CN104560912B - Esterase and its encoding gene and application - Google Patents
Esterase and its encoding gene and application Download PDFInfo
- Publication number
- CN104560912B CN104560912B CN201510005882.5A CN201510005882A CN104560912B CN 104560912 B CN104560912 B CN 104560912B CN 201510005882 A CN201510005882 A CN 201510005882A CN 104560912 B CN104560912 B CN 104560912B
- Authority
- CN
- China
- Prior art keywords
- cyano group
- esterase
- acid ethyl
- carboxyethyls
- gene
- 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.)
- Active
Links
- 108090000371 Esterases Proteins 0.000 title claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 42
- -1 carboxyethyl Chemical group 0.000 claims abstract description 19
- 230000014509 gene expression Effects 0.000 claims abstract description 9
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 5
- 238000010931 ester hydrolysis Methods 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 16
- 230000007062 hydrolysis Effects 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 8
- 230000000284 resting effect Effects 0.000 claims description 7
- 239000006285 cell suspension Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000007806 chemical reaction intermediate Substances 0.000 claims description 2
- 239000013598 vector Substances 0.000 claims description 2
- 238000012805 post-processing Methods 0.000 claims 1
- 125000000143 2-carboxyethyl group Chemical group [H]OC(=O)C([H])([H])C([H])([H])* 0.000 abstract description 9
- 241000589516 Pseudomonas Species 0.000 abstract description 5
- 125000004093 cyano group Chemical group *C#N 0.000 abstract 3
- LFLSVOVPJVCWKQ-UHFFFAOYSA-N ethyl 2-methylhexanoate Chemical compound CCCCC(C)C(=O)OCC LFLSVOVPJVCWKQ-UHFFFAOYSA-N 0.000 abstract 3
- 239000000758 substrate Substances 0.000 description 41
- 210000004027 cell Anatomy 0.000 description 24
- 239000000047 product Substances 0.000 description 17
- 238000001514 detection method Methods 0.000 description 11
- 239000007789 gas Substances 0.000 description 10
- AYXYPKUFHZROOJ-ZETCQYMHSA-N pregabalin Chemical compound CC(C)C[C@H](CN)CC(O)=O AYXYPKUFHZROOJ-ZETCQYMHSA-N 0.000 description 10
- 229960001233 pregabalin Drugs 0.000 description 10
- 230000029087 digestion Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000006114 decarboxylation reaction Methods 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000012459 cleaning agent Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 208000019901 Anxiety disease Diseases 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 208000012839 conversion disease Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000004494 ethyl ester group Chemical group 0.000 description 2
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 230000006340 racemization Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000000707 stereoselective effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 101000600880 Arabidopsis thaliana Deaminated glutathione amidase, chloroplastic/cytosolic Proteins 0.000 description 1
- 102100021851 Calbindin Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- VUOGVGBBHBPFGM-UHFFFAOYSA-N Ethyl 5-methylhexanoate Chemical class CCOC(=O)CCCC(C)C VUOGVGBBHBPFGM-UHFFFAOYSA-N 0.000 description 1
- 101000898082 Homo sapiens Calbindin Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108010033272 Nitrilase Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 101001021643 Pseudozyma antarctica Lipase B Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000223258 Thermomyces lanuginosus Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000011982 enantioselective catalyst Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 230000001037 epileptic effect Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- IAHFWCOBPZCAEA-UHFFFAOYSA-N succinonitrile Chemical compound N#CCCC#N IAHFWCOBPZCAEA-UHFFFAOYSA-N 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of esterase and its encoding gene and application, the amino acid sequence of the esterase is as shown in SEQ ID NO.2.Present invention clone from pseudomonad Pseudomonas CGMCC NO.4184 obtains esterase gene, the characteristics of esterase obtained after the gene expression has high expression quantity, high selectivity and chiral selectivity；Engineering bacteria comprising esterase gene is applied to the methylhexanoic acid ethyl ester hydrolysis of 3 cyano group of catalysis 2 carboxyethyls of rac 5 and prepared in the methylhexanoic acid ethyl ester of 2 carboxyethyls (S) 3 cyano group 5 by the present invention, the conversion ratio of reaction can be controlled when more than 50%, you can obtain the methylhexanoic acid ethyl ester of the carboxyethyl of high-purity 2 (S) 3 cyano group 5 not being hydrolyzed.
