CN110066835A

CN110066835A - A kind of application of lipase in resolution of racemic 2- bromo ethyl isovalerate

Info

Publication number: CN110066835A
Application number: CN201910217266.4A
Authority: CN
Inventors: 郑建永; 吴鹏; 汪钊; 章银军; 张梦婕
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang Yingwodi Biotechnology Co ltd
Priority date: 2019-03-21
Filing date: 2019-03-21
Publication date: 2019-07-30
Anticipated expiration: 2039-03-21
Also published as: CN110066835B

Abstract

The present invention provides a kind of lipase to split the application in 2- bromo ethyl isovalerate, and the amino acid sequence of the lipase is as shown in SEQ ID NO.1, and encoding gene is as shown in SEQ ID NO.2.Lipase gene of the present invention can connect building with expression vector and obtain the intracellular expression recombinant plasmid containing the gene, then convert into coli strain, obtain recombination bacillus coli；The recombination bacillus coli isolates and purifies acquisition recombinant lipase through clasmatosis；The recombinant lipase has the ability of catalysis resolution of racemic 2- bromo ethyl isovalerate, available product (R) -2- bromo ethyl isovalerate, enantiomeric excess value > 99% and conversion ratio reach 50.8%, the mass yield of product (R) -2- bromo ethyl isovalerate reaches 96.4%, and splits high-efficient.

Description

A kind of application of lipase in resolution of racemic 2- bromo ethyl isovalerate

(1) technical field

The invention belongs to biocatalysis technology fields, are related to a kind of lipase stereoselectivity catalyzing hydrolysis fractionation 2- bromo Ethyl isovalerate enantiomer synthesizes the application of (R) -2- bromo ethyl isovalerate enantiomer of single configuration.

(2) background technique

Taufluvalinate is the pyrethroid of non-cyclopropane shuttle acids, has structure similar with fenvalerate.It is total There are four optical isomers.A pair of of active isomer that industrial goods are made of dextronic acid and racemization cyanalcohol, the desinsection with wide spectrum are living Property tool tag and stomach poison function, there are also food refusals and repellent activity.Its virulence and fenvalerate can quite effectively prevent cotton, fruit On the crops such as tree, vegetables, corn, tealeaves, tobacco including Lepidoptera, coleoptera, Homoptera and Diptera, including Main Harmful Worm.In addition it can also prevent and treat for example blood red homalocephalus bee of livestock pest, sheep nose fly horn fly, stable fly, dog comb head flea etc..It is most in document to use Raw material D-Val synthesizes to obtain taufluvalinate, although D-Val method can directly obtain chiral product, price ratio Costly.And the raceme of taufluvalinate is synthesized from the more cheap 2- bromo ethyl isovalerate of price, then pass through chirality It splits to obtain the active isomer of industrial goods application, production cost can be reduced, increased economic efficiency.However we provide one kind Lipase carrys out catalyzing hydrolysis and splits 2- bromo ethyl isovalerate efficiently to produce the effective ways of (R) -2- bromo ethyl isovalerate, Chiral product can be directly obtained, cost is thus greatly reduced.

Lipase (Lipase, EC 3.1.1.3) full name is Lipase (Triacylglycerol It acylhydrolase), is current research a kind of hydrolase most with industrial applications.Lipase can be catalyzed ester hydrolysis, ester closes At, alcoholysis, acidolysis, ester transesterification and ammonolysis reaction.Lipase is widely present in the various organisms of nature, especially micro- In biology and animal vegetable tissue.Animal tallow enzyme is primarily present in the organ-tissues such as pancreas, such as Pig Liver Esterase and pig pancreas fat Enzyme, but due to the factors such as animal tallow enzymatic activity is lower, enzyme extraction purification higher cost and raw material sources are limited, limit its Application in industry.Microbial lipase abundance, it is many kinds of, it is not influenced by factors such as seasonal climates, microorganism is raw The long producing enzyme period is shorter, and has that organic solvent-resistant, that Substratspezifitaet is strong, catalytic selectivity is high and catalytic activity is high etc. is special Point, thus microbe-derived lipase has higher industrial application value.Most of commercial lipases currently on the market All obtained by the fermentation of the microcultures such as bacterium, fungi and yeast.Novo Nordisk company, Denmark, Amano company, Japan A variety of commercialization enzyme preparations is developed with Major Enzymes manufacturers such as Genencor companies, the U.S..These enzyme preparation companies, which utilize, to be divided Sub- renovation technique is transformed microbial lipase, improves the zymologic properties such as enzyme activity, stability and stereoselectivity, with full The demand of foot difference industrial circle application.Thus building lipase gene engineering bacteria is to which mass production recombinant lipase is with ten Divide significance.

