CN109628544A

CN109628544A - A kind of lipase is splitting the application in N- acetyl-DL- methionine methyl ester

Info

Publication number: CN109628544A
Application number: CN201910057292.5A
Authority: CN
Inventors: 郑建永; 吴鹏; 章银军; 孙杰; 汪钊
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-01-22
Filing date: 2019-01-22
Publication date: 2019-04-16
Anticipated expiration: 2039-01-22
Also published as: CN109628544B

Abstract

The present invention provides a kind of lipase to split the application in N- acetyl-DL- methionine methyl ester, 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 engineering bacteria；The recombination bacillus coli isolates and purifies acquisition recombinant lipase through clasmatosis；The ability that there is the recombinant lipase catalysis to split N- acetyl-DL- methionine methyl ester, can obtain N- acetyl-L-Methionine methyl esters, enantiomeric excess value > 99% and conversion ratio reach 51.2%.

Description

A kind of lipase is splitting the application in N- acetyl-DL- methionine methyl ester

(1) technical field

The invention belongs to biocatalysis technology fields, are related to a kind of lipase stereoselectivity catalyzing hydrolysis fractionation N- second N- acetyl-l-methionine methyl esters enantiomer application of acyl-DL- methionine methyl ester enantiomer synthesis single configuration.

(2) background technique

N- acetyl-l-methionine (N-Ac-L-Met) is a kind of valuable nutritional supplement, by l-methionine (L- Met chemical acetylation) generates, and passes through the fractionation of acylase catalyzed N- acetyl-DL- methionine (N-Ac-DL-Met) Preparation.DL-Met is the material for N- acetyl-DL- methionine methyl ester (N-Ac-DL-MetOMe) production, is closed by chemistry At with low cost large-scale production.The hydrolysis of enzymatic N- acetyl-DL- methionine methyl ester has been proposed for N-Ac-L- Met production.In this process, N-Ac-L-Met and D-Met will be produced, they can be used as the achirality synthetic or original of drug Material.But since the enantio-selectivity of reaction is poor, the low yield of N-Ac-L-Met is not developed effective so far Solution.Therefore, we provide a kind of Lipase catalyzed hydrolysis, and N-Ac-DL-MetOMe to be split efficiently to produce N-Ac-L- The effective ways of MetOMe.Enzymatic Resolution can utilize High level of stereoselectivity, the site, regioselectivity of enzyme, catalytic chemistry synthesis A certain enantiomer in racemic modification or derivative is hydrolyzed to obtain high yield, highly selective single enantiomer, has Selectivity is high, reaction condition is mild, reaction is more stable and is easy to industrialized feature.Meanwhile Enzymatic Resolution can solve chemistry Synthesis easily causes environmental pollution, a large amount of invalid or even environmentally harmful enantiomers is led to the problem of, for protecting the nature of the mankind Environment and health have particularly important meaning.

Lipase (Lipase, EC 3.1.1.3) full name is Lipase (Triacyl-glycerol 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 N- acetyl-DL- methionine first in bioanalysis Application in ester can obtain high purity N-acetyl-L- first using lipase with efficient separating N- acetyl-DL- methionine methyl ester Methyllanthionine methyl esters improves yield.

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 2) in N- acetyl-DL- methionine methyl ester, As shown in SEQ ID NO.1, the nucleotides sequence of encoding gene is classified as shown in SEQ ID NO.2 the fat enzyme amino acid sequence, Specific application method are as follows: with the engineering bacteria of the enzyme coding gene of fat containing stereoselectivity it is fermented culture obtain wet thallus or Wet thallus freeze-dried powder is catalyst, using N- acetyl-DL- methionine methyl ester as substrate, using 7.0 buffer of pH as reaction medium, Resolution reaction is carried out under the conditions of 25-45 DEG C, 600-800rpm, after fully reacting, reaction solution is isolated and purified, obtains N- second Acyl-l-methionine methyl esters.The catalyst amount is calculated as 10g/L with buffer volume, and the Final substrate concentrations are with buffer Volume is calculated as 5-20g/L.

Further, preferred reaction time 2-60min, more preferable reaction condition is 35 DEG C, 800rpm reacts 10min.

