CN109321494A - Intermediate gram Lyu Wall bacterium ZJB-17004 and its application - Google Patents

Intermediate gram Lyu Wall bacterium ZJB-17004 and its application Download PDF

Info

Publication number
CN109321494A
CN109321494A CN201811153827.0A CN201811153827A CN109321494A CN 109321494 A CN109321494 A CN 109321494A CN 201811153827 A CN201811153827 A CN 201811153827A CN 109321494 A CN109321494 A CN 109321494A
Authority
CN
China
Prior art keywords
phenylacetyl
hydroxyl
phosphoryl
reaction
zjb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201811153827.0A
Other languages
Chinese (zh)
Other versions
CN109321494B (en
Inventor
柳志强
郑裕国
康雪梅
张晓健
金利群
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University of Technology ZJUT
Original Assignee
Zhejiang University of Technology ZJUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University of Technology ZJUT filed Critical Zhejiang University of Technology ZJUT
Priority to CN201811153827.0A priority Critical patent/CN109321494B/en
Publication of CN109321494A publication Critical patent/CN109321494A/en
Application granted granted Critical
Publication of CN109321494B publication Critical patent/CN109321494B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/20—Aspartic acid; Asparagine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/222—Phenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/225—Tyrosine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to one plant of centre gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004 and its applications; it reaches 98% to catalysis N- phenylacetyl-DL- amino acids production l-amino acid enantio-selectivity value, reaches 99% to catalysis 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid preparation 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid enantio-selectivity value.