Description
Technical field
The present invention relates to genetic engineering field, more particularly to a kind of esterase and its encoding gene and application.
Background technology
2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters be Chemoenzymatic synthesis Pregabalin (Pregabalin,
Abbreviation PGB, trade name) important chiral intermediate.Pregabalin is a kind of neurotransmitter γ-aminobutyric acid
(GABA) three isobutyl group substituents, it has been applied to treat a variety of central nervous systems as antiepileptic of new generation
System disorders, including neurogenic pain, sociability anxiety disorder, general hair anxiety disorder and auxiliary therapy limitation portion
Divide epileptic attack etc..Due to the medicine compared to other it is similar treatment epilepsy medicaments there is obvious advantage, by Pfizer
Produce simultaneously list marketingAnnual sales amount was up to respectively in gesture soaring year by year at 2011,2012 and 2013
36.9 hundred million, 41.6 hundred million and 46.0 hundred million dollars.Due to only having the Pregabalin of S configurations that there is pharmacological activity, therefore, optical voidness is obtained
Intermediate 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acids ethyl ester effectively reduce taking dose, improve treatment window, subtract
Have great importance in terms of side effect caused by light nonactive enantiomer.
Preparing Pregabalin mainly has two class methods：One class is using asymmetric catalyst, chiral ligand or with chirality
Compound is the dissymmetric synthesis of raw material, and another kind of is in racemization using chemical chiral resolution agent or biology enzyme
The racemate resolution method that mesosome or racemic Pregabalin are split.Dissymmetric synthesis is because needing expensive chiral examination
Agent, special equipment, harsh reaction condition etc., its application receives obvious limitation.Chemical resolution method equally has
The defect being difficult to avoid that, for example reactions steps are numerous and diverse, and labor intensity is big, and complex operation, yield is low, and environmental pollution is larger etc..Phase
Than under, biological resolution rule can be prevented effectively from these problems, and it is simply gentle to possess operating condition, and enantioselectivity is strong, cost
It is low, advantages of environment protection, therefore with great potentiality to be exploited and wide application prospect.
At present, many biological catalysis prepare the approach of Pregabalin and appeared in the newspapers.For example, with high vigor and high mapping
(conversion ratio is 45% to body selectivity, product enantiomeric excess value eepFor nitrilase AtNit1 98%), it can realize efficiently
Ground stereo selective hydrolysis isobutyl group succinonitrile, obtains intermediate (3S) -3- cyano group -5- methylhexanoic acids.From the false silk ferment in the South Pole
Female lipase CALB is also applied to stereoselective syntheses chiral intermediate (S)-or (R)-isobutyl group-glutarate (is received
Rate is 96%, ee values for 95.5%).The consideration of Kernel-based methods efficiency and product cost, these paths can not meet system at present
The requirement on industrial application of standby Pregabalin.
An other paths, i.e. Pfizer utilize commercial fatty enzymeOptionally hydrolysis 2- carboxyethyls-
(S) -3- cyano group -5- methylhexanoic acid ethyl esters, then be successfully realized the industrialized production that chemo-enzymatic process prepares Pregabalin.The party
The sodium salt of 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acids (conversion ratio is 45%-50%, ee values ＞ 98%) obtained by method,
(S)-Pregabalin can be finally made by subsequent chemical reaction step.This process route realizes the true as early as possible of chiral centre
The racemization of vertical and 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acid ethyl esters is recycled, and significantly reduces raw material dosage
With discarded object yield, the E factors are made to be down to 8 by the 86 of the first generation (classical resolution) route.Based on this bioanalysis disconnecting route,
Other enzymes for acting on 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters or microorganism are developed and reported successively.Such as one
The bacterial strain KM8 that strain soil sieve is obtained is through ultraviolet and dimethyl suflfate (DES) mutagenesis, its conversion ratio and eepValue be respectively increased to
76.1% and 92.6%.Another plant of bacterium Morgarella morganii ZJB-09203 and therefrom purify obtained esterase also by
Applied to selective hydrolysis 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters, 1.5M is hydrolyzed when using whole-cell catalyst
During substrate, its conversion ratio and eepValue is respectively 45.3% and 95%.From Thermomyces lanuginosus fat
Enzyme Lip is by Fixedpoint mutation modified, and enzyme activity is largely increased, realized under 1M substrate input amounts 42.4% conversion ratio and
98% eepValue.However, the Enzymatic Resolution of above-mentioned route can not all obtain the S type products of more high-optical-purity (ee ＞ 99%).
