CN108396016A - A kind of esterase PHE21 and its encoding gene and the application in prepared by the secondary butyl ester of chiral acetic - Google Patents

A kind of esterase PHE21 and its encoding gene and the application in prepared by the secondary butyl ester of chiral acetic Download PDF

Info

Publication number
CN108396016A
CN108396016A CN201810114144.8A CN201810114144A CN108396016A CN 108396016 A CN108396016 A CN 108396016A CN 201810114144 A CN201810114144 A CN 201810114144A CN 108396016 A CN108396016 A CN 108396016A
Authority
CN
China
Prior art keywords
phe21
esterase
sec
butyl acetate
application
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
CN201810114144.8A
Other languages
Chinese (zh)
Other versions
CN108396016B (en
Inventor
胡云峰
王依龙
张云
孙爱君
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.)
South China Sea Institute of Oceanology of CAS
Original Assignee
South China Sea Institute of Oceanology of CAS
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 South China Sea Institute of Oceanology of CAS filed Critical South China Sea Institute of Oceanology of CAS
Priority to CN201810114144.8A priority Critical patent/CN108396016B/en
Publication of CN108396016A publication Critical patent/CN108396016A/en
Application granted granted Critical
Publication of CN108396016B publication Critical patent/CN108396016B/en
Expired - Fee Related 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
    • 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)
    • 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
    • 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
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A kind of application the invention discloses esterase PHE21 and its encoding gene and in prepared by the secondary butyl ester of chiral acetic.The present invention is cloned into an esterase gene PHE21 from marine pseudomonas (Pseudomonadaceae oryzihabitans HUP022), its nucleotide sequence is as shown in SEQ ID NO.1, overall length is 966bp, the amino acid sequence of its esterase PHE21 encoded includes 321 amino acid altogether as shown in SEQ ID NO.2.Using the esterase PHE21 of recombinant expression as catalyst, on the one hand it directly can prepare (S) sec-butyl acetate that optical purity is more than 98% by catalytic hydrolysis reaction, on the other hand can also be catalyzed transesterification reaction and prepare (R) sec-butyl acetate that optical purity is more than 64%.Esterase PHE21 has very big application prospect in biological medicine and field of fine chemical.

