorganism shown in the table 1-1, react on 20 ℃ and carried out 3 hours, when using microorganism shown in the table 1-2, react on 20 ℃ and carried out 1 hour.(output (mM) of α-AMP) is shown in table 1-1 and 1-2 to 2-L-aspartyl-L-phenylalanine-β-methyl esters.It is to be noted, under all situations that uses described microorganism, all do not detect β-AMP.

Table 1-1

Microorganism	α-AMP (mM)
Microorganism	α-AMP (mM)	Aeromonas hydrophila (Aeromonas hydrophila) ATCC 13136	1.55
Wei Nielande vinelandii (Azotobacter vinelandii) IFO 3741	0.15	Aeromonas hydrophila (Aeromonas hydrophila) ATCC 13136	1.55
Wei Nielande vinelandii (Azotobacter vinelandii) IFO 3741	0.15	Alcaligenes faecalis (Alcaligenes faecalis) FERM P-8460	0.37
Little bar tyrothricin (Brevibacterium minutiferuna) FERM BP-8277	0.10	Alcaligenes faecalis (Alcaligenes faecalis) FERM P-8460	0.37
	0.10	The excellent bacillus of little Huang (Corynebacterium flavescens) ATCC 10340	0.26
Intestinal bacteria (Escherichia coli) FERM BP-8276	3.68		0.26
Intestinal bacteria (Escherichia coli) FERM BP-8276	3.68	Short steady bacillus (Empedobacter brevis) ATCC 14234	6.31
Flavobacterium resinovorum (Flavobacterium resinovorum) ATCC 14231	0.62	Short steady bacillus (Empedobacter brevis) ATCC 14234	6.31
	0.62	Tree-shaped microbacterium (Microbacterium arborescens) ATCC 4348	0.08
Xie Shi propionibacterium (Propionibacterium shermanii) BERM BP-8100	3.41		0.08
	3.41	Tyrothricin (Brevibacillus parabrevis) ATCC 8185	0.08
Honeycomb series bacillus (Paenibacillus alvei) IFO 14175	0.09	Tyrothricin (Brevibacillus parabrevis) ATCC 8185	0.08
Honeycomb series bacillus (Paenibacillus alvei) IFO 14175	0.09	Pseudomonas fragi (Pseudomonas fragi) IFO 3458	0.84
Ge Shi Serratia (Serratia grimesii) ATCC 14460	0.47	Pseudomonas fragi (Pseudomonas fragi) IFO 3458	0.84
Ge Shi Serratia (Serratia grimesii) ATCC 14460	0.47	Germ oligotrophy unit cell (Stenotrophomonas maltophilia) ATCC 13270	0.18
Sphingobacterium kind (Sphingobacterium sp.) FERM BP-8124	5.97		0.18
Sphingobacterium kind (Sphingobacterium sp.) FERM BP-8124	5.97	Lilac grey streptomycete (Streptomyces lavendulae) NRRL B-1305	0.89
Xanthomonas maltophilia (Xanthomonas maltophilia) FERM BP-5568	0.40		0.89
	0.40	Intend Weir yeast (Williopsis saturnus) IFO 0895	0.05
Candida magnoliae (Candida magnoliae) IFO 0705	0.26	Intend Weir yeast (Williopsis saturnus) IFO 0895	0.05
Candida magnoliae (Candida magnoliae) IFO 0705	0.26	Ground mould (Geotrichum amycelium) CBS 152.25	0.19
Ground mould (Geotrichum amycelium) IFO 0905	0.06	Ground mould (Geotrichum amycelium) CBS 152.25	0.19
Ground mould (Geotrichum amycelium) IFO 0905	0.06	Saccharomyces unisporus (Saccharomyces unisporus) IFO 0724	0.07
There is spore torula (Torulaspora delbrueckii) IFO 0422 in Dell	0.04	Saccharomyces unisporus (Saccharomyces unisporus) IFO 0724	0.07
	0.04	The west is pichia spp (Pichia ciferrii) IFO 0905 not	0.06

Table 1-2

Microorganism	α-AMP (mM)	Microorganism	α-AMP (mM)
Microorganism	α-AMP (mM)	Microorganism	α-AMP (mM)	Phagocyte bacterium (Cellulophaga lytica) NBRC 14961	tr	Tongue spiral bacterium (Spirosoma linguale) DSMZ 74	0.15
Poisonous Weeks Salmonella (Weeksella virosa) NBRC 16016	tr	Big knee bacterium (Flectobacillus major) DSMZ 103	0.68	Phagocyte bacterium (Cellulophaga lytica) NBRC 14961	tr	Tongue spiral bacterium (Spirosoma linguale) DSMZ 74	0.15
Poisonous Weeks Salmonella (Weeksella virosa) NBRC 16016	tr	Big knee bacterium (Flectobacillus major) DSMZ 103	0.68	Separate Vitrum AB soil bacillus (Pedobacter heparinus) NBRC 12017	0.07	Bacillus (Tenacibaculum maritimum) ATCC 43398 is subdued in the ocean	tr
Wandering peachiness bacillus (Persicobacter diffluens) NBRC 15940	tr	The red thermophilic salt bacterium in ocean (Rhodotermus marinus) DSMZ 4252	0.06		0.07		tr
	tr		0.06	Gentle bacterium (Flexithrix dorotheae) NBRC 15987 that sends out of many trailing plants silk	2.47	Zobellia galactanivorans DSMZ 12802	0.42
Pine bites chitin bacterium (Chitinophaga pinensis) NBRC 15968	0.08	Muricauda ruestringensis DSMZ 13258	0.51		2.47	Zobellia galactanivorans DSMZ 12802	0.42
	0.08	Muricauda ruestringensis DSMZ 13258	0.51	Sea round bacteria (Cyclobacterium marinum) ATCC 25205	0.91	Salegentibacter salegens DSMZ 5424	tr
Soft ancient character shape bacterium (Runella slithyformis) ATCC 29530 wafts	0.07	Shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116	0.02	Sea round bacteria (Cyclobacterium marinum) ATCC 25205	0.91	Salegentibacter salegens DSMZ 5424	tr
	0.07	Shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116	0.02	Death hot line bacterium (Thermonema lapsum) ATCC 43542 of dwelling	tr	Cytophaga hutchinsonii (Cytophaga hutchinsonii) NBRC 15051	tr
The cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359	0.09	Sliding bacterium (Marinilabilia salmonicolor) NBRC 15948 in salmon colour sea	0.02		tr	Cytophaga hutchinsonii (Cytophaga hutchinsonii) NBRC 15051	tr
	0.09		0.02	Cool ice-cold bacillus (Gelidibacter algens) ATCC 700364	0.07	Connect and rely Wen bacterium (Lewinella cohaerens) ATCC 23123	0.33
Dyadobacter fermentans ATCC 700827	0.04	Big corrupt spirobacteria (Saprospira grandis) ATCC 231195	0.03	Cool ice-cold bacillus (Gelidibacter algens) ATCC 700364	0.07		0.33
Dyadobacter fermentans ATCC 700827	0.04	Big corrupt spirobacteria (Saprospira grandis) ATCC 231195	0.03	Happiness flare up look bacillus (Flammeovirga aprica) NBRC 15941	0.08	Water constraint bacillus (Haliscomenobacter hydrossis) ATCC 27775	tr

Reference example 1

Produce the microorganism of β-L-aspartyl-L-phenylalanine-α-methyl esters

With microorganism shown in the method cultivation table 2 that is similar to embodiment 1 table 1 bacterium.After cultivation is finished, microorganism cells is separated with nutrient solution, and in containing the 0.1M borate buffer solution (pH9.0) of 10mM EDTA, be suspended into 100g/L (in the microorganism cells weight in wet base) by centrifugal.In these microorganisms 0.1ml microorganism cells suspension separately, add 0.1ml and contain 10mM EDTA, 100mM hydrochloric acid L-aspartic acid-α, the 100mM borate buffer solution (pH9.0) of senecioate and 200mM L-phenylalanine, making total amount is 0.2ml, then in 30 ℃ of reactions 2 hours.(output (mM) of β-AMP) is shown in table 2 to β-L-aspartyl-L-phenylalanine-α-methyl esters in the case.It is to be noted, in all described microorganisms, all do not detect α-AMP.