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

Further, the catalyst is prepared as follows: by the engineering bacteria of the enzyme coding gene of fat containing stereoselectivity (preferably e. coli bl21) is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀To 0.4-0.6 (preferably 0.5), add IPTG to end Concentration 0.02mM, 30 DEG C of culture 10-12h, bacterium solution 8000rpm, 4 DEG C of centrifugation 10min collect thallus, then are washed with PBS buffer solution Thallus 2 times, 8000rpm, 4 DEG C centrifugation 10min, collect thallus, and freeze-drying obtains the thick enzyme powder of lipase, i.e. wet thallus freeze-dried powder；Institute LB culture medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 5g/L is stated, solvent is 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 4mM HCl acid Change to pH 2.0, is then extracted with isometric ethyl acetate, separatory funnel isolates organic phase, and (preferably separatory funnel has been isolated Machine phase, water phase are extracted with ethyl acetate again, merge organic phase, twice through pure water by organic phase), then through pure water two Secondary, saturation NaCl is washed twice, and the organic phase obtained after washing is dried with anhydrous magnesium sulfate, after removing water, then will be had Machine is mutually rotated to doing, and obtains final product N- acetyl-l-methionine methyl esters.

Stereoselectivity lipase of the present invention, amino acid sequence is as shown in SEQ ID NO.1.

MTINYHELETSHGRIAVRESEGEGAPLLMIHGNSSSGAVFAPQLEGEIGKKWRVISPDLPGHGKSSDA IDPEHSYSMEGYADAMTEVMQKLGIADAVVFGWSLGGHIGIEMIARYPEMRGLMITGTPPVAREEVGQGFKSGPDM ALAGQEVFSERDVDSYARSTCGEPFEASLLDIVARTDGRARRIMFEKFGNGTGGNQRDIVAQAKLPIAVVNGRDEP FVELDFVSKVKFGNLWEGKTHVIDNAGHAPFRETPAIFDRYLMRFLSDCT IG。

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 invention further relates to the encoding genes of the stereoselectivity lipase, specifically, the e. coli codon Encoding gene nucleotide sequence after optimization is as shown in SEQ ID NO.2:

CCATGGGCATGACCATCAACTATCACGAACTGGAGACCTCTCATGGTCGCATTGCCGTGCGCGAAAGC GAAGGTGAAGGTGCCCCGCTGCTGATGATTCATGGCAACAGCAGCAGCGGTGCCGTGTTTGCACCGCAGCTGGAGG GCGAGATCGGCAAGAAATGGCGTGTGATTAGCCCGGATTTACCGGGTCATGGCAAAAGCAGCGATGCCATTGACCC GGAACATAGCTACAGCATGGAAGGCTATGCCGATGCCATGACCGAAGTGATGCAGAAGCTGGGCATTGCCGATGCC GTGGTGTTTGGTTGGTCTTTAGGCGGTCATATTGGCATCGAAATGATCGCCCGCTATCCGGAAATGCGCGGTTTAA TGATTACCGGTACCCCGCCGGTTGCCCGTGAAGAAGTTGGCCAAGGTTTTAAAAGCGGCCCGGATATGGCACTGGC CGGTCAAGAAGTGTTCAGCGAGCGCGATGTGGATAGTTATGCCCGCAGCACTTGTGGTGAACCGTTCGAAGCCTCT TTACTGGATATTGTTGCACGCACCGATGGTCGTGCACGCCGCATCATGTTCGAAAAGTTTGGCAACGGCACCGGTG GCAATCAGCGTGACATTGTGGCCCAAGCTAAACTGCCGATCGCCGTTGTGAATGGTCGCGATGAGCCGTTTGTTGA GCTGGACTTCGTGAGCAAGGTGAAGTTCGGCAATCTGTGGGAGGGCAAGACCCACGTGATTGATAATGCCGGTCAT GCCCCGTTTCGTGAAACACCGGCCATTTTCGATCGCTATTTAATGCGCTTTCTGAGCGATTGCACCATTGGTCTCG AG。

Particularity of the AG due to nucleotide sequence, the variant of polynucleotides shown in any SEQ ID NO.2, as long as itself and this Polynucleotides have 70% or more homology and function having the same, belong to the column of the scope of the present invention.The multicore The variant of thuja acid refers to a kind of polynucleotide sequence changed with one or more nucleotide.The variant of this polynucleotides can be with It is the variant that the allelic variant naturally occurred or non-natural occur, including substitution variants, Deletion variants and insertion become Allosome.As known in the art, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleosides Acid 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 and the engineering bacteria.