(3) summary of the invention

It is an object of the present invention to provide a kind of stereoselectivity lipase to split in 2- bromo ethyl isovalerate in bioanalysis Using can directly obtain chiral product, substantially reduce cost.

The technical solution adopted by the present invention is that:

The present invention provides a kind of lipase and is splitting the application (as shown in Figure 1) in 2- bromo ethyl isovalerate, the rouge For the amino acid sequence of fat enzyme as shown in SEQ ID NO.1, coding gene sequence is lipase gene source shown in SEQ ID NO.2 From aspergillus oryzae WZ007 (Aspergillus oryzaeWZ007), which is preserved in China typical culture collection center, address: Wuhan, China, Wuhan University, 430072, deposit number: CCTCC No:M 206105, preservation date: on October 8th, 2006, Relevant bacteria species preservation information is had submitted in previously applying for a patent (Chinese patent CN 101186938) and discloses its heredity money Source source.The present invention carries out the excavation of aimed aliphatic enzyme and gene on the basis of this patent application.Specific application method Are as follows: from this aspergillus oryzae WZ007, mRNA is extracted through reverse transcription and obtains cDNA, the aspergillus oryzae genome checked according to NCBI On the basis of GenBank:NWUI02000090.1 base sequence, design upstream primer is 5'-CCATGGGCATGAAGATTCAA AAACTCATTCTGTACAGTCT-3' and downstream primer are 5'-CTCGAGTTAATATGCATACTTTGCAATGTCAGGC-3', PCR is carried out, obtains base gene order SEQ ID NO.2 segment, sequence verification is correct, which is connected to carrier pET- 28b (+), conversion to Rosetta competence, the thick enzyme that the wet thallus that fermented culture obtains extracts is catalyst, with 2- bromo Ethyl isovalerate is substrate, using 7.0 phosphate buffer of pH as reaction medium, under the conditions of 20-45 DEG C, 800-1000rpm into After fully reacting, reaction solution is isolated and purified for row resolution reaction, obtains product (R) -2- bromo ethyl isovalerate.The catalysis Agent dosage is calculated as 15-30g/L (preferably 30g/L) with buffer volume, and the Final substrate concentrations are calculated as 5- with buffer volume 10g/L (preferably 6g/L).

Further, preferred reaction time 3-7h, more preferable reaction condition is 35 DEG C, 1000rpm reacts 5h.

Further, the buffer is pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄Buffer solution.

Further, the catalyst is prepared as follows: by engineering bacteria (the preferably large intestine bar of fatty enzyme coding gene Bacterium Rosetta) it is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀To 0.4-0.6, add IPTG to final concentration 0.02mM, 30 DEG C of trainings 10-12h, bacterium solution 8000rpm, 4 DEG C of centrifugation 10min are supported, collects thallus, then with PBS buffer solution washing thalline 2 times, 8000rpm, 4 DEG C centrifugation 10min, collect thallus, freeze-drying, obtain the thick enzyme powder of lipase；LB culture medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 5g/L, solvent are deionized water, and pH value is natural.

Further, the method that reaction solution of the present invention isolates and purifies are as follows: after reaction, reaction solution is carried out with 4mMHCl It is acidified to pH 2.0, is then extracted with isometric ethyl acetate, separatory funnel isolates organic phase, then twice through pure water, Saturation NaCl is washed twice, dry, and revolving obtains product (R) -2- bromo ethyl isovalerate.