Table 2

Microorganism	β-AMP(mM)
Microorganism	β-AMP(mM)	Hafnia alvei (Hafnia alvei) ATCC 9760	0.30
Klebsiella pneumonia (Klebsiella pneumoniae) ATCC 8308	0.26	Hafnia alvei (Hafnia alvei) ATCC 9760	0.30

Embodiment 2

The purifying of enzyme in the short steady bacillus (Empedobacter brevis)

The 50ml substratum (pH6.2) that contains 5g glucose, 5g ammonium sulfate, 1g potassium primary phosphate, 3g dipotassium hydrogen phosphate, 0.5g sal epsom, 10g yeast extract and 10g peptone among the 1L is transferred to 500ml Sakaguchi shakes in the bottle, and in 115 ℃ of sterilizations 15 minutes.Then with (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center in short steady bacillus (Empedobacter brevis) the bacterial strain FERM of 16 hours 2ml of 30 ℃ of cultivations BP-8113 in same medium, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, 2002) inoculate this substratum, then in 30 ℃ with 120 times/minute shaking culture 16 hours.

Subsequently, on ice or in 4 ℃ of steps after carrying out centrifugation.Centrifugal gained nutrient solution is collected microorganism cells.Behind 50mM Tris-HCl damping fluid (pH8.0) cleaning 16g microorganism cells, it is suspended in the 40ml same buffer, carry out 45 minutes ultrasonic disruptions with 195 watts again and handle.Centrifugal then (10,000rpm, 30 minutes) this ultrasonic disruption liquid to remove broken cell debris, obtains the supernatant liquor of ultrasonic disruption liquid.The supernatant liquor that makes this ultrasonic disruption liquid is then removed indissolvable component by ultracentrifugation (50,000rpm, 30 minutes) to 50mM Tris-HCl damping fluid (pH8.0) dialysed overnight, obtains the soluble constituent into the supernatant liquor form.The gained soluble constituent is splined on the Q-Sepharose HP post (Amersham production) of using Tris-HCl damping fluid (pH8.0) pre-equilibration, by not collecting active ingredient in the adsorbed components.This active ingredient to 50mM acetate buffer (pH4.5) dialysed overnight, is then removed indissolvable component by centrifugation (10,000rpm, 30 minutes), obtain dialysis component into the supernatant liquor form.Then this dialysis component is splined on Mono S post (Amersham production), with the same buffer gradient elution enzyme that contains the linear concentration of 0-1M NaCl with 50mM acetate buffer (pH4.5) pre-equilibration.The component that contains the minimum level foreign protein in the active ingredient is splined on the Superdex 200pg post (Amersham production) of using 50mM acetate buffer (pH4.5) pre-equilibration that contains 1M NaCl, make the same buffer (pH4.5) that contains 1M NaCl carry out gel-filtration, obtain active component solution through this post.Carry out after these steps,, confirm to be used for one-tenth peptase of the present invention homogeneous purifying according to The results in electrophoresis.The enzyme yield of aforementioned purge process is 12.2%, and purification degrees is 707 times.

Embodiment 3

Use short steady bacillus (Empedobacter brevis) enzyme component to produce 2-L-aspartyl-L-phenylalanine-β-methyl esters

The 10 μ l Mono S component enzymes (about 20U/ml) that will obtain in embodiment 2 add 190 μ l and contain 105.3mM hydrochloric acid L-aspartic acid-α, in the borate buffer solution (pH9.0) of senecioate, 210.5mM L-phenylalanine and 10.51mM EDTA, react in 20 ℃.(production process of α-AMP) is shown in table 3 to 2-L-aspartyl-L-phenylalanine-β-methyl esters.It is to be noted that verified, that does not add enzyme batch does not almost form 2-L-aspartyl-L-phenylalanine-β-methyl esters.

In addition, the 10 μ l Mono S component enzymes (about 20U/ml) that will obtain in embodiment 2 add 190 μ l and contain in the borate buffer solution (pH9.0) of each 105.3mM hydrochloric acid L-aspartic acid-α-methyl esters and hydrochloric acid L-aspartic acid-beta-methyl esters, 210.5mM L-phenylalanine and 10.51mM EDTA, react in 20 ℃.As a result, do not observe the corresponding peptide of formation.

Table 3

Reaction times (minute)	α-the AMP (mM) that produces
Reaction times (minute)	α-the AMP (mM) that produces	30 60 120	23.0 42.1 61.7

Embodiment 4

The purifying of the enzyme of Sphingobacterium kind (Sphingobacterium sp.)

Use substratum shown in the embodiment 2, in the mode identical with embodiment 2, (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to cultivate Sphingobacterium kind (Sphingobacterium sp.) bacterial strain FERM BP-8124, preservation mechanism address: Chuo Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation day: on July 22nd, 2002).Step after the centrifugation or in carrying out on ice or in 4 ℃.Centrifugal (10,000rpm, 15 minutes) gained nutrient solution is collected microorganism cells.Behind 20mM Tris-HCl damping fluid (pH7.6) cleaning 2g microorganism cells, it is suspended in the 8ml same buffer, carry out 45 minutes ultrasonic disruptions with 195W and handle.Centrifugal then (10,000rpm, 30 minutes) this ultrasonic disruption liquid to remove broken cell debris, obtains the supernatant liquor of ultrasonic disruption liquid.The supernatant liquor that makes this ultrasonic disruption liquid is then removed indissolvable component by ultracentrifugation (50,000rpm, 30 minutes) to 20mM Tris-HCl damping fluid (pH7.6) dialysed overnight, obtains the soluble constituent into the supernatant liquor form.The gained soluble constituent is splined on the Q-SepharoseHP post (Amersham production) of using Tris-HCl damping fluid (pH7.6) pre-equilibration, by not collecting active ingredient in the adsorbed components.This active ingredient to 20mM acetate buffer (pH5.0) dialysed overnight, is then removed indissolvable component by centrifugation (10,000rpm, 30 minutes), obtain dialysis component into the supernatant liquor form.Then this dialysis component is splined on the SP-Sepharose HP post (Amersham production) of usefulness 20mM acetate buffer (pH5.0) pre-equilibration, to obtain active ingredient, wherein enzyme usefulness contains the linear concentration gradient wash-out of 0-1M NaCl same buffer.

Embodiment 5

Use Sphingobacterium kind (Sphingobacterium sp.) enzyme component to produce 2-L-aspartyl-L-phenylalanine-β-methyl esters and 2-L-aspartyl-L-phenylalanine-β-ethyl ester

Producing 2-L-aspartyl-L-phenylalanine-β-methyl esters (during α-AMP), the 15 μ l SP-Sephrose HP component concentrated solutions (about 15U/ml) that will obtain in embodiment 4 join 185 μ l and contain 108.1mM hydrochloric acid L-aspartic acid-α, in the borate buffer solution (pH9.0) of senecioate, 216.2mM L-phenylalanine and 10.8mM EDTA, react in 20 ℃.Equally, producing 2-L-aspartyl-L-phenylalanine-β-ethyl ester (during α-AEP), the 10 μ l SP-Sephrose HP component concentrated solutions (about 15U/ml) that will obtain in embodiment 4 join 190 μ l and contain 52.6mM hydrochloric acid L-aspartic acid-α, in the borate buffer solution (pH9.0) of β-diethyl ester, 105.2mM L-phenylalanine and 10.8mM EDTA, react in 20 ℃.The forming process of AMP or AEP is shown in table 4.It is to be noted, verified, batch almost do not form AMP or AEP what add enzyme.For the formation of AEP, describe with AMP numerical value that standard substance obtain.