Description

A kind of esterase PHE21 and its encoding gene and in prepared by the secondary butyl ester of chiral acetic Using
Technical field：
The invention belongs to biochemical industries and biotechnology, and in particular to a kind of esterase PHE21 and its encoding gene and Application in prepared by the secondary butyl ester of chiral acetic.
Background technology：
Chipal compounds be it is a kind of be made of same atoms, a kind of compound of stereochemical structure mirror image each other, different mappings Body often shows completely different physiological activity and toxicity.As 2 enantiomers of benzmorphan have analgesic activity, but Meeting habituation after the isomers of (-) is taken, and the isomers of (+) then will not；" reaction stops " of R types is pregnant woman's antalgesic and analgesic Medicine, and " reaction stops " of S types then has teratogenesis to fetus；The d-isomer of antimicrobial Ofloxacin can damage Liver and kidney function, but Left-handed Ofloxacin drug effect is very high, and toxicity is minimum, deeply welcomed by the people after various countries list.So in synthesis hand During property drug, the optical purity of precursor raw material is very important link.
Chiral sec-butyl alcohol and its ester derivative are important chiral chemistry product and resolving agent, industrially there is very big need It asks, is widely used in perfume industry, medical industry, detergent industry and fine chemistry industry industry.If chiral butanol and its ester are as one Kind environment-friendly type solvent, is not only synthesizing chiral compound and is especially some anti-tubercular drugs (such as ethambutol) and some antihyperalgesics The important key intermediate of (such as labetalol), and it is also widely used for the solvent of the chemical products such as nitrate paint, acrylic paint With the pesticides industry such as emulsifiable concentrates for agricultural chemicals agent.
The synthesis of chipal compounds mainly has：(1) chemical method, that is, utilize enantiomer physics and chemical property difference into Row separation.Usually there are salting out method, inclusion method and Combinatorial resolution.The disadvantage is that the nature difference between requiring enantiomer is big, Applicable compound is few.(2) Chromatographic resolution, this method are using the difference of the chiral enantiomer adsorption property of filler come real Existing, the disadvantage is that equipment is expensive, popularization is poor.(3) synthetic method is closed by designing reaction to carry out the chemistry of chipal compounds At.This method disadvantage is that reaction process is usually relatively violent, expends energy, and using a large amount of toxic organic in reacting Solvent.(4) biological enzyme, biological lipase/esterase have the high specificity of three-dimensional site areas and substrate, and react item The advantages that part is mild, side reaction is few, optical purity is high and environmental pollution is small, therefore develop the fat with optical selective Enzyme/esterase has great importance.
Invention content：
The present invention's is directed to deficiency in the prior art, provides a kind of new esterase PHE21 and its encoding gene and in hand Property sec-butyl acetate prepare in application.
Present invention exploitation from one plant of marine pseudomonas (Pseudomonadaceae oryzihabitans HUP022) A kind of new esterase PHE21 and its encoding gene PHE21, construct recombinant expression carrier and genetic engineering containing PHE21 Bacterium obtains esterase PHE21 after culturing gene engineering bacteria, can be applied to prepare the secondary butyl ester of chiral acetic.
The first purpose of the invention is to provide a kind of esterase PHE21, and amino acid sequence is as shown in SEQ ID NO.2.
Second object of the present invention is to provide the esterase gene PHE21 of the esterase PHE21 described in coding a kind of.
It is preferred that the nucleotide sequence of the esterase gene PHE21 is as shown in SEQ ID NO.1.
The present invention also provides a kind of recombinant expression carriers containing the esterase gene PHE21.The expression vector, It is preferred that pET28a (+) carrier.
The present invention also provides a kind of genetic engineering bacteriums containing the esterase gene PHE21.The genetic engineering bacterium, It is preferred that e. coli bl21 (DE3).
Third object of the present invention is to provide the esterase PHE21 in preparation (S)-sec-butyl acetate or (R)-acetic acid Application in secondary butyl ester.
It is preferred that the application is esterase PHE21 is prepared (S)-sec-butyl acetate in fractionation (±)-sec-butyl acetate In application.
Further preferably, the application is：It takes esterase PHE21 in the buffer solution that pH is 6.0~9.0, adds (±)-sec-butyl acetate, is reacted, and (S)-sec-butyl acetate is obtained.
The buffer solution, preferably citric acid-sodium citrate buffer solution, phosphate buffer, Tris-HCl buffer solutions and One kind in glycine-NaOH buffer.
It is preferred that the application is esterase PHE21 is prepared (R)-acetic acid in the transesterification reaction of catalysis (±)-sec-butyl alcohol Application in secondary butyl ester.
Further preferably, the application is：It takes esterase PHE21 in reaction medium, adds (±)-sec-butyl alcohol and acyl Base donor, is reacted, and (R)-sec-butyl acetate is obtained.
The reaction medium, preferably Isosorbide-5-Nitrae-dioxane, tetrahydrofuran, the tert-butyl alcohol, isopropanol, dichloromethane, first One kind in benzene, hexamethylene, n-hexane, normal heptane and isooctane.
One kind in the acry radical donor, preferably acetyl vinyl acetate, acetyl isopropyl enester and ethyl acetate.
One plant of marine pseudomonas that the esterase gene PHE21 of the present invention is screened in the sample of deep-sea (Pseudomonadaceae oryzihabitans HUP022), is stored in Chinese Academy of Science Nanhai Ocean Research Institute laboratory. The method of present invention bioinformatic analysis, from the pseudomonad (Pseudomonadaceae of gene order-checking Oryzihabitans HUP022) in screening obtain esterase gene PHE21, overall length is 966bp (close from initiation codon to terminating Numeral), encode 321 amino acid.By cloning the esterase gene PHE21 of the esterase PHE21 of encoding mature and connecting table E. coli bl21 (DE3) is converted afterwards up to carrier pET-28a (+), is cultivated and after induced expression, the esterase recombinantly expressed PHE21.Esterase PHE21 can be used for preparing the secondary butyl ester of chiral acetic, using the esterase PHE21 of recombinant expression as catalyst, one Aspect directly can prepare (S)-sec-butyl acetate that optical purity is more than 98% by catalytic hydrolysis reaction, on the other hand can also urge Change transesterification reaction and prepares (R)-sec-butyl acetate that optical purity is more than 64%.Esterase PHE21 is in biochemical industry and biological medicine Equal fields have very big application value.