Description

Intermediate gram Lyu Wall bacterium ZJB-17004 and its application
(1) technical field
The present invention relates to bacterial strain --- centre gram Lyu Wall bacterium (Kluyvera that one plant produces hydroamidase Intermedia) ZJB-17004, and its prepare chiral amino acid in catalysis N- phenylacetyl group amino acid and derive with catalytic amino acid The N- phenylacetyl group substituent of object prepares the application in chiral amino acid derivative.
(2) background technique
L-glufosinate-ammonium chemical name are as follows: 4- [hydroxyl (methyl) phosphono]-L- high lactamine, is a kind of knot of Pidolidone Structure analog can inhibit glutamine synthelase (GS) active, accumulate ammonia in plant, and the accumulation of high concentration ammonia blocks plant Photorespiration, the destruction of chloroplast structure and the vesiculation of matrix, while Amino acid synthesis is obstructed, and leads to damaged membrane And cell death, to kill weeds.It, with broad spectrum activity, is second-biggest-in-the-world genetically modified crops herbicide-tolerant.
L-glufosinate-ammonium synthetic method has chemical synthesis and biological synthesis process at present.Chemical method mainly passes through following 4 kinds of sides The chiral centre of formula building L-glufosinate-ammonium: 1) utilizing chiral auxiliary reagent, and chiral induction constructs chiral centre；2) with native amino Acid is chiral source, and conversion obtains L-glufosinate-ammonium；3) asymmetric catalysis synthesis being catalyzed by chiral catalyst；4) disappear outside chiral resolution Revolve body.But chemical method synthesis L-glufosinate-ammonium process is more, yield is lower, and chiral reagent is with high costs, and the quantity of three wastes generated is big, It handles relatively difficult.Biological rule has stringent stereoselectivity, mild reaction condition, higher yield and is easily isolated The advantages such as purifying, therefore, bioanalysis produce L-glufosinate-ammonium technology and are worth with highly important industrialization development.
Bioanalysis production L-glufosinate-ammonium has protease hydrolytic bialaphos according to substrate specificity difference, passes through phosphodiesterase I, amidase I and glutaminase substep synergistic effect keep optical activity to hydrolyze L-3- acetylaminohydroxyphenylarsonic acid 4- (hydroxymethyl phosphono Base) butyramide, the fractionation bialaphos ethyl ester of alpha -chymotrypsin, deacetylase fractionation N- acetyl-glufosinate-ammonium, amidase Split 2- amino -4- (hydroxymethyl phosphono)-butyramide, ester hydrolase hydrolyzes L-glufosinate-ammonium-N- carboxylic acid anhydrides, nitrile hydratase water Solve glufosinate-ammonium substrate containing nitrile, transaminase-catalyzed 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid etc..
The chemical method of synthesis L-glufosinate-ammonium mainly has at present: 1) chiral adjuvant revulsion, with (S) -2- hydroxyl -3- pinane Ketone is chiral adjuvant preparation, and yield 51%, e.e. value is 79%；Using D-Val methyl esters as chiral adjuvant, -78 are needed DEG C low-temp reaction, yield 51%, e.e. value are 93.5%.2) natural amino acid chiral source method, using L-Glu as chiral source, E.e. value is 99.4%；L-Methionine is chiral source, and total recovery 42.3%, e.e. value is 93.5%, needs to use hypertoxic iodine Methane.3) dissymmetric synthesis, asymmetric catalytic hydrogenation --- catalyst amount is big and third level natural division is expensive；It is asymmetric Strecker reaction --- use hypertoxic cyanide；Asymmetric Michael reaction --- catalyst amount is big, and product yield and E.e. it is worth low.4) racemic modification Split Method, the method highest yield 86%, e.e. value highest 99%, but D- glufosinate-ammonium after fractionation It is unconverted, it wastes raw material.
2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid system is split with hydroamidase selective catalysis Important effective component 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid of standby L-glufosinate-ammonium, to realize industrialized route L-glufosinate-ammonium synthesis provides foundation.
The effect of chiral amino acid and its derivative in pharmaceutical developments is increasing, and current pharmaceutical developments are chiral pure The requirement of degree is higher and higher.Chiral amino acid production process has the techniques such as chemical resolution and Enzymatic Resolution.Wherein Enzymatic Resolution because For its mild condition, high specificity, pollution is small, has a clear superiority compared with chemical resolution.Wherein hydroamidase selective catalysis is torn open N- phenylacetyl group amino acids production chiral amino acid is divided to have broad application prospects.
(3) summary of the invention
The object of the present invention is to provide the bacterial strains that one kind can generate hydroamidase --- intermediate gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004, and its in catalysis N- phenylacetyl-DL- amino acid derivativges (2-N- phenylacetyl Base -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid) prepare important effective component l-amino acid derivative (the 2- ammonia of L-glufosinate-ammonium Base -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid) or catalysis N- phenylacetyl-DL- amino acid preparation l-amino acid in application, L-glufosinate-ammonium effective component is produced for chemo-enzymatic process and l-amino acid provides new enzyme source, promotes Green Chemistry catalysis Development.
The technical solution adopted by the present invention is that:
In a first aspect, the present invention provides one plant of production hydroamidase bacterial strain --- centre gram Lyu Wall bacterium (Kluyvera Intermedia) ZJB-17004, is preserved in China typical culture collection center, deposit number CCTCC No:M2018033, Preservation date on January 15th, 2018, address: the Chinese Wuhan Wuhan University, 430072.The bacterial strain is by Zhejiang Polytechnical University's biology Graduate School of Engineering is separated from soil and is obtained, and has the ability for catalyzing and synthesizing L-glufosinate-ammonium through detecting.
Second aspect, the present invention provide a kind of centre gram Lyu Wall bacterium ZJB-17004 and split N- in microorganism catalysis Phenylacetyl-DL- amino acid derivativges (preferably 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid) prepare L- grass In the important effective component l-amino acid derivative (preferably 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid) of ammonium phosphine Using the application are as follows: using the wet thallus after the intermediate gram fermented culture of Lyu Wall bacterium ZJB-17004 as catalyst, with N- benzene Acetyl-DL-amino acid derivative (preferably 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid) is substrate, with Buffer (preferably ammonium hydroxide) is the reaction system that reaction medium constitutes that pH value is 8.5, and at 25-55 DEG C, 100-200rpm is (preferably 30-40 DEG C, 150rpm) under the conditions of carry out conversion reaction, after reaction, it is derivative that reaction solution is isolated and purified to obtain l-amino acid Object (preferably 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid).In the reaction system, catalyst amount is with wet thallus Weight is calculated as 10-200g/L, preferably 50g/L, and the substrate is initially added final concentration of 10-500mM, preferably 100mM.It is described anti- Answer system that can also be made of the fermentation liquid and substrate that gram Lyu Wall bacterium ZJB-17004 fermented culture in centre obtains, pH8.5； Wherein final concentration 10-200g/L of the wet thallus in entire reaction system in fermentation liquid, preferably 50g/L, substrate is in reaction system In final concentration 10-500mM, preferably 100mM.
The method that the reaction solution isolates and purifies are as follows: after reaction, reaction solution is extracted with dichloromethane, by water layer tune PH to 1.0-5.0 (preferably 2.5) carries out ion-exchange chromatography with the speed loading of 1-6.0Bv/h (preferably 4Bv/h), first spends Ion water washing, then eluted with the ammonium hydroxide of 0.2-4.5M (preferably 1M) with 0.5-3.0Bv/h (preferably 2Bv/h), with dip dyeing The filter paper of 0.2% ninhydrin solution detects eluent, and filter paper purpling shows that eluent contains 2- amino -4- [hydroxyl (methyl) Phosphoryl]-L- butyric acid, the eluent containing target components is collected, vacuum distillation takes crystal with methanol dissolving-recrystallization to paste It is dry, obtain l-amino acid derivative (preferably 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid).
The third aspect, the present invention also provides a kind of centre gram Lyu Wall bacterium ZJB-17004 in catalysis N- phenylacetyl- DL- amino acid prepares the application of l-amino acid, and the application is obtained with the fermented culture of centre gram Lyu Wall bacterium ZJB-17004 Wet thallus be catalyst, using N- phenylacetyl-DL- amino acid as substrate, with buffer (preferably ammonium hydroxide) be reaction medium constituted The transformation system of pH8.5 carries out conversion reaction under the conditions of 25-55 DEG C, 100-200rpm (preferably 30-40 DEG C, 150rpm), instead After answering, reaction solution is isolated and purified, and obtains l-amino acid.In the transformation system, catalyst amount is with wet thallus weight It is calculated as 20-300g/L (preferably 20-60g/L), the substrate is initially added final concentration of 50-500mM (preferably 100mM).It is described N- phenylacetyl-DL- amino acid is one of following: N- phenylacetyl-DL-Alanine, N- phenylacetyl-DL-serine, N- phenylacetyl- DL- glutamic acid, N- phenylacetyl-DL- tyrosine, N- phenylacetyl-DL- threonine, N- phenylacetyl-DL-proline, N- phenylacetyl- DL- aspartic acid, N- phenylacetyl-DL- methionine, N- phenylacetyl-DL-leucine, N- phenylacetyl-DL- isoleucine, N- benzene Acetyl-DL-phenylalanine.The method that the reaction solution isolates and purifies are as follows: after reaction, reaction solution methylene chloride is extracted It takes, by water layer tune pH to 1.0-5.0 (preferably 2.5), ion exchange is carried out with the speed loading of 1-6.0Bv/h (preferably 4Bv/h) Chromatography, is first washed with deionized, then carried out with the ammonium hydroxide of 0.2-4.5M (preferably 1M) with 0.5-3.0Bv/h (preferably 2Bv/h) Elution detects eluent with the filter paper of the ninhydrin solution of dip dyeing 0.2%, and filter paper purpling black shows that eluent contains L- amino Acid, collects the eluent containing target components, and vacuum distillation to paste is taken crystal dry, obtained L- ammonia with methanol dissolving-recrystallization Base acid.