Therefore, left not by water by enantioselective hydrolysis 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acids ethyl ester
The high-optical-purity 2- carboxyethyls of solution-(S) -3- cyano group -5- methylhexanoic acid ethyl esters, which are that a kind of tool is potential, splits selection.
The content of the invention
The invention provides a kind of esterase and its encoding gene and application, the esterase have high expression quantity, high selectivity and
The characteristics of chiral selectivity.
The invention provides a kind of esterase, amino acid sequence is as shown in SEQ ID NO.2.
The esterase (being named as EstZF172) is to clone to obtain from pseudomonad Pseudomonas CGMCC NO.4184
, by 1146 base compositions, esterase hydrolyzed generation acid and alcohol can be catalyzed.
Present invention also offers a kind of gene of the esterase described in coding.
Preferably, the base sequence of described gene is as shown in SEQ ID NO.1.
The invention provides a kind of expression cassette, recombinant vector and transformant for including the gene.
Present invention also offers a kind of application of described esterase in catalysis ester-type hydrolysis.
Specifically, described esterase prepares 2- carboxylics in catalysis rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acids ethyl ester hydrolysis
Application in ethyl-(S) -3- cyano group -5- methylhexanoic acid ethyl esters.
Present invention also offers the method that one kind prepares 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters, including with
Lower step：
(1) the resting cell suspension of the engineering bacteria comprising the gene is prepared；
(2) it is hydrolyzed instead toward addition rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters in resting cell suspension
Should, it is post-treated, obtain 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters.
Described hydrolysis is shown below：
The reaction of catalysis rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester hydrolysis of the present invention, be actually
By 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acids of the R configurations in rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters
Ethyl ester hydrolysis obtains 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acids and ethanol, so that being left in reaction system optically pure
The 2- carboxyethyls of S configurations-(S) -3- cyano group -5- methylhexanoic acid ethyl esters；After hydrolysis terminates, reaction solution is centrifuged to remove body
Cell in system, appropriate NaCl is added into centrifuged supernatant, and is extracted repeatedly repeatedly with ethyl acetate, and extract is merged
Afterwards, vacuum revolving is carried out at 35 DEG C and removes extractant, separation obtains 2- carboxyethyls-(S) -3- cyano group -5- methyl of high-purity
Ethyl hexanoate.
In the preparation process of 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters of whole S configurations, rac-2- carboxylics
The usage ratio of ethyl -3- cyano group -5- methylhexanoic acids ethyl ester and resting cell, and hydrolysis temperature and ion buffer solution
PH value can all have an impact to the conversion ratio of the esterase catalyzed hydrolysis.Preferably, rac-2- carboxyethyl -3- cyanogen
The mass ratio of base -5- methylhexanoic acids ethyl ester and resting cell is 20~60: 1；The temperature of the hydrolysis is 20~55 DEG C；Water
The pH value for solving course of reaction intermediate ion buffer solution is 5~10.5.
Compared with prior art, the invention has the advantages that：
(1) present invention clone from pseudomonad Pseudomonas CGMCC NO.4184 obtains esterase gene, the gene
The characteristics of esterase obtained after expression has high expression quantity, high selectivity and chiral selectivity；
(2) present invention by the engineering bacteria comprising esterase gene be applied to catalysis rac-2- carboxyethyl -3- cyano group -5- methyl oneself
Hydrolysis of ethyl acetate is prepared in 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters, when hydrolysis conversion ratio slightly above
When 50%, you can obtain high-purity 2- carboxyethyls-(the S) -3- cyano group -5- methylhexanoic acid ethyl esters not being hydrolyzed；Especially, when anti-
It is 127.5g L to answer rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acid ethyl esters concentration in system-1When, reacting conversion ratio after 7h is
52.3% (i.e. yield up to more than 47%), now ees＞ 99%.