Table 4

Reaction times (minute)	α-the AMP (mM) that produces	α-the AEP (mM) that produces
Reaction times (minute)	α-the AMP (mM) that produces	α-the AEP (mM) that produces	30	25.8	7.5
60	40.7	13.3	30	25.8	7.5
60	40.7	13.3	120	56.0	20.6
180	61.8	-	120	56.0	20.6

Embodiment 6

Derive from the separation of the one-tenth peptidase genes of short steady bacillus (Empedobacter brevis)

Hereinafter will be elaborated into the separation of peptidase genes.(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to use short steady bacillus (Empedobacterbrevis) bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, 2002) as microorganism.During isolated genes, intestinal bacteria JM-109 is as the host, and pUC118 is as carrier.

(1) produces the PCR primer according to the internal amino acid sequence of determining

Origin comes from short steady bacillus (Empedobacter brevis) bacterial strain FERM BP-8113, and (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 thing shifts day: one-tenth peptase lysyl endopeptidase digestion product on July 8th, 2002) is determined aminoacid sequence (SEQ ID NO:1 and 2) with the Edman edman degradation Edman, produces respectively to have the mix primer of base sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.

(2) acquisition of microorganism cells

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center will to lack steady bacillus (Empedobacter brevis) bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, 2002) (contain 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l NaCl and 20g/l agar, pH7.0) go up in 30 ℃ and cultivated 24 hours at the CM2G nutrient agar.One loopful gained microorganism cells is inoculated the 500mlSakaguchi that contains 50ml CM2G liquid nutrient medium (the aforementioned substratum that does not contain agar) shake in the bottle, in 30 ℃ of shaking culture.

(3) obtain chromosomal DNA from microorganism cells

Centrifugal (12,000rpm, 4 ℃, 15 minutes) 50ml nutrient solution is collected microorganism cells.Then, use QIAGEN Genomic-Tip System (QIAGEN),, obtain chromosomal DNA by microorganism cells according to the step of describing in its specification sheets.

(4) obtain to contain the dna fragmentation that part becomes peptidase genes by PCR

Use LA-Taq (Takara Shuzo production), (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to obtain to contain short steady bacillus (Empedobacter brevis) the bacterial strain FERM of part BP-8113 by the PCR method, preservation mechanism address: ChuoDai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, the 2002) dna fragmentation of Lai Yuan one-tenth peptidase genes.Use the primer of base sequence then with SEQ ID NO:3 and 4, to (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center by short steady bacillus (Empedobacter brevis) bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, 2002) chromosomal DNA of Huo Deing carries out the PCR reaction.

Under the following conditions, use Takara PCR thermal cycler PERSONAL (TakaraShuzo production) to carry out 30 round-robin PCR reactions.

94 ℃ 30 seconds

52 ℃ 1 minute

72 ℃ 1 minute

After reaction is finished, be splined on 0.8% agarose electrophoresis with 3 μ l reaction solutions.The result confirms the dna fragmentation amplification of about 1.5 kilobase (kb).

(5) clone into peptidase genes by gene library

Become peptidase genes in order to obtain total length, use the dna fragmentation that in the PCR step, increases to carry out DNA hybridization as probe.The step of DNA hybridization is set forth in molecular cloning, and the 2nd edition, Cold Spring Harbor Press (1989).

The about 1.5kb dna fragmentation that separates the amplification of PCR step by 0.8% agarose electrophoresis.Cut out target stripe and purifying then.Use DIG High Prime (Boehringer-Mannheim production), according to the method for the use DIG High Prime (Boehringer-Mannheim production) that describes in its specification sheets, with this dna fragmentation of probe digoxin (digoxinigen) mark.

By reacting the steady bacillus of weak point (Empedobacter brevis) chromosomal DNA that the complete digestion embodiment of the invention 6 steps (3) obtain, product electrophoresis on 0.8% sepharose afterwards 16 hours in 37 ℃ with restriction enzyme HindIII.Behind the electrophoresis, electrophoretic chromosomal DNA is transferred on the positively charged nylon leaching film (Roche Diagnostics production) by sepharose, then comprises alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, adds the digoxin labelled probe of preparation as mentioned above in 50 ℃ of prehybridizations, and in 50 ℃ of hybridization 16 hours.Subsequently, with the 2 * SSC that contains 0.1%SDS in room temperature rinsing filter membrane 20 minutes.In addition, again twice usefulness 0.1 * SSC of containing 0.1%SDS in 65 ℃ of rinsing filter membranes 15 minutes.

Use DIG Nucleotide detection kit (Boehringer-Mannheim production), use step, detect band with described probe hybridization according to the DIG Nucleotide detection kit of describing in its specification sheets (Boehringer-Mannheim production).The result detects and can be approximately 4kb with the band of described probe hybridization.

Then, the 5 μ g chromosomal DNAs that prepare with the HindIII complete digestion embodiment of the invention 6 steps (3).DNA by the about 4kb of 0.8% agarose electrophoresis separation then uses Gene Clean II test kit (Funakoshi production) purify DNA, and DNA is dissolved among the 10 μ lTE.Then 4 these products of μ l are mixed with pUC118 HindIII/BAP (Takara Shuzo production), use 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), with transformed into escherichia coli.Then it is coated on the suitable solid medium and creates chromosomal dna library.

Become peptidase genes for obtaining complete total length, use aforementioned probe to screen chromosomal dna library by colony hybridization.The step of colony hybridization is set forth in molecular cloning, and the 2nd edition, ColdSpring Harbor Press (1989).

The colony lift of chromosomal dna library to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization) (Roche Diagnostics production), is then comprised alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, adds aforementioned digoxin labelled probe in 37 ℃ of prehybridizations, then in 50 ℃ of hybridization 16 hours.Subsequently, with the 2 * SSC that contains 0.1%SDS in room temperature rinsing filter membrane 20 minutes.In addition, again twice usefulness 0.1 * SSC of containing 0.1%SDS in 65 ℃ of rinsing filter membranes 15 minutes.

Use DIG Nucleotide detection kit (Boehringer-Mannheim production),, detect bacterium colony with described label probe hybridization according to the DIG Nucleotide detection kit of describing in its specification sheets (Boehringer-Mannheim production) operation instruction.The result confirms to have in the bacterium colony two bacterial strains and label probe hybridization.

(6) lack the base sequence of the one-tenth peptidase genes in steady bacillus (Empedobacter brevis) source

Use Wizard Plus Minipreps dna purification system (Promega production), two strain microorganism cellss by above-mentioned confirmation and label probe hybridization prepare the plasmid that e. coli jm109 has, and the part and near the base sequence of mensuration and described probe generation hybridization.Use CEQ DTCS-Quick Start test kit (Beckman-Coulter production), carry out sequencing reaction according to the method that its specification sheets is described.In addition, use CEQ 2000-XL (Beckman-Coulter production) to carry out electrophoresis.

The result confirms that the proteic open reading-frame (ORF) that coding contains into peptase internal amino acid sequence (SEQ ID NO:1 and 2) exists really, therefore confirms that this open reading-frame (ORF) is into the encoding gene of peptase.Total length becomes the base sequence of peptidase genes and the SEQ ID NO:5 that corresponding amino acid sequence is shown in sequence table.The homology of the open reading-frame (ORF) that obtains with the BLASTP programanalysis; found that and two kinds of enzyme homologies: the alpha-Amino acid ester hydrolase that is presented on the amino acid sequence level with Acetobacter pasteurianus (Acetobacter pasteurianus) has 34% homology (referring to Appl.Environ.Microbiol.; 68 (1); 211-218 (2002)); glutaryl-7ACA acylase with side spore bacillus brevis (Brevibacillus laterosporum) on amino acid sequence level has 26% homology (referring to J.Bacteriol.; 173 (24), 7848-7855 (1991)).