N- phenylacetyl-DL- the amino acid is prepared as follows: amino acid and NaOH are added in distilled water, 4 DEG C of ice baths Under the conditions of to be stirred well to solution colorless and transparent, phenyllacetyl chloride is added dropwise, after being added dropwise to complete, continues 4 DEG C of condition of ice bath reaction 2h, then Room temperature (25-30 DEG C) is stirred to react 5h to solution in colorless and transparent, and HCl is added to adjust pH to 1.5-5.5 (preferably 2-4), is precipitated solid Body filters drying, obtains N- phenylacetyl-DL- amino acid；The amino acid and NaOH molar ratio are 1:1~7 (preferably 1:1.5- 2.5)；The amino acid and phenyllacetyl chloride molar ratio are 1:0.1~2 (preferably 1:0.5-1.0), the distilled water volume dosage with Amino acid masses are calculated as 3-10ml/g；The amino acid is one of following: alanine, serine, glutamic acid, tyrosine, Soviet Union's ammonia Acid, valine, aspartic acid, methionine, leucine, isoleucine, phenylalanine.
The wet thallus of the fermented acquisition of centre gram Lyu of the present invention Wall bacterium ZJB-17004 is prepared as follows:
(1) inclined-plane culture:
Centre gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004 is seeded to slant medium, at 30 DEG C 48h is cultivated, inclined-plane thalline is obtained；The slant medium is final concentration of: lactose 1-12g/L, peptone 1-10g/L, Na2HPO4 0.5-5.3g/L, K2HPO40.5-6.0g/L, Na2EDTA 0.01-0.3g/L, MnSO4
0.0001-0.001g/L, MgSO40.001-0.002g/L, ZnSO40.001-0.01g/L, FeSO4 0.001- 0.03g/L, agar 20g/L, solvent are deionized water, pH value 6.5-7.5；It is preferred that slant medium final concentration forms are as follows: cream Sugared 8g/L, peptone 6g/L, Na2HPO43.2g/L, K2HPO41.5g/L, Na2EDTA0.06g/L, MnSO40.002g/L, MgSO40.001g/L, ZnSO40.002g/L, FeSO40.01g/L, agar 20g/L, solvent are deionized water, pH value 6.8.
(2) seed culture
It is seeded to seed culture medium from one oese thallus of inclined-plane thalline picking, is cultivated at 30 DEG C for 24 hours, obtains seed liquor； The seed culture medium final concentration composition are as follows: lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L, K2HPO4 0.5-6.0g/L, Na2EDTA 0.01-0.3g/L, MnSO40.0001-0.001g/L, MgSO40.001-0.002g/L, ZnSO4 0.001-0.01g/L, FeSO40.001-0.03g/L, solvent are deionized water, pH value 6.5-7.5；Preferred seed culture medium Final concentration composition are as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L, K2HPO41.5g/L, Na2EDTA 0.06g/L, MnSO40.002g/L, MgSO40.001g/L, ZnSO40.002g/L, FeSO40.01g/L, solvent are deionized water, pH value It is 6.8.
(3) fermented and cultured
Seed liquor is seeded in fermentation medium with the inoculum concentration of volumetric concentration 1~10% (preferably 1%), 30 DEG C, After 150rpm shaken cultivation 60h, it is centrifuged 10min at 12000g, collects wet thallus；The fermentation medium final concentration composition Are as follows: lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L, K2HPO40.5-6.0g/L, Na2EDTA 0.01- 0.3g/L, MnSO40.0001-0.001g/L, MgSO40.001-0.002g/L, ZnSO40.001-0.01g/L, FeSO4 0.001-0.03g/L, solvent are deionized water, pH value 6.5-7.5；It is preferred that fermentation medium final concentration forms are as follows: lactose 8g/ L, peptone 6g/L, Na2HPO43.2g/L, K2HPO41.5g/L, Na2EDTA 0.06g/L, MnSO40.002g/L, MgSO4 0.001g/L, ZnSO40.002g/L, FeSO40.01g/L, solvent are deionized water, pH value 6.8.
The buffer that pH value of the present invention is 8.5 is constituted are as follows: citrate buffer solution (100mM, pH 4.0-6.0), phosphoric acid Buffer (100mM, pH 6.0-8.0), Tris-HCl (100mM, pH 7.0-9.0), borate buffer (100mM, pH 9.0- 10.5), ammonium hydroxide buffer (substrate solution pH is adjusted as 7.0-10.0 with ammonium hydroxide), sieve Er Shi buffer (100mM, pH10.5- 12.0)；Preferred buffer composition are as follows: substrate solution pH is adjusted as 8.5 with ammonium hydroxide.
The beneficial effects are mainly reflected as follows: the present invention provides the bacterial strains that one plant can generate hydroamidase -- Centre gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004, the present invention also provides utilize intermediate gram Lyu Wall bacterium The fermented culture of ZJB-17004 obtains wet thallus and is catalyzed 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphinylidyne as biocatalyst Base]-DL- butyric acid prepare L-glufosinate-ammonium important effective component 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid side Method reaches 49.5% to 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl] butyric acid conversion ratio, and product L-glufosinate-ammonium reaches E.e.%=99.9%, the present invention also provides obtain wet thallus using the fermented culture of centre gram Lyu Wall bacterium ZJB-17004 As the method for biocatalyst catalysis N- phenylacetyl-DL- amino acid preparation l-amino acid, conversion ratio can reach 49%, produce Object chiral amino acid can reach e.e.%=99-99.9%, split preparation process green high-efficient, and bacterial strain is easy to cultivate collection and answers With catalytic reaction condition is mild, with important application prospects.
(4) Detailed description of the invention:
Fig. 1 is the concentration standard curve of OPA/NAC- glufosinate-ammonium under high-throughput screening method.
Fig. 2 be pre-column derivatization reversed-phase high performance liquid chromatography under 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid and The liquid phase result map of 2- amino -4- [hydroxyl (methyl) phosphoryl]-D- butyric acid.
Fig. 3 is the Electronic Speculum sight for screening gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004 among obtained strains Examine result.
(5) specific embodiment:
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This: ultrapure water of the present invention is UP water, refers to that resistivity reaches the water of 18M Ω * cm (25 DEG C).Ammonium hydroxide of the present invention is to contain The aqueous solution of ammonia 25%~28%.Room temperature of the present invention is 25-30 DEG C.Ice bath described in the embodiment of the present invention is 4 DEG C.This hair Bright 2% ninhydrin solution preparation method are as follows: 2g ninhydrin and 0.08g stannous chloride are dissolved in 100mL ultrapure water, then stirred Filtering, takes filtrate to be kept in dark place.
Embodiment 1: the screening of centre gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004
1, primary dcreening operation
The present invention is total to 80 parts of soil sampling from soil sampling in all parts of the country.The specific method of screening: it weighs 1g soil sample and is placed in 10mL In 0.85% physiological saline, is stood after rocking, take supernatant into enriched medium, in 30 DEG C, 150r/min shaken cultivation 2-3 days.It takes 1mL pregnant solution to be added in the fresh enriched medium of 50mL, is isolated and purified after being so repeated 3 times.
Bromothymol blue filter paper is selected to detect the bacterium colony for the substrate that can degrade as instruction filter paper.Its principle are as follows: contain mesh Enzyme bacterium colony using degradation substrate after, acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) can be generated, to make to indicate The pH value of filter paper part changes.Indicator Bromothymol blue color change interval is (yellow-blue) 5.8-7.6, therefore can be utilized Yellow is presented in the periphery of bacterial colonies of chlorinated organics, and unavailable, and periphery of bacterial colonies plate color is still dark green.
Pregnant solution is diluted into 10 gradients with sterile 0.85% physiological saline step by step, chooses 10-4、10-5、10-6、10-7、10-8、10-9、10-10The dilution of five gradients respectively takes 0.1mL to be uniformly coated on plate screening culture medium, 30 DEG C of constant temperature incubations 48h.The aseptic filter paper for soaking the bromthymol blue containing 10% (instruction filter paper) is laid on plate, picking indicates on filter paper The corresponding colony inoculation of yellow is shown as into 96 orifice plates containing growth medium, is put into 30 DEG C, 180rpm shaking table constant temperature During which culture detects OD600Light absorption value, draw growth curve, when strain growth arrive logarithmic phase when, switching the 300 fresh hairs of μ l Ferment culture medium, remaining bacterium solution sealing preservation is spare as strain in 4 DEG C of refrigerators after being inoculated with 96 orifice plates.Contain fermentation after switching 96 orifice plate of culture medium sufficiently grows it and producing enzyme in 30 DEG C, 180rpm shaking table constant temperature incubation 48h, obtains bacteria suspension.
2, OPA/NAC high flux screening
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, 1ml step 1 is added The bacteria suspension of preparation adjusts solution ph to 8.5 with ammonium hydroxide, constitutes 10mL reaction system, make Final substrate concentrations 50mM, in 30 DEG C, the isothermal reaction of 180rpm shaking table for 24 hours.For gained conversion fluid respectively with corresponding 10 times of the dilution of ultrapure water, 4 DEG C of ice baths use OPA/NAC High-throughput screening method is detected, and the higher bacterial strain of relative fluorescence is filtered out.
OPA/NAC high-throughput screening method: the 40 μ l of ice bath reaction solution after dilution is drawn to 96 hole fluorescence detections with the volley of rifle fire Plate, the A liquid after ice bath is added, vibrates 30s in microplate reader, adds 100 μ l ultrapure waters, 30s is vibrated again, in λex=340nm； λem=455nm measures fluorescence intensity level, gained fluorescence intensity level and 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid mark Directrix curve, which compares, can be obtained the concentration of contained 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid in sample, then can be with The yield for calculating 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid obtains bacterial strain catalysis and splits 2-N- phenylacetyl group - 4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid prepares important effective component 2- amino -4- [hydroxyl (methyl) phosphorus of L-glufosinate-ammonium Acyl group]-L- butyric acid conversion ratio.