Fat enzyme amino acid sequence of the invention is as shown in SEQ ID NO.1:

MKIQKLILYKFANELTASGLIPSKALTCLNGYAIIDLNSIHRHNPSPENLSIYPYKAKSDAPYSIAENT LRAGIHIPRSFSHKRDKKIPVLLVPGTAVPAAITFYFNFGKLRRALPESELVWIDLPQASLDDIQLSAEYVAYALNY VSALTSSKIAVISWSQGALDIQWALKYWPSTRGVVNDFIAISPDFHGTIVKWLVCPLLNDLACTPSIWQQGWDANFI QALRSQGGDSAYVPTTTIYSSFDKIVRPMSGENASARLLDYRGVGVSNNHLQTICANNAAGGLYTHEGVLYNPLAWA LTVDALLHDGPSNITRIDTQKICEQVLPPYLELTDMLGTEALLLVALAKILTYSPKVSGEPDIAKYAY

Due to the particularity of amino acid sequence, the segment of any polypeptide containing amino acid sequence shown in SEQ ID NO.1 Or its variant, such as its examples of conservative variations, bioactive fragment or derivative, as long as the segment of the polypeptide or polypeptide variants with it is aforementioned Amino acid sequence homology belongs to the column of the scope of the present invention 90% or more and enzymatic activity having the same.Specifically , the change may include the missing of amino acid, insertion or replacement in amino acid sequence；Wherein, the conservative of variant is changed Become, the amino acid replaced has structure similar with original acid or chemical property, such as replaces isoleucine with leucine, becomes Body can also have non-conservation change, such as replace glycine with tryptophan.

The segment of albumen of the present invention, derivative or the like, which refer to, is kept substantially protease phase of the present invention Same biological function or active albumen, can be following state: (I) one or more amino acid residues are guarded or non-guarantor Amino acid residue (preferably conservative amino acid residues) substitution is kept, and the amino acid replaced can be and may not be by losing Pass codon coding；(II) some group on one or more amino acid residues is replaced by other groups；(III) at soft-boiled eggs It is white to be merged with another compound (for example extending the compound of protein half-life, such as polyethylene glycol)；(IV) additional amino Acid sequence is integrated into mature albumen and the protein sequence (sequence or proprotein sequence as being used to purify this albumen) that is formed.

The albumen can be recombinant protein, native protein or synthetic proteins, can be the product of pure natural purifying, or Chemically synthesized product, or using recombinant technique from protokaryon or eucaryon host (such as: bacterium, yeast, higher plant, insect and Mammalian cell) in generate.According to host used in recombinant production scheme, albumen of the invention can be glycosylated.This The albumen of invention can also include or not include the methionine residues originated.

The encoding gene nucleotide sequence of lipase of the present invention is as shown in SEQ ID NO.2:

ATGAAAATCCAGAAACTGATTTTATATAAATTTGCAAACGAGCTCACCGCATCAGGCCTGATACCTTCA AAGGCACTGACATGCCTGAATGGCTACGCGATCATTGACCTCAATTCGATTCACCGTCACAACCCATCCCCAGAAAA CTTGAGCATATACCCATACAAAGCAAAGAGCGATGCGCCTTACTCCATTGCGGAAAATACTCTCCGGGCTGGAATTC ACATTCCTAGATCATTCTCACACAAACGGGACAAGAAAATACCGGTGCTCCTGGTACCAGGAACTGCTGTTCCTGCG GCCATAACCTTTTATTTCAACTTTGGCAAGTTGAGGAGAGCCTTACCCGAAAGTGAGCTAGTTTGGATCGATCTCCC TCAGGCCTCGTTGGATGACATTCAATTGAGCGCTGAATATGTCGCCTATGCGCTCAATTATGTTTCCGCTTTGACCT CATCTAAGATTGCCGTGATTTCTTGGTCCCAAGGTGCACTGGATATTCAGTGGGCACTGAAGTACTGGCCATCTACC CGGGGCGTCGTCAACGACTTCATTGCTATCAGCCCCGACTTCCATGGAACAATAGTCAAGTGGCTGGTATGCCCTCT ATTAAATGATTTAGCCTGTACTCCTTCGATTTGGCAGCAAGGGTGGGATGCAAACTTTATACAGGCCTTGCGGAGCC AAGGGGGAGACTCTGCCTATGTCCCCACGACCACCATTTACTCTTCATTCGACAAGATTGTGCGACCCATGAGTGGG GAAAATGCTTCTGCTCGGCTACTGGACTACAGGGGGGTGGGCGTTTCCAATAACCATCTACAGACCATCTGCGCAAA TAATGCCGCTGGTGGCCTCTATACGCATGAAGGCGTGTTGTACAACCCTCTTGCATGGGCCCTTACTGTTGACGCAC TCCTTCACGATGGGCCTAGCAATATTACGCGAATCGATACCCAGAAGATCTGTGAACAAGTACTTCCGCCATATCTC GAATTAACTGATATGCTGGGAACCGAAGCCCTCCTTTTAGTAGCCCTAGCAAAGATTCTTACATATTCCCCGAAAGT CTCCGGTGAGCCTGACATTGCAAAGTATGCATATTAA

