CN106754788B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754788B
CN106754788B CN201710006572.4A CN201710006572A CN106754788B CN 106754788 B CN106754788 B CN 106754788B CN 201710006572 A CN201710006572 A CN 201710006572A CN 106754788 B CN106754788 B CN 106754788B
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CN106754788A (en
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蔡宇杰
王亚红
曹憬
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)

Abstract

The present invention relates to it is a kind of from morganella morganii rub root subspecies (Morganella morganii subsp.morganii) D-ALPHA-Hydroxypropionic acid oxidase gene acquisition and its clonal expression, belong to bioengineering field.Its substrate specificity is disclosed, while the D-ALPHA-Hydroxypropionic acid oxidizing ferment can aoxidize (R)-alpha-hydroxy acid ester, can be applied to the preparation of optical voidness (S)-alpha-hydroxy acid ester.

Description

A kind of oxidizing ferment and its application
Technical field
A kind of D-ALPHA-Hydroxypropionic acid oxidizing ferment of clonal expression of the present invention, and disclose its nucleotide sequence and amino acid sequence and enzyme Property and application are learned, industrial microorganism field is belonged to.
Background technique
D-ALPHA-Hydroxypropionic acid oxidizing ferment (D-lactate oxidase) is a kind of alpha-hydroxy acid oxidizing ferment with FAD (FMN) for coenzyme (being traditionally referred to as D-ALPHA-Hydroxypropionic acid oxidizing ferment).D-ALPHA-Hydroxypropionic acid oxidizing ferment can be used in biosensor measuring the content of lactic acid, or It aoxidizes D-ALPHA-Hydroxypropionic acid and produces pyruvic acid.Also there is the preparation (Chinese patent 201210109290.4) for being used for optical voidness alpha-hydroxy acid
So far, in edwardsiella tarda (Edwardsiella tarda) and zymomonas mobilis D-ALPHA-Hydroxypropionic acid oxidizing ferment is had found in (Zymomonas mobilis) etc..(Kalnenieks U,Galinina N, Bringer- Meyer S,et al.Membrane D-lactate oxidase in Zymomonas mobilis: evidence for a branched respiratory chain[J].FEMS microbiology letters,1998, 168(1):91-97)
The present invention rubs in root subspecies (Morganella morganii subsp.morganii) from morganella morganii for the first time Clonal expression goes out a kind of novel D-ALPHA-Hydroxypropionic acid oxidizing ferment, which can not only aoxidize (R)-alpha-hydroxy acid, but also can aoxidize (R)- Alpha-hydroxy acid ester, reaction back reaction compared with the reaction that the lactic dehydrogenase that NAD (NADP) is coenzyme participates in is atomic weak, can apply In the preparation of optical voidness (S)-alpha-hydroxy acid ester and (S)-alpha-hydroxy acid.
Summary of the invention
The present invention rubs from morganella morganii, and root subspecies (Morganella morganii subsp.morganii) are middle to clone It has obtained a kind of disclosing it using colibacillus engineering heterogenous expression by the gene of the D-ALPHA-Hydroxypropionic acid oxidizing ferment of coenzyme of FAD Relevant enzymatic property, and carried out application study.
Technical scheme is as follows:
1, bacterial strain
The source bacterial strain of D-ALPHA-Hydroxypropionic acid oxidase gene of the present invention are as follows: Morganella morganii subsp. Morganii ATCC 258297 is purchased from U.S. ATCC strain library.
2, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract 258297 phage gene group total DNA of Morganella morganii subsp.morganii ATCC.If It counts specific primer and amplifies D-ALPHA-Hydroxypropionic acid oxidase gene overall length encoder block sequence using PCR method.And construction recombination plasmid.
3, D-ALPHA-Hydroxypropionic 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment
Influence of the pH to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention is studied by substrate of D-ALPHA-Hydroxypropionic acid.
Influence of the temperature to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention is studied by substrate of D-ALPHA-Hydroxypropionic acid.