Brief description of the drawings
Fig. 1 is that recombinant plasmid pET30a-estZF172 expression plasmids build figure.
Fig. 2 is target stripe PCR primer electrophoresis result figure；
1：Target stripe；2：DNA marker.
Fig. 3 is restructuring pET30a-estZF172 expression plasmid digestion verification electrophoretograms；
1：Nde I single endonuclease digestions；2：Xho I single endonuclease digestions；3：Nde I/Xho I double digestions；4：DNA marker.
Fig. 4 is recombinant protein induced expression figure；
1：There is no the blank control of foreign gene；2：The full cells of BL21-pET30a-estZF172 (IPTG inductions)；
3：BL21-pET30a-estZF172 breaks and precipitated after born of the same parents；4：BL21-pET30a-estZF172 breaks supernatant after born of the same parents；
5：Protein marker.
Fig. 5 is 2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester and 2- carboxyethyl -3- cyano group -5- methylhexanoic acid decarboxylation things
Chiral gas chromatography analysis collection of illustrative plates；
Peak 1 is 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acid decarboxylation things (11.46min)；
Peak 2 is 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid decarboxylation things (12.53min)；
Peak 3 is 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acids ethyl esters (40.66min)；
Peak 4 is 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acids ethyl esters (41.86min).
Fig. 6 be the hydrolysis of esterase catalyzed rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester prepare 2- carboxyethyl-(S) -
The conditional curve of 3- cyano group -5- methylhexanoic acid ethyl esters.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited only to
This.
Experimental method in the present invention is conventional method unless otherwise instructed, for details, reference can be made to the volume such as J. Pehanorm Brookers
Write《Molecular Cloning:A Laboratory guide》.
Restriction enzyme NdeI, Xho I and T4 DNA ligases used in case study on implementation of the present invention are purchased from
TaKaRa；Genome extracts kit, plasmid extraction kit, that DNA recovery purifying kits are purchased from Axygen Hangzhou is limited
Company；E.coli DH5 α, E.coli BL21 (DE3), plasmid pET-30a (+) are purchased from Novagen companies；DNA marker、
Protein Marker, agarose electrophoresis reagent are purchased from Beijing Quanshijin Biotechnology Co., Ltd.Above reagent user
Method refers to catalogue.Esterase gene of the present invention is cloned from pseudomonad Pseudomonas CGMCC NO.4184 and obtained,
The strain is purchased from China General Microbiological culture presevation administrative center, and preserving number is：CGMCC NO.4184.
The detection method of following case study on implementation design is as follows：
1. gas phase detection method：Gas chromatographicanalyzer uses 9790 model gassy systems of Zhejiang Fu Li companies.Chromatogram
Post is chirality gas phase post Astec CHIRALDEXTMCapillary G-TA (30m × 0.25mm × 0.12 μm), detector is
Fid detector, testing conditions are：Injector and detector temperature are 250 DEG C, and column temperature is 135 DEG C, and carrier gas (N2) flow velocity is
30mL/min, air velocity is 300mL/min, and hydrogen flow rate is 30mL/min.After present invention reaction terminates, the μ of product 180 is taken
L, adds the hydrochloric acid terminating reaction that 40 μ L concentration are 1mol/L, adds appropriate sodium chloride to saturation, and add the acetic acid of 3 times of volumes
Ethyl ester is extracted.After centrifugation, supernatant anhydrous sodium sulfate drying takes 0.3 μ L sample detections, obtain 2- carboxyethyls-(R)-
3- cyano group -5- methylhexanoic acids, 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acids, 2- carboxyethyls-(R) -3- cyano group -5- methyl oneself
The gas phase figure of acetoacetic ester and 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters.