Description of the drawings：
Fig. 1 is the NCBI blast comparing results of esterase gene PHE21.
Fig. 2 is influences of the pH to enzyme kinetics Hydrolysis Resolution sec-butyl acetate.
Fig. 3 is influence of the temperature to enzyme kinetics Hydrolysis Resolution sec-butyl acetate.
Fig. 4 is influence of the concentration of substrate to enzyme kinetics Hydrolysis Resolution sec-butyl acetate.
Fig. 5 is influence of the enzyme concentration to enzyme kinetics Hydrolysis Resolution sec-butyl acetate.
Fig. 6 is influence of the reaction time to enzyme kinetics Hydrolysis Resolution sec-butyl acetate.
Fig. 7 is that esterase PHE21 splits (±)-sec-butyl acetate reaction GC figures；A is sample (±)-sec-butyl acetate gas phase Figure, B are that esterase PHE21 splits the gas phase figure after (±)-sec-butyl acetate reaction 3h, and wherein S represents (S)-sec-butyl acetate, R Represent (R)-sec-butyl acetate.
Fig. 8 is influence of the reaction time to transesterification reaction.
Fig. 9 is esterase PHE21 catalysis (±)-sec-butyl alcohol transesterification reaction GC figures；A is sample (±)-sec-butyl acetate gas phase Figure, B are the gas phase figure that esterase PHE21 is catalyzed after (±)-sec-butyl alcohol transesterification reaction 4h, and wherein S represents (S)-sec-butyl acetate, R Represent (R)-sec-butyl acetate.
Specific implementation mode：
The following examples are further illustrations of the invention, rather than limiting the invention.
The experimental method not indicated specifically in following Examples, can conventionally carry out, or be given birth to according to product used Produce the operation instruction of manufacturer.The materials, reagents and the like used in the following examples can pass through commercial sources unless otherwise specified It obtains.
One plant of marine pseudomonas that the esterase gene PHE21 of the present invention is screened in the sample of deep-sea (Pseudomonadaceae oryzihabitans HUP022), the bacterium are stored in Chinese Academy of Science Nanhai Ocean Research Institute's reality Test room.
Embodiment 1：Esterase gene PHE21 design of primers and open reading frame boundary determine
The genomic DNA for extracting pseudomonad (Pseudomonadaceae oryzihabitans HUP022), through sequencing Verify it is errorless after, genome is annotated using bioinformatics means, analyzes esterase gene therein, it is determined that wherein ester The open reading frame of enzyme gene PHE21, for nucleotide sequence as shown in SEQ ID NO.1, overall length is 966bp (from initiation codon Son arrives terminator codon), the amino acid sequence of the esterase PHE21 of coding is as shown in SEQ ID NO.2, totally 321 amino acid； The gene is a completely new esterase gene, and the maximum similarity with other esterase gene sequences is 96% (Fig. 1).According to analysis Obtained esterase gene PHE21 sequences, design primer are as follows：Forward primer：5′-CACGAATTCATGCCCGACGTCTTCGCGC G-3 ', underscore part are EcoRI restriction enzyme sites；Reverse primer：5′-CCGCTCGAGTTAGGCGGCCTGCTGCAGATGC- 3 ', underscore part is Xho I restriction enzyme sites.
Embodiment 2：The clone of esterase gene PHE21 and vector construction
2.1PCR amplification
Primer (the forward primer that embodiment 1 is designed：5′-CACGAATTCATGCCCGACGTCTTCGCGCG-3 ', reversely Primer：5′-CCGCTCGAGTTAGGCGGCCTGCTGCAGATGC-3 ') supreme marine growth Engineering Co., Ltd synthetic primer is sent, The primer of synthesis is diluted to 10 μM using TE, pseudomonad (Pseudomonadaceae oryzihabitans HUP022) Total DNA establishes reaction system as shown in table 1 as DNA profiling：
1 PCR reaction systems of table
Use following PCR amplification program amplification esterase gene PHE21：A.94 DEG C denaturation 3min；DEG C b.94 denaturation 30s, 55 ~65 DEG C of annealing 0.5min, 72 DEG C of extension 1min carry out 30 cycles；C.72 DEG C extension 10min, is cooled to 10 DEG C.
By PCR product in 1% Ago-Gel, electrophoresis 20min, is placed in gel imaging system and sees under 120V voltages It examines.Recycle the band of 984bp or so.PCR product is recycled according to the method for plastic recovery kit, uses the 20 sterile washings of μ L It is de-, obtain the PCR product of purifying recycling.
2.2 digestion
The PCR product of purifying recycling is subjected to double digestion, digestion time 1h using following system.Digestion system is：EcoRI 2 μ L, XhoI 2 μ L, DNA<The distilled water of 0.3 μ g, sterilizing add to 30 μ L.Purifying recycling obtains the PCR by double digestion after digestion Product.
The double digestion of plasmid pET-28a (+)：Picking contains the bacillus coli DH 5 alpha single bacterium colony of plasmid pET-28a (+), mistake Night cultivates.Plasmid is extracted using plasmid extraction kit, following system double digestion, digestion time 1h are pressed with BamHI and XhoI.Enzyme The system of cutting is：22 μ L of μ L, XhoI of EcoRI, Plasmid DNA<The distilled water of 1 μ g, sterilizing add to 20 μ L.Purifying is recycled after digestion To pET-28a (+) carrier by double digestion.
The restriction enzyme that above-mentioned double digestion uses is the quick restriction endonuclease of Thermo companies production, the purifying after digestion Recycling uses nucleic acid purification QIAquick Gel Extraction Kit (Magen, Hipure Gel Pure DNA Micro Kit), plasmid extraction reagent Box is the Plasmid Miniprep Kit of Shanghai Jierui Biology Engineering Co., Ltd, and operating method presses its operation instructions.
2.3 connection
By the PCR product of process double digestion and pET-28a (+) carrier according to 3:1 molar ratio is attached.Connection makes T4 ligases are purchased from Beijing Quanshijin Biotechnology Co., Ltd, and it is 5U/5 μ L linked systems to connect the enzyme amount used, even Jointing temp is 25 DEG C, Connection Time 30min.Thus connection product is obtained.
2.4 conversions and screening
Take 5 μ L connection products in 50 μ L escherichia coli DH5a competent cells, ice bath 30min, after in 42 DEG C of water-baths It is added 500 μ L LB liquid mediums after heat shock 90s, ice bath 2min, under 37 DEG C of 200rpm rotating speeds, is incubated culture 1h.It takes a certain amount of Bacterium solution be coated on the LB tablets containing 100 μ g/mL kanamycins, cultivate picking individual colonies after 20h.Single bacterium falls within 5mL LB cultures Plasmid is extracted after being incubated overnight in base, carries out double digestion verification, endonuclease bamhi identical with gene size is positive colony.
2.5 gene nucleotide series measure
The correct positive colony of screening is sent to Shanghai Mei Ji biological medicines Co., Ltd and is sequenced, sequencing result and ester Enzyme gene PHE21 nucleotide sequences are compared, and confirmation is by esterase gene PHE21 (its nucleotide sequence such as SEQ ID NO.1 It is shown) it is inserted into pET-28a (+) plasmid, as a result completely correct rear confirmation obtains the pET-28a with esterase gene PHE21 (+) plasmid (is named as pET-28a (+)-PHE21), can be used for carrying out next step experiment.