The A liquid: N-acetyl-L-cysteine (0.448g) and o-phthalaldehyde (0.185g) are dissolved in the anhydrous second of 10mL Borate buffer (140mM, pH 9.5) is added under condition of ice bath and is settled to 50mL, saves, can be reserved for 3 days under condition of ice bath for alcohol, N-acetyl-L-cysteine final concentration 0.313mol/L in institute's A liquid, o-phthalaldehyde final concentration 0.138mol/L.
2- amino -4- [hydroxyl (methyl) the phosphoryl]-L- butyric acid standard curve making method: it is prepared with ultrapure water 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid standard solution of 5g/L, is diluted to 3 concentration gradients: 0.01~ 0.1g/L (0.01,0.02,0.04,0.06,0.08,0.1g/L), 0.1~1.0g/L (0.1,0.2,0.4,0.6,0.8,1.0g/ L), (1.0,2.0,3.0,4.0,5.0g/L) 1.0~5.0g/L, the standard solution 40 of various concentration gradient is drawn with the volley of rifle fire respectively μ l is separately added into the A liquid that the prepared ice bath of 40 μ l saves, 30s is vibrated in microplate reader to 96 hole porous fluorescent detection plates, then 100 μ l ultrapure waters are separately added into, then vibrate 30s, in λex=340nm；λem=455nm measures fluorescence intensity level, with gained Fluorescence intensity level is ordinate, and corresponding mark product concentration is abscissa, makes standard curve.The standard of 3 concentration ranges is made altogether Curve, it is known that linear good (such as Fig. 1), R when sample concentration is 0.01~0.1g/L2=0.9978, show that this method has very High accuracy and applicability.
3, high performance liquid chromatography secondary screening
Step 2 is filtered out into strain inoculated to slant medium, after 30 DEG C of culture 48h, is saved in 4 DEG C of refrigerators.
By the strain inoculated being stored on inclined-plane into seed culture medium, cultivated for 24 hours at 30 DEG C.Seed liquor is dense with volume The inoculum concentration of degree 1% is seeded in fermentation medium, and 30 DEG C, 150rpm shaken cultivation 60h.It is centrifuged 5min at 12000g, receives Collect wet thallus.Wet thallus is taken, substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid and ammonium hydroxide is added, is used Ammonium hydroxide adjusts pH value of solution 8.5, constitutes 10mL reaction system, wet thallus final concentration 50g/L, Final substrate concentrations 50mM are placed in 30 DEG C Shaking bath converts for 24 hours.1mL conversion fluid is taken, supernatant is taken to carry out HPLC analysis after centrifuge separation, one plant of catalysis is finally obtained and lives The higher wild strain of power, and this bacterial strain is denoted as bacterial strain ZJB-17004.
High performance liquid chromatography (HPLC) secondary screening method are as follows: by above-mentioned conversion fluid centrifuging and taking supernatant, dilute 10 times with ultrapure water Afterwards, it takes 200 μ l to clean 1.5mL EP to manage, 200 μ l of derivatization reagent is added, after 30 DEG C of reaction 5min, it is ultrapure that 600 μ l are added After water mixes, after (0.22 μm) of syringe filter membrane filtering, it is put into HPLC and is detected, obtain 2- amino -4- [hydroxyl in reaction solution (methyl) phosphoryl]-butyric acid concentration.
The derivatization reagent: 0.1g o-phthalaldehyde, 0.12g N- phenylacetyl-L-cysteine, with the anhydrous second of 10mL After alcohol dissolution, 50mL is settled to borate buffer (100mM, pH 9.8).
The HPLC condition: it wears peace U3000 high performance liquid chromatograph and is equipped with fluorescence detector in the U.S.；Wear peace C18Silicone hydroxyl Filled column (250mm × 4.6mm)；Mobile phase is ammonium acetate (50mM pH 4.7) solution containing 10% pure methanol, and column temperature control exists 35 DEG C, fluorescence exciting wavelength λex=350nm, emission wavelength lambdaem=450nm, sample volume are 10 μ l.With this condition, 2- amino- The appearance time of 4- [hydroxyl (methyl) phosphoryl]-L- butyric acid and 2- amino -4- [hydroxyl (methyl) phosphoryl]-D- butyric acid is 7.800min, 9.290min (such as Fig. 2).
Enriched medium composition: 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid 1g/L, glucose 5g/L, sodium chloride 1g/L, K2HPO4·3H2O 0.8g/L, KH2PO43.3g/L, MgSO4·7H2O 0.2g/L, solvent be go from Sub- water, pH 7.
Plate screening culture medium composition: 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid 1g/L, grape Sugared 5g/L, sodium chloride 1g/L, K2HPO4·3H2O 0.8g/L, KH2PO43.3g/L, MgSO4·7H2O 0.2g/L, agar 20g/ L, solvent are deionized water, pH 7.
Growth medium composition: sodium chloride 10g/L, peptone 10g/L, yeast powder 5g/L, solvent are deionized water.Inclined-plane Culture medium, seed culture medium, fermentation medium composition are the same as embodiment 3.Embodiment 2: bacterial strain ZJB-17004 identification
1, Morphological Identification:
The embodiment of the present invention 1 screens obtained bacterial strain ZJB-17004 on solid medium, 37 DEG C of shapes after culture 24 hours At round or subcircular, quality is soft, and surface is more smooth, flat, neat in edge, glossy opaque milky bacterium colony, diameter 2-4mm.Gram's staining observation: pink rod-short, no gemma.Solid medium composition: sodium chloride 10g/L, peptone 10g/L, yeast powder 5g/L, agar 20g/L, solvent are deionized water.
2, Physiology and biochemistry is identified:
94 kinds of phenotypes are carried out to bacterial strain ZJB-17004 using Biolog (GEN III) automatic microbe identification systems to test, Including 71 kinds of utilization of carbon source situation detections and 23 kinds of chemosensitivity detections: by strain inoculated in BUG plating medium (BIOLOG UNIVERSAL GROWTH AGAR), 33 DEG C constant temperature incubation 2 days, the thallus on plate is washed down with aseptic cotton carrier, It is mixed with inoculation liquid (IF-A), bacteria suspension is made, adjusted with nephelometer to 91%T/IF-A.Bacterium is hanged with 8 hole electric plus liquid devices Liquid is added in respectively in each hole of III micropore identification plate of BiologGEN, every 100 μ L of hole.Micropore identification plate is placed on 33 DEG C of incubators In, respectively culture 12h, for 24 hours, placed it in after 36h, 48h and read result on Biolog readout instrument.
Bacterial strain ZJB-17004 analyzes Metabolic Fingerprinting through Biolog readout instrument, and bacterial strain ZJB-17004 can utilize more by force 65 kinds of carbon Source cannot utilize other 2 kinds of carbon sources or Utilization ability is weaker；Bacterial strain ZJB-17004 is sensitive to 23 kinds of chemical substances.Biolog It is as shown in Table 1 and Table 2 that system provides 48h qualification result.
Utilization ability of the 1. bacterial strain ZJB-17004 of table to 71 kinds of carbon sources on III plate of BiologGEN
Chemosensitivity of the 2. bacterial strain ZJB-17004 of table to 23 kinds of chemical substances on III plate of BiologGEN
3, molecular biology identification:
Using the total DNA of bacterial strain ZJB-17004 as template, using primer P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3' expands the 16S rDNA gene of bacterial strain, after gene product is connected with carrier T, It entrusts the raw work in Shanghai to expand and be sequenced the bacterium 16S rDNA, obtains 16S rDNA sequence (the SEQ ID NO.1 institute of the bacterial strain Show) after, the 16S rDNA gene order of related strain in GenBank is retrieved with BLAST on the website NCBI, and carry out homology It compares.Bacterial strain ZJB-17004 and 256 homology highest (homology, 99%, based of Kluyvera intermedia bacterial strain On 16S ribosomal RNA gene), according to Microbial Genetics identity principle, it is higher than based on 16S rDNA homology 95%, identification bacterium substantially belongs to control bacterium.Therefore, this experimental identification bacterial strain ZJB-17004 is intermediate gram Lyu Wall bacterium (Kluyvera intermedia), it is quasi- to be named as centre gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004, it protects It is hidden in China typical culture collection center, deposit number CCTCC No:M 2018033, preservation date on January 15th, 2018, Address: the Chinese Wuhan Wuhan University, 430072.
It is any that the one or more nucleotide of the progress of nucleotide sequence shown in SEQ ID NO.1 in nucleotides sequence list are taken In generation, lacks or is inserted into resulting nucleotide sequence, as long as having 90% or more homology with the sequence, belongs to of the invention Protection scope.
Embodiment 3: the preparation of wet thallus
(1) inclined-plane culture:
Centre gram Lyu Wall bacterium ZJB-17004 is seeded to slant medium, in 30 DEG C of culture 48h, obtains inclined-plane thalline；
The slant medium is final concentration of: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L, K2HPO41.5g/ L, Na2EDTA0.06g/L, MnSO40.002g/L, MgSO40.001g/L, ZnSO40.002g/L, FeSO40.01g/L, agar For 20g/L, solvent is deionized water, pH value 6.8.
(2) seed culture
It is seeded to seed culture medium from one oese thallus of inclined-plane thalline picking, is cultivated at 30 DEG C for 24 hours, obtains seed liquor；
The seed culture medium final concentration composition are as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L, K2HPO4 1.5g/L, Na2EDTA 0.06g/L, MnSO40.002g/L, MgSO40.001g/L, ZnSO40.002g/L, FeSO4 0.01g/L, solvent are deionized water, pH value 6.8.
(3) fermented and cultured
Seed liquor is seeded in fermentation medium with the inoculum concentration of volumetric concentration 1%, 30 DEG C, 150rpm shaken cultivation After 60h, it is centrifuged 10min at 12000g, collects wet thallus；
The fermentation medium final concentration composition are as follows: lactose 8g/L, peptone 6g/L, Na2HPO43.2g/L, K2HPO4 1.5g/L, Na2EDTA 0.06g/L, MnSO40.002g/L, MgSO40.001g/L, ZnSO40.002g/L, FeSO4 0.01g/ L, solvent are deionized water, pH value 6.8.
Embodiment 4: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