(7) the one-tenth peptidase genes of originating the short steady bacillus of expression in escherichia coli (Empedobacter brevis)

Use the oligonucleotide of listing among the SEQ ID NO:7 and 8 as primer, by the target gene district on the promoter region that carries out trp operon on the pcr amplification intestinal bacteria W3110 chromosomal DNA, the dna fragmentation that obtains is connected to pGEM-Teasy carrier (Promega production).Transform e. coli jm109 then in this connection solution, by the bacterial strain of selecting to have target plasmid in the amicillin resistance bacterial strain, the direction of insertion of the trp promotor of inserting in the described target plasmid is opposite with the direction of lac promotor.Next step handles the dna fragmentation that this plasmid obtains to contain the trp promotor with EcoO109I/EcoRI, and the EcoO109I/EcoRI that is connected to pUC19 (Takara production) handles product.Connect solution transformed into escherichia coli JM109 with this then, by those bacterial strains of selecting to have target plasmid in the amicillin resistance bacterial strain.Next step will handle dna fragmentation that plasmid obtains and be connected by the dna fragmentation that contains the rmB terminator of handling pKK223-3 (Amersham Pharmacia production) acquisition with HindIII/HincII with HindIII/PvuII.Connect damping fluid transformed into escherichia coli JM109 with this then, by the bacterial strain of selecting to have target plasmid in the amicillin resistance bacterial strain, with 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 Dai-6,1-1Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: chromosomal DNA on July 8th, 2002) is as template, the oligonucleotide that SEQ ID NO:9 and 10 lists is as primer, by the pcr amplification target gene.Handle this dna fragmentation with NdeI/PstI then, the dna fragmentation that obtains is handled product with the NdeI/PstI of pTrpT be connected.Transform solution transformed into escherichia coli JM109 with this then, by the bacterial strain of selecting to have target plasmid in the amicillin resistance bacterial strain, with this plasmid called after pTrpT_Gtg2.

The e. coli jm109 that will have a pTrpT_Gtg2 in containing the LB substratum of 100mg/l penbritin in 30 ℃ of inoculation culture 24 hours.1ml gained nutrient solution is seeded in the 500ml Sakaguchi that 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 and 100mg/l penbritin) shakes in the bottle, then cultivated 24 hours in 25 ℃.Alpha-L-aspartyl-the phenylalanine of nutrient solution-β-methyl esters forms the active 0.11U/ml that is, confirms the escherichia coli expression clone gene.And the transformant that only imports pTrpT does not in contrast detect activity.

The prediction of signal sequence

Using Signal Pv 1.1 programs (referring to Protein Engineering, Vol.12, No.1, the 3-9 page or leaf, 1999) during the described aminoacid sequence of analytical sequence table SEQ ID NO:6, predicted amino acid ordinal number 1-22 works the signal effect that is secreted in the pericentral siphon, and estimates that maturation protein is positioned at the downstream of amino acid ordinal number 23.

Excretory confirms

The e. coli jm109 that will have a pTrpT_Gtg2 in containing the LB substratum of 100mg/l penbritin in 30 ℃ of inoculation culture 24 hours.1ml gained nutrient solution is seeded in the 500ml Sakaguchi that 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 and 100mg/l penbritin) to be shaken in the bottle, then in 25 ℃ of cultivations 24 hours, to obtain the cultured microorganism cell.

Use the 20g/dl sucrose solution cultured microorganism cell grade to be become pericentral siphon component and cytoplasm fraction by the osmotic shock method.The microorganism cells that immerses in the 20g/dl sucrose solution is immersed 5mM MgSO ₄In the aqueous solution.The centrifugal supernatant liquor is called pericentral siphon component (" Pe ").In addition, the centrifugal pellet resuspended, and carry out ultrasonic disruption.Product is called cytoplasm fraction (" Cy ").The known zwischenferment that is present in the tenuigenin is active in indicator, confirm that tenuigenin separates.By (containing 1mM glucose 6-phosphoric acid, 0.4mM NADP, 10mM MgSO to reaction soln ₄With 50mM Tris-Cl (pH8)) in add appropriate amount enzyme carry out this detections in 30 ℃, the then generation by detection 340nm absorbance detection NADPH.

When the activity of the cell-free extract that will prepare separately was decided to be 100%, Fig. 1 had shown the enzyme amount in pericentral siphon component and the cytoplasm fraction.The pericentral siphon component is not sneaked into the zwischenferment activity, and this shows that the pericentral siphon component not mix with cytoplasm fraction.The pericentral siphon component reclaims 2-L-aspartyl-L-phenylalanine-β-methyl esters of about 60%, and (that α-AMP) forms is active, confirms as using Signal Pv1.1 program to predict that by aminoacid sequence Ala-Gln-forms enzyme secretion in pericentral siphon.

Embodiment 7

The separation of the one-tenth peptidase genes in Sphingobacterium kind (Sphingobacterium sp.) source

Hereinafter will be elaborated into the separation of peptidase genes.(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to use Sphingobacterium kind (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 day: on July 22nd, 2002) as microorganism.During isolated genes, bacillus coli DH 5 alpha is as the host, and pUC118 is as carrier.

(1) acquisition of microorganism cells

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center with Sphingobacterium kind (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 day: on July 22nd, 2002) (contain 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l NaCl and 20g/l agar, pH7.0) go up and cultivated 24 hours in 25 ℃ at the CM2G nutrient agar.One loopful gained microorganism cells is inoculated the 500ml Sakaguchi that contains 50ml CM2G liquid nutrient medium (the aforementioned substratum that does not contain agar) shake in the bottle, then in 25 ℃ of shaking culture.

(2) acquisition of chromosomal DNA in the microorganism cells

(3) obtain probe dna fragment by PCR

Use LA-Taq (Takara Shuzo production), (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to obtain to contain short steady bacillus (Empedobacter brevis) the bacterial strain FERM of part BP-8113 by the PCR method, preservation mechanism address: ChuoDai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, the 2002) dna fragmentation of Lai Yuan one-tenth peptidase genes.Use primer then with SEQ ID NO:3 and 4 base sequences, to (preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center by short steady bacillus (Empedobacter brevis) bacterial strain FERM BP-8113, preservation mechanism address: Chuo Dai-6,1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international preservation thing shifts day: on July 8th, 2002) chromosomal DNA of Huo Deing carries out the PCR reaction.

Under the following conditions, use Takara PCR thermal cycler-PERSONAL (TakaraShuzo) to carry out 30 round-robin PCR reactions.

94 ℃ 30 seconds

52 ℃ 1 minute

72 ℃ 1 minute

After reaction is finished, with 0.8% agarose electrophoresis on the 3 μ l reaction solutions.Result's confirm to increase dna fragmentation of about 1.5kb.

(4) clone into peptidase genes by gene library

Become peptidase genes in order to obtain total length, use the dna fragmentation that in aforementioned PCR step, increases to carry out DNA hybridization as probe.The operation of DNA hybridization is set forth in molecular cloning, and the 2nd edition, Cold Spring Harbor Press (1989).

The about 1.5kb dna fragmentation that separates aforementioned PCR step amplification by 0.8% agarose electrophoresis.Cut out target stripe and purifying then.Use DIG High Prime (Boehringer-Mannheim production),, use this dna fragmentation of probe digoxigenin labeled according to the method that its specification sheets is described.

Sphingobacterium kind (Sphingobacteriumsp.) chromosomal DNA that the embodiment of the invention 7 steps (2) obtain and restriction enzyme SacI were reacted 16 hours in 37 ℃, with the described DNA of complete digestion, product electrophoresis on 0.8% sepharose afterwards.The sepharose of electrophoretic chromosomal DNA after by electrophoresis is transferred on the positively charged nylon leaching film (Roche Diagnostics production), then comprises alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, added the digoxin labelled probe of preparation as mentioned above in 37 ℃ of prehybridizations, in 37 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim),, detect band with described probe hybridization according to the step of describing in its specification sheets.The result successfully detects the band that is approximately 3kb with described probe hybridization.

Chromosomal DNA with the preparation of the SacI complete digestion 5 μ g embodiment of the invention 7 steps (2).DNA by the about 3kb of 0.8% agarose gel electrophoresis separation uses Gene CleanII test kit (Funakoshi production) purify DNA, and DNA is dissolved among the 10 μ l TE.Then 4 μ l gained solution are mixed with pUC118, use dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation, before mixing, handled 30 minutes in 37 ℃ wherein said pUC118 with alkaline phosphatase (intestinal bacteria C75), in 50 ℃ of processing 30 minutes, react 16 hours with complete digestion in 37 ℃ again with SacI.Mix this ligation liquid of 5 μ l and 100 μ l bacillus coli DH 5 alpha competent cells (Takara Shuzo production), with transformed into escherichia coli.Then it is coated on the suitable solid medium and creates chromosomal dna library.

