CN106754789B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754789B
CN106754789B CN201710006577.7A CN201710006577A CN106754789B CN 106754789 B CN106754789 B CN 106754789B CN 201710006577 A CN201710006577 A CN 201710006577A CN 106754789 B CN106754789 B CN 106754789B
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alpha
ester
acid
hydroxy acid
leu
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CN106754789A (en
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蔡宇杰
沈天成
冯佳婷
白亚军
郑晓晖
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Zhuohong Chaoyuan Biotechnology Zhengzhou Co ltd
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03015—(S)-2-Hydroxy-acid oxidase (1.1.3.15)

Abstract

The present invention relates to a kind of acquisition of L- alpha-hydroxy acid oxidase gene from extension brevibacterium (Brevibacterium linens) and its clonal expressions, belong to bioengineering field.Its substrate specificity is disclosed, while the L- alpha-hydroxy acid oxidizing ferment can aoxidize (S)-alpha-hydroxy acid ester, can be applied to the preparation of optical voidness (R)-alpha-hydroxy acid ester.

Description

A kind of oxidizing ferment and its application
Technical field
A kind of L- alpha-hydroxy acid oxidizing ferment of clonal expression of the present invention, and disclose its nucleotide sequence and amino acid sequence and Zymologic property and application belong to industrial microorganism field.
Background technique
L- alpha-hydroxy acid oxidizing ferment (L- α-hydroxyacid oxidase) is a kind of dehydrogenase with FMN (FAD) for coenzyme (Aliphatic l-α-hydroxyacid oxidase from rat livers purification and Properties. Biochimica et Biophysica Acta (BBA)-Enzymology 1968,167:9-22), usually It include glycolate oxidase (glycolate oxidase) (Preparation and some properties of crystalline glycolic acid oxidase of spinach.J.Biol.Chem.1958,231(1):135–57)、 Pfansteihl oxidizing ferment (L-lactate oxidase) (Conversion of L-lactate oxidase to a long chain alpha- hydroxyacid oxidase by site-directed mutagenesis of alanine 95to glycine.J Biol Chem.1996 8；271(45):28300-28305).It can be used for measuring lactic acid in biosensor Content, or oxidation Pfansteihl produce pyruvic acid.Also there is the preparation (Chinese patent for being used for optical voidness alpha-hydroxy acid 201210109290.4)
So far, in pseudomonas putida (Pseudomonas putida), aerococcus viridans (Aerococcus viridians), streptococcus (Streptococcus sp.), Pediococcus (Pediococcus Sp.), Lactococcus lactis (Lactococus lactis), edwardsiella tarda (Edwardsiella tarda), shame dirt branch Bacillus (Mycobacterium smegmatis), zymomonas mobilis (Zymomonas mobilis) and peroxidating acetobacter Clonal expression has obtained Pfansteihl oxidizing ferment in bacteriums such as (Acetobacter peroxidans).Pfansteihl oxidizing ferment is also one It is detected in a little fungies, such as geotrichum candidum (Geotrichum candidum) and Yarrowia lipolytica (Yarrowia lipolytica).(Search for Lactate Oxidase Producer Microorganisms, Applied Biochemistry and Microbiology,2007,43(2)178–181)
The clonal expression from extension brevibacterium (Brevibacterium linens) goes out a kind of novel L- to the present invention for the first time Alpha-hydroxy acid oxidizing ferment, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, but also can aoxidize (S)-alpha-hydroxy acid ester, can be applied to optics The preparation of pure (R)-alpha-hydroxy acid ester and (R)-alpha-hydroxy acid.
Summary of the invention
Present invention clone from extension brevibacterium (Brevibacterium linens) has obtained a kind of L- alpha-hydroxy acid oxidation The gene of enzyme discloses its relevant enzymatic property, and carried out application study using colibacillus engineering heterogenous expression.
Technical scheme is as follows:
1, bacterial strain
The source bacterial strain of L- alpha-hydroxy acid oxidase gene of the present invention are as follows: Brevibacterium linens ATCC 8377, Purchased from U.S.'s ATCC strain library.
2, the clone of L- alpha-hydroxy acid oxidase gene
Extract 8377 phage gene group total DNA of Brevibacterium linens ATCC.Specific primer is designed, is answered With PCR method, alpha-hydroxy acid oxidase gene overall length encoder block sequence is amplified.And construction recombination plasmid.