Due to the particularity of nucleotide sequence, the variant of polynucleotides shown in any SEQ ID NO.2, as long as it is more with this Nucleotide has 70% or more homology and function having the same, belongs to the column of the scope of the present invention.The multicore glycosides The variant of acid refers to a kind of polynucleotide sequence changed with one or more nucleotide.The variant of this polynucleotides can be The variant that the allelic variant or non-natural naturally occurred occurs, including substitution variants, Deletion variants and insertion variation Body.As known in the art, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide Substitution, missing or insertion, but not from substantially change its encode amino acid function.

In addition, the polynucleotides that can hybridize with polynucleotide sequence shown in SEQ ID NO:2 are (at least homologous with 50% Property, preferably there is 70% homology), it, especially under strict conditions can be with institute of the present invention also in the column of the scope of the present invention State the polynucleotides of nucleotide sequence hybridization." stringent condition " refers to: (1) under compared with low ionic strength and higher temperature Hybridization and elution, such as 0.2SSC, 0.1%SDS, 60 DEG C；Or (2) hybridize Shi Jiayong denaturant, such as 50% (v/v) formamide, 0.1% calf serum, 0.1%Ficoll, 42 DEG C；Or (3) only the homology between two sequences is at least 95% or more, more Just hybridize when being well 97% or more.Also, egg shown in the albumen of interfertile polynucleotide encoding and SEQ ID NO:1 It is white to have identical biological function and activity.

The invention further relates to the recombinant vectors for containing the encoding gene, and converted using the recombinant vector Recombination engineering bacteria.

Compared with prior art, the beneficial effects are mainly reflected as follows: the present invention provides a kind of lipase genes Nucleotide sequence；The lipase gene can connect building with expression vector and obtain the intracellular expression recombinant plasmid containing the gene, then Conversion obtains recombination bacillus coli into coli strain, and recombination bacillus coli or recombinant lipase is recycled to urge for biology Agent catalysis fractionation 2- bromo ethyl isovalerate, available product (R) -2- bromo ethyl isovalerate, enantiomeric excess value > 99% and conversion ratio reach 50.8%, the mass yield of product (R) -2- bromo ethyl isovalerate reaches 96.4%.The present invention is raw The chiral synthetic reaction of object catalysis has mild condition, high-efficient, chemo-selective, regioselectivity and the enantiomer of height The advantages that selective, and biocatalysis process has the characteristics that nontoxic, pollution-free and low energy consumption, is a kind of environmental-friendly conjunction At method.

(4) Detailed description of the invention

Fig. 1 is the reaction schematic diagram that lipase enantioselective hydrolysis splits 2- bromo ethyl isovalerate；

Fig. 2 is racemic 2- bromo ethyl isovalerate standard specimen gas chromatogram；

Fig. 3 is the gas chromatogram of the lipase-catalyzed 2- bromo ethyl isovalerate hydrolysis 5h of embodiment 4.

(5) specific embodiment

The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This, the transformation in the method that those skilled in the art are made according to these embodiments is all contained in guarantor of the invention It protects in range.

1 aspergillus oryzae WZ007 of embodiment fermentation, cDNA preparation, cloning lipase gene and engineering bacteria building

1, aspergillus oryzae WZ007 fermentation condition: slant medium (g/L): peeled potatoes 200, sucrose 20, agar 20 are molten Agent is deionized water, and pH is natural.Fermentation medium (g/L): peptone 20, olive oil 1% (V/V), KH₂PO₄1, MgSO₄ 0.5, NaCl 0.5, solvent is deionized water, pH 7.0.

Aspergillus oryzae WZ007 (Aspergillus oryzaeWZ007) is seeded to slant medium, 30 DEG C of culture 6-8d, Obtain slant strains.Aspergillus oryzae WZ007 is preserved in China typical culture collection center, address: Wuhan, China, Wuhan University, 430072, deposit number: CCTCC No:M 206105, preservation date: on October 8th, 2006, in patent application CN It is disclosed in 101186938A.

Slant strains are taken to be seeded in the 500mL shaking flask of the fermentation medium containing 100mL, at 30 DEG C of temperature, revolving speed 200rpm Constant-temperature table culture 12h, seed liquor is made.The 30L fermentor that 1L seed liquor is poured into the fermentation medium containing 18L is sent out Ferment culture, wherein temperature and revolving speed are controlled respectively in 30 DEG C and 200rpm, and fermented and cultured 48h collects wet thallus.

2, prepared by aspergillus oryzae WZ007 cDNA: taking the above-mentioned aspergillus oryzae wet thallus of 1g, uses the TransZol of Quan Shi King Company Up Plus RNA Kit kit extracts mRNA, reuses the ReverTra Ace qPCR RT Kit of TOYOBO company, efficiently Synthesize to rate the cDNA template suitable for Realtime PCR.

3, the clone of Aspergillus oryzae lipase gene: the aspergillus oryzae genome checked according to NCBI

On the basis of GenBank:NWUI02000090.1 base sequence, design upstream primer is

5'-CCATGGGCATGAAGATTCAAAAACTCATTCTGTACAGTCT-3' and downstream primer are 5'-CTCGAGT TAATATGCATACTTTGCAATGTCAGGC-3' carries out PCR, obtains nucleotides sequence and be classified as SEQ ID using cDNA as template Genetic fragment shown in NO.2, sequence verification are correct.The coding protein amino acid sequence of genetic fragment shown in SEQ ID NO.2 is Shown in SEQ ID NO.1.

4, genetic fragment shown in the SEQ ID NO.2 building of colibacillus engineering: is connected to carrier pET-28b (+) converts to Escherichia coli Rosetta competence, obtains the colibacillus engineering (being denoted as engineering bacteria M5) of fatty enzyme.

The fermented and cultured of the colibacillus engineering (M5) of the fatty enzyme of embodiment 2

The colibacillus engineering (M5) that embodiment 1 obtains is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀Most 0.5 (probably culture 2h), adds IPTG to final concentration 0.02mM, 30 DEG C of culture 10-12h.300mL bacterium solution 8000rpm, 4 DEG C of centrifugations 10min collects thallus, then with PBS buffer solution washing thalline 2 times, 8000rpm, 10min collect thallus.The thallus of collection is frozen The thick enzyme powder of lipase is obtained after dry, is denoted as lipase M5,4 DEG C of refrigerators is put in and saves.LB culture medium composition: tryptone 10g/L, Yeast powder 5g/L, NaCl 5g/L, solvent are deionized water, and pH is natural.

The reaction of 3 enzymatic of embodiment fractionation 2- bromo ethyl isovalerate

The thick enzyme powder of lipase of 2 method of 0.03g embodiment preparation is weighed in 2mL EP pipe, addition 1mL PB (pH 7.0, It 0.2mM) is used as reaction dissolvent, 0.006g substrate 2- bromo ethyl isovalerate is then added and is set so that thallus is not added as blank control 3h is reacted in 35 DEG C, 1000rpm constant temperature blending instrument.After reaction, reaction solution is acidified through 4mM HCl, adds 1mL acetic acid Ethyl ester, turbula shaker vibrate 2min, sufficiently extract, be centrifuged (1200rpm, 3min), obtain organic phase.Take 700 μ L acetic acid second Ester layer by the stereoselectivity of gas chromatographic detection thallus and enzymatic hydrolysis activity, obtain the enantiomeric excess value of substrate > 99% and conversion ratio reach 50.8%.