Additional description is needed, decarboxylic reaction can occur under gas phase condition for 2- carboxyethyl -3- cyano group -5- methylhexanoic acids,
Generate 3- cyano group -5- methylhexanoic acid ethyl esters.Therefore product peak tie substance actually 2- carboxyethyls -3- cyano group -5- methylhexanoic acids is de-
Carboxylic thing, i.e. 3- cyano group -5- methylhexanoic acid ethyl esters.
The conversion ratio of substrate rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters is calculated as follows：
Substrate conversion efficiency (%)=(initial substrate concentration-residue concentration of substrate)/initial substrate concentration × 100%.
Calculate the enantiomeric excess value of substrate and product respectively as follows：
Substrate enantiomer excessive value ees(%)=[(AS, s-AR, s)/(AS, s+AR, s)] × 100%.
Product enantiomeric excess value eep(%)=[(AR, p-AS, p)/(AR, p+AS, p)] × 100%.
Wherein, AS, s, AR, s, AS, p, AR, pRespectively gas-chromatography measures (S)-substrate, (R)-substrate, (S)-product and
(R) peak area value of-product.
2. sequencing：Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced.
The structure of the recombinant plasmid of the esterase gene of embodiment 1
(1) clone of esterase gene
According to pseudomonad (Pseudomonas) CGMCC NO.4184 genomic dna sequences, pair of primers, primer are designed
Sequence is as follows：
Sense primer is：5’-GGGAATTCCATATGCAGATCCAGGGTCACTATGAGCTGAAGTTCG-3 ', (such as SEQ
Shown in ID NO.3), wherein dashed part is Nde I restriction enzyme sites；
Anti-sense primer is：5’-CCCCTCGAGAAGGCAAGTGCCAAGAACGCGTACCA-3 ', (such as SEQ ID NO.4 institutes
Show), wherein dashed part is Xho I restriction enzyme sites.
Using pseudomonad (Pseudomonas sp.) CGMCC NO.4184 genomic DNAs template, enter performing PCR amplification,
PCR reaction systems and reaction condition are as follows：
PCR amplification system：
PCR amplification conditions：
1) pre-degeneration：95℃ 5min；
2) it is denatured：98℃ 10s；Annealing：59℃ 15s；Extension：72℃ 20s；Circulate 30 times altogether；
3) extend：72℃ 10min；
4) 4 DEG C of preservation 2.0h.
After PCR amplifications terminate, amplified production is detected with 1.0% agarose gel electrophoresis, as a result electrophoresis as shown in Figure 2
Figure, wherein, swimming lane 1 is the purpose fragment of the long 1146bp comprising restriction enzyme site.Purpose fragment is sequenced, purpose piece is confirmed
The sequence of section is as shown in SEQ NO.1.
(2) structure of expression vector
The purpose fragment is subjected to cleaning recovery with PCR cleaning agents box, product a is recycled.Recovery product a is used
Nde I and Xho I digestion 3h under the conditions of 37 DEG C, are cleaned with PCR cleaning agents box and reclaimed, be recycled product b.By pET-
30a (+) carrier digestion 3h under the conditions of 37 DEG C with Nde I and Xho I, is cleaned with PCR cleaning agents box and reclaimed, be recycled production
Thing c.Digestion products b and c are mixed, T4 ligases are added, 16 DEG C connect overnight, obtain linked system d.Esterase base will be connected to
The plasmid of cause is named as pET30a-estZF172.Linked system is as shown in table 1 below：
The pET30a-estZF172 recombinant expression plasmid linked systems of table 1
By linked system d Transformed E .coli BL21 (DE3) competent cell, it is coated on and receives mycin containing 50 μ g/mL cards
On LB flat boards, picking Colony Culture obtains recombinant cell after 37 DEG C of culture 16-24h.The recombinant cell of acquisition is extracted into plasmid
It is sequenced, confirms that it has the sequence as shown in SEQ NO.1.