Embodiment 3：High efficient expressions of the esterase gene PHE21 in e. coli bl21 (DE3)
It is prepared by 3.1 e. coli bl21s (DE3) competent cell
1, a small amount of e. coli bl21 (DE3) strain is accessed in 5mL LB test tube liquid, 37 DEG C are shaken training, 250rpm overnight；
2, e. coli bl21 (DE3) bacterium solution after shaking training overnight is inoculated into 300ml by the inoculum concentration of 1% volume ratio In LB shaking flasks, 37 DEG C are shaken training 3-4h (>=300rpm)；
3, cultured shaking flask in ice water is rapidly cooled to 0 DEG C, dispenses the centrifuge tube (50mL) being pre-chilled to ice, ice is set Several minutes；
4,4 DEG C, 4000rpm centrifuges 10min and recycles cell, removes supernatant；
5, the CaCl of ice-cold 10mL 0.1M2Cell is resuspended, 4 DEG C, 4000rpm centrifuges 10min and recycles cell；
6,5 are repeated, with the CaCl of 10mL 0.1M2Cell, ice bath 1h or more is resuspended；
7,4 DEG C, 4000rpm centrifuges 10min and recycles cell；
8, the 0.1M CaCl of 50mL stock cultures 2mL 15%DMSO containing volume fraction2Be resuspended, be sub-packed in 1.5mL from Heart pipe, 100 μ L are often managed, -80 DEG C of preservations.Thus e. coli bl21 (DE3) competent cell is obtained.
3.2 conversion
0.5~1 μ L of pET-28a (+)-PHE21 plasmids and 50 μ L e. coli bl21s (DE3) obtained in Example 2 Competent cell mixes, and 500 μ L LB Liquid Cultures are added in ice bath 30min after 42 DEG C of water-baths heat shock 45s, ice bath 2min Base, 37 DEG C of 200rpm cultivate 1h.It is coated with the kanamycins LB tablets of 50 μ g/mL after culture centrifugation, list was selected after cultivating 15h Bacterium.Thus the e. coli bl21 (DE3) containing pET-28a (+)-PHE21 is obtained.
Embodiment 4：The expression and purifying of esterase PHE21
4.1 protein induced
The 37 DEG C of cultures in LB culture mediums of e. coli bl21 (DE3) containing pET-28a (+)-PHE21 are to OD600 0.5 or so, add IPTG to final concentration 0.2mM, 20 DEG C of 16 hours of culture.300mL bacterium solutions 4000rpm, 4 DEG C centrifuge 10min, receive Collect thalline, with PBS buffer solution washing thalline 2 times, 4000rpm, 10min collect thalline.With 30mL (50mM, pH7.4) Tris- Thalline is resuspended in HCl buffer solutions, and ultrasonic 400w, super 5s stop 5s, broken 10min, 4 DEG C, 10000rmp centrifugation 10min, in collection Clearly.The supernatant of collection is lyophilized with freeze dryer to obtain the thick enzyme powders of esterase PHE21, -80 DEG C of refrigerators is put in and preserves.4.2 esterase PHE21 Purifying
Esterase PHE21 (the i.e. esters that the supernatant collected in step 4.1 is carried out purifying to purify with nickel ion affinity chromatograph column The pure enzyme solutions of enzyme PHE21, a concentration of 10mg/mL), the albumen size about 37kD of purifying meets theory expectation.Specific embodiment is such as Under：5 column volumes are eluted using the imidazoles of 10mM, 30mM imidazoles elutes 30 column volumes, finally uses 100~1000mM imidazoles 5 column volumes are eluted, intermediate 3.5mL is collected.Desalination is carried out with desalting column SephadexG25, concrete operation method is public with reference to GE The operation manual of department carries out.
Embodiment 5：Applications of the esterase PHE21 in fractionation (±)-sec-butyl acetate
5.1 enzyme kinetics Hydrolysis Resolution sec-butyl acetates obtain optically pure (S)-sec-butyl acetate
Enzyme kinetics Hydrolysis Resolution sec-butyl acetate obtain optically pure (S)-sec-butyl acetate by cosolvent type and The influence of all many conditions of ratio, reaction temperature, rotating speed etc..Above-mentioned parameter is optimized in the present invention, and reaction mixture is： In the Na of 500 μ L50mM, pH7.52HPO4/NaH2PO4In buffer solution, be added final concentration 0.01g/L esterase PHE21 (i.e. plus Enter the pure enzyme solutions of esterase PHE21 made from the step 4.2 of 1mL/L), (±)-sec-butyl acetate of 0.02M, in 37 DEG C, 200rpm items 120min is reacted under part.
Concrete analysis condition is：Gas chromatograph is stood using good fortune, is furnished with chiral column (30m × 0.25mm Cyclosil B Chirl column) and hydrogen ion flame detector.Instrumental conditions are set as：220 DEG C of injector temperature, detector temperature 250 DEG C, carrier gas N2, flow velocity 1.2mL/min analyzed using gradient increased temperature：80 DEG C holding 1min, 15 DEG C/min, 120 DEG C 1min is kept, 10 DEG C/min to 220 DEG C, keeps 1min.
Influences of the 5.2pH to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
Different buffer solutions is prepared, these buffer solutions have different pH, and as shown in table 2, concentration is 50mM：
The pH of the different buffer systems of table 2
By the buffer solution (Na described in determination condition in 5.12HPO4/NaH2PO4Buffer) according to the buffer solution in table 2 It is replaced respectively, measures influence (Fig. 2) of the buffer solution of different pH to esterase PHE21 Hydrolysis Resolution sec-butyl acetates, tie Fruit illustrates that in the buffer solution that pH is 8, esterase PHE21 is best to the fractionation effect of substrate (±)-sec-butyl acetate.(S)- The enantiomeric excess value and conversion ratio of sec-butyl acetate are respectively 70% and 41.2%.
Influence of 5.3 temperature to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
In pH8.0,50mM Na2HPO4/NaH2PO4As buffer solution, it is placed in by the reaction mixture in 5.1 different The experiment for splitting sec-butyl acetate, under the conditions of different reaction temperatures, (S)-acetic acid are hydrolyzed at temperature (25~50 DEG C) The enantiomeric excess value and conversion ratio of secondary butyl ester are as shown in Figure 3.As a result illustrate, when reaction temperature is 35 DEG C, (S)-acetic acid is secondary The enantiomeric excess value highest of butyl ester is 80%.When reaction temperature is higher than 40 DEG C or less than 30 DEG C, (S)-sec-butyl acetate Enantiomeric excess value can all decline, illustrate that temperature has a very big impact the optical selective of esterase PHE21.
The influence of 5.4 organic solvents and surfactant to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
In the experiment of Hydrolysis Resolution sec-butyl acetate, by the reaction mixture in 5.1 by esterase PHE21 and substrate (±)-sec-butyl acetate is placed in pH8.0,50mM Na2HPO4/NaH2PO4In buffer solution, under conditions of temperature is 35 DEG C, 11 kinds of different organic solvents are added and resolution reaction is hydrolyzed in 5 kinds of different surfactants, the results are shown in Table 3.Knot Fruit shows that 11 kinds of different organic solvents in experiment and 5 kinds of surfactants can generate the stereoselectivity of esterase PHE21 Different degrees of inhibition illustrates that organic solvent and surfactant can generate the stereoselectivity of esterase PHE21 larger shadow It rings.