The substrate specificity of D-ALPHA-Hydroxypropionic acid oxidizing ferment is analyzed: substrate used has D-ALPHA-Hydroxypropionic acid, glycolic, D- phenyllactic acid, D- pairs Hydroxyphenyl lactic acid, D- tartaric acid, D-malic acid, D- mandelic acid, D- danshensu.
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, D-ALPHA-Hydroxypropionic 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.(R) Alpha-hydroxy in-alpha-hydroxy acid ester, which is dehydrogenated, is oxidized to corresponding 2-ketoacid Ester, (S)-alpha-hydroxy acid ester are not oxidized.
Product (S)-alpha-hydroxy acid ester optical purity is evaluated by enantiomeric excess value (%e.e):
Enantiomeric excess value %e.e=[(SS-SR)/(SS+SR)] × 100%
(S)-alpha-hydroxy acid ester yield (%)=(SS/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 bright usefulness is delivered: from Morganella morganii subsp.morganii ATCC 258297 Clonal expression goes out a kind of novel D-ALPHA-Hydroxypropionic acid oxidizing ferment, which can aoxidize (R)-alpha-hydroxy acid and (R)-alpha-hydroxy acid ester, can be used for Prepare with scale chiral purity (S)-alpha-hydroxy acid ester has important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of D-ALPHA-Hydroxypropionic acid oxidase gene of the present invention and colibacillus engineering construct.
1, the extraction of Morganella morganii subsp.morganii ATCC 258297DNA
258297 bacterial strain of Morganella morganii subsp.morganii ATCC is cultivated in LB culture medium 12h, 12,000rmp/min centrifugation 10min obtain thallus, press using bacterial genomes DNA extraction agent box (TaKaRa company) It is operated according to it and extracts phage gene group total 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 D-ALPHA-Hydroxypropionic acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCATGATCAACAATCAACAACCTGACG 3'
Primer 2: 5'GCCGTCTAGATTACTGATTATACTTTCCGCAGCCA 3'
(2) PCR amplification
With two primers synthesized above, with Morganella morganii subsp.morganii ATCC 258297 Genomic DNA be template carry 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 the gene order of the enzyme after sending Hua Da gene sequencing, as shown in SEQ ID NO:1.According to the base The amino acid sequence obtained by sequence 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 D-ALPHA-Hydroxypropionic 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.Using D-ALPHA-Hydroxypropionic acid as substrate, by substrate and pH It is lauched bath 15min in 30-60 DEG C of different temperature condition for 6.0 phosphate buffer, measures the enzyme activity of D-ALPHA-Hydroxypropionic acid oxidizing ferment, The optimal reactive temperature for determining enzyme is 45 DEG C.
Embodiment 4
The present embodiment is the optimum pH of D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.Using D-ALPHA-Hydroxypropionic acid as substrate, by substrate in pH 3-9, the enzyme activity of 45 DEG C of water-bath 15min measurement enzymes, as a result, it has been found that D- lactate oxidase enzyme activity highest under the conditions of 6.0 pH.
Embodiment 5
The present embodiment is the response characteristic of D-ALPHA-Hydroxypropionic acid oxidizing ferment and different substrates of the present invention, is listed in table 1.