Embodiment 2 builds restructuring E.coli BL21 (DE3) induced expression esterase
Recombinant cell with pET30a-estZF172 plasmids is inoculated in 5mL LB fluid nutrient mediums (containing 50g/
ML cards receive mycin) in, overnight incubation under the conditions of 37 DEG C and 200rpm.Above-mentioned culture is inoculated in 1% ratio and contained
In 50mLLB fluid nutrient mediums (receiving mycin containing 50g/mL cards), cultivated under the conditions of 37 DEG C and 200rpm to OD600Reach 0.6-
When 0.8, IPTG to final concentration of 0.5mmol/L is added.Fiber differentiation under 18 DEG C and 200rpm.12,000 × g centrifugations are received after 12h
Collect thalline, washed twice with the Tris-HCl buffer solutions (300mM, pH 8.5) of precooling, the wet cell of gained is stored in 4 DEG C.
Embodiment 3
In the ratio of 0.085 ‰ (0.085g stem cells/L) by corresponding wet cell 14mL Tris-HCl buffer solutions
(300mM, pH 8.5) is resuspended, and is divided into 7 parts, every part of 2mL is separately added into substrate rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acids
Ethyl ester so that rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester relative to rac-2- carboxyethyl -3- cyano group -5- methyl oneself
The mass concentration of acetoacetic ester and cell re-suspension liquid total amount is 5g/L.It is 20 DEG C, 30 DEG C, 35 DEG C that 7 parts of solution are respectively put into temperature,
During 40 DEG C, 45 DEG C, 50 DEG C and 55 DEG C of rotating speed is 200rpm constant-temperature table, after 1h with gas phase detection method detection substrate and
The concentration of product, its retention time in gas phase are as shown in figure 5, peak 1 is 2- carboxyethyls-(R) -3- cyano group -5- methylhexanoic acids
Decarboxylation thing (11.46min)；Peak 2 is 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid decarboxylation things (12.53min)；Peak 3 is 2-
Carboxyethyl-(R) -3- cyano group -5- methylhexanoic acids ethyl esters (40.66min)；Peak 4 be 2- carboxyethyls-(S) -3- cyano group -5- methyl oneself
Acetoacetic ester (41.86min).Calculate conversion ratio and ees, as a result such as table 1.From table 1 it was found from data, under the conditions of 20~35 DEG C,
Conversion ratio is raised and raised with temperature, and at 35 DEG C, substrate conversion efficiency is 53.5%, eesHigher than 99%；In 35~55 DEG C of conditions
Under, with the rise of reaction temperature, because enzyme is easily inactivated at high temperature, cause substrate conversion efficiency to decline on the contrary, eesAlso therewith
Decline.
The esterase of table 1 hydrolyzes the reaction conversion of rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters at different temperatures
Rate and eesValue
Embodiment 4
In the ratio of 0.085 ‰ (0.085g stem cells/L) by corresponding wet cell respectively with the Tris- of the different pH value of 2mL
HCl buffer solutions (300mM) are resuspended, and pH is respectively 5.0,6.0,7.0,8.0,8.5,9.0,10.0, is separately added into substrate rac-2-
Carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters so that rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters are relative to rac-
The mass concentration of 2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester and cell re-suspension liquid total amount is 5g/L.7 parts of solution are put into
35 DEG C, in 200rpm constant-temperature table, with the concentration of gas phase detection method detection substrate and product after 1h, calculate conversion ratio and
ees, as a result such as table 2.From table 2 it was found from data, under the conditions of pH 5.0~8.5, conversion ratio is raised and risen with pH of cushioning fluid
Height, in pH 8.5, substrate conversion efficiency is 52.9%, eesHigher than 99%；Under the conditions of pH 8.5~10.0, with pH of buffer
The rise of value, because in the stronger environment of alkalescence, the stability of enzyme is not good, and substrate causes substrate to convert easily from hydrolyzing
Rate declines on the contrary, eesAlso decline therewith.