3, L- alpha-hydroxy acid Oxidase Expression and purifying
Recombinant plasmid is imported in E.coli BL21 (DE3), inducing expression.Crude enzyme liquid is obtained after bacterial cell disruption, after purification It is freeze-dried spare.
4, the characterization analysis of L- alpha-hydroxy acid oxidizing ferment
Influence of the pH to L- alpha-hydroxy acid oxidizing ferment enzyme activity of the present invention is studied by substrate of Pfansteihl.
Influence of the temperature to L- alpha-hydroxy acid oxidizing ferment enzyme activity of the present invention is studied by substrate of Pfansteihl.
The substrate specificity of L- alpha-hydroxy acid oxidizing ferment is analyzed: substrate used has Pfansteihl, glycolic, L- phenyllactic acid, L- Tartaric acid, L MALIC ACID, L- para hydroxybenzene lactic acid, L- danshensu, L- mandelic acid.
Enzyme activity determination method are as follows: according to Characterization of a Lactate Oxidase from a Strain of Gram Negative Bacterium from Soil, Applied Biochemistry and Biotechnology,56, 1996,278-288.The method carries out.
5, L- alpha-hydroxy acid oxidizing ferment splits the alpha-hydroxy acid ester of mixed
The method of resolution of alpha-carboxylic esters (alpha-hydroxy esters) are as follows: take 0.1 gram of purified enzyme in 50 mL tri- In the bottle of angle, it is added dissolved in the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, is converted in 30 DEG C, 150rpm shaking bath 16h, liquid-phase chromatographic analysis supernatant after conversion.(S) Alpha-hydroxy in-alpha-hydroxy acid ester, which is dehydrogenated, is oxidized to corresponding 2-ketoacid Ester, (R)-alpha-hydroxy acid ester are not oxidized.
Product (R)-alpha-hydroxy acid ester optical purity is evaluated by enantiomeric excess value (%e.e):
Enantiomeric excess value %e.e=[(SR-SS)/(SR+SS)] × 100%
(R)-alpha-hydroxy acid ester yield (%)=(SR/S0) × 100%
S in formulaRFor the peak area of (R)-enantiomer after reaction, SSFor reaction after (S)-enantiomer liquid chromatogram peak area, S0For the sum of the liquid chromatogram peak area of (R)-and (S)-enantiomer before reaction.
Product measures liquid phase chromatogram condition are as follows: Chiralcel OD-H chiral column (4.6 × 250mm), mobile phase volume ratio For n-hexane: isopropanol: trifluoroacetic acid=80:20:0.1, flow velocity 0.5mL/min, 25 DEG C of column temperature, Detection wavelength 210nm, 20 μ L of sample volume.
The alpha-hydroxy acid ester is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, benzene cream Isopropyl propionate, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene isopropyl lactate, mandelic acid norbornene ester, almond isopropyl propionate, Radix Salviae Miltiorrhizae Plain asarum alcohol ester, lactic acid norbornene ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
The alpha-hydroxy acid ester, according to Chinese patent 200610042787.3,201410180490.8, 201410175950.8 the method synthesis announced with 20140699506.6.
Originally deliver bright usefulness: clone has obtained one kind from Brevibacterium linens ATCC 8377 L- alpha-hydroxy acid oxidizing ferment, the enzyme can aoxidize (S)-alpha-hydroxy acid and (S)-alpha-hydroxy acid ester, can be used for prepare with scale chiral purity (R)-alpha-hydroxy acid ester has important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of L- alpha-hydroxy acid oxidase gene of the present invention and colibacillus engineering construct.
1, the extraction of Brevibacterium linens ATCC 8377DNA
8377 bacterial strain of Brevibacterium linens ATCC is cultivated into 12h, 12,000 rmp/ in LB culture medium Min centrifugation 10min obtains thallus, operates using bacterial genomes DNA extraction agent box (TaKaRa company) according to it and extracts bacterium Body genome DNA, it is spare to put refrigerator.
2, prepared by E. coli competent
(1) inoculation E.coli DH5 α and BL21 (DE3) is respectively in the 250mL shaking flask containing 20mL LB culture medium, and 37 DEG C, 200rpm/min overnight incubation.
(2) it is inoculated in 50mL LB culture medium by 1% inoculum concentration, 37 DEG C of cultures to OD600About 0.6 (about 2~3h).