Become peptidase genes for obtaining total length, use aforementioned probe to screen chromosomal dna library by colony hybridization.The step of colony hybridization is set forth in molecular cloning, and the 2nd edition, Cold SpringHarbor Press (1989).

The colony lift of chromosomal dna library to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization, Roche Diagnostics produces), is then comprised alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, adds aforementioned digoxin labelled probe in 37 ℃ of prehybridizations, then in 37 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim production),, detect bacterium colony with described label probe hybridization according to the operation instruction of describing in its specification sheets.The result confirms to have in the bacterium colony six bacterial strains and label probe hybridization.

(5) base sequence of the one-tenth peptidase genes in Sphingobacterium kind (Sphingobacterium sp.) source

Use Wizard Plus Minipreps dna purification system (Promega production), by confirming that six strain microorganism cellss with label probe hybridization prepare the plasmid that bacillus coli DH 5 alpha has, the part and near the base sequence of hybridization takes place with definite and described probe.Use CEQDTCS-Quick Start test kit (Beckman-Coulter production), carry out sequencing reaction according to the method that its specification sheets is described.In addition, use CEQ 2000-XL (Beckman-Coulter production) to carry out electrophoresis.

The result discloses the open reading-frame (ORF) that is encoded into peptase and exists really.The total length base sequence and the corresponding amino acid sequence of the one-tenth peptidase genes in Sphingobacterium kind (Sphingobacterium sp.) source are shown in SEQ ID NO:11.The one-tenth peptase in Sphingobacterium kind (Sphingobacterium sp.) source on amino acid sequence level with short steady bacillus (Empedobacter brevis) source become peptase 63.5% homology (using the BLASTP program determination).

(6) expression of one-tenth peptidase genes in intestinal bacteria in Sphingobacterium kind (Sphingobacterium sp.) source

(preservation mechanism: Independent Administrative Leged Industrial Technology Complex Inst speciallys permit biological sustenance center to use Sphingobacterium kind (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 day: chromosomal DNA on July 22nd, 2002) is as template, use the oligonucleotide of listing among the SEQ ID NO:13 and 14 as primer, by the pcr amplification 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 solution transformed into escherichia coli JM109 with this then, by the bacterial strain of selecting to have target plasmid in the amicillin resistance bacterial strain.With this plasmid called after pTrpT_Sm_aet.

The e. coli jm109 that one loopful is had pTrpT_Sm_aet is inoculated in the common test tube that contains 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 and 100mg/l penbritin), cultivates 20 hours in 25 ℃.It is 0.53U/ml that the α of nutrient solution-AMP produces activity, confirms the escherichia coli expression clone gene.And the transformant that only contains pTrpT does not in contrast detect activity.

The prediction of signal sequence

Using Signal Pv1.1 program (Protein Engineering, Vol.12, No.1, the 3-9 page or leaf, 1999) during the aminoacid sequence of the described SEQ ID of analytical sequence table NO:12, predicted amino acid ordinal number 1-20 works the signal effect that is secreted in the pericentral siphon, and estimates that maturation protein is positioned at the downstream of amino acid ordinal number 21.

The confirmation of signal sequence

The e. coli jm109 that one loopful is had pTrpT_Sm_aet is inoculated in the common test tube that 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 and 100mg/l penbritin), in 25 ℃ of main cultivations 20 hours.

Hereinafter, the step after the centrifugation or in carrying out on ice or in 4 ℃.After cultivation is finished, microorganism cells is separated with nutrient solution, clean, be suspended in the same buffer then with 100mM phosphate buffered saline buffer (pH7) by centrifugal.Then, with 195W microorganism cells is carried out 20 minutes ultrasonic disruptions and handle, centrifugal (12,000rpm, 30 minutes) ultrasonic disruption liquid to remove broken cell debris, obtains soluble constituent.The gained soluble constituent is splined on the CHT-II post (Biorad production) of using 100mM phosphate buffered saline buffer (pH7) pre-equilibration, uses the 500mM phosphate buffered saline buffer with linear concentration gradient wash-out enzyme.Active ingredient is mixed with the 2M ammonium sulfate and the 100mM phosphate buffered saline buffer of 5 times of volumes, gained solution is splined on the Resource-PHE post (Amersham production) with 2M ammonium sulfate and 100mM phosphate buffered saline buffer pre-equilibration, with linear concentration gradient wash-out enzyme, obtain active component solution with 2-0M ammonium sulfate.Confirm that the described one-tenth peptase that these steps are produced is that the electrophoresis homogeneous is pure.

When using the Edman edman degradation Edman to measure the aminoacid sequence of aforementioned one-tenth peptase, obtain the aminoacid sequence of SEQID NO:15, confirm as using Signal Pv1.1 program to be predicted that maturation protein is positioned at the downstream of amino acid ordinal number 21.

Embodiment 8

Separate the separation of the one-tenth peptidase genes in Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 sources

Hereinafter will be elaborated into the separation of peptidase genes.The microorganism of using be separate Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan).During isolated genes, e. coli jm109 is as the host, and pUC118 is as carrier.

(1) acquisition of microorganism cells

To separate Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka 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/lNaCl and 20g/l agar, pH7.0) go up and cultivated 24 hours at the CM2G nutrient agar in 25 ℃.One loopful gained microorganism cells is inoculated the 500ml Sakaguchi that contains 50mlCM2G liquid nutrient medium (the aforementioned substratum that does not contain agar) shake in the bottle, then in 25 ℃ of shaking culture.

(2) acquisition of chromosomal DNA in the microorganism cells

Centrifugal 50ml nutrient solution (12,000rpm, 4 ℃, 15 minutes) is collected microorganism cells.Then, use QIAGEN Genomic-Tip System (QIAGEN),, obtain chromosomal DNA by microorganism cells according to the step of describing in its specification sheets.

(3) obtain probe dna fragment by PCR

Use LA-Taq (Takara Shuzo production), obtain to contain part by the PCR method and separate Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) dna fragmentation of Lai Yuan one-tenth peptidase genes.Use primer then with SEQ ID NO:15 and 16 base sequences, to by separating Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute, preservation mechanism address: 2-17-85Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) chromosomal DNA of Huo Deing carries out the PCR reaction.Separate the about 1kb dna fragmentation that increases by the PCR method by 0.8% agarose electrophoresis.Cut out target stripe and purifying then.Use DIG High Prime (Boehringer-Mannheim production),, use probe digoxigenin labeled dna fragmentation according to the method that its specification sheets is described.

(4) clone into peptidase genes by gene library

Become peptidase genes for obtaining total length, use the dna fragmentation that in aforementioned PCR step, increases to carry out DNA hybridization as probe.The operation of DNA hybridization is set forth in molecular cloning, and the 2nd edition, Cold Spring Harbor Press (1989).

Make and separate Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute, preservation mechanism address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) chromosomal DNA and restriction enzyme HindIII were in 37 ℃ of reactions 16 hours, with the described DNA of complete digestion, product electrophoresis on 0.8% sepharose afterwards.The sepharose of electrophoretic chromosomal DNA after by electrophoresis is transferred on the positively charged nylon leaching film (Roche Diagnostics production), then comprises alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, added the digoxin labelled probe of preparation as mentioned above in 50 ℃ of prehybridizations, in 50 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim),, detect band with described probe hybridization according to the step of describing in its specification sheets.The band that the result successfully detects with described probe hybridization is approximately 5kb.