(1) substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds implementation The wet thallus 0.3g of 3 method of example preparation, and pH 8.5 is adjusted with ammonium hydroxide, 10mL reaction system is constituted, Final substrate concentrations are made 50mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours.It takes 1mL conversion fluid into ep pipe, takes supernatant real after centrifuge separation It applies method described in example 1 and carries out HPLC analysis detection.The result shows that ZJB-17004 can make substrate 2-N- phenylacetyl group -4- [hydroxyl Base (methyl) phosphoryl]-DL- butyric acid converts to obtain product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid, conversion ratio It is 49.9%, the optical purity of product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid is 99.9%.
(2) conversion fluid of the amino of 2- containing product -4- [hydroxyl (methyl) the phosphoryl]-L- butyric acid obtained enzymic catalytic reaction It is separated, extracts product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
1) it extracts:
Separatory funnel is added in obtained conversion fluid, while isometric methylene chloride is added, stands 3h after rocking, from point Liquid funnel lower end releases organic layer, pours out aqueous layer from upper end.Obtaining aqueous layer is containing product 2- amino -4- [hydroxyl (first Base) phosphoryl]-L- butyric acid aqueous solution.
2) product separation is carried out with anion exchange resin
1. resin pre-processes
Ion exchange resin 201 × 7 is impregnated with 50 DEG C of warm water, expand resin sufficiently and remove fine particle (inclination or Person's floatation)；The NaOH aqueous solution soaking 3h for first using 1.0M, is washed with deionized water to neutrality, then is soaked with the HCL aqueous solution of 1.0M It is washed with deionized water after bubble 3h to neutrality, then uses the NaOH aqueous solution soaking 3h of 1.0M, be converted into OH-Form is finally spent Ion is washed to neutral spare.
2. filling column
Using wet method dress post (internal diameter 1.5cm, high 40cm), the deionization of 13cm height is first first added in ion exchange column Water, then 30mL wet resin 201 × 7 is taken to be packed into glass, 50mL deionized water is added, slowly stirs, the resin of suspension pours into In ion exchange column, its natural subsidence is allowed, keep it uniform in pillar endocrine, should not have apparent line of demarcation and bubble to generate Deng.
3. loading, elution
The resulting aqueous solution of step 1) is adjusted to pH 2.5, the above column flow rate 4.0Bv/h carries out loading, at regular intervals It takes out efflux progress liquid phase detection to be first washed with deionized when absorption reaches peak, then is carried out with the ammonium hydroxide of 1.0M Elution, elution speed 2.0Bv/h are detected with the filter paper of the ninhydrin solution of dip dyeing 0.2%, and filter paper purpling color table is bright to be washed De- liquid contains target substance 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid, collects target components eluent, Mei Geyi The section time detects the content of 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid contained therein.It is spent after elution Ion water washing ion exchange column, and convert the resin to OH-For separating next time.
4. purifying
Eluent is evaporated under reduced pressure to obtain clear yellow viscous substance, and methanol dissolution is added, and stirring is recrystallized to give white under ice bath Solid filters up to product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
With the conversion fluid 1L of step (1) reaction condition preparation after the above method isolates and purifies, 2- amino -4- [hydroxyl is obtained Base (methyl) phosphoryl]-L- butyric acid solid 4.9g, efficiency of pcr product 98.9%, optical purity 99.9%.
Embodiment 5 is anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 0.5g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 100mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by Method described in embodiment 1 carries out HPLC analysis detection and obtains conversion ratio 49.9%.1L conversion fluid is prepared under same reaction condition, is pressed After method isolates and purifies in embodiment 4, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 9.3g is obtained, product obtains Rate 93.9%, optical purity 99.9%.
Embodiment 6: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 0.6g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 200mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by Method described in embodiment 1 carries out HPLC analysis detection and obtains conversion ratio 49.9%.1L conversion fluid is prepared under same reaction condition, is pressed After method isolates and purifies in embodiment 4, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 19.2g, product are obtained Yield 96.4%, optical purity 99.9%.
Embodiment 7: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 2g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 200mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours, takes 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by Method described in embodiment 1 carries out HPLC analysis detection and obtains conversion ratio 49.9%.1L conversion fluid is prepared under same reaction condition, is pressed After method isolates and purifies in embodiment 4, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 19.4g, product are obtained Yield 97.4%, optical purity 99.9%.
Embodiment 8: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 0.5g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 50mM, is placed in 40 DEG C of shaking baths, and 150rpm is converted for 24 hours, takes 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by Method described in embodiment 1 carries out HPLC analysis detection and obtains conversion ratio 49.9%.1L conversion fluid is prepared under same reaction condition, is pressed After method isolates and purifies in embodiment 4, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 4.87g, product are obtained Yield 98.4%, optical purity 99.9%.
Embodiment 9: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 2g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 200mM, is placed in 50 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by Method described in embodiment 1 carries out HPLC analysis detection and obtains conversion ratio 49.9%.1L conversion fluid is prepared under same reaction condition, is pressed After method isolates and purifies in embodiment 4, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 19.4g, product are obtained Yield 97.4%, optical purity 99.9%.
Embodiment 10: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL-Alanine
6.0g alanine and 4.6g NaOH are added in 30ml distilled water, and it is colourless to be stirred well to solution under condition of ice bath It is bright, 5.0g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h in colorless and transparent, adds HCl to adjust pH to 2.0 or so to solution, and white solid is precipitated, and filters drying and obtains white solid and is N- phenylacetyl-DL-Alanine 13g.
Substrate N- phenylacetyl-DL-Alanine is dissolved in ammonium hydroxide, adds the wet thallus 0.6g of 3 method of embodiment preparation, is used Ammonium hydroxide adjusts pH 8.5, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into EP pipe, supernatant is taken to carry out after centrifuge separation by method described in embodiment 1 HPLC analysis detection obtains conversion ratio 49.8%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product l-Alanine 4.25g, efficiency of pcr product 95.5%, optical purity 99.9% are obtained.
Embodiment 11: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL- threonine
6.0g threonine and 4.6g NaOH are added in 30ml distilled water, and it is colourless to be stirred well to solution under condition of ice bath It is bright, 5.0g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h in colorless and transparent, adds HCl to adjust pH to 2.0 or so to solution, and white solid is precipitated, and filters drying and obtains white solid and is N- phenylacetyl-DL- threonine 10.35g.
Substrate N- phenylacetyl-DL- threonine is dissolved in ammonium hydroxide, the wet thallus 3g of 3 method of embodiment preparation is added, uses ammonia Water tune pH 8.5 constitutes 100mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into EP pipe, supernatant is taken to carry out after centrifuge separation by method described in embodiment 1 HPLC analysis detection obtains conversion ratio 49.5%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product L-threonine 5.54g, efficiency of pcr product 93.1%, optical purity 99.9% are obtained.
Embodiment 12: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL- tyrosine
3.619g DL- tyrosine and 1.8g NaOH are added in 25ml distilled water, are stirred well to solution under condition of ice bath It is bright, 2.875g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is creamy white, and continues condition of ice bath and reacts 2h, then stirring at normal temperature After reacting 5h, HCl is added to adjust pH to 2.0 or so, a large amount of white solids are precipitated, filtering drying and obtaining white solid is N- benzene second Acyl-DL- tyrosinase 15 .5g.
Substrate N- phenylacetyl-DL- tyrosine is dissolved in ammonium hydroxide, adds the wet thallus 0.3g of 3 method of embodiment preparation, is used Ammonium hydroxide adjusts pH 8.5, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into EP pipe, supernatant is taken to carry out after centrifuge separation by method described in embodiment 1 HPLC analysis detection obtains conversion ratio 49.0%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product l-tyrosine 8.72g, efficiency of pcr product 96.3%, optical purity 99.9% are obtained.
Embodiment 13: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL-phenylalanine
3.3g DL-phenylalanine and 1.8g NaOH are added in 25ml distilled water, are sufficiently stirred under condition of ice bath, solution is saturating It is bright, 2.875g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is creamy white, and continuation condition of ice bath reaction 2h, then stirring at normal temperature are anti- After answering 5h, HCl is added to adjust pH to 4.0 or so, a large amount of white solids are precipitated, filtering drying and obtaining white solid is N- benzene second Acyl-DL-phenylalanine 5.2g.