(3) bacterium solution is transferred in the centrifuge tube of 50mL pre-cooling, places 30min, 8000rpm/min, 4 DEG C of centrifugations on ice 5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl of 5mL pre-cooling is added2Solution makes thallus suspend, and places 20min on ice, 8000rpm/min, 4 DEG C of centrifugation 5min.It is repeated 2 times.
(5) supernatant is abandoned, the 0.1mol/L CaCl of 1.5mL pre-cooling is added2Solution (contains 15% glycerol), gently suspension thalline, Then the packing of 100 μ L bacterium solutions is added by each centrifuge tube (1.5mL), -70 DEG C of Storage in refrigerator are spare.
3, the clone of L- alpha-hydroxy acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCATGAAACGTCGACTGCCTGATCTC 3'
Primer 2: 5'GCCGTCTAGAGAGTCGGCCGGCAGCTCC 3'
(2) PCR amplification
With two primers synthesized above, the genomic DNA with Brevibacterium linens ATCC 8377 is Template carries out PCR amplification.
Amplification system in this step are as follows:
Amplification program are as follows:
98 DEG C, 10min
98 DEG C, 10sec；55 DEG C, 15sec；72 DEG C, 2min reacts 30 circulations
72 DEG C, 10min
PCR product obtains this gene order after sending Hua Da gene sequencing, as shown in SEQ ID NO:1.According to the gene The amino acid sequence that sequence obtains is as shown in SEQ ID NO:2.
(3) double digestion and connection
II plasmid of pCold and PCR product are subjected to double digestion, digestion system are as follows: 10 × cut buffer, 3 μ l, DNA 4 Each 0.5 μ l of μ l, enzyme BamHI and XbaI, 2 μ l of sterile water totally 30 μ l.Double digestion 1h under 37 DEG C of water-baths.DNA fragmentation is cloned into On II carrier of pCold, and it is transformed into E.coli DH5 α competent cell.Linked system: 10 × DNA ligase buffer 2.5 μ l, 8 μ l of DNA fragmentation, 2 μ l, T4DNA ligase of carrier DNA 1 μ l, 11.5 μ l of sterile water totally 25 μ l.Under 16 DEG C of water-baths Connect 12h-16h.
(4) it converts
Step:
1 is added 100 μ l DH5 α competent bacterias in linked system, light to mix, ice bath 30min.
2 are put into 42 DEG C of water-baths of preheating, place 90s and carry out heat shock processing.
3 ice bath 2min immediately.
4 are added the not antibiotic LB culture solution of 1ml, and 37 DEG C of culture 1h make thallus recover.
5 are uniformly coated on thallus on antibiotic LB plate.
6 cultures are grown fine for 24 hours.It chooses single colonie and carries out bacterium colony PCR, recombinant plasmid is extracted in nucleic acid electrophoresis verifying.It will recombination Plasmid imports in BL21 E. coli competent, saves backup.
Embodiment 2
The present embodiment is the inducing expression of L- alpha-hydroxy acid oxidizing ferment of the present invention and isolates and purifies.
1, plus 500 μ l recombination bacterium solution is into 50ml LB culture solution.37 DEG C of culture 2.5h stand 0.5h at 15 DEG C.Again plus 20 The IPTG of μ l 0.5M, cold-induction culture is for 24 hours at 15 DEG C.Fermentation liquid is centrifuged (8000rmp/min, 10min) and obtains bacterium Body redissolves thallus with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (20mmol/L, pH 7.0), and Ultrasonic Cell Disruptor is broken, Centrifugation (8000rmp/min, 10 min) collects supernatant and obtains crude enzyme liquid.
2, the crude enzyme liquid for obtaining step 1 carries out ni-sepharose purification using the operation of 150 protein purification system of AKTA avant, Elution process are as follows: all put the tetra- root canal road A1, A2, B1, B2 into water, system flow 20ml/min flow velocity is set, carry out Exhaust.Then system flow 1ml/min, flow path (column position 3), delta pressure are set 0.3, pre-pressure 0.5, Gradient 0, inset A1, fill pillar after water droplet uniformly flows out, balance ten minutes it A1 is put into conjunction in liquid afterwards, B1 is put into eluent, then primary, balance 20 minutes is exhausted, then loading crude enzyme liquid, With high concentration imidazole buffer (solution locating for B1) gradient elution destination protein of 500mM, the albumen that will be adsorbed on ion column Elute the enzyme purified.Enzyme after purification is freeze-dried spare.