Separate Vitrum AB soil bacillus (Pedobacterheparinus) IFO 12017 chromosomal DNAs with HindIII complete digestion 5 μ g.DNA by the about 5kb of 0.8% agarose gel electrophoresis separation uses Gene Clean II test kit (Funakoshi production) purify DNA, and DNA is dissolved among the 10 μ l TE.Then 4 μ l gained solution are mixed with pUC118HindIII/BAP, use dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l escherichia coli jm109 competent cells (Takara Shuzo production), with transformed into escherichia coli.Then it is coated on the suitable solid medium and creates chromosomal dna library.

The colony lift of chromosomal dna library to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization, Roche Diagnostics produces), is then carried out alkaline denaturation, neutralization and immobilization and handles.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, adds aforementioned digoxin labelled probe in 37 ℃ of prehybridizations, then in 37 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim production),, detect bacterium colony with described label probe hybridization according to the operation instruction of describing in its specification sheets.The result has observed its bacterium colony of bacterial strain and label probe hybridization.

(5) separate the base sequence of the one-tenth peptidase genes in Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 source

Prepare the plasmid that e. coli jm109 has by the bacterium colony that confirms and label probe is hybridized, and the part and near the base sequence of mensuration and described probe generation hybridization.Use CEQDTCS-Quick Start test kit (Beckman-Coulter production), carry out sequencing reaction according to the method that its specification sheets is described.In addition, use CEQ 2000-XL (Beckman-Coulter production) to carry out electrophoresis.

The result discloses the open reading-frame (ORF) that is encoded into peptase and exists really.Separate Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute; 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) the total length base sequence of Lai Yuan one-tenth peptidase genes and corresponding amino acid sequence are shown in SEQ ID NO:17.

Embodiment 9

Separate the expression of one-tenth peptidase genes in intestinal bacteria in Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 sources

Vitrum AB soil bacillus (Pedobacter heparinus) IFO 12017 (preservation mechanism: Osaka fermentation research institute is separated in use; 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, chromosomal DNA Japan) are as template, and the oligonucleotide that SEQ ID NO:19 and 20 lists is as primer, by the pcr amplification target gene.Handle this dna fragmentation with NdeI/HindIII, the dna fragmentation that obtains is handled product with the NdeI/HindIII of pTrpT be connected.Transform solution transformed into escherichia coli JM109 with this then, by the bacterial strain of selecting to have target plasmid in the amicillin resistance bacterial strain.This plasmid called after pTrpT_Ph_aet.

The e. coli jm109 that one loopful is had pTrpT_Ph_aet is inoculated in the common test tube that contains 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 and 100mg/l penbritin), in 25 ℃ of main cultivations 20 hours.It is 0.01U/ml that the α of nutrient solution-AMP produces activity, has confirmed the escherichia coli expression clone gene thus.And the transformant that only imports pTrpT does not in contrast detect activity.

Embodiment 10

The separation of the one-tenth peptidase genes in shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 sources

Hereinafter will be elaborated into the separation of peptidase genes.The microorganism of using is shaping bacillus (Taxeobacter gelupurpurascens) DSMZ a 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany).During isolated genes, e. coli jm109 is as the host, and pUC118 is as carrier.

(1) acquisition of microorganism cells

With shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: MascheroderWeg 1b, 38124 Braunschweig, Germany) (contain 50g/l glucose, 10g/l yeast extract, 10g/l peptone, 5g/l NaCl and 20g/l agar, pH7.0) go up and cultivated 24 hours at the CM2G nutrient agar in 25 ℃.One loopful gained microorganism cells is inoculated the 500ml Sakaguchi that contains 50ml CM2G liquid nutrient medium (the aforementioned substratum that does not contain agar) shake in the bottle, then in 25 ℃ of shaking culture.

(2) acquisition of chromosomal DNA in the microorganism cells

(3) obtain probe dna fragment by PCR

Use LA-Taq (Takara Shuzo production), obtain to contain part shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center by the PCR method, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) dna fragmentation of Lai Yuan one-tenth peptidase genes.Use primer then with SEQ ID NO:21 and 16 base sequences, to by shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124Braunschweig, Germany) chromosomal DNA of Huo Deing carries out the PCR reaction.Separate the about 1kb dna fragmentation that increases by the PCR method by 0.8% agarose electrophoresis.Cut out target stripe and purifying then.Use DIG High Prime (Boehringer-Mannheim production),, use probe digoxigenin labeled dna fragmentation according to the method that its specification sheets is described.

(4) clone into peptidase genes by gene library

Make shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanism: German microorganism and cell culture preservation center, preservation mechanism address: MascheroderWeg 1b, 38124 Braunschweig, Germany) chromosomal DNA and restriction enzyme PstI were in 37 ℃ of reactions 16 hours, with the described DNA of complete digestion, product electrophoresis on 0.8% sepharose afterwards.The sepharose of electrophoretic chromosomal DNA after by electrophoresis is transferred on the positively charged nylon leaching film (Roche Diagnostics production), then comprises alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, added the digoxin labelled probe of preparation as mentioned above in 50 ℃ of prehybridizations, in 50 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim),, detect band with described probe hybridization according to the step of describing in its specification sheets.The result successfully detects the band that is approximately 5kb with described probe hybridization.

With PstI complete digestion 5 μ g shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 (preservation mechanisms: German microorganism and cell culture preservation center, preservation mechanism address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) chromosomal DNA.DNA by the about 5kb of 0.8% agarose gel electrophoresis separation uses GeneClean II test kit (Funakoshi production) purify DNA, and DNA is dissolved among the 10 μ l TE.Then 4 μ l gained solution are mixed with pUC118 PstI/BAP (Takara Shuzo production), use dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l escherichia coli jm109 competent cells (TakaraShuzo production), with transformed into escherichia coli.Then it is coated on the suitable solid medium and creates chromosomal dna library.

The colony lift of chromosomal dna library to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization) (Roche Diagnostics production), is then carried out alkaline denaturation, neutralization and immobilization and handles.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, adds aforementioned digoxin labelled probe in 37 ℃ of prehybridizations, then in 37 ℃ of hybridization 16 hours.Subsequently, with containing 1 * SSC of 0.1%SDS in 60 ℃ of rinsing filter membranes.

(5) base sequence of the one-tenth peptidase genes in shaping bacillus (Taxeobacter gelupurpurascens) DSMZ 11116 sources

Prepare the plasmid that e. coli jm109 has by the bacterial strain that confirms and label probe is hybridized, and the part and near the base sequence of mensuration and described probe generation hybridization.Use CEQDTCS-Quick Start test kit (Beckman-Coulter production), carry out sequencing reaction according to the method that its specification sheets is described.In addition, use CEQ 2000-XL (Beckman-Coulter production) to carry out electrophoresis.

The result discloses the open reading-frame (ORF) that is encoded into peptase and exists really.The total length base sequence and the corresponding amino acid sequence of the one-tenth peptidase genes in shaping bacillus (Taxeobactergelupurpurascens) DSMZ 11116 sources are shown in SEQ ID NO:22.

Embodiment 11

The separation of the one-tenth peptidase genes in round bacteria (Cyclobacterium marinum) ATCC 25205 sources, sea

Hereinafter will be elaborated into the separation of peptidase genes.The microorganism of using is extra large round bacteria (Cyclobacterium marinum) ATCC a 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America).During isolated genes, e. coli jm109 is as the host, and pUC118 is as carrier.

(1) acquisition of microorganism cells

With extra large round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: 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 NaCl and 20g/l agar, pH7.0) go up and cultivated 24 hours at the CM2G nutrient agar in 25 ℃.One loopful gained microorganism cells is inoculated the 500ml Sakaguchi that contains 50ml CM2G liquid nutrient medium (the aforementioned substratum that does not contain agar) shake in the bottle, then in 25 ℃ of shaking culture.

(2) acquisition of chromosomal DNA in the microorganism cells

(3) obtain probe dna fragment by PCR

Use LA-Taq (Takara Shuzo production), contain the dna fragmentation of the one-tenth peptidase genes in round bacteria (Cyclobacterium marinum) ATCC 25205 sources, part sea by the acquisition of PCR method.Use primer then with SEQ ID NO:15 and 16 base sequences, to by extra large round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the United States of America) chromosomal DNA that obtains carries out the PCR reaction.Separate the about 1kb dna fragmentation that increases by the PCR method by 0.8% agarose electrophoresis.Cut out target stripe and purifying then.Use DIG High Prime (Boehringer-Mannheim production),, use probe digoxigenin labeled dna fragmentation according to the method that its specification sheets is described.