Substrate N- phenylacetyl-DL-phenylalanine is dissolved in ammonium hydroxide, adds the wet thallus 0.4g of 3 method of embodiment preparation, PH 8.5 is adjusted with ammonium hydroxide, constitutes 10mL reaction system, wherein final concentration of 100mM is added in substrate, 30 DEG C of shaking baths are placed in, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into EP pipe, supernatant is taken to carry out after centrifuge separation by method described in embodiment 1 HPLC analysis detection obtains conversion ratio 49.2%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product L-phenylalanine 7.82g, efficiency of pcr product 94.7%, optical purity 99.9% are obtained.
Embodiment 14: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL-leucine
3.92g leucine and 3.2g NaOH are added in 30ml distilled water, and it is bright to be stirred well to solution under condition of ice bath, 6.2g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h Afterwards, add HCl to adjust pH to 2.0 or so, a large amount of white solids are precipitated, filtering drying and obtaining white solid is N- phenylacetyl-DL- Leucine 7.43g.
Substrate N- phenylacetyl-DL-leucine is dissolved in ammonium hydroxide, adds the wet thallus 0.6g of 3 method of embodiment preparation, is used Ammonium hydroxide adjusts pH 8.5, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into EP pipe, supernatant is taken to carry out after centrifuge separation by method described in embodiment 1 HPLC analysis detection obtains conversion ratio 49.4%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product L-Leu 5.94g, efficiency of pcr product 90.6%, optical purity 99.9% are obtained.
Embodiment 15: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL- isoleucine
7.84g isoleucine and 6.4g NaOH are added in 60ml distilled water, are sufficiently stirred under condition of ice bath, and solution is bright, 5.78g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h Afterwards, HCl is added to adjust PH to 2.0 or so, solution becomes colorless transparent, is precipitated a small amount of white solid, and filtering drying, to obtain white solid Body is N- phenylacetyl-DL- isoleucine 7.28g.
Substrate N- phenylacetyl-DL- isoleucine is dissolved in ammonium hydroxide, adds the wet thallus 0.5g of 3 method of embodiment preparation, PH 8.5 is adjusted with ammonium hydroxide, constitutes 10mL reaction system, wherein final concentration of 100mM is added in substrate, 30 DEG C of shaking baths are placed in, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into EP pipe, supernatant is taken to carry out after centrifuge separation by method described in embodiment 1 HPLC analysis detection obtains conversion ratio 49.5%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product l-Isoleucine 6.12g, efficiency of pcr product 93.4%, optical purity 99.9% are obtained.
Embodiment 16: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL-serine
6.0g serine and 4.6g NaOH are added in 20ml distilled water, and it is colourless to be stirred well to solution under condition of ice bath It is bright, 5.2g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction After 5h, solution adds HCl to adjust PH to 2.0 or so in colorless and transparent, and white solid is precipitated, and filters drying and obtains white solid i.e. For N- phenylacetyl-DL-serine 11g.
Substrate N- phenylacetyl-DL-serine is dissolved in ammonium hydroxide, the wet thallus 0.5g for adding the preparation of 3 method of embodiment is used It is 8.5 that ammonium hydroxide, which adjusts pH value of solution, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of water-baths and shakes Bed, 150rpm are converted for 24 hours.Take 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by method described in embodiment 1 into Row HPLC analysis detection obtains conversion ratio 49.2%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with leaching The filter paper of the ninhydrin solution of dye 0.2% is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product Serine 4.86g, efficiency of pcr product 92.5%, optical purity 99.9% are obtained.
Embodiment 17: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL- methionine
8.94g methionine and 7.2g NaOH are added in 120ml distilled water, and it is saturating to be stirred well to solution under condition of ice bath It is bright, 10.3g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is transparent, continues condition of ice bath and reacts 2h, then stirring at normal temperature reaction After 5h, HCl is added to adjust PH to 2.0 or so, a large amount of white solids are precipitated, decompression filters drying and obtains N- phenylacetyl-DL- first sulphur ammonia Sour 14.8g.
Substrate N- phenylacetyl-DL- methionine is dissolved in ammonium hydroxide, adds the wet thallus of 3 method of embodiment preparation 0.45g, adjusting pH value of solution with ammonium hydroxide is 8.5 composition 10mL reaction systems, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C shaking bath, 150rpm are converted for 24 hours.It takes 1mL conversion fluid into EP pipe, takes supernatant by institute in embodiment 1 after centrifuge separation It states method progress HPLC analysis detection and obtains conversion ratio 49.3%.1L conversion fluid is prepared under same reaction condition, by side in embodiment 4 Method (is detected, filter paper purpling black shows that eluent contains target substance) with the filter paper of the ninhydrin solution of dip dyeing 0.2% After isolating and purifying, product L-methionine 7.03g, efficiency of pcr product 94.4%, optical purity 99.9% are obtained.
Embodiment 18: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL-proline
9.36g valine and 7.2g NaOH are added in 60ml distilled water, and it is bright to be stirred well to solution under condition of ice bath, 14.24g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in yellowish, slightly muddy, continuation condition of ice bath reaction 2h, then room temperature After being stirred to react 5h, solution bleach is transparent, and HCl is added to adjust PH to 2.0 or so, and a large amount of white solids are precipitated, and suction filtration is dried It is N- phenylacetyl-DL-proline 17.3g to white solid.
Substrate N- phenylacetyl-DL-proline is dissolved in ammonium hydroxide, adds the wet thallus 0.2g of 3 method of embodiment preparation, is used It is 8.5 that ammonium hydroxide, which adjusts pH value of solution, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of water-baths and shakes Bed, 150rpm are converted for 24 hours.Take 1mL conversion fluid into EP pipe, taken after centrifuge separation supernatant by method described in embodiment 1 into Row HPLC analysis detection obtains conversion ratio 49.2%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with leaching The filter paper of the ninhydrin solution of dye 0.2% is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product Valine 5.6g, efficiency of pcr product 95.7%, optical purity 99.9% are obtained.
Embodiment 19: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL- glutamic acid
5g glutamic acid and 4.6g NaOH are added in 30ml distilled water, and it is bright to be stirred well to solution under condition of ice bath, are added dropwise After 5.735g phenyllacetyl chloride, after being added dropwise to complete, solution is transparent, continues condition of ice bath and reacts 2h, then stirring at normal temperature reacts 5h, add HCl adjusts pH to 2.0 or so, and solution becomes cloudy, and a large amount of white solids are precipitated in 4 DEG C of refrigerator cold-storages after overnight, and decompression filters drying Obtain N- phenylacetyl-DL- glutamic acid 8.1g.
Substrate N- phenylacetyl-DL- glutamic acid is dissolved in ammonium hydroxide, adds the wet thallus 0.4g of 3 method of embodiment preparation, is used It is 8.5 that ammonium hydroxide, which adjusts pH value of solution, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of water-baths and shakes Bed, 150rpm are converted for 24 hours.Take 1mL conversion fluid into ep pipe, taken after centrifuge separation supernatant by method described in embodiment 1 into Row HPLC analysis detection obtains conversion ratio 49.1%.1L conversion fluid is prepared under same reaction condition, by method in embodiment 4 (with leaching The filter paper of the ninhydrin solution of dye 0.2% is detected, and filter paper purpling black shows that eluent contains target substance) it isolates and purifies Afterwards, product Pidolidone 7.12g, efficiency of pcr product 96.8%, optical purity 99.9% are obtained.
Sequence table
<110>Zhejiang Polytechnical University
<120>intermediate gram Lyu Wall bacterium ZJB-17004 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1400
<212> DNA
<213>intermediate gram Lyu Wall bacterium (Kluyvera intermedia)
<400> 1
cgccctcccg aaggttaagc tacctacttc ttttgcaccc actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgtagcattc tgatctacga ttactagcga 120
ttccgacttc atggagtcga gttgcagact ccaatccgga ctacgacata ctttatgagg 180
tccgcttgct ctcgcgaggt cgcttctctt tgtatatgcc attgtagcac gtgtgtagcc 240
ctactcgtaa gggccatgat gacttgacgt catccccacc ttcctccagt ttatcactgg 300
cagtctcctt tgagttcccg gccgaaccgc tggcaacaaa ggataagggt tgcgctcgtt 360
gcgggactta acccaacatt tcacaacacg agctgacgac agccatgcag cacctgtctc 420
agagttcccg aaggcaccaa agcatctctg ctaagttctc tggatgtcaa gagtaggtaa 480
ggttcttcgc gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540
attcatttga gttttaacct tgcggccgta ctccccaggc ggtcgactta acgcgttagc 600
tccggaagcc acgcctcaag ggcacaacct ccaagtcgac atcgtttacg gcgtggacta 660
ccagggtatc taatcctgtt tgctccccac gctttcgcac ctgagcgtca gtctttgtcc 720
agggggccgc cttcgccacc ggtattcctc cagatctcta cgcatttcac cgctacacct 780
ggaattctac ccccctctac aagactctag cctgccagtt tcgaatgcag ttcccaggtt 840
gagcccgggg atttcacatc cgacttgaca gaccgcctgc gtgcgcttta cgcccagtaa 900
ttccgattaa cgcttgcacc ctccgtatta ccgcggctgc tggcacggag ttagccggtg 960
cttcttctgc gagtaacgtc aatcgctgca gttattaact acagcgcctt cctcctcgct 1020
gaaagtactt tacaacccga aggccttctt catacacgcg gcatggctgc atcaggcttg 1080
cgcccattgt gcaatattcc ccactgctgc ctcccgtagg agtctggacc gtgtctcagt 1140
tccagtgtgg ctggtcatcc tctcagacca gctagggatc gtcgcctagg tgagccgtta 1200
ccccacctac tagctaatcc catctgggca catccgatgg tgtgaggccc gaaggtcccc 1260
cactttggtc ttgcgacgtt atgcggtatt agctaccgtt tccagtagtt atccccctcc 1320
atcgggcagt ttcccagaca ttactcaccc gtccgccact cgtcacccaa gagcaagctc 1380
tctgtgctac cgttcgactg 1400