Activity of the table 1D-lactate oxidase 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 (S)-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> 1746
<212> DNA
<213> Morganella morganii subsp. morganii ATCC 258297
<400> 1
atgatcaaca atcaacaacc tgacggcgca cgtcttattc aggcactgac agatattgtc 60
agtcacaaac acattttaac agagccgcgt aagacggagc gataccgcaa aggcttccgt 120
tccggagaag gcagcgcgct ggcggtggtg ttcccgggca cgctgtatga actgtggcag 180
gtgtttaaag ccgctgttga agctgacaga attgtgatca tgcaggcggc caataccggt 240
ctgaccgaag gctccacccc gagcggggat gattatgacc gcgaaattat tattatcagc 300
acactgcgcc tggataaaat tcaggtactg acaaaacagc agcaggtagt ggcactgccg 360
ggcagcaccc tgtggcacct ggaaaaagtc ctgaaaccgc tgggtcgtga gccgcattca 420
gtgatcggtt cttcctgtat cggggcttcc gtggtcggcg ggatttgtaa taactccggc 480
ggttcactgg tgcagcgcgg tcccgcttat accgaactgg cgctgtacgg ccgcgttaat 540
gcggacggca gcgcagaact ggttaaccat ctggggattg atttaggatc cacgccggaa 600
gaaattctga attgtctgga ataccgtcag tatcaggatg cggatattga ggacacggat 660
aaaccggcct ctgaccatga ttatcatcag cgtatccgtg atgtggatgc ggacacccca 720
tcccgtttca acgccgacga acgccgcctg tttgaagcct ccggctgtgc cggtaagctg 780
gtggtgtttg cagtgcgcct tgataccttc ccgtctcagg gagagagcaa agttttctat 840
atcggcacca atacgccttc tgtgctggaa gatatccgcc gtcatattct tgcggagttt 900
gagcatctgc cggtggccgg tgaatatatg caccgcgaat gttatgacat cgcaaaggtc 960
tacggcaaag acagcttcct gatgattgat aaactcggca ctgagaaaat gccgaagctg 1020
ttcactatca aagggcggat ggatgctgtt ttcaataaaa tcccgctgct gccgaaaaat 1080
ctgattgacc gcacaatgca gttattcagt catctgtggc cgaaccatct gccgcgccgg 1140
atggaagagt accgcgataa atatgagcat cacctgatgc tgaaaatggc cggagagggg 1200
attgatgaag cccgtaactg gctgagcact ttctttgaca atgcagaggg tgctttcttt 1260
gagtgtgatg ccaaagaggg ggcggatgct ttcctgcacc gctttgtggc cgccggtgcc 1320
gccatccgct atcacgcggt caacagcagc aaagtggaag atatcctcgc actggatatt 1380
gcactgcgcc gtaatgacca ggcgtggctg gaagtcctgc cgccggaaat tgagagcaaa 1440
ctggtgcata aactctacta cggacatttt ttatgccacg ttttccatca ggactatatt 1500
gtgaagaaag gcgtggatcc gcatgcgctg aaagaggaaa tgctggaaat cctgaacacg 1560
cgcggggcgg aatatccggc agagcataat gtcggccatc tgtataaagc gaaacctcag 1620
ctgaaagcct tctataaagc caccgatccg accaacaccc tgaacccggg actgggcaaa 1680
accagtaaat taaaatattg gggggaaggg cacgaaggat gtggctgcgg aaagtataat 1740
cagtaa 1746
<210> 2
<211> 581
<212> PRT
<213> Morganella morganii subsp. morganii ATCC 258297
<400> 2
Met Ile Asn Asn Gln Gln Pro Asp Gly Ala Arg Leu Ile Gln Ala Leu
1 5 10 15
Thr Asp Ile Val Ser His Lys His Ile Leu Thr Glu Pro Arg Lys Thr
20 25 30
Glu Arg Tyr Arg Lys Gly Phe Arg Ser Gly Glu Gly Ser Ala Leu Ala
35 40 45
Val Val Phe Pro Gly Thr Leu Tyr Glu Leu Trp Gln Val Phe Lys Ala
50 55 60
Ala Val Glu Ala Asp Arg Ile Val Ile Met Gln Ala Ala Asn Thr Gly
65 70 75 80
Leu Thr Glu Gly Ser Thr Pro Ser Gly Asp Asp Tyr Asp Arg Glu Ile
85 90 95
Ile Ile Ile Ser Thr Leu Arg Leu Asp Lys Ile Gln Val Leu Thr Lys