The beneficial effects are mainly reflected as follows: the present invention provides a kind of stereoselectivity lipase, the lipase Gene can connect building with expression vector and obtain the intracellular expression recombinant plasmid containing the gene, then convert to coli strain In, recombination bacillus coli is obtained, recombination bacillus coli is recycled or recombinant lipase is that biological catalyst splits N- acetyl- DL- methionine methyl ester, can be generated N- acetyl-l-methionine methyl esters, and enantiomeric excess value > 99% and conversion ratio reach 51.2%.Common l-methionine synthetic method is amino-acylase Split Method at present, utilizes aminoacylates enzymatic N- acetyl- The reaction of DL- methionine stereoselectivity acyl hydrolase obtains l-methionine, downstream separation l-methionine and N- acetyl-D- Methionine needs column chromatography for separation, and separation costs are high.The method of the present invention separates simpler compared with amino-acylase Split Method It is single, enzymatic product can be obtained by solvent extraction.The chiral synthetic reaction of biocatalysis of the present invention has mild condition, effect The advantages that rate is high, the chemo-selective of height, regioselectivity and enantio-selectivity, and biocatalysis process has nothing It is malicious, pollution-free and the features such as low energy consumption, it is a kind of environmental-friendly synthetic method.

(4) Detailed description of the invention

Fig. 1 is the reaction schematic diagram that recombinant lipase enantioselective hydrolysis splits N- acetyl-DL- methionine methyl ester；

Fig. 2 is the reaction schematic diagram of organic synthesis N- acetyl-DL- methionine methyl ester；

Fig. 3 is N- acetyl-DL- methionine methyl ester standard specimen gas chromatogram；

Fig. 4 is the gas chromatogram of lipase-catalyzed N- acetyl-DL- methionine methyl ester hydrolysis 10min.

(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 chemical synthesis substrate N- acetyl-DL- methionine methyl ester of embodiment

5g N- acetyl-DL- methionine and 5mL methanol (analysis is pure, and methanol is excessive) is taken to be put into 250mL round-bottomed flask, 20mL toluene is added as reaction dissolvent, adds the concentrated sulfuric acid (mass concentration 98%) of 125uL as catalyst.Pass through oil bath At 80 DEG C of magnetic stirring apparatus, 6-8h is reacted under the conditions of 600rpm.The reaction solution obtained after reaction, first with saturation Na₂CO₃It washes It washs, removes unreacted acid therein, then extracted with isometric ethyl acetate, separatory funnel separates organic phase and water Phase, water phase are extracted with ethyl acetate twice, merge organic phase, then twice through pure water, and saturation NaCl is washed twice, and will wash The organic phase obtained afterwards is dried with anhydrous magnesium sulfate, after removing water, then organic phase is rotated to dry, is obtained more pure production Object N- acetyl-DL- methionine methyl ester (as shown in Figure 1), yield 80%.

2 enzymatic of embodiment splits the lipase screening of N- acetyl-DL- methionine methyl ester

Weighing the separate sources screened in 0.01g table 1 has the wet thallus of freeze-drying of lipase hydrolysis activated protein Bacterium powder is added 1mL PB (pH 7.0,0.2mM) and is used as reaction dissolvent, 0.01g substrate N- second is then added in 2mL EP pipe Acyl-DL- methionine methyl ester is placed in 35 DEG C, reacts 10min in 800rpm constant temperature blending instrument so that thallus is not added as blank control. After reaction, reaction solution is acidified through 2mM HCl, adds 1mL ethyl acetate, and turbula shaker vibrates 2min, sufficiently extracts, It is centrifuged (1200rpm, 3min), obtains organic phase.Take three-dimensional selection of the 700 μ L ethyl acetate layers by gas chromatographic detection thallus Property and enzymatic hydrolysis activity, the results are shown in Table 1, screen obtain substrate enantiomeric excess value > 99% and conversion ratio reach 51.2% microbial strains.Final choice be originated from thiophene anthropi (Ochrobactrum thiophenivorans, GenBank:WP_094505884.1 protein sequence), by obtaining gene after escherichia expression system codon optimization Nucleotide sequence shown in sequence SEQ ID NO.2, coding protein amino acid sequence are shown in SEQ ID NO.1.The segment is connected It is connected on pET28b carrier and obtains cloning vector pET28b-lip and converted in Escherichia coli Escherichia coli In BL21, recombination bacillus coli is obtained, e. coli bl21 (F1) is denoted as.To recombination plasmid order-checking, and using software to sequencing As a result it is analyzed, which contains the open reading frame (SEQ ID NO.2) of an a length of 819bp.