Claims (10)

1. gram Lyu Wall bacterium (Kluyvera intermedia) ZJB-17004 among, is preserved in China typical culture collection The heart, deposit number CCTCC No:M 2018033, preservation date on January 15th, 2018, address: Wuhan, China Wuhan is big It learns, 430072.
2. intermediate gram Lyu Wall bacterium ZJB-17004 is in catalysis 2-N- phenylacetyl group -4- [hydroxyl (first described in a kind of claim 1 Base) phosphoryl]-DL- butyric acid prepares the application in 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
3. application as claimed in claim 2, it is characterised in that the application is fermented with centre gram Lyu Wall bacterium ZJB-17004 It is catalyst that culture, which obtains wet thallus, using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid as substrate, with slow Fliud flushing is the reaction system that reaction medium constitutes pH8.5, and conversion reaction is carried out under the conditions of 25-55 DEG C, 100-200rpm, is reacted After, reaction solution is isolated and purified to obtain 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
4. application as claimed in claim 3, it is characterised in that in the reaction system, catalyst amount is in terms of wet thallus weight For 10-200g/L, substrate is initially added final concentration of 10-500mM.
5. application as claimed in claim 3, it is characterised in that the method that the reaction solution isolates and purifies are as follows: after reaction, Reaction solution is extracted with dichloromethane, by water layer tune pH to 1.0-5.0, ion exchange is carried out with the speed loading of 1-6.0Bv/h Chromatography, is first washed with deionized, then eluted with the ammonium hydroxide of 0.2-4.5M with 0.5-3.0Bv/h, collects and contains target components Eluent, vacuum distillation with methanol dissolving-recrystallization, taken crystal dry, obtains 2- amino -4- [hydroxyl (methyl) to paste Phosphoryl]-L- butyric acid.
6. intermediate gram Lyu Wall bacterium ZJB-17004 described in a kind of claim 1 prepares L- in catalysis N- phenylacetyl-DL- amino acid The application of amino acid.
7. application as claimed in claim 6, it is characterised in that the application is with centre gram Lyu Wall bacterium ZJB-17004 through sending out The wet thallus that ferment culture obtains is that catalyst is constituted using N- phenylacetyl-DL- amino acid as substrate by reaction medium of buffer The transformation system of pH8.5 carries out conversion reaction under the conditions of 25-55 DEG C, 100-200rpm, and after reaction, reaction solution is through dividing From purifying, N- phenylacetyl-l-amino acid is obtained.
8. the use as claimed in claim 7, it is characterised in that in the transformation system, catalyst amount is in terms of wet thallus weight For 20-300g/L, the substrate is initially added final concentration of 50-500mM.
9. application as claimed in claim 7, it is characterised in that the N- phenylacetyl-DL- amino acid is one of following: N- benzene second Acyl-DL-Alanine, N- phenylacetyl-DL-serine, N- phenylacetyl-DL- glutamic acid, N- phenylacetyl-DL- tyrosine, N- benzene second Acyl-DL- threonine, N- phenylacetyl-DL-proline, N- phenylacetyl-DL- aspartic acid, N- phenylacetyl-DL- methionine, N- Phenylacetyl-DL-leucine, N- phenylacetyl-DL- isoleucine, N- phenylacetyl-DL-phenylalanine.
10. the use as claimed in claim 7, it is characterised in that the following method preparation of wet thallus: (1) inclined-plane culture: will Centre gram Lyu Wall bacterium ZJB-17004 is seeded to slant medium, in 30 DEG C of culture 48h, obtains inclined-plane thalline；The inclined-plane training It is final concentration of to support base: lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L, K2HPO40.5-6.0g/L, Na2EDTA0.01-0.3g/L, MnSO40.0001-0.001g/L, MgSO40.001-0.002g/L, ZnSO4 0.001- 0.01g/L, FeSO40.001-0.03g/L, agar 20.0g/L, solvent are deionized water, pH value 6.5-7.5；
(2) seed culture: being seeded to seed culture medium from one oese thallus of inclined-plane thalline picking, cultivates for 24 hours, obtains at 30 DEG C Seed liquor；The seed culture medium final concentration composition are as follows: lactose 1-12g/L, peptone 1-10g/L, Na2HPO4 0.5-5.3g/ L, K2HPO40.5-6.0g/L, Na2EDTA0.01-0.3g/L, MnSO40.0001-0.001g/L, MgSO4 0.001- 0.002g/L, ZnSO40.001-0.01g/L, FeSO40.001-0.03g/L, solvent are deionized water, pH value 6.5-7.5；
(3) fermented and cultured: seed liquor being seeded in fermentation medium with the inoculum concentration of volumetric concentration 1~10%, 30 DEG C, After 150rpm shaken cultivation 60h, it is centrifuged 10min at 12000g, collects wet thallus；The fermentation medium final concentration composition: Lactose 1-12g/L, peptone 1-10g/L, Na2HPO40.5-5.3g/L, K2HPO40.5-6.0g/L, Na2EDTA 0.01- 0.3g/L, MnSO40.0001-0.001g/L, MgSO40.001-0.002g/L, ZnSO40.001-0.01g/L, FeSO4 0.001-0.03g/L, solvent are deionized water, pH value 6.5-7.5.
CN201811153827.0A 2018-09-30 2018-09-30 Kluyveromyces intermedia ZJB-17004 and application thereof Active CN109321494B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811153827.0A CN109321494B (en) 2018-09-30 2018-09-30 Kluyveromyces intermedia ZJB-17004 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811153827.0A CN109321494B (en) 2018-09-30 2018-09-30 Kluyveromyces intermedia ZJB-17004 and application thereof