Specific gas phase analysis condition: Agilent6890 gas chromatograph, BGB-175 chiral capillary chromatographic column are used (30.0m × 0.25mm × 0.25um), fid detector；Testing conditions are column temperature from 100 DEG C of initial temperature (constant temperature keeps 5min) It is warming up to 120 DEG C, 2 DEG C/min of heating rate, 250 DEG C of injector temperature, 250 DEG C of detector temperature, air mass flow and hydrogen flowing quantity Respectively 300mL/min and 30mL/min.Carrier gas is high-purity N₂, stigma pressure 98.7Kpa；Make-up gas flow 30.0mLmin^-1； Split ratio 30:1, sampling volume 1uL.As shown in the result of GC, (R) -2- bromo ethyl isovalerate and (S) -2- bromo isoamyl The retention time of acetoacetic ester is respectively 8.58min and 8.75min, sees Fig. 2.

The effect that the different lipase hydrolysis of embodiment 4 split 2- bromo ethyl isovalerate compares

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the difference fat of final concentration 30g/L is added Enzyme (the thick enzyme powder of lipase of the preparation of embodiment 2, Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Amano Lipase PS IM), 0.006g substrate 2- bromo ethyl isovalerate reacts under the conditions of 1000rpm in 35 DEG C of constant temperature blending instrument 5h extracts reaction solution enantiomeric excess value and conversion ratio by 1 method of embodiment detection (R) -2- bromo ethyl isovalerate, as a result sees Shown in table 1.The results show that after 35 DEG C of reaction 5h, lipase Novozym 435, Lipozyme TL IM, Lipozyme RM IM, PS IM are active without Hydrolysis Resolution to two kinds of configurations of substrate 2- bromo ethyl isovalerate, then above four kinds of commercial fat enzymes To substrate 2- bromo ethyl isovalerate all without enantio-selectivity, and when the thick enzyme powder catalysis reaction of lipase, product (R)- Ee value > 99% of 2- bromo ethyl isovalerate, conversion ratio 50.8%, shown in gas-chromatography Fig. 3.

The Hydrolysis Resolution effect of the different lipase-catalyzed 2- bromo ethyl isovalerates of table 1 compares

Influence of 5 reaction time of embodiment to enzyme kinetics Hydrolysis Resolution 2- bromo ethyl isovalerate

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the embodiment 2 of final concentration 30g/L is added The thick enzyme powder of lipase of method preparation, 0.006g2- bromo ethyl isovalerate, in 35 DEG C of constant temperature blending instrument, under the conditions of 1000rpm It reacts different time (3~6h), extracts reaction solution the enantiomeric excess by 1 method of embodiment detection (R) -2- bromo ethyl isovalerate Value and conversion ratio, the results are shown in Table shown in 2.

The result shows that the enantiomeric excess value of product (R) -2- bromo ethyl isovalerate has reached highest, right after reaction 5h Reflect body excessive value > 99%, conversion ratio 50.8%, when reacted between be greater than 5h after, product (R) -2- bromo ethyl isovalerate Enantiomeric excess value is gradually increasing, and conversion ratio is also increasing.

Influence of 2 reaction time of table to enzymic catalytic reaction

Influence of 6 reaction temperature of embodiment to enzyme kinetics Hydrolysis Resolution 2- bromo ethyl isovalerate

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the embodiment 2 of final concentration 30g/L is added The thick enzyme powder of the lipase of preparation, 0.006g 2- bromo ethyl isovalerate, in constant temperature blending instrument different temperatures (20-45 DEG C), 5h is reacted under the conditions of 1000rpm, extracts reaction solution the enantiomeric excess by 1 method of embodiment detection (R) -2- bromo ethyl isovalerate Value and conversion ratio, the results are shown in Table shown in 3.

As a result illustrate, when reaction temperature is 35 DEG C, the enantiomeric excess value highest of (R) -2- bromo ethyl isovalerate, Ee value > 99%.When reaction temperature is higher than 35 DEG C or is lower than 35 DEG C, the conversion ratio of (R) -2- bromo ethyl isovalerate and mapping Body excessive value can all decline, and illustrate that temperature has a very big impact the optical selective of lipase M5.