The influence of 3 organic solvent of table and surfactant to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
Influence of 5.5 concentration of substrate to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
Under the conditions of optimal reactive temperature (35 DEG C) and (8.0) optimal reaction pH, exist respectively by the reaction mixture in 5.1 Substrate (±)-sec-butyl acetate of various concentration is added in reaction system, test concentration of substrate is to esterase PHE21 Hydrolysis Resolution second The influence of the secondary butyl ester of acid, reaction result are as shown in Figure 4.As a result illustrate, when concentration of substrate is 0.04M, esterase PHE21 hydrolysis The effect of (±)-sec-butyl acetate is best, the enantiomeric excess value ＞ 91% of product (S)-sec-butyl acetate, and conversion ratio is 48.9%, concentration of substrate is higher, and it is poorer to split effect.
Influence of 5.6 enzyme concentrations to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
Under the conditions of optimal reactive temperature (35 DEG C) and (8.0) optimal reaction pH, by being added in the reaction mixture in 5.1 The pure enzyme solution of various concentration is added in substrate (±)-sec-butyl acetate (0.04M) of optimum concentration in the reaction system respectively, surveys The most suitable enzyme concentration of esterase PHE21 Hydrolysis Resolutions (±)-sec-butyl acetate (0.04M) is tried, reaction result is as shown in Figure 5.As a result Illustrate, when enzyme concentration is 0.016g/L, the effect of Hydrolysis Resolution (±)-sec-butyl acetate (0.04M) has reached best, product (S) the enantiomeric excess value ＞ 92% of-sec-butyl acetate, conversion ratio 49.7%, when enzyme concentration is more than 0.03g/L, product (S) enantiomeric excess value and conversion ratio of-sec-butyl acetate are basically unchanged.
Influence of 5.7 reaction time to enzyme kinetics Hydrolysis Resolution sec-butyl acetate
(35 DEG C, pH 8.0,50mM Na of the reaction temperature under optimum reaction conditions2HPO4/NaH2PO4It is molten as buffering Liquid, a concentration of 0.016g/L of substrate (±)-sec-butyl acetate a concentration of 0.04M, esterase PHE21), by the reaction condition in 5.1 Lower reaction different time (40~200min) respectively, the results are shown in Figure 6.The result shows that after reaction 180min, product (S)-second The enantiomeric excess value of the secondary butyl ester of acid has reached highest, enantiomeric excess value ＞ 98%, yield 83.6%, when reacted between it is big After 180min, the enantiomeric excess value and conversion ratio of product (S)-sec-butyl acetate are basically unchanged.It can be more than interior for 24 hours (S)-sec-butyl acetate (Fig. 7) of 98% optical purity.
Embodiment 6：Applications of the esterase PHE21 in catalysis (±)-sec-butyl alcohol transesterification reaction prepares (R)-sec-butyl acetate
Under optimal conditions, i.e., in 3mL reaction systems, 1.7 × 10 are added6The thick enzyme powders of esterase PHE21 of U/L, (±)-sec-butyl alcohol of the nytril 0mM of 30mM, remaining is hexamethylene, and in 45 DEG C, transesterification is carried out under the conditions of 200rpm Reaction, the results are shown in Figure 8, when reacted between be 4 hours when, the enantiomeric excess value of reaction product (R)-sec-butyl acetate and Conversion ratio is optimal, and respectively 64% and 43%.In (R)-sec-butyl acetate that optical purity can be obtained and be more than 64% interior for 24 hours (Fig. 9).
Concrete analysis condition is：Gas chromatograph is stood using good fortune, is furnished with chiral column (30m × 0.25mm Cyclosil B Chirl column) and hydrogen ion flame detector.Instrumental conditions are set as：220 DEG C of injector temperature, detector temperature 250 DEG C, carrier gas N2, flow velocity 1.2mL/min analyzed using gradient increased temperature：80 DEG C holding 1min, 15 DEG C/min, 120 DEG C 1min is kept, 10 DEG C/min to 220 DEG C, keeps 1min.
Sequence table
<110>Chinese Academy of Science Nanhai Ocean Research Institute
<120>A kind of esterase PHE21 and its encoding gene and the application in prepared by the secondary butyl ester of chiral acetic
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 966
<212> DNA
<213>Marine pseudomonas (Pseudomonadaceae oryzihabitans HUP022)
<400> 1
atgcccgacg tcttcgcgcg cctcactccc gaggtggccg gcctgctggc gcagctggcc 60
gcccacccgc agccgccgct ggacagcctc accccggcgc aggcgcgcgc cgcctatgtg 120
cagctgaatc gtgccctcgg cctgccgcca gcgccggtga agcgggtgga ggcgctcagc 180
gctccggggc cgctggggcc catcccgctg cgcctctacc gcccctatgc ggaagacgat 240
cgaccgcgcc cgctggtgat ctatctgcac ggcggtggct gggtgatcgg cgacctggac 300
agccatgatt cgctctgccg gcagatcgcg atcggaaccg gctatgcggt gctggcggtg 360
cactatgccc tcgcccccga gcatccggca ccggcgagcc ccgacgatgt gctggccgcc 420
ctgcactggc tggccgaggc cggcagcgcc ttccacctcg atctcgaccg catcgccgtg 480
gccggcgaca gcgccggtgg cgggctggcg gccatggccg cattgtacct gcgcgaagga 540
ccgctcaggc tcaaggccca ggtgctgatc taccctggcg tcgacaacac gcccgaggcc 600
tggaatcacc cctcgcggat cgagaacgcc caggtcccac cgctgacccg gccaatgatg 660
gattactttt cggggatgta tctccaggac gtggacaccc gcgacccgcg cgtctcgccg 720
ctttatgccg cctcccacgc cgacctgccg ccggcgctga tcttcggcgc cgaatgcgac 780
gccctgcgcg acgatgcccg cctctatgcc caggcgctga tcggcgccgg caacgccgtg 840
gagtaccacg agttgccggg catgatccat ggcttcatcg agatgctcgg ggtgctgccc 900
agcgtgcgct ggaccctggc gcggatgaat gcctttctgc gcgagcatct gcagcaggcc 960
gcctga 966
<210> 2
<211> 321
<212> PRT
<213>Marine pseudomonas (Pseudomonadaceae oryzihabitans HUP022)
<400> 2
Met Pro Asp Val Phe Ala Arg Leu Thr Pro Glu Val Ala Gly Leu Leu
1 5 10 15
Ala Gln Leu Ala Ala His Pro Gln Pro Pro Leu Asp Ser Leu Thr Pro
20 25 30
Ala Gln Ala Arg Ala Ala Tyr Val Gln Leu Asn Arg Ala Leu Gly Leu
35 40 45
Pro Pro Ala Pro Val Lys Arg Val Glu Ala Leu Ser Ala Pro Gly Pro
50 55 60
Leu Gly Pro Ile Pro Leu Arg Leu Tyr Arg Pro Tyr Ala Glu Asp Asp
65 70 75 80
Arg Pro Arg Pro Leu Val Ile Tyr Leu His Gly Gly Gly Trp Val Ile
85 90 95
Gly Asp Leu Asp Ser His Asp Ser Leu Cys Arg Gln Ile Ala Ile Gly
100 105 110
Thr Gly Tyr Ala Val Leu Ala Val His Tyr Ala Leu Ala Pro Glu His
115 120 125
Pro Ala Pro Ala Ser Pro Asp Asp Val Leu Ala Ala Leu His Trp Leu
130 135 140
Ala Glu Ala Gly Ser Ala Phe His Leu Asp Leu Asp Arg Ile Ala Val
145 150 155 160
Ala Gly Asp Ser Ala Gly Gly Gly Leu Ala Ala Met Ala Ala Leu Tyr
165 170 175
Leu Arg Glu Gly Pro Leu Arg Leu Lys Ala Gln Val Leu Ile Tyr Pro
180 185 190
Gly Val Asp Asn Thr Pro Glu Ala Trp Asn His Pro Ser Arg Ile Glu
195 200 205
Asn Ala Gln Val Pro Pro Leu Thr Arg Pro Met Met Asp Tyr Phe Ser
210 215 220
Gly Met Tyr Leu Gln Asp Val Asp Thr Arg Asp Pro Arg Val Ser Pro
225 230 235 240
Leu Tyr Ala Ala Ser His Ala Asp Leu Pro Pro Ala Leu Ile Phe Gly
245 250 255
Ala Glu Cys Asp Ala Leu Arg Asp Asp Ala Arg Leu Tyr Ala Gln Ala
260 265 270
Leu Ile Gly Ala Gly Asn Ala Val Glu Tyr His Glu Leu Pro Gly Met
275 280 285
Ile His Gly Phe Ile Glu Met Leu Gly Val Leu Pro Ser Val Arg Trp
290 295 300
Thr Leu Ala Arg Met Asn Ala Phe Leu Arg Glu His Leu Gln Gln Ala
305 310 315 320
Ala