Publications (2)

Publication Number Publication Date
CN109321494A true CN109321494A (en) 2019-02-12
CN109321494B CN109321494B (en) 2020-06-23

Family

ID=65266562

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811153827.0A Active CN109321494B (en) 2018-09-30 2018-09-30 Kluyveromyces intermedia ZJB-17004 and application thereof

Country Status (1)

Country Link
CN (1) CN109321494B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110331153A (en) * 2019-06-24 2019-10-15 浙江工业大学 A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application
CN113215048A (en) * 2021-05-21 2021-08-06 中国农业科学院农业资源与农业区划研究所 Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100842450B1 (en) * 2007-02-16 2008-07-01 대한민국 -2 Kluyvera sp. CL-2 and Method of solubilizing insoluble-phosphate immobilized in soils by using the same
KR101136480B1 (en) * 2010-10-28 2012-04-19 주식회사 두산에코비즈넷 Novel kluyvera intermedia ds-ebn-gbi and microbial agent comprising the same
WO2018140778A1 (en) * 2017-01-26 2018-08-02 Manus Bio, Inc. Metabolic engineering for microbial production of terpenoid products

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100842450B1 (en) * 2007-02-16 2008-07-01 대한민국 -2 Kluyvera sp. CL-2 and Method of solubilizing insoluble-phosphate immobilized in soils by using the same
KR101136480B1 (en) * 2010-10-28 2012-04-19 주식회사 두산에코비즈넷 Novel kluyvera intermedia ds-ebn-gbi and microbial agent comprising the same
WO2018140778A1 (en) * 2017-01-26 2018-08-02 Manus Bio, Inc. Metabolic engineering for microbial production of terpenoid products

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
杜乐妍等: "眼镜蛇中间克吕沃菌的分离鉴定及药敏试验", 《动物医学进展》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110331153A (en) * 2019-06-24 2019-10-15 浙江工业大学 A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application
CN110331153B (en) * 2019-06-24 2021-04-30 浙江工业大学 Kluyveromyces tyrosol lyase mutant and application thereof
CN113215048A (en) * 2021-05-21 2021-08-06 中国农业科学院农业资源与农业区划研究所 Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof

Also Published As

Publication number Publication date
CN109321494B (en) 2020-06-23

Similar Documents

Publication Publication Date Title
CN109321493A (en) The lysine of resistance to boron bacillus ZJB-17007 and its application
Tsygankov et al. Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction
CN106434434B (en) One plant of strains A rthrobacter woluwensis for producing dimethylbenzene monooxygenase and its application
CN110527646A (en) Tropical bacillus WZZ018 and its application
CN106591179B (en) Methyl packing bacterium and its prepare the application on (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt in selective fractionation
CN109504634B (en) Bacillus WZZ006 and application thereof
CN109321494A (en) Intermediate gram Lyu Wall bacterium ZJB-17004 and its application
Tang et al. Biocatalytic preparation of (S)-phenyl glycidyl ether using newly isolated Bacillus megaterium ECU1001
CN104263713A (en) Tistrella mobilis, halohydrin dehalogenase, gene, vector, recombinant strain and application of halohydrin dehalogenase
CN105112385A (en) Recombinant esterase, coding gene, carrier, engineering bacterium and application
CN101240254B (en) Organic solvent resisting proteinase high-yield bacterium, gene and application of the organic solvent resisting proteinase
CN109321487A (en) Pseudomonad zjut126 and the application in production L-glufosinate-ammonium
CN109321490A (en) Clostridium perfringen ZJB-17003 and its application
CN109182201A (en) Enterobacter amnigenus I type ZJB-17002 of biology and its application
CN109182200A (en) Spindle lysine bacillus ZJB-17006 and its application
CN109321497A (en) Long lysine bacillus ZJB-17009 and its application
KR100339723B1 (en) Process for production of amide compounds using microorganism
CN109321492A (en) Leclercia adecarboxylata ZJB-17008 and its application
JPH066069B2 (en) Method for producing (x) -trans-cyclopropanecarboxylic acid
CN109321491A (en) Xiangfang enterobacteria ZJB-17001 and its application
CN109321495A (en) Kluyvera cryocrescens ZJB-17005 and its application
CN109321496A (en) Citrobacter freundii ZJB-17010 and its application
CN100547067C (en) Brevibacterium epidermidis ZJB-07021 and the preparation (S)-2, the application in the 2-dimethyl-cyclopropane carboxamide
CN101701243A (en) Method for producing R-mandelic acid and derivates thereof by biocatalysis
WO1994020635A1 (en) Enantioselective hydrolysis of ketoprofen esters by beauveria bassiana and enzymes derived therefrom

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant