CN110358752A

CN110358752A - A kind of Aspergillus oryzae lipase and preparing the application in Bu Waxitan chiral intermediate

Info

Publication number: CN110358752A
Application number: CN201910605913.9A
Authority: CN
Inventors: 郑建永; 李琪; 汪钊; 章银军
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-07-05
Filing date: 2019-07-05
Publication date: 2019-10-22
Anticipated expiration: 2039-07-05
Also published as: CN110358752B

Abstract

The present invention provides a kind of Aspergillus oryzae lipase and splitting the application for preparing Bu Waxitan intermediate, the amino acid sequence of the Aspergillus oryzae lipase is as shown in SEQ ID NO.1, the dry mycelium obtained after the wet thallus obtained using fermented and cultured is freeze-dried is catalyst, racemic 2- (2- tert-butoxy -2- oxoethyl) methyl valerate is substrate, using pH7.0 phosphate buffer solution as reaction medium, at 20-60 DEG C, it carries out splitting preparation Bu Waxitan intermediate R-2- (2- tert-butoxy -2- oxoethyl) valeric acid under the conditions of 1000rpm, mass yield reaches 49.5%, product R-2- (2- tert-butoxy -2- oxoethyl) valeric acid enantiomeric excess value > 99% after esterification.

Description

A kind of Aspergillus oryzae lipase and preparing the application in Bu Waxitan chiral intermediate

(1) technical field

The invention belongs to biological enzyme fields, are related to a kind of lipase stereoselectivity catalyzing hydrolysis racemic 2- (2- Tert-butoxy -2- oxoethyl) methyl valerate enantiomer preparation Bu Waxitan intermediate R-2- (2- tert-butoxy -2- oxo second Base) valeric acid application.

(2) background technique

Bu Waxitan (brivaracetam, trade name Briviact), (2S) -2- [(4R) -2- oxo -4- propyl -1- pyrrole Cough up alkyl] butyramide, also referred to as UCB 34714, it is a kind of new antiepileptic drugs (AED) developed by Belgian UCB. S.A. (BE) Bruxelles Belgium, point Do not suffered from as a adjuvant therapy medicaments for 16 years old and the above epilepsy in January, 2016 and 2 months by EMA and FDA approval listing The treatment of person's partial seizures.Bu Waxitan belongs to the IIIth generation new antiepileptic drugs, is current most situation of selling well antiepileptic The structural derivative of Levetiracetam.Bu Waxitan is a kind of protrusion vacuolar protein 2A ligand of highly selective high-affinity, Antiepileptic action is generated by number of mechanisms, main mechanism be by with maincenter synaptic vesicle proteins 2A (synaptic Vesicle protein 2A, SV2A) combine influence synaptic function, and the inhibiting effect to Voltage-gated Sodium Channels It is produced.Because of its good tolerability with good safety and pharmacokinetics, especially central nervous system, it is always Research hotspot in epilepsy therapy possesses very big market application prospect.

Bu Waxitan reports that synthetic route is more at present, and main includes being torn open by chiral chromatographic column preparation, chiral resolving agent Point and the methods of asymmetric syntheses.Chiral column chromatography preparation method document report is more, Yu Chenggong etc. to its synthetic method into Summary is gone, these methods need chiral column chromatography to split, and separation preparation cost is high, and production scale is limited, is not able to satisfy industrialization The requirement of production；The chiral resolution agent method of the reports such as Wang Chunyan has selected (R)-(+)-α-phenylethylamine cheap and easy to get to tear open Divide agent, de (diastereomeric excess percentage) value is high, the disadvantage is that yield is low after splitting, has more than 50% diastereoisomer damage It loses.Liu Jiuzhi etc. is studied in the method for asymmetric syntheses to prepare, but is unfavorable for industry using column chromatographic purification methods in the process Production.In summary several chemical synthesis process can see chemical synthesis process approach there is yields low, at high cost, step Various, the problems such as product purity is lower, seriously polluted.Document report splits preparation Bu Waxitan intermediate R- using protease 2- (2- tert-butoxy -2- oxoethyl) valeric acid (Schule A, Merschaert A, Szczepaniak C, et al.A Biocatalytic Route to the Novel Antiepileptic Drug Brivaracetam[J].Organic Process Research&Development, 2016,20 (9)), biocatalysis has reaction mildly, the few advantage of by-product. Since Bu Waxitan patent will expire in 2021, existing market demand is increasingly increased, but Bu Waxitan synthetic route at present There is certain limitation causes its current bulk pharmaceutical chemicals market price odd high, therefore progress Bu Waxitan synthesis technology and related object Matter research, development cost is cheap, is suitble to industrialization amplification technique route, prepares high-optical-purity Bu Waxitan, has great Significance of scientific research and economic value.The present invention prepares cloth watt using the lipase-catalyzed stereo selective hydrolysis reaction fractionation newly screened Western smooth chiral intermediate R-2- (2- tert-butoxy -2- oxoethyl) valeric acid, catalyst are easy to get at low cost, ee value and conversion ratio Height, route short operation are easy.

(3) summary of the invention

The invention aims to solve the deficiency of existing synthetic method, by screening yielding lipase microorganism, one is provided The stereoselectivity lipase of kind Cheap highly effective splits RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate in bioanalysis In application.

The technical solution adopted by the present invention is that:

The present invention provides a kind of Aspergillus oryzae lipase, the Aspergillus oryzae lipase amino acid sequence such as SEQ ID NO.1 institute Show, the fat enzyme coding gene nucleotide sequence is shown in SEQ ID NO.2.

The lipase gene is originated from aspergillus oryzae WZ007 (Aspergillus oryzaeWZ007), which is preserved in China Type Tissue Collection, address: Wuhan, China, Wuhan University, 430072, deposit number: CCTCC No:M 206105, Preservation date: related bacterium is had submitted in previously applying for a patent (Chinese patent CN 101186938A) on October 8th, 2006 Kind preservation information simultaneously discloses its genetic resources source.The present invention extracts mRNA and obtains through reverse transcription from aspergillus oryzae WZ007 CDNA, on the basis of the aspergillus oryzae genome GenBank:XM_023233508.1 base sequence checked according to NCBI, in design Trip primer is 5'-CCATGGGCATGACAGCACACGAAGCCCTGA-3' and downstream primer is 5'- CTCGAGCTAATGTAACGCAACCCGAATATGC-3' carries out PCR, obtains base gene order, and sequencing result is SEQ ID NO.2 segment.

The invention further relates to the recombinant vectors containing Aspergillus oryzae lipase encoding gene, and are turned using the recombinant vector Change obtained recombination engineering bacteria, gene shown in SEQ ID NO.2 is connected to carrier pET-28b (+) by the engineering bacteria, is turned Change to Escherichia coli Rosetta competent cell, obtains genetic engineering bacterium.

The present invention provides a kind of Aspergillus oryzae lipase and is splitting RS-2- (2- tert-butoxy -2- oxoethyl) valeric acid Methyl esters prepares the application in Bu Waxitan intermediate R-2- (2- tert-butoxy -2- oxoethyl) valeric acid (as shown in Figure 1, product For Bu Waxitan chiral intermediate), concrete application method are as follows: obtain the fermented culture of the engineering bacteria of the gene containing Aspergillus oryzae lipase The thick enzyme powder of lipase obtained after the wet thallus freeze-drying obtained is catalyst, with racemic RS-2- (2- tert-butoxy -2- oxygen For ethyl) methyl valerate be substrate, using pH7.0 phosphate buffer solution as reaction medium, under the conditions of 20-60 DEG C, 1000rpm into After fully reacting, reaction solution is isolated and purified for row resolution reaction, prepares Bu Waxitan intermediate R-2- (2- tert-butoxy- 2- oxoethyl) valeric acid.The catalyst amount is calculated as 20g/L-100g/L (preferably 50g/L), the bottom with buffer volume The final concentration of 1%-3% of object (V/V) (preferably 1.75%).

Further, preferred reaction time 5min-180min, more preferable reaction condition is 45 DEG C, 1000rpm reacts 90min。

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

Further, the catalyst is prepared as follows: (preferably by the engineering bacteria of the encoding gene containing Aspergillus oryzae lipase Escherichia coli Rosetta) it is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀Most 0.4-0.6 adds IPTG to final concentration 0.02mM, 30 DEG C of culture 10-12h, bacterium solution 8000rpm, 4 DEG C of centrifugation 10min collect thallus, then with PBS buffer solution washing thalline 2 times, 8000rpm, 4 DEG C centrifugation 10min collect thallus, are lyophilized (- 80 DEG C of vacuum freeze drier cold-trap, vacuum degree 20Pa), obtain To the thick enzyme powder of lipase；LB culture medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 5g/L, solvent is deionization Water, pH value are natural.

Further, the method that reaction solution of the present invention isolates and purifies are as follows: after reaction, reaction solution first uses 2M NaOH Isometric unreacted S-2- of hexamethylene extraction and separation (2- tert-butoxy -2- oxoethyl) is added in aqueous solution tune pH to 9.0 Methyl valerate；Then water phase is acidified to pH 2.0 with 4M HCL aqueous solution, isometric ethyl acetate extraction, separatory funnel is added Isolate organic phase, organic phase again through pure water twice, saturation NaCl is washed twice, dry, obtains product R-2- (the tertiary fourth of 2- Oxygroup -2- oxoethyl) valeric acid.

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

MTAHEALNPIHPSVLPHLDPVFIKLYNENVANTPNKPIDLAILRSKYSVLYSYGTGPAPDPARIYDAT VPGYNGDLIPVRVYEPSSPGPWPVHIDFHGGGMHSFPLLYPRIRRLMPTAGWGLGDLDTEAHICKHLSVKADVCVI DIGYRLVPEQPFPIGIQDSFAALEYIHAQGASKFNIDTTRISLGGVSAGGNIALIVAHLARDASIPLKLVAVGTPV IDDISKYASASESPYPSVQQMEHAPTLNWARLKWFDNLKWESLSSDVGLRKEQLDKISWYANAMNAPSFTNLPKTV IYTAGCDPLRDEGEAYAMKLVEGGNEVTLKRFEGVPHPFMHMDNDLWQAKEFIDKTAAHIRVALH。

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:

ATGACAGCACACGAAGCCCTGAACCCTATCCACCCGTCCGTCCTGCCTCATTTGGACCCCGTCTTTAT CAAACTCTACAATGAAAATGTCGCCAACACCCCCAACAAGCCCATAGACTTGGCCATTCTTCGATCAAAATATTCC GTGTTATATTCTTATGGTACCGGGCCAGCCCCCGATCCAGCTAGAATATACGATGCAACCGTGCCGGGATATAATG GCGATTTGATTCCAGTGCGAGTATACGAGCCATCGTCTCCGGGGCCTTGGCCGGTGCATATTGATTTTCATGGCGG TGGTATGCACTCCTTCCCTCTCTTGTATCCTCGGATCAGAAGACTAATGCCGACCGCAGGCTGGGGCCTTGGCGAC CTCGACACTGAAGCTCATATCTGCAAGCATCTGTCCGTCAAAGCGGACGTTTGTGTAATCGACATTGGTTACCGGC TGGTCCCAGAACAGCCGTTTCCCATTGGCATCCAAGACTCCTTTGCTGCCCTGGAATACATTCATGCCCAGGGCGC TTCCAAGTTCAACATTGACACGACCCGCATCTCCCTTGGCGGTGTCTCAGCTGGAGGAAACATCGCCCTGATCGTG GCCCACCTTGCAAGGGATGCCAGCATCCCTCTGAAACTCGTCGCGGTGGGCACACCCGTCATTGACGATATCTCCA AGTACGCCTCTGCAAGCGAGTCTCCATACCCCTCTGTCCAACAGATGGAGCACGCGCCCACTCTCAACTGGGCCAG GTTGAAGTGGTTCGATAATCTCAAATGGGAAAGCCTTTCCAGCGATGTGGGTTTGAGGAAGGAGCAATTAGATAAA ATCAGCTGGTATGCGAATGCAATGAATGCGCCTAGTTTCACCAACCTACCCAAGACGGTGATCTACACTGCTGGCT GTGATCCGCTACGAGATGAAGGAGAGGCGTACGCAATGAAGCTAGTGGAGGGTGGCAATGAAGTTACGCTTAAAAG GTTTGAAGGCGTGCCACACCCTTTCATGCATATGGATAACGACTTATGGCAGGCGAAGGAGTTTATAGACAAGACG GCTGCGCATATTCGGGTTGCGTTACATTAG。

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.

Compared with prior art, the beneficial effects are mainly reflected as follows: the present invention provides a kind of aspergillus oryzae fat Enzyme, which can connect building with expression vector and obtain the intracellular expression recombinant plasmid containing the gene, then convert Into coli strain, recombination bacillus coli is obtained, recombination bacillus coli is recycled or recombinant lipase is biocatalyst It is catalyzed resolution of racemic 2- (2- tert-butoxy -2- oxoethyl) methyl valerate, available product R-2- (2- tert-butoxy - 2- oxoethyl) valeric acid, enantiomeric excess value > 99% and conversion ratio reach 49.88%, product R-2- (2- tert-butoxy -2- oxygen For ethyl) mass yield of valeric acid reaches 49.5%.Current existing research method such as chiral chromatographic column preparation, chiral resolving agent It splits and the conversion ratio of asymmetric syntheses etc. is 20%-30%, yield is generally in 10%-30%.The conversion that the present invention obtains Rate is 49.88%, 20%-30% higher than existing method；Mass yield is 49.5%, 20%-40% higher than existing method.This hair The chiral synthetic reaction of bright biocatalysis has a mild condition, high-efficient, the chemo-selective of height, regioselectivity and right The advantages that body is selective is reflected, and biocatalysis process has the characteristics that nontoxic, pollution-free and low energy consumption, is a kind of environmental-friendly Synthetic method.

(4) Detailed description of the invention

The reaction of Fig. 1 lipase enantioselective hydrolysis fractionation RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Schematic diagram；

The liquid chromatogram of Fig. 2 RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate；

The liquid phase of the lipase-catalyzed RS-2- of Fig. 3 (2- tert-butoxy -2- oxoethyl) methyl valerate hydrolysis 90min Chromatogram；

Fig. 4 R-2- (2- tert-butoxy -2- oxoethyl) liquid chromatogram of valeric acid through esterification reaction of organic acid.

(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, MgSO₄0.5, NaCl 0.5, solvent are deionized water, pH 7.0.

Aspergillus oryzae (Aspergillusoryzae) CCTCC No:M 206105 is seeded to slant medium, 30 DEG C of cultures 6-8d obtains 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 pours into the fermentation medium containing 18L is fermented 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 CCTCC No:M 206105cDNA: taking the above-mentioned aspergillus oryzae wet thallus of 1g, uses Quan Shi King Company TransZol Up Plus RNA Kit kit extract mRNA, reuse the ReverTra Ace qPCR of TOYOBO company RT Kit, expeditiously synthesis is suitable for the cDNA template of Realtime PCR.

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

On the basis of GenBank:XM_023233508.1 base sequence, separately designing upstream primer is 5'- CCATGGGCATGACAGCACACGAAGCCCTGA-3' and downstream primer are 5'- CTCGAGCTAATGTAACGCAACCCGAATATGC-3' carries out PCR, obtains nucleotides sequence and be classified as SEQ using cDNA as template Genetic fragment shown in ID NO.2, sequence verification are correct.The coding protein amino acid sequence of genetic fragment shown in SEQ IDNO.2 For 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 M16) of fatty enzyme.

The fermented and cultured of the colibacillus engineering (M16) of the fatty enzyme of embodiment 2

The colibacillus engineering (M16) that embodiment 1 obtains is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀To 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, collects thallus.The thallus of collection is made The thick enzyme powder of lipase is obtained after (- 80 DEG C of cold-trap, vacuum degree 20Pa) of vacuum freeze drier freeze-dryings, is denoted as lipase M16, 4 DEG C of refrigerators are put in save.LB culture medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 5g/L, solvent is deionization Water, pH are natural.

The reaction of 3 enzymatic of embodiment fractionation RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate

The lipase M16 of 2 method of 0.05g embodiment preparation is weighed in 2mL EP pipe, addition 1mL PB (pH7.0, It 0.2mM) is used as reaction dissolvent, 17.5uL substrate RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate is then added, with Lipase is not blank control, is placed in 45 DEG C, reacts 90min in 1000rpm constant temperature blending instrument.After reaction, reaction solution 2M NaOH aqueous solution tune pH to 9.0 is first used, isometric hexamethylene, turbula shaker oscillation sufficiently extraction, centrifugation is added (10000rpm, 5min) obtains the organic phase containing S-2- (2- tert-butoxy -2- oxoethyl) methyl valerate, takes 500uL through true Sky is dry, and 1mL is added and flows phased soln, living using the stereoselectivity and enzymatic hydrolysis of high performance liquid chromatography detection thallus Property.Lipase hydrolysis resolution reaction, specific for hydrolysis RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate obtain institute Bu Waxitan intermediate R-2- (2- tert-butoxy -2- oxoethyl) valeric acid needed；S-2- (2- tert-butoxy -2- oxygen is not hydrolyzed For ethyl) methyl valerate, so only showing S-2- (2- tert-butoxy -2- oxoethyl) methyl valerate on liquid chromatogram Peak.

Specific liquid phase analysis condition: 1525 type liquid chromatograph of liquid chromatograph Waters is used；Daicel CHIRALPAK AD-H (5 μm, 4.6 × 250mm) chiral chromatographic column, ultraviolet light Detection wavelength 220nm, mobile phase n-hexane: different Propyl alcohol=20:1 (V/V)；Flow velocity is 0.5mL/min, 30 DEG C of column temperature, 10 μ L of sample volume.S-2- (2- tert-butoxy -2- oxo second Base) methyl valerate standard items and R-2- (2- tert-butoxy -2- oxoethyl) methyl valerate standard items respectively in 9.87min and 12.471min appearance (as shown in Figure 2).After enzyme hydrolysis resolution reaction, S-2- (2- tert-butoxy -2- oxoethyl) valeric acid is obtained The liquid chromatogram (as shown in Figure 3) of methyl esters, conversion ratio 49.68%, substrate ee value > 99%.

4 reaction temperature of embodiment is to enzyme kinetics Hydrolysis Resolution RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Influence

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the embodiment 2 of final concentration 50g/L is added Lipase M16,17.5 μ L RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate of method preparation, in constant temperature blending instrument Different temperatures (20-60 DEG C) reacts 90min under the conditions of 1000rpm, extracts reaction solution and detects S-2- (the tertiary fourth of 2- by 3 method of embodiment Oxygroup -2- oxoethyl) methyl valerate enantiomeric excess value and conversion ratio, the results are shown in Table 1.

As a result illustrate, when reaction temperature is 45 DEG C, the mapping of S-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Body excessive value highest, ee value > 99%.When reaction temperature is higher than 45 DEG C or is lower than 45 DEG C, catalytic conversion and enantiomer Excessive value can all decline, and illustrate that temperature has a very big impact the catalytic activity of lipase M16.

Influence of 1 reaction temperature of table to reaction

5 reaction time of embodiment is to enzyme kinetics Hydrolysis Resolution RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Influence

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, 2 side of final concentration 50g/L embodiment is added Lipase M16,17.5 μ LRS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate of method preparation, in constant temperature blending instrument 45 DEG C, different time (5min-180min) is reacted under the conditions of 1000rpm, extracts reaction solution and detects R-2- (uncle 2- by 3 method of embodiment Butoxy -2- oxoethyl) methyl valerate enantiomeric excess value and conversion ratio, the results are shown in Table shown in 2.

The result shows that after reaction 90min, the enantiomer of product S-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Excessive value has reached highest, enantiomeric excess value > 99%, conversion ratio 49.88%, when reacted between be greater than 90min, conversion ratio Increase, the enantiomeric excess value that will lead to product R-2- (2- tert-butoxy -2- oxoethyl) valeric acid reduces.

The influence that 2 reaction time of table reacts enzymic catalytic reaction

The different lipase hydrolysis of embodiment 6 split the effect ratio of RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Compared with

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the difference fat of final concentration 50g/L is added Enzyme (lipase M16, Novozym 435, the Lipozyme TL IM, Lipozyme RM IM of the preparation of 2 method of embodiment), 17.5 μ L RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerate reacts under the conditions of 1000rpm in 45 DEG C of constant temperature blending instrument 90min extracts reaction solution the enantiomer mistake by 3 method of embodiment detection R-2- (2- tert-butoxy -2- oxoethyl) methyl valerate Magnitude and conversion ratio, the results are shown in Table shown in 3.After 45 DEG C of reaction 90min, Novozym 435 is to RS-2- (2- tert-butoxy -2- Oxoethyl) two kinds of configurations of methyl valerate can be carried out and hydrolyze to a certain degree, but not have enantioselectivity to two kinds of configurations of substrate Property, and LipozymeTL IM, LipozymeRM IM do not have hydrolysing activity and enantio-selectivity to substrate.When the thick enzyme of lipase Powder M16 has good stereoselectivity.

The fractionation effect of different lipase-catalyzed RS-2- (2- tert-butoxy -2- oxoethyl) methyl valerates of table 3

The separation and Extraction and esterification of 7 product R-2- of embodiment (2- tert-butoxy -2- oxoethyl) valeric acid

10mL pH 7.0, the Na of 0.2mM are added in 50mL round-bottomed flask₂HPO₄/NaH₂PO₄Buffer solution weighs The lipase M16 of 2 method of 0.5g embodiment preparation, adds the RS-2- (2- tert-butoxy -2- oxygen of final concentration 1.75% (V/V) For ethyl) methyl valerate, stream plus drop reaction are carried out with 200mMNaOH aqueous solution, control reaction pH maintains 7.0, in magnetic force 45 DEG C of blender, 90min is reacted under the conditions of 600rpm.After reaction, the 2M NaOH aqueous solution tune pH of reaction solution first is extremely 9.0, unreacted (the S) -2- of isometric hexamethylene extraction and separation (2- tert-butoxy -2- oxoethyl) methyl valerate is added； Then it is acidified to pH 2.0 with 4M HCL aqueous solution, isometric ethyl acetate extraction is added, separatory funnel is isolated organic phase, had Twice, saturation NaCl is washed twice machine Xiang Zaijing pure water, dry, obtains final product, and the 0.071g that weighs, product R-2- The mass yield of (2- tert-butoxy -2- oxoethyl) valeric acid reaches 49.5%.

The esterification of R-2- (2- tert-butoxy -2- oxoethyl) valeric acid: by gained R-2- (2- tert-butoxy -2- oxo Ethyl) valeric acid, is dissolved with the DMF of 1mL iodomethane containing 10uL, the K of 0.02g is added₂CO₃, in 30 DEG C, 800rpm constant temperature blending instrument Middle reaction 3h mutually carries out liquid phase analysis, liquid chromatogram with sufficiently being extracted after 500 μ L pure waters with 1mL n-hexane in centrifuging and taking As shown in figure 4, obtaining enantiomeric excess value > 99% of R-2- (2- tert-butoxy -2- oxoethyl) valeric acid.