The screening of table 1 has the lipase of hydrolysing activity

Lipase	Conversion ratio (%)	Ee value (%)	Protein series source
				L1	56.6	0	Aspergillus oryzae IF04202
L2	62.7	0	Aspergillus oryzae IF04202
				F1	51.2	>99	Ochrobactrum thiophenivorans
F2	100	0	Rhodococcus erythropolis
				F3	100	0	Ochrobactrum anthropi
A2	0	0	Aspergillus luchuensis
				A3	0	0	Penicillium subrubescens
M1	0	0	Aspergillus flavus NRRL3357
				M2	63.5	0	Aspergillus oryzae 3.042
M4	0	0	Aspergillus oryzae RIB40

Specific gas phase analysis condition: Agilent6890 gas chromatograph, BGB-174 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 3min) It is warming up to 200 DEG C (constant temperature keeps 2min), 5 DEG C/min of heating rate, 250 DEG C of injector temperature, 250 DEG C of detector temperature, air Flow and hydrogen flowing quantity are respectively 300mL/min and 40mL/min.Carrier gas is high-purity N₂, stigma pressure 93.5Kpa；Make-up gas flow 25.0mL·min^-1；Split ratio 50:1, sampling volume 1uL.As shown in the result of GC, N- acetyl group-l-methionine methyl esters It is respectively 23.9min and 24.0min (as shown in Figure 3) with N- acetyl group-D-Met methyl esters retention time.

3 Lipase protein inductive condition of embodiment

The e. coli bl21 (F1) that embodiment 2 obtains is seeded in LB culture medium, 37 DEG C of culture OD₆₀₀It is (big to 0.5 General culture 2h), add IPTG to final concentration 0.02mM, 30 DEG C of culture 10-12h.300mL bacterium solution 8000rpm, 4 DEG C of centrifugation 10min, Thallus is collected, then with PBS buffer solution washing thalline 2 times, 8000rpm, 10min, collects wet thallus.The wet thallus of collection is frozen Dry machine is lyophilized to obtain the thick enzyme powder of lipase F1, is put in 4 DEG C of refrigerators and saves.LB culture medium composition: tryptone 10g/L, yeast powder 5g/L, NaCl 5g/L, solvent are water, and pH is natural.

The effect of the different lipase-catalyzed N- acetyl-DL- methionine methyl ester Hydrolysis Resolutions of embodiment 4 compares

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the difference fat of final concentration 10g/L is added Enzyme (embodiment 2 prepare the thick enzyme powder of lipase F1, Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Lipase PS IM), 0.01g N- acetyl-DL- methionine methyl ester reacts under the conditions of 800rpm in 35 DEG C of constant temperature blending instrument 10min is extracted reaction solution and is detected N- acetyl-l-methionine methyl esters enantiomeric excess value and conversion ratio, knot by 1 method of embodiment Fruit is shown in Table 2.The results show that lipase 435, TL IM, RM IM and PS IM do not have substrate after 35 DEG C of reaction 10min There is apparent Hydrolysis Resolution activity, and when lipase F1 is catalyzed and reacts, product N- acetyl-l-methionine methyl esters ee value > 99%, conversion ratio is 51.2% (as shown in Figure 4).

The Hydrolysis Resolution effect of the different lipase-catalyzed N- acetyl-DL- methionine methyl esters of table 2 compares

Influence of 5 reaction time of embodiment to enzyme kinetics Hydrolysis Resolution N- acetyl-DL- methionine methyl ester

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the embodiment 3 of final concentration 10g/L is added The thick enzyme powder of lipase F1 of preparation, 0.02g N- acetyl-DL- methionine methyl ester, in 35 DEG C of constant temperature blending instrument, 800rpm condition Lower reaction different time (2min~60min) extracts reaction solution and detects N- acetyl-l-methionine methyl esters by 2 method of embodiment Enantiomeric excess value and conversion ratio, the results are shown in Table shown in 3.

The result shows that product N- acetyl-l-methionine methyl esters enantiomeric excess value has reached most after reaction 10min Height, enantiomeric excess value > 99%, conversion ratio 51.2%, when reacted between be greater than 10min, product N- acetyl-l-methionine The enantiomeric excess value of methyl esters is basically unchanged, and conversion ratio is increasing.

Influence of 3 reaction time of table to enzymic catalytic reaction

Influence of 6 reaction temperature of embodiment to enzyme kinetics Hydrolysis Resolution N- acetyl-DL- methionine methyl ester

In pH 7.0, the Na of 0.2mM₂HPO₄/NaH₂PO₄In buffer solution 1mL, the embodiment 3 of final concentration 10g/L is added The thick enzyme powder of lipase F1 of preparation, 0.02gN- acetyl-DL- methionine methyl ester, in constant temperature blending instrument different temperatures (25-45 DEG C), same time 10min is reacted under the conditions of 800rpm, is extracted reaction solution and is detected N- acetyl-l-methionine by 2 method of embodiment The enantiomeric excess value and conversion ratio of methyl esters, the results are shown in Table shown in 4.

As a result illustrate, when reaction temperature is 35 DEG C, N- acetyl-l-methionine methyl esters enantiomeric excess value highest, Its ee value > 99%.When reaction temperature is higher than 35 DEG C or is lower than 35 DEG C, N- acetyl-l-methionine methyl esters enantiomer mistake Magnitude can all decline, and illustrate that temperature has a very big impact the optical selective of lipase F1.

Influence of 4 reaction temperature of table to reaction

7 product N- acetyl of embodiment-l-methionine methyl esters separation and Extraction

20mL pH 7.0 is added in 50mL round-bottomed flask, 0.2mM PB buffer solution weighs the preparation of 0.2g embodiment 3 The thick enzyme powder of lipase F1, add the N- acetyl-DL- methionine methyl ester of final concentration 10g/L, flowed with 50mM NaOH Add drop reaction, control reaction pH maintains 7.0, at 35 DEG C of magnetic stirring apparatus, 2h is reacted under the conditions of 600rpm.After reaction Obtained reaction solution is acidified pH to 2.0 with 4mM HCl, is then extracted with isometric ethyl acetate, separatory funnel separation Organic phase out, water phase are extracted with ethyl acetate again, merge organic phase, twice through pure water by organic phase, saturation NaCl washing Twice, the organic phase obtained after washing is dried with anhydrous magnesium sulfate, after removing water, then organic phase is rotated to dry, is obtained It to final product, and weighs, product N- acetyl-l-methionine methyl esters yield reaches 92.3%, purity 100%.

Sequence table

<110>Zhejiang Polytechnical University

<120>a kind of lipase is splitting the application in N- acetyl-DL- methionine methyl ester

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 272

<212> PRT

<213>unknown (Unknown)