Claims (10)

1. a kind of esterase PHE21, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of esterase gene PHE21 of coding esterase PHE21 described in claim 1.
3. esterase gene PHE21 according to claim 2, which is characterized in that the nucleotide of the esterase gene PHE21 Sequence is as shown in SEQ ID NO.1.
4. applications of the esterase PHE21 described in claim 1 in preparation (S)-sec-butyl acetate or (R)-sec-butyl acetate.
5. application according to claim 4, which is characterized in that the application is esterase PHE21 in fractionation (±)-acetic acid The application in (S)-sec-butyl acetate is prepared in secondary butyl ester.
6. application according to claim 5, which is characterized in that the application is：Take esterase PHE21 in pH be 6.0- In 9.0 buffer solution, (±)-sec-butyl acetate is added, is reacted, obtains (S)-sec-butyl acetate.
7. application according to claim 6, which is characterized in that the buffer solution be citric acid-sodium citrate buffer solution, One kind in phosphate buffer, Tris-HCl buffer solutions and glycine-NaOH buffer.
8. application according to claim 4, which is characterized in that the application is esterase PHE21 in catalysis (±)-Zhong Ding The application in (R)-sec-butyl acetate is prepared in the transesterification reaction of alcohol.
9. application according to claim 8, which is characterized in that the application is：Take esterase PHE21 in reaction medium In, (±)-sec-butyl alcohol and acry radical donor are added, is reacted, (R)-sec-butyl acetate is obtained.
10. application according to claim 9, which is characterized in that the reaction medium is Isosorbide-5-Nitrae-dioxane, tetrahydrochysene furan It mutters, one kind in the tert-butyl alcohol, isopropanol, dichloromethane, toluene, hexamethylene, n-hexane, normal heptane and isooctane；The acyl Base donor is one kind in acetyl vinyl acetate, acetyl isopropyl enester and ethyl acetate.
CN201810114144.8A 2018-02-05 2018-02-05 Esterase PHE21, encoding gene thereof and application thereof in preparation of chiral sec-butyl acetate Expired - Fee Related CN108396016B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810114144.8A CN108396016B (en) 2018-02-05 2018-02-05 Esterase PHE21, encoding gene thereof and application thereof in preparation of chiral sec-butyl acetate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810114144.8A CN108396016B (en) 2018-02-05 2018-02-05 Esterase PHE21, encoding gene thereof and application thereof in preparation of chiral sec-butyl acetate