Influence of 3 reaction temperature of table to reaction

The separation and Extraction of 7 product of embodiment (R) -2- bromo ethyl isovalerate

20mLpH 7.0,0.2mM Na are added in 50mL round-bottomed flask₂HPO₄/NaH₂PO₄Buffer solution weighs 0.3g The thick enzyme powder of lipase prepared by embodiment 2, adds the 2- bromo ethyl isovalerate of final concentration 5g/L, is carried out with 50mM NaOH Stream plus drop reaction, control reaction pH maintain 7.0, at 35 DEG C of magnetic stirring apparatus, 6h are reacted under the conditions of 600rpm.Reaction terminates The reaction solution obtained afterwards is carried out being acidified to pH 2.0 with 4mM HCl, is then extracted with isometric ethyl acetate, separatory funnel Organic phase being isolated, then twice through pure water, saturation NaCl is washed twice, and dry, revolving obtains final product, and weigh, The mass yield of product (R) -2- bromo ethyl isovalerate reaches 96.4%.

Sequence table

<110>Zhejiang Polytechnical University

<120>application of a kind of lipase in resolution of racemic 2- bromo ethyl isovalerate

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 368

<212> PRT

<213>aspergillus oryzae (Aspergillus oryzae)

<400> 1

Met Lys Ile Gln Lys Leu Ile Leu Tyr Lys Phe Ala Asn Glu Leu Thr

1 5 10 15

Ala Ser Gly Leu Ile Pro Ser Lys Ala Leu Thr Cys Leu Asn Gly Tyr

20 25 30

Ala Ile Ile Asp Leu Asn Ser Ile His Arg His Asn Pro Ser Pro Glu

35 40 45

Asn Leu Ser Ile Tyr Pro Tyr Lys Ala Lys Ser Asp Ala Pro Tyr Ser

50 55 60

Ile Ala Glu Asn Thr Leu Arg Ala Gly Ile His Ile Pro Arg Ser Phe

65 70 75 80

Ser His Lys Arg Asp Lys Lys Ile Pro Val Leu Leu Val Pro Gly Thr

85 90 95

Ala Val Pro Ala Ala Ile Thr Phe Tyr Phe Asn Phe Gly Lys Leu Arg

100 105 110

Arg Ala Leu Pro Glu Ser Glu Leu Val Trp Ile Asp Leu Pro Gln Ala

115 120 125

Ser Leu Asp Asp Ile Gln Leu Ser Ala Glu Tyr Val Ala Tyr Ala Leu

130 135 140

Asn Tyr Val Ser Ala Leu Thr Ser Ser Lys Ile Ala Val Ile Ser Trp

145 150 155 160

Ser Gln Gly Ala Leu Asp Ile Gln Trp Ala Leu Lys Tyr Trp Pro Ser

165 170 175

Thr Arg Gly Val Val Asn Asp Phe Ile Ala Ile Ser Pro Asp Phe His

180 185 190

Gly Thr Ile Val Lys Trp Leu Val Cys Pro Leu Leu Asn Asp Leu Ala

195 200 205

Cys Thr Pro Ser Ile Trp Gln Gln Gly Trp Asp Ala Asn Phe Ile Gln

210 215 220

Ala Leu Arg Ser Gln Gly Gly Asp Ser Ala Tyr Val Pro Thr Thr Thr

225 230 235 240

Ile Tyr Ser Ser Phe Asp Lys Ile Val Arg Pro Met Ser Gly Glu Asn

245 250 255

Ala Ser Ala Arg Leu Leu Asp Tyr Arg Gly Val Gly Val Ser Asn Asn

260 265 270

His Leu Gln Thr Ile Cys Ala Asn Asn Ala Ala Gly Gly Leu Tyr Thr

275 280 285

His Glu Gly Val Leu Tyr Asn Pro Leu Ala Trp Ala Leu Thr Val Asp

290 295 300

Ala Leu Leu His Asp Gly Pro Ser Asn Ile Thr Arg Ile Asp Thr Gln

305 310 315 320

Lys Ile Cys Glu Gln Val Leu Pro Pro Tyr Leu Glu Leu Thr Asp Met

325 330 335

Leu Gly Thr Glu Ala Leu Leu Leu Val Ala Leu Ala Lys Ile Leu Thr

340 345 350

Tyr Ser Pro Lys Val Ser Gly Glu Pro Asp Ile Ala Lys Tyr Ala Tyr

355 360 365

<210> 2

<211> 1107

<212> DNA

<213>aspergillus oryzae (Aspergillus oryzae)