Embodiment 3
The present embodiment is the optimum temperature of L- alpha-hydroxy acid oxidizing ferment of the present invention.Using Pfansteihl as substrate, by substrate with The phosphate buffer that pH is 6.0 is lauched bath 15min in 30-60 DEG C of different temperature condition, measures the enzyme of L- alpha-hydroxy acid oxidizing ferment It is living, determine that the optimal reactive temperature of enzyme is 40 DEG C.
Embodiment 4
The present embodiment is the optimum pH of L- alpha-hydroxy acid oxidizing ferment of the present invention.Using Pfansteihl as substrate, substrate is existed PH 3-9, the enzyme activity of 40 DEG C of water-bath 15min measurement enzymes, as a result, it has been found that L- alpha-hydroxy acid oxidizing ferment enzyme activity is most under the conditions of 6.0 pH It is high.
Embodiment 5
The present embodiment is that L- alpha-hydroxy acid oxidizing ferment of the present invention is listed in table 1 from the response characteristic of different substrates.
Activity of the 1 L- alpha-hydroxy acid oxidizing ferment of table to different substrates
Embodiment 6
Various racemic ' alpha '-carboxylic esters are split according to the method in summary of the invention, as a result as shown in the table:
Table 2 splits the effect of various racemic ' alpha '-carboxylic esters
As can be seen from the above table, when the reaction time is abundant, available all kinds of optically pure (R)-α-hydroxy acids of height The optics specificity of ester, the enzyme is very good.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of oxidizing ferment and its application
<130> No
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1254
<212> DNA
<213> Brevibacterium linens ATCC 8377
<400> 1
atgaaacgtc gactgcctga tctcaaagag cttgccccct tgctgcagtt cgacctgcct 60
cgcctcgacc gggtcgccgc caggctggaa gcggccgcag acatctggga cctccgccgc 120
atcgccaagc gcgtgactcc tacagccccg ttcgactatg tcgacggtgc cgcactggac 180
gagcggacat tggcgaagaa ccgggacgct ctgggcaacg tcgagcttct tcctcgcatc 240
ctccacggag tcgacgcccc ggatacgtcg acgacgatcg ccggcgcccg tgttcgactg 300
cccttcggca tcgctccgac cggttacaca cgcatgatgc actccgaagg cgaagtcgcc 360
ggagtgcgtg cggccgcgaa ggccggaatc ccgttctcgc tgtcgacgat gggaacgacc 420
tcggtcgaag acgtcgcgca ggcggcaccg gattccaccc ggtggttcca gctctacctg 480
tggaaggacc gacagcgcag cctcgacctg atccagcgag ccgccgccag cggatacaag 540
actctgctgg tcaccgtcga caccccgatc actggccagc gtctgcgcga tcaccgcaac 600
ggtctgacga tcccgccccg gctgacgctg ggaacgattc tcgacgcctc ctaccggccc 660
ggctggtggt tcaacttcct caccaccgaa ccgccgaagt acgcgtcact gtcgaacacc 720
tctcagtccc tggcggagat gacgcagacg atgttcgacc cgacccttga cctcgaggac 780
ctgcggtgga tccgcgaaca gtggaacgga cgactgttcg tcaagggcat tctcaccgcc 840
gacgatgccc gacgtgccca atccgtcgga gccgatgggc tcgtcgtgtc caaccacggc 900
ggtcgccaac tcgatcgggc cccggactcg ctgacctcac tggccgaagt ccgcgcggag 960
gtcgggccgg agatggagct gatcttcgac tccgggatca tgtccggcac cgatgtcgtc 1020
gccgcgctgt gtgccggagc ggacttcgtg ctcatcggtc gggcgtatct atacgggctc 1080
atggccggcg gtcagcgagg cgtcgagagg gccatcgcac tcatccaaca ggagatcctc 1140
accgcgatgg ggctcatggg tgcccgcagc atctctgatc tcggccccga actcgttcgt 1200