Sequence table

<110>Zhejiang Polytechnical University

<120>a kind of Aspergillus oryzae lipase and the application in Bu Waxitan chiral intermediate is being prepared

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 361

<212> PRT

<213>aspergillus oryzae (Aspergillus oryzae)

<400> 1

Met Thr Ala His Glu Ala Leu Asn Pro Ile His Pro Ser Val Leu Pro

1 5 10 15

His Leu Asp Pro Val Phe Ile Lys Leu Tyr Asn Glu Asn Val Ala Asn

20 25 30

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

35 40 45

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

50 55 60

Tyr Asp Ala Thr Val Pro Gly Tyr Asn Gly Asp Leu Ile Pro Val Arg

65 70 75 80

Val Tyr Glu Pro Ser Ser Pro Gly Pro Trp Pro Val His Ile Asp Phe

85 90 95

His Gly Gly Gly Met His Ser Phe Pro Leu Leu Tyr Pro Arg Ile Arg

100 105 110

Arg Leu Met Pro Thr Ala Gly Trp Gly Leu Gly Asp Leu Asp Thr Glu

115 120 125

Ala His Ile Cys Lys His Leu Ser Val Lys Ala Asp Val Cys Val Ile

130 135 140

Asp Ile Gly Tyr Arg Leu Val Pro Glu Gln Pro Phe Pro Ile Gly Ile

145 150 155 160

Gln Asp Ser Phe Ala Ala Leu Glu Tyr Ile His Ala Gln Gly Ala Ser

165 170 175

Lys Phe Asn Ile Asp Thr Thr Arg Ile Ser Leu Gly Gly Val Ser Ala

180 185 190

Gly Gly Asn Ile Ala Leu Ile Val Ala His Leu Ala Arg Asp Ala Ser

195 200 205

Ile Pro Leu Lys Leu Val Ala Val Gly Thr Pro Val Ile Asp Asp Ile

210 215 220

Ser Lys Tyr Ala Ser Ala Ser Glu Ser Pro Tyr Pro Ser Val Gln Gln

225 230 235 240

Met Glu His Ala Pro Thr Leu Asn Trp Ala Arg Leu Lys Trp Phe Asp

245 250 255

Asn Leu Lys Trp Glu Ser Leu Ser Ser Asp Val Gly Leu Arg Lys Glu

260 265 270

Gln Leu Asp Lys Ile Ser Trp Tyr Ala Asn Ala Met Asn Ala Pro Ser

275 280 285

Phe Thr Asn Leu Pro Lys Thr Val Ile Tyr Thr Ala Gly Cys Asp Pro

290 295 300

Leu Arg Asp Glu Gly Glu Ala Tyr Ala Met Lys Leu Val Glu Gly Gly

305 310 315 320

Asn Glu Val Thr Leu Lys Arg Phe Glu Gly Val Pro His Pro Phe Met

325 330 335

His Met Asp Asn Asp Leu Trp Gln Ala Lys Glu Phe Ile Asp Lys Thr

340 345 350

Ala Ala His Ile Arg Val Ala Leu His

355 360

<210> 2

<211> 1086

<212> DNA

<213>aspergillus oryzae (Aspergillus oryzae)