(4) clone into peptidase genes by gene library

Make extra large round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) chromosomal DNA and restriction enzyme PstI or HincII were in 37 ℃ of reactions 16 hours, with the described DNA of complete digestion, product electrophoresis on 0.8% sepharose afterwards.The sepharose of electrophoretic chromosomal DNA after by electrophoresis is transferred on the positively charged nylon leaching film (Roche Diagnostics production), then comprises alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, added the digoxin labelled probe of preparation as mentioned above in 50 ℃ of prehybridizations, in 50 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim),, detect band with described probe hybridization according to the step of describing in its specification sheets.The result successfully detects in the PstI digestion product 7kb band with described probe hybridization, and successfully detects in the HincII digestion product 2kb band with described probe hybridization.

With PstI or HincII complete digestion 5 μ g sea round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) chromosomal DNA.The DNA that separates about 7kb or 2kb by 0.8% agarose gel electrophoresis.Use Gene Clean II test kit (Funakoshi production) purify DNA, DNA is dissolved among the 10 μ l TE.4 μ l gained solution are mixed with pUC118 PstI/BAP (Takara Shuzo production) or pUC118 HincII/BAP (Takara Shuzo production), use dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l escherichia coli jm109 competent cells (Takara Shuzo production), with transformed into escherichia coli.Then it is coated on the suitable solid medium and creates chromosomal dna library.

The colony lift of chromosomal dna library to nylon leaching film (nylon membrane that is used for bacterium colony and plaque hybridization) (Roche Diagnostics production), is then carried out alkaline denaturation, neutralization and immobilization and handles.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, adds aforementioned digoxin labelled probe in 37 ℃ of prehybridizations, then in 37 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim production),, detect bacterium colony with described label probe hybridization according to the operation instruction of describing in its specification sheets.The result observes respectively its bacterium colony of bacterial strain and label probe hybridization.

(5) base sequence of the one-tenth peptidase genes in extra large round bacteria (Cyclobacterium marinum) ATCC 25205 sources

Prepare the plasmid that e. coli jm109 has by each bacterial strain that confirms and label probe is hybridized, and the part and near the base sequence of mensuration and described probe generation hybridization.Use CEQ DTCS-Quick Start test kit (Beckman-Coulter production), carry out sequencing reaction according to the method that its specification sheets is described.In addition, use CEQ 2000-XL (Beckman-Coulter production) to carry out electrophoresis.

The result discloses the open reading-frame (ORF) that is encoded into peptase and exists really.Sea round bacteria (Cyclobacterium marinum) ATCC 25205 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America) the total length base sequence and the corresponding amino acid sequence of one-tenth peptidase genes in source be shown in SEQ ID NO:24.

Embodiment 12

The separation of the one-tenth peptidase genes in the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 sources

Hereinafter will be elaborated into the separation of peptidase genes.The microorganism of using is the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC a 700359 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America).During isolated genes, e. coli jm109 is as the host, and pUC118 is as carrier.

(1) acquisition of microorganism cells

With the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: 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 NaCl and 20g/l agar, pH7.0) go up and cultivated 24 hours at the CM2G nutrient agar in 25 ℃.One loopful gained microorganism cells is inoculated the 500ml Sakaguchi that contains 50ml CM2G liquid nutrient medium (the aforementioned substratum that does not contain agar) shake in the bottle, then in 10 ℃ of shaking culture.

(2) acquisition of chromosomal DNA in the microorganism cells

(3) obtain probe dna fragment by PCR

Use LA-Taq (Takara Shuzo production), obtain to contain the cold snake bacterium in part Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection by the PCR method, preservation mechanism address: P.O.Box 1549, Manassas, VA20110, the United States of America) dna fragmentation of the one-tenth peptidase genes in source.Use primer then with SEQ ID NO:15 and 16 base sequences, to by the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, the United States of America) chromosomal DNA that obtains carries out the PCR reaction.Separate the about 1kb dna fragmentation that increases by the PCR method by 0.8% agarose electrophoresis.Cut out target stripe and purifying then.Use DIG High Prime (Boehringer-Mannheim production),, use probe digoxigenin labeled dna fragmentation according to the method that its specification sheets is described.

(4) clone into peptidase genes by gene library

Make the chromosomal DNA of the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 and limiting enzyme EcoRI in 37 ℃ of reactions 16 hours, with the described DNA of complete digestion, product electrophoresis on 0.8% sepharose afterwards.The sepharose of electrophoretic chromosomal DNA after by electrophoresis is transferred on the positively charged nylon leaching film (Roche Diagnostics production), then comprises alkaline denaturation, neutralization and immobilized processing.Use EASY HYB (Boehringer-Mannheim production) to hybridize.Filter membrane after 1 hour, added the digoxin labelled probe of preparation as mentioned above in 50 ℃ of prehybridizations, in 50 ℃ of hybridization 16 hours.Subsequently, with the 1 * SSC that contains 0.1%SDS in twice of 60 ℃ of rinsing filter membrane.

Use DIG Nucleotide detection kit (Boehringer-Mannheim),, detect band with described probe hybridization according to the step of describing in its specification sheets.The result successfully detects the band that is approximately 7kb with described probe hybridization.

With the EcoRI complete digestion 5 μ g Christian Breton cold snake bacterium in lake (Psychroserpensburtonensis) ATCC 700359 chromosomal DNAs.DNA by the about 7kb of 0.8% agarose gel electrophoresis separation uses Gene Clean II test kit (Funakoshi production) purify DNA, and DNA is dissolved among the 10 μ l TE.Then 4 μ l gained solution are mixed with pUC118EcoRI/BAP (Takara Shuzo production), use dna ligation kit Ver.2 (Takara Shuzo production) to carry out ligation.Mix this ligation liquid of 5 μ l and 100 μ l escherichia coli jm109 competent cells (Takara Shuzo production), with transformed into escherichia coli.Then it is coated on the suitable solid medium and creates chromosomal dna library.

(5) base sequence of the one-tenth peptidase genes in the cold snake bacterium in Christian Breton lake (Psychroserpens burtonensis) ATCC 700359 sources

The result discloses the open reading-frame (ORF) that is encoded into peptase and exists really.The cold snake bacterium in Christian Breton lake (psychroserpens burtonensis) ATCC 700359 (preservation mechanism: American type culture collection, preservation mechanism address: P.O.Box 1549, Manassas, VA 20110, theUnited States of America) the total length base sequence of one-tenth peptidase genes in source is shown in SEQ ID NO:26 together with corresponding amino acid sequence.

Although set forth the present invention with specific embodiments, with complete clearly open, but claims are therefore not limited, but should regard the basic theories that comprises that enforceable all modifications of those skilled in the art and variation scheme, these modifications and variations schemes belong to this paper fully and proposed as.

Sequence table independence text

SEQ ID NO:3: synthetic primer 1

SEQ ID NO:4: synthetic primer 2

SEQ ID NO:5: become the peptidase-encoding gene

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: become the peptidase-encoding gene

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 in soil Bacillaceae (Pedobacter) source

SEQ ID NO:20: the primer 2 that is used to make up the aet expression vector in soil Bacillaceae (Pedobacter) source

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

Sequence table

＜110〉Ajincomoto Co., Inc (AJINOMOTO CO.LTD.)