ggcctgcccc aggcaccgaa cacagatcag gagctgccgg ccgactccat gtga 1254
<210> 2
<211> 417
<212> PRT
<213> Brevibacterium linens ATCC 8377
<400> 2
Met Lys Arg Arg Leu Pro Asp Leu Lys Glu Leu Ala Pro Leu Leu Gln
1 5 10 15
Phe Asp Leu Pro Arg Leu Asp Arg Val Ala Ala Arg Leu Glu Ala Ala
20 25 30
Ala Asp Ile Trp Asp Leu Arg Arg Ile Ala Lys Arg Val Thr Pro Thr
35 40 45
Ala Pro Phe Asp Tyr Val Asp Gly Ala Ala Leu Asp Glu Arg Thr Leu
50 55 60
Ala Lys Asn Arg Asp Ala Leu Gly Asn Val Glu Leu Leu Pro Arg Ile
65 70 75 80
Leu His Gly Val Asp Ala Pro Asp Thr Ser Thr Thr Ile Ala Gly Ala
85 90 95
Arg Val Arg Leu Pro Phe Gly Ile Ala Pro Thr Gly Tyr Thr Arg Met
100 105 110
Met His Ser Glu Gly Glu Val Ala Gly Val Arg Ala Ala Ala Lys Ala
115 120 125
Gly Ile Pro Phe Ser Leu Ser Thr Met Gly Thr Thr Ser Val Glu Asp
130 135 140
Val Ala Gln Ala Ala Pro Asp Ser Thr Arg Trp Phe Gln Leu Tyr Leu
145 150 155 160
Trp Lys Asp Arg Gln Arg Ser Leu Asp Leu Ile Gln Arg Ala Ala Ala
165 170 175
Ser Gly Tyr Lys Thr Leu Leu Val Thr Val Asp Thr Pro Ile Thr Gly
180 185 190
Gln Arg Leu Arg Asp His Arg Asn Gly Leu Thr Ile Pro Pro Arg Leu
195 200 205
Thr Leu Gly Thr Ile Leu Asp Ala Ser Tyr Arg Pro Gly Trp Trp Phe
210 215 220
Asn Phe Leu Thr Thr Glu Pro Pro Lys Tyr Ala Ser Leu Ser Asn Thr
225 230 235 240
Ser Gln Ser Leu Ala Glu Met Thr Gln Thr Met Phe Asp Pro Thr Leu
245 250 255
Asp Leu Glu Asp Leu Arg Trp Ile Arg Glu Gln Trp Asn Gly Arg Leu
260 265 270
Phe Val Lys Gly Ile Leu Thr Ala Asp Asp Ala Arg Arg Ala Gln Ser
275 280 285
Val Gly Ala Asp Gly Leu Val Val Ser Asn His Gly Gly Arg Gln Leu
290 295 300
Asp Arg Ala Pro Asp Ser Leu Thr Ser Leu Ala Glu Val Arg Ala Glu
305 310 315 320
Val Gly Pro Glu Met Glu Leu Ile Phe Asp Ser Gly Ile Met Ser Gly
325 330 335
Thr Asp Val Val Ala Ala Leu Cys Ala Gly Ala Asp Phe Val Leu Ile
340 345 350
Gly Arg Ala Tyr Leu Tyr Gly Leu Met Ala Gly Gly Gln Arg Gly Val
355 360 365
Glu Arg Ala Ile Ala Leu Ile Gln Gln Glu Ile Leu Thr Ala Met Gly
370 375 380
Leu Met Gly Ala Arg Ser Ile Ser Asp Leu Gly Pro Glu Leu Val Arg
385 390 395 400
Gly Leu Pro Gln Ala Pro Asn Thr Asp Gln Glu Leu Pro Ala Asp Ser
405 410 415
Met

Claims (2)

1. a kind of method of resolution of alpha-carboxylic esters (alpha-hydroxy esters), which is characterized in that the method are as follows: take pure 0.1 gram of the enzyme changed is added in 50mL triangular flask dissolved in the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, in 30 DEG C, 16h is converted in 150rpm shaking bath, liquid-phase chromatographic analysis supernatant after conversion；The enzyme is from extension brevibacterium The L- alpha-hydroxy acid oxidizing ferment of (Brevibacterium linens), amino acid sequence are shown in SEQ ID NO:2；Described Alpha-hydroxy acid ester is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, to hydroxyl Phenyllactic acid norbornene ester, para hydroxybenzene isopropyl lactate, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu are thin Octanol ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
2. the method according to claim 1, wherein the nucleotides sequence of the L- alpha-hydroxy acid oxidizing ferment is classified as SEQ Shown in ID NO:1.
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