<400> 2

atgacagcac acgaagccct gaaccctatc cacccgtccg tcctgcctca tttggacccc 60

gtctttatca aactctacaa tgaaaatgtc gccaacaccc ccaacaagcc catagacttg 120

gccattcttc gatcaaaata ttccgtgtta tattcttatg gtaccgggcc agcccccgat 180

ccagctagaa tatacgatgc aaccgtgccg ggatataatg gcgatttgat tccagtgcga 240

gtatacgagc catcgtctcc ggggccttgg ccggtgcata ttgattttca tggcggtggt 300

atgcactcct tccctctctt gtatcctcgg atcagaagac taatgccgac cgcaggctgg 360

ggccttggcg acctcgacac tgaagctcat atctgcaagc atctgtccgt caaagcggac 420

gtttgtgtaa tcgacattgg ttaccggctg gtcccagaac agccgtttcc cattggcatc 480

caagactcct ttgctgccct ggaatacatt catgcccagg gcgcttccaa gttcaacatt 540

gacacgaccc gcatctccct tggcggtgtc tcagctggag gaaacatcgc cctgatcgtg 600

gcccaccttg caagggatgc cagcatccct ctgaaactcg tcgcggtggg cacacccgtc 660

attgacgata tctccaagta cgcctctgca agcgagtctc catacccctc tgtccaacag 720

atggagcacg cgcccactct caactgggcc aggttgaagt ggttcgataa tctcaaatgg 780

gaaagccttt ccagcgatgt gggtttgagg aaggagcaat tagataaaat cagctggtat 840

gcgaatgcaa tgaatgcgcc tagtttcacc aacctaccca agacggtgat ctacactgct 900

ggctgtgatc cgctacgaga tgaaggagag gcgtacgcaa tgaagctagt ggagggtggc 960

aatgaagtta cgcttaaaag gtttgaaggc gtgccacacc ctttcatgca tatggataac 1020

gacttatggc aggcgaagga gtttatagac aagacggctg cgcatattcg ggttgcgtta 1080

cattag 1086

Claims

1. a kind of Aspergillus oryzae lipase, it is characterised in that the amino acid sequence of the lipase is as shown in SEQ ID NO.1.

2. Aspergillus oryzae lipase encoding gene as described in claim 1, it is characterised in that the nucleotide sequence of the encoding gene As shown in SEQ ID NO.2.

3. a kind of engineering bacteria of the building of Aspergillus oryzae lipase encoding gene described in claim 2.

4. Aspergillus oryzae lipase described in a kind of claim 1 is in resolution of racemic 2- (2- tert-butoxy -2- oxoethyl) valeric acid Methyl esters prepares the application in Bu Waxitan chiral intermediate.

5. application as claimed in claim 4, it is characterised in that the method for the application are as follows: to encode base containing Aspergillus oryzae lipase The thick enzyme powder of lipase after the wet thallus freeze-drying that the fermented culture of the engineering bacteria of cause obtains is biocatalyst, with racemic 2- (2- tert-butoxy -2- oxoethyl) methyl valerate be substrate, using pH7.0 buffer as reaction medium, 20-60 DEG C, Resolution reaction is carried out under the conditions of 1000rpm, after fully reacting, reaction solution is isolated and purified, and obtains Bu Waxitan intermediate R-2- (2- tert-butoxy -2- oxoethyl) valeric acid.

6. application as claimed in claim 5, it is characterised in that the catalyst amount is calculated as 20g/L- with buffer volume 100g/L, the final concentration of 1%-3% of substrate volume.

7. application as claimed in claim 5, it is characterised in that reaction time 5min-180min.

8. application as claimed in claim 5, it is characterised in that the buffer is pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄ Buffer solution.

9. application as claimed in claim 5, it is characterised in that the catalyst is prepared as follows: aspergillus oryzae fat will be contained The engineering bacteria of enzyme coding gene is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀Most 0.4-0.6 adds IPTG to final concentration 0.02mM, 30 DEG C of culture 10-12h, bacterium solution 8000rpm, 4 DEG C of centrifugation 10min collect thallus, then with PBS buffer solution washing thalline 2 times, 8000rpm, 4 DEG C centrifugation 10min, collect thallus, and freeze-drying obtains the thick enzyme powder of lipase.

10. application as claimed in claim 5, it is characterised in that the reaction solution isolates and purifies the method for preparing product are as follows: anti- After answering, isometric hexamethylene extraction and separation unreacted is added in the 2M NaOH aqueous solution tune pH to 9.0 of reaction solution first S-2- (2- tert-butoxy -2- oxoethyl) methyl valerate；Then pH 2.0, the bodies such as addition are acidified to 4M HCL aqueous solution Product ethyl acetate extraction, separatory funnel isolate organic phase, then twice through pure water, saturation NaCl is washed twice, dry, obtain To R-2- (2- tert-butoxy -2- oxoethyl) valeric acid.