＜120〉production method of the production method of 2-L-aspartyl-L-phenylalanine-β-ester and 2-L-aspartyl-L-phenylalanine-α-methyl esters

<130>PAMA-15899

<150>JP2003-016764

<151>2003-01-24

<150>JP2003-201819

<151>2003-07-25

<150>US60/491,546

<151>2003-08-01

<160>27

<170>PatentIn Ver.2.1

<210>1

<211>9

<212>PRT

＜213〉lack steady bacillus (Empedobacter brevis)

<220>

＜223〉contriver: horizontal crux three (YOKOZEKI, Kenzo)

The contriver: big wild thin satin (OHNO, Ayako)

The contriver: former sincere one (HARA, Seiichi)

The contriver: Ah portion skilful (ABE, Isao)

<400>1

Leu Phe Thr Ala Ile Tyr Gln Pro Lys

1 5

<210>2

<211>9

<212>PRT

＜213〉lack steady bacillus (Empedobacter brevis)

<400>2

Thr Asn Val Thr Tyr Thr Met Pro Asp

1 5

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer 1

<400>3

ttyacngcna thtaycarcc 20

<210>4

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic primer 2

<400>4

tcnggcatng trtangtnac rtt 23

<210>5

<211>2024

<212>DNA

＜213〉lack steady bacillus (Empedobacter brevis)

<220>

<221>CDS

<222>(61)..(1908)

＜223〉become the peptidase-encoding gene

<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 bacillus (Empedobacter brevis)

<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〉artificial sequence

<220>

＜223〉description of artificial sequence: the synthetic primer that is used to prepare pTrpT

<400>7

gtatcacgag gccctagctg tggtgtcatg gtcggtgatc 40

<210>8

<211>40

<212>DNA

＜213〉artificial sequence

<220>

<400>8

ttcggggatt ccatatgata ccctttttac gtgaacttgc 40

<210>9

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the synthetic primer that is used to prepare pTrpT_Gtg2

<400>9

gggaattcca tatgaaaaaa ttaacattaa aagtaact 38

<210>10

<211>36

<212>DNA

＜213〉artificial sequence

<220>

<400>10

gggggctgca gtacttgtac ggtttcgccc gataaa 36

<210>11

<211>1935

<212>DNA

＜213〉Sphingobacterium kind (Sphingobacterium sp.)

<220>

<221>CDS

<222>(61)..(1917)

＜223〉become peptidase genes

<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 kind (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〉artificial sequence

<220>

＜223〉description of artificial sequence: the synthetic primer that is used to prepare pTrpT_Sm_aet

<400>13

gggaattcca tatgaaaaat acaatttcgt 30

<210>14

<211>29

<212>DNA

＜213〉artificial 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 (Pedobacter heparinus)

<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 Gln 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

Val Phe Gln Thr Glu Pro Leu Ser Ser Asp Leu Thr Leu Thr Gly Pro

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 (Pedobacter heparinus)

<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 LysIle 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 expression vector in soil Bacillaceae (Pedobacter) source

<400>19

gggaattcca tatgactcct ttcaaatcgt tctccttc 38

<210>20

<21l>30

<212>DNA

＜213〉artificial

<220>

＜223〉be used to make up the primer 2 of the expression vector in soil Bacillaceae (Pedobacter) source

<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〉shaping bacillus (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 ProIle

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 SerIle 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〉shaping bacillus (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 (Cyclobacterium marinum)

<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 (Cyclobacterium marinum)

<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〉the cold snake bacterium in Christian Breton lake (Psychroserpens 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 IIe 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

<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 Gln 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

1. method of producing 2-L-aspartyl-L-phenylalanine-β-ester, described method comprises that use can be connected to L-aspartic acid-α with L-phenylalanine selectivity by peptide bond, the enzyme in the α of β-diester-ester site or contain enzyme material, by L-aspartic acid-α, β-diester and L-phenylalanine generate 2-L-aspartyl-L-phenylalanine-β-ester.

2. 2-L-aspartyl-L-phenylalanine-the β of claim 1-ester production method, wherein said enzyme or contain enzyme material and be selected from following one or both or multiple: can L-phenylalanine selectivity be connected to L-aspartic acid-α by peptide bond, the microorganisms cultures in the α of β-diester-ester site, separate microorganism cells product from the microorganism cells of described culture and the described microorganism of handling.

3. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism belong to and are selected from following genus: Aeromonas (Aeromonas); Azotobacter (Azotobacter); Alcaligenes (Alcaligenes); brevibacterium sp (Brevibacterium); Corynebacterium (Corynebacterium); Escherichia (Escherichia); steady Bacillaceae (Empedobacter); Flavobacterium (Flavobacterium); Microbacterium (Microbacterium); propiono-bacterium (Propionibacterium); bacillus brevis belongs to (Brevibacillus); series bacillus belongs to (Paenibacillus); Rhodopseudomonas (Pseudomonas); serratia (Serratia); oligotrophy zygosaccharomyces (Stenotrophomonas); Sphingobacterium (Sphingobacterium); streptomyces (Streptomyces); xanthomonas (Xanthomonas); intend Weir yeast belong (Williopsis); mycocandida (Candida); Geotrichum (Geotrichum); Pichia (Pichia); yeast belong (Saccharomyces); spore torulopsis (Torulaspora) is arranged; Cytophaga (Cellulophaga); Weeks Bordetella (Weeksella); soil Bacillaceae (Pedobacter); peachiness Bacillaceae (Persicobacter); Flexithrix (Flexithrix); bite chitin Pseudomonas (Chitinophaga); round bar Pseudomonas (Cyclobacterium); ancient character shape Pseudomonas (Runella); hot line Pseudomonas (Thermonema) dwells; cold snake Pseudomonas (Psychroserpens); ice-cold Bacillaceae (Gelidibacter); Dyadobacter; heat color Bacillaceae (Flammeovirga); Spirosoma (Spirosoma); knee Pseudomonas (Flectobacillus); Flexibacter (Tenacibaculum); red thermophilic salt Pseudomonas (Rhodotermus); Zobellia; Muricauda; Salegentibacter; shaping Bacillaceae (Taxeobacter); Cytophage (Cttophaga); the sliding Pseudomonas (Marinilabilia) in sea; rely Wen Pseudomonas (Lewinella); Saprospira (Saprospira) and constraint Bacillaceae (Haliscomenobacter).

4. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism are to express following (A) or (B) proteinic conversion microorganism:

5. the production method of the 2-L-aspartyl-L-phenylalanine-β of claim 2-ester, wherein said microorganism are to express following (C) or (D) proteinic conversion microorganism:

6. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism are to express following (E) or (F) proteinic conversion microorganism:

7. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism are to express following (G) or (H) proteinic conversion microorganism:

8. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism are to express following (I) or (J) proteinic conversion microorganism:

9. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism are to express following (K) or (L) proteinic conversion microorganism:

10. 2-L-aspartyl-L-phenylalanine-the β of claim 2-ester production method, wherein said microorganism are to express following (M) or (N) proteinic conversion microorganism:

11. the 2-L-aspartyl-L-phenylalanine-β of claim 2-ester production method, wherein said microorganism is to express following (O) or (P) proteinic conversion microorganism:

12. the 2-L-aspartyl-L-phenylalanine-β of claim 2-ester production method, wherein said microorganism is to express following (Q) or (R) proteinic conversion microorganism:

13. the 2-L-aspartyl-L-phenylalanine-β of claim 2-ester production method, wherein said microorganism is to express following (S) or (T) proteinic conversion microorganism:

14. the 2-L-aspartyl-L-phenylalanine-β of claim 2-ester production method, wherein said microorganism is to express following (U) or (V) proteinic conversion microorganism:

15. the 2-L-aspartyl-L-phenylalanine-β of claim 2-ester production method, wherein said microorganism is to express following (W) or (X) proteinic conversion microorganism:

16. the 2-L-aspartyl-L-phenylalanine-β of claim 1-ester production method, wherein said enzyme are selected from least a in following (A)-(X):

17. the production method of 2-L-aspartyl-L-phenylalanine-α-methyl esters, described method comprises: the reactions steps of utilizing the synthetic 2-L-aspartyl-L-phenylalanine-β of the 2-L-aspartyl-L-phenylalanine-β-ester production method of any one-methyl esters among the claim 1-16; And the reactions steps that 2-L-aspartyl-L-phenylalanine-β-methyl esters is changed into 2-L-aspartyl-L-phenylalanine-α-methyl esters.