100 105 110
Gln Gln Gln Val Val Ala Leu Pro Gly Ser Thr Leu Trp His Leu Glu
115 120 125
Lys Val Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser
130 135 140
Ser Cys Ile Gly Ala Ser Val Val Gly Gly Ile Cys Asn Asn Ser Gly
145 150 155 160
Gly Ser Leu Val Gln Arg Gly Pro Ala Tyr Thr Glu Leu Ala Leu Tyr
165 170 175
Gly Arg Val Asn Ala Asp Gly Ser Ala Glu Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly Ser Thr Pro Glu Glu Ile Leu Asn Cys Leu Glu Tyr
195 200 205
Arg Gln Tyr Gln Asp Ala Asp Ile Glu Asp Thr Asp Lys Pro Ala Ser
210 215 220
Asp His Asp Tyr His Gln Arg Ile Arg Asp Val Asp Ala Asp Thr Pro
225 230 235 240
Ser Arg Phe Asn Ala Asp Glu Arg Arg Leu Phe Glu Ala Ser Gly Cys
245 250 255
Ala Gly Lys Leu Val Val Phe Ala Val Arg Leu Asp Thr Phe Pro Ser
260 265 270
Gln Gly Glu Ser Lys Val Phe Tyr Ile Gly Thr Asn Thr Pro Ser Val
275 280 285
Leu Glu Asp Ile Arg Arg His Ile Leu Ala Glu Phe Glu His Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Glu Cys Tyr Asp Ile Ala Lys Val
305 310 315 320
Tyr Gly Lys Asp Ser Phe Leu Met Ile Asp Lys Leu Gly Thr Glu Lys
325 330 335
Met Pro Lys Leu Phe Thr Ile Lys Gly Arg Met Asp Ala Val Phe Asn
340 345 350
Lys Ile Pro Leu Leu Pro Lys Asn Leu Ile Asp Arg Thr Met Gln Leu
355 360 365
Phe Ser His Leu Trp Pro Asn His Leu Pro Arg Arg Met Glu Glu Tyr
370 375 380
Arg Asp Lys Tyr Glu His His Leu Met Leu Lys Met Ala Gly Glu Gly
385 390 395 400
Ile Asp Glu Ala Arg Asn Trp Leu Ser Thr Phe Phe Asp Asn Ala Glu
405 410 415
Gly Ala Phe Phe Glu Cys Asp Ala Lys Glu Gly Ala Asp Ala Phe Leu
420 425 430
His Arg Phe Val Ala Ala Gly Ala Ala Ile Arg Tyr His Ala Val Asn
435 440 445
Ser Ser Lys Val Glu Asp Ile Leu Ala Leu Asp Ile Ala Leu Arg Arg
450 455 460
Asn Asp Gln Ala Trp Leu Glu Val Leu Pro Pro Glu Ile Glu Ser Lys
465 470 475 480
Leu Val His Lys Leu Tyr Tyr Gly His Phe Leu Cys His Val Phe His
485 490 495
Gln Asp Tyr Ile Val Lys Lys Gly Val Asp Pro His Ala Leu Lys Glu
500 505 510
Glu Met Leu Glu Ile Leu Asn Thr Arg Gly Ala Glu Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Leu Tyr Lys Ala Lys Pro Gln Leu Lys Ala Phe
530 535 540
Tyr Lys Ala Thr Asp Pro Thr Asn Thr Leu Asn Pro Gly Leu Gly Lys
545 550 555 560
Thr Ser Lys Leu Lys Tyr Trp Gly Glu Gly His Glu Gly Cys Gly Cys
565 570 575
Gly Lys Tyr Asn Gln
580

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 the root that rubs from morganella morganii The D-ALPHA-Hydroxypropionic acid oxidizing ferment of subspecies (Morganella morganii subsp.morganii), amino acid sequence are SEQ ID Shown in NO:2；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, lactic acid norbornene ester, mandelic acid norbornene ester, mandelic acid Isopropyl ester, danshensu asarum alcohol 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment is classified as SEQ ID Shown in NO:1.
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