<400> 2

atgaaaatcc agaaactgat tttatataaa tttgcaaacg agctcaccgc atcaggcctg 60

ataccttcaa aggcactgac atgcctgaat ggctacgcga tcattgacct caattcgatt 120

caccgtcaca acccatcccc agaaaacttg agcatatacc catacaaagc aaagagcgat 180

gcgccttact ccattgcgga aaatactctc cgggctggaa ttcacattcc tagatcattc 240

tcacacaaac gggacaagaa aataccggtg ctcctggtac caggaactgc tgttcctgcg 300

gccataacct tttatttcaa ctttggcaag ttgaggagag ccttacccga aagtgagcta 360

gtttggatcg atctccctca ggcctcgttg gatgacattc aattgagcgc tgaatatgtc 420

gcctatgcgc tcaattatgt ttccgctttg acctcatcta agattgccgt gatttcttgg 480

tcccaaggtg cactggatat tcagtgggca ctgaagtact ggccatctac ccggggcgtc 540

gtcaacgact tcattgctat cagccccgac ttccatggaa caatagtcaa gtggctggta 600

tgccctctat taaatgattt agcctgtact ccttcgattt ggcagcaagg gtgggatgca 660

aactttatac aggccttgcg gagccaaggg ggagactctg cctatgtccc cacgaccacc 720

atttactctt cattcgacaa gattgtgcga cccatgagtg gggaaaatgc ttctgctcgg 780

ctactggact acaggggggt gggcgtttcc aataaccatc tacagaccat ctgcgcaaat 840

aatgccgctg gtggcctcta tacgcatgaa ggcgtgttgt acaaccctct tgcatgggcc 900

cttactgttg acgcactcct tcacgatggg cctagcaata ttacgcgaat cgatacccag 960

aagatctgtg aacaagtact tccgccatat ctcgaattaa ctgatatgct gggaaccgaa 1020

gccctccttt tagtagccct agcaaagatt cttacatatt ccccgaaagt ctccggtgag 1080

cctgacattg caaagtatgc atattaa 1107

Claims

1. a kind of application of lipase in resolution of racemic 2- bromo ethyl isovalerate, it is characterised in that the ammonia of the lipase Base acid sequence is as shown in SEQ ID NO.1.

2. application as described in claim 1, it is characterised in that the nucleotide sequence of the encoding gene of the lipase such as SEQ Shown in ID NO.2.

3. application as described in claim 1, it is characterised in that the method for the application are as follows: with the work of fatty enzyme coding gene The thick enzyme powder of lipase after the wet thallus freeze-drying that the fermented culture of journey bacterium obtains is biocatalyst, with racemic 2- bromo isoamyl Acetoacetic ester is substrate, using pH7.0 buffer as reaction medium, carries out resolution reaction under the conditions of 20-45 DEG C, 800-1000rpm, After fully reacting, reaction solution is isolated and purified, obtains (R) -2- bromo ethyl isovalerate.

4. application as claimed in claim 3, it is characterised in that the catalyst amount is calculated as 15-30g/L with buffer volume, The Final substrate concentrations are calculated as 5-10g/L with buffer volume.

5. application as claimed in claim 3, it is characterised in that reaction time 3-7h.

6. application as claimed in claim 3, it is characterised in that the reaction condition is 35 DEG C, 1000rpm reacts 5h.

7. application as claimed in claim 3, it is characterised in that the buffer is pH7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄ Buffer solution.

8. application as claimed in claim 3, it is characterised in that the catalyst is prepared as follows: fatty enzyme is encoded The engineering bacteria of gene is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀To 0.4-0.6, add IPTG to final concentration 0.02mM, 30 DEG C 10-12h, bacterium solution 8000rpm, 4 DEG C of centrifugation 10min are cultivated, collects thallus, then with PBS buffer solution washing thalline 2 times, 8000rpm, 4 DEG C of centrifugation 10min, collect thallus, and freeze-drying obtains the thick enzyme powder of lipase.

9. application as described in claim 1, it is characterised in that the method that the reaction solution isolates and purifies are as follows: after reaction, Reaction solution is acidified to pH2.0 with 4mM HCl, is then extracted with isometric ethyl acetate, separatory funnel isolates organic phase, then passes through Twice, saturation NaCl is washed twice pure water, and dry, revolving obtains final product (R) -2- bromo ethyl isovalerate.