The reaction that the Recombinant esterase of table 2 hydrolyzes rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters under different pH turns
Rate and eesValue
Embodiment 5
According to the difference of concentration of substrate, substrate/cell quality ratio of appropriate fixation is selected, i.e.,：When substrate quality is dense
When spending for 5g/L and 30g/L, it is 0.085 ‰ (0.085g stem cells/L) that cell concentration is added per 5g/L substrates；When substrate quality is dense
When spending for 60g/L and 80g/L, it is 0.10 ‰ that cell concentration is added per 5g/L substrates；When substrate mass concentration is 150g/L and 200g/
During L, it is 0.16 ‰ that cell concentration is added per 5g/L substrates；When substrate mass concentration is 350g/L and 500g/L, per 5g/L substrates
It is 0.24 ‰ to add cell concentration.Corresponding wet cell is resuspended with 2mL Tris-HCl buffer solutions (300mM, pH 8.5) respectively,
It is separately added into different amounts of substrate rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acid ethyl esters so that rac-2- carboxyethyl -3- cyanogen
Base -5- methylhexanoic acids ethyl ester is relative to rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester and the matter of cell re-suspension liquid total amount
It is respectively 5g/L, 30g/L, 60g/L, 80g/L, 150g/L, 200g/L, 350g/L, 500g/L to measure concentration.8 parts of solution are put into
35 DEG C, in 200rpm constant-temperature table, with the concentration of gas phase detection method detection substrate and product after 8h, calculate conversion ratio and
ees, as a result such as table 3.From table 3 it was found from data, by controlling the mass ratio of substrate and the cell of input, it can realize anti-
Answer after 12h, 5g/L~127.5g/L substrate is all by successful conversion, eesThe level higher than 99% is reached, and conversion ratio is controlled
Below 62%.When concentration of substrate is relatively low, ee has been realized within the shorter time due to reactingsHigher than 99%, therefore during reaction
Between continue to extend to 12h more substrates can be made slowly to be converted, cause reaction conversion ratio to improve, 2- carboxyethyls-(S) -3- cyano group -
The yield of 5- methylhexanoic acid ethyl esters declines., it is necessary to adjust cell addition to improve response inhabitation situation when concentration of substrate is higher,
eesAlso changed the time required to higher than 99%, required time extends in general.
The esterase of table 3 hydrolyzes the reaction of rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters under different concentration of substrate
Conversion ratio and eesValue
Embodiment 6
In the ratio of 3.26 ‰ (3.26g stem cells/L) by corresponding wet cell 5mL Tris-HCl buffer solutions
(300mM, pH8.5) is resuspended, and adds substrate rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters so that rac-2- carboxylic second
Base -3- cyano group -5- methylhexanoic acids ethyl ester is relative to rac-2- carboxyethyls -3- cyano group -5- methylhexanoic acids ethyl ester and cell re-suspension liquid
The mass concentration of total amount is 127.5g/L, is put into 35 DEG C of water-baths, using magnetic agitation, is sampled in first 2 hours every 0.5h,
Sampled afterwards every 1h, with the concentration of gas phase detection method detection substrate and product, calculate conversion ratio and ees, as a result such as Fig. 6.
As can be seen from Figure 6, with the increase in reaction time, the conversion ratio of substrate is continuously increased, and conversion ratio is up to 52.3%, ee after 7hs＞
99%, the concentration for finally giving 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters is about 60.8g/L.
Claims (10)
1. a kind of esterase, it is characterised in that amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of gene for encoding the esterase described in claim 1.
3. gene as claimed in claim 2, it is characterised in that the base sequence of described gene is as shown in SEQ ID NO.1.
4. a kind of include the expression cassette of gene, recombinant vector and transformant described in Claims 2 or 3.
5. application of the esterase as claimed in claim 1 in catalysis ester-type hydrolysis.
6. esterase as claimed in claim 1 prepares 2- in catalysis rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acids ethyl ester hydrolysis
Application in carboxyethyl-(S) -3- cyano group -5- methylhexanoic acid ethyl esters.
7. the method that one kind prepares 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters, comprises the following steps：
(1) the resting cell suspension for including the engineering bacteria of gene described in claim 2 is prepared；
(2) reaction is hydrolyzed toward addition rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters in resting cell suspension, passes through
Post processing, obtains 2- carboxyethyls-(S) -3- cyano group -5- methylhexanoic acid ethyl esters.