Publications (2)

Publication Number Publication Date
CN108396016A true CN108396016A (en) 2018-08-14
CN108396016B CN108396016B (en) 2021-05-25

Family

ID=63095184

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810114144.8A Expired - Fee Related CN108396016B (en) 2018-02-05 2018-02-05 Esterase PHE21, encoding gene thereof and application thereof in preparation of chiral sec-butyl acetate

Country Status (1)

Country Link
CN (1) CN108396016B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111018663A (en) * 2019-12-02 2020-04-17 苏州凯瑞医药科技有限公司 Nepitastat important intermediate and enzyme catalytic synthesis method thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006012277A2 (en) * 2004-06-30 2006-02-02 Amr Technology, Inc. Biocatalytic process for preparing enantiomerically enriched pramipexole
CN1950080A (en) * 2004-04-09 2007-04-18 ARYx医疗有限公司 Novel compounds for treatment of cardiac arrhythmia and methods of use
CN104140959A (en) * 2014-07-14 2014-11-12 中国科学院南海海洋研究所 Novel esterase as well as coding gene and application of esterase
CN105132394A (en) * 2015-09-24 2015-12-09 中国科学院南海海洋研究所 LIPASE 6 as well as encoding gene and application thereof
CN105296446A (en) * 2015-11-09 2016-02-03 中国科学院南海海洋研究所 Lipase L-1 and coding gene and application thereof
CN105543191A (en) * 2016-02-24 2016-05-04 中国科学院南海海洋研究所 Esterase PHE21 and encoding gene and application thereof
CN105802935A (en) * 2016-05-05 2016-07-27 中国科学院南海海洋研究所 Esterase PHE14 as well as encoding gene and application thereof
CN106244569A (en) * 2016-07-27 2016-12-21 中国科学院南海海洋研究所 A kind of esterase EstC10 and encoding gene thereof and application
CN106818805A (en) * 2016-12-27 2017-06-13 东莞市联洲知识产权运营管理有限公司 A kind of natural acetylcholinesteraseinhibitors inhibitors and its pesticidal applications

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1950080A (en) * 2004-04-09 2007-04-18 ARYx医疗有限公司 Novel compounds for treatment of cardiac arrhythmia and methods of use
WO2006012277A2 (en) * 2004-06-30 2006-02-02 Amr Technology, Inc. Biocatalytic process for preparing enantiomerically enriched pramipexole
CN104140959A (en) * 2014-07-14 2014-11-12 中国科学院南海海洋研究所 Novel esterase as well as coding gene and application of esterase
CN105132394A (en) * 2015-09-24 2015-12-09 中国科学院南海海洋研究所 LIPASE 6 as well as encoding gene and application thereof
CN105296446A (en) * 2015-11-09 2016-02-03 中国科学院南海海洋研究所 Lipase L-1 and coding gene and application thereof
CN105543191A (en) * 2016-02-24 2016-05-04 中国科学院南海海洋研究所 Esterase PHE21 and encoding gene and application thereof
CN105802935A (en) * 2016-05-05 2016-07-27 中国科学院南海海洋研究所 Esterase PHE14 as well as encoding gene and application thereof
CN106244569A (en) * 2016-07-27 2016-12-21 中国科学院南海海洋研究所 A kind of esterase EstC10 and encoding gene thereof and application
CN106818805A (en) * 2016-12-27 2017-06-13 东莞市联洲知识产权运营管理有限公司 A kind of natural acetylcholinesteraseinhibitors inhibitors and its pesticidal applications

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
YILONG WANG，等: "Functional Characterization of a Robust Marine Microbial Esterase and Its Utilization in the Stereo-Selective Preparation of Ethyl (S)-3-Hydroxybutyrate", 《APPL BIOCHEM BIOTECHNOL》 *
YILONG WANG等: "Utilization of deep-sea microbial esterase PHE21 to generate chiral sec-butyl acetate through kinetic resolutions", 《CHIRALITY》 *
王博: "具有手性选择性酯酶/脂肪酶的筛选、催化特点及应用研究", 《中国博士学位论文全文数据库基础科学辑》 *
王跃军等: "超嗜热羧酸酯酶的性质和应用研究", 《中国生物工程杂志》 *
许建和等: "《生物催化工程》", 31 October 2008, 华东理工大学出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111018663A (en) * 2019-12-02 2020-04-17 苏州凯瑞医药科技有限公司 Nepitastat important intermediate and enzyme catalytic synthesis method thereof

Also Published As

Publication number Publication date
CN108396016B (en) 2021-05-25

Similar Documents

Publication Publication Date Title
CN106755209A (en) A kind of method that enzyme process prepares β nicotinamide mononucleotides
CN105543191B (en) A kind of esterase PHE21 and its encoding gene and application
CN112280761B (en) Recombinant transaminase, mutant of recombinant transaminase and application of mutant
CN114134134B (en) L-threonine aldolase mutant and application thereof in synthesis of L-syn-p-methylsulfonyl phenylserine
CN104673809B (en) A kind of malate dehydrogenase gene and its recombinant expression carrier
CN108285895A (en) A kind of esterase EstC11 and its encoding gene and application
CN109468290A (en) A kind of carbonyl reduction enzyme mutant, expression vector, engineering bacteria and its application
CN109929822B (en) Aspergillus oryzae lipase mutant and application thereof
CN105802935B (en) A kind of esterase PHE14 and its encoding gene and application
CN108396016A (en) A kind of esterase PHE21 and its encoding gene and the application in prepared by the secondary butyl ester of chiral acetic
CN106544336A (en) A kind of nitrile hydratase improved by aliphatic dintrile regioselectivity
CN105132394B (en) A kind of lipase LIPASE6 and its encoding gene and application
CN105238769B (en) A kind of lipase LIPASE7 and its encoding gene and application
CN110004120A (en) A kind of recombination aldehyde Ketoreductase mutant and application
CN105950595B (en) (-)-gamma-lactam enzyme, gene, mutant, carrier and its preparation and application
CN106544328B (en) Sulfoxide reductase and application and preparation method thereof
CN110951711B (en) Esterase with activity of degrading chiral ester and coding gene and application thereof
CN111100851A (en) Alcohol dehydrogenase mutant and application thereof in synthesis of chiral diaryl alcohol compound
CN105969751B (en) Beta-glucosidase gene and application thereof
CN109971730A (en) A kind of monoamine oxidase from aspergillus niger is used for the preparation of chiral amine intermediates
CN106085985B (en) A kind of esterase WDEst9 and its encoding gene and application
CN107475218A (en) A kind of esterase B ae02030 and its encoding gene and application
CN105296446B (en) A kind of lipase L-1 and its encoding gene and application
CN110563852A (en) Efficient and simple enzyme immobilization method
CN105349507B (en) A kind of lipase LIPDa6 and its encoding gene and application

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
CB02 Change of applicant information
CB02 Change of applicant information

Address after: No.1119 Haibin Road, Nansha District, Guangzhou City, Guangdong Province

Applicant after: SOUTH CHINA SEA INSTITUTE OF OCEANOLOGY, CHINESE ACADEMY OF SCIENCES

Address before: 510301 No. 164 West Xingang Road, Guangdong, Guangzhou

Applicant before: SOUTH CHINA SEA INSTITUTE OF OCEANOLOGY, CHINESE ACADEMY OF SCIENCES

GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20210525