<400> 1

Met Thr Ile Asn Tyr His Glu Leu Glu Thr Ser His Gly Arg Ile Ala

1 5 10 15

Val Arg Glu Ser Glu Gly Glu Gly Ala Pro Leu Leu Met Ile His Gly

20 25 30

Asn Ser Ser Ser Gly Ala Val Phe Ala Pro Gln Leu Glu Gly Glu Ile

35 40 45

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

50 55 60

Ser Ser Asp Ala Ile Asp Pro Glu His Ser Tyr Ser Met Glu Gly Tyr

65 70 75 80

Ala Asp Ala Met Thr Glu Val Met Gln Lys Leu Gly Ile Ala Asp Ala

85 90 95

Val Val Phe Gly Trp Ser Leu Gly Gly His Ile Gly Ile Glu Met Ile

100 105 110

Ala Arg Tyr Pro Glu Met Arg Gly Leu Met Ile Thr Gly Thr Pro Pro

115 120 125

Val Ala Arg Glu Glu Val Gly Gln Gly Phe Lys Ser Gly Pro Asp Met

130 135 140

Ala Leu Ala Gly Gln Glu Val Phe Ser Glu Arg Asp Val Asp Ser Tyr

145 150 155 160

Ala Arg Ser Thr Cys Gly Glu Pro Phe Glu Ala Ser Leu Leu Asp Ile

165 170 175

Val Ala Arg Thr Asp Gly Arg Ala Arg Arg Ile Met Phe Glu Lys Phe

180 185 190

Gly Asn Gly Thr Gly Gly Asn Gln Arg Asp Ile Val Ala Gln Ala Lys

195 200 205

Leu Pro Ile Ala Val Val Asn Gly Arg Asp Glu Pro Phe Val Glu Leu

210 215 220

Asp Phe Val Ser Lys Val Lys Phe Gly Asn Leu Trp Glu Gly Lys Thr

225 230 235 240

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

245 250 255

Ile Phe Asp Arg Tyr Leu Met Arg Phe Leu Ser Asp Cys Thr Ile Gly

260 265 270

<210> 2

<211> 830

<212> DNA

<213>unknown (Unknown)

<400> 2

ccatgggcat gaccatcaac tatcacgaac tggagacctc tcatggtcgc attgccgtgc 60

gcgaaagcga aggtgaaggt gccccgctgc tgatgattca tggcaacagc agcagcggtg 120

ccgtgtttgc accgcagctg gagggcgaga tcggcaagaa atggcgtgtg attagcccgg 180

atttaccggg tcatggcaaa agcagcgatg ccattgaccc ggaacatagc tacagcatgg 240

aaggctatgc cgatgccatg accgaagtga tgcagaagct gggcattgcc gatgccgtgg 300

tgtttggttg gtctttaggc ggtcatattg gcatcgaaat gatcgcccgc tatccggaaa 360

tgcgcggttt aatgattacc ggtaccccgc cggttgcccg tgaagaagtt ggccaaggtt 420

ttaaaagcgg cccggatatg gcactggccg gtcaagaagt gttcagcgag cgcgatgtgg 480

atagttatgc ccgcagcact tgtggtgaac cgttcgaagc ctctttactg gatattgttg 540

cacgcaccga tggtcgtgca cgccgcatca tgttcgaaaa gtttggcaac ggcaccggtg 600

gcaatcagcg tgacattgtg gcccaagcta aactgccgat cgccgttgtg aatggtcgcg 660

atgagccgtt tgttgagctg gacttcgtga gcaaggtgaa gttcggcaat ctgtgggagg 720

gcaagaccca cgtgattgat aatgccggtc atgccccgtt tcgtgaaaca ccggccattt 780

tcgatcgcta tttaatgcgc tttctgagcg attgcaccat tggtctcgag 830

Claims

1. a kind of lipase is splitting the application in N- acetyl-DL- methionine methyl ester, 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 such as SEQ ID of the fat enzyme coding gene Shown in 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 wet thallus or wet thallus freeze-dried powder that the fermented culture of journey bacterium obtains are catalyst, using N- acetyl-DL- methionine methyl ester the bottom of as Object carries out resolution reaction, fully reacting under the conditions of 25-45 DEG C, 600-800rpm using 7.0 buffer of pH as reaction medium Afterwards, reaction solution is isolated and purified, obtains N- acetyl-l-methionine methyl esters.

4. application as claimed in claim 3, it is characterised in that the catalyst amount is calculated as 10g/L with buffer volume, institute It states Final substrate concentrations and 5-20g/L is calculated as with buffer volume.

5. application as claimed in claim 3, it is characterised in that reaction time 2-60min.

6. application as claimed in claim 3, it is characterised in that the reaction condition is 35 DEG C, 800rpm reacts 10min.

7. application as claimed in claim 3, it is characterised in that the buffer is the Na of pH 7.0,0.2mM₂HPO₄/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 wet thallus, and freeze-drying obtains wet thallus freeze-dried powder.

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 pH 2.0 with 4mM HCl, is then extracted with isometric ethyl acetate, and separatory funnel isolates organic phase, then Twice through pure water, saturation NaCl is washed twice, and the organic phase obtained after washing is dried with anhydrous magnesium sulfate, is removed After water, then organic phase rotated to dry, obtains product N- acetyl-l-methionine methyl esters.