8. preparation method as claimed in claim 7, it is characterised in that rac-2- carboxyethyl -3- cyano group -5- methylhexanoic acid ethyl esters
Mass ratio with resting cell is 20~60: 1.
9. preparation method as claimed in claim 7, it is characterised in that the temperature of the hydrolysis is 20~55 DEG C.
10. preparation method as claimed in claim 7, it is characterised in that the pH value of hydrolysis reaction intermediate ion buffer solution is 5
~10.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510005882.5A CN104560912B (en) | 2015-01-06 | 2015-01-06 | Esterase and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510005882.5A CN104560912B (en) | 2015-01-06 | 2015-01-06 | Esterase and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104560912A CN104560912A (en) | 2015-04-29 |
| CN104560912B true CN104560912B (en) | 2017-09-08 |
Family
ID=53077992
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510005882.5A Active CN104560912B (en) | 2015-01-06 | 2015-01-06 | Esterase and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104560912B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119224B (en) * | 2016-07-27 | 2019-12-10 | 中国科学院南海海洋研究所 | Esterase EstP00714 and coding gene and application thereof |
| CN112301017A (en) * | 2020-12-01 | 2021-02-02 | 江苏美科生物科技有限公司 | Lipase derived from pseudomonas and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100354422C (en) * | 2005-01-07 | 2007-12-12 | 上海医药工业研究院 | Ester hydrolase and its gene and recombinant enzyme |
| CN101985609B (en) * | 2010-10-26 | 2012-06-13 | 浙江大学 | Pseudomonas and method for performing microbial transformation on pregabalin intermediate by using same |
-
2015
- 2015-01-06 CN CN201510005882.5A patent/CN104560912B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104560912A (en) | 2015-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2019154249A1 (en) | Nitrilase mutant, construction method therefor, and application thereof | |
| CN102834515B (en) | New hydrolase protein | |
| CN103627685B (en) | Higher-activity partial glyceride lipase mutant and application thereof | |
| CN104774825B (en) | Nitrilase mutants and its application | |
| CN104212784B (en) | Restructuring nitrilase, encoding gene, mutant, engineering bacteria and application | |
| CN113249365B (en) | L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonylbenzserine | |
| TW200914610A (en) | Isopropyl alcohol-producing bacteria and method for producing isopropyl alcohol using the same | |
| CN103525784B (en) | Partial glyceride lipase mutant, plasmids, recombination strains, preparation methods and applications | |
| CN107653233A (en) | A kind of improved transaminase, its encoding gene and the genetic engineering bacterium for expressing the enzyme | |
| EP2840135B1 (en) | Cis-epoxysuccinate hydrolase-encoding gene, polypeptide encoded by the gene, and related application thereof | |
| CN109182298B (en) | Recombinant lipase mutant, engineering bacterium and application | |
| CN104745547B (en) | A kind of epoxide hydrolase mutant, engineering bacteria and its application | |
| CN104560912B (en) | Esterase and its encoding gene and application | |
| CN106244569B (en) | Esterase EstC10, and coding gene and application thereof | |
| CN104031899B (en) | A kind of lipase and application thereof | |
| CN104404011B (en) | Amidase and its encoding gene and application | |
| CN105296513B (en) | A kind of ocean esterase and its encoding gene E22 and application | |
| CN105734028A (en) | Epoxide hydrolase mutant and application thereof | |
| CN112175919A (en) | Lactone hydrolase mutant and application thereof | |
| CN107177569A (en) | A kind of Kidney bean epoxide hydrolase and its heterogenetic expression method | |
| CN113025601B (en) | Optimized expression and application of nitrilase promoter | |
| CN106119224B (en) | Esterase EstP00714 and coding gene and application thereof | |
| CN105238769B (en) | A kind of lipase LIPASE7 and its encoding gene and application | |
| CN105132394B (en) | A kind of lipase LIPASE6 and its encoding gene and application | |
| CN105543192B (en) | A kind of esterase B SE01701 and its encoding gene and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |