CN106754778B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754778B
CN106754778B CN201710006540.4A CN201710006540A CN106754778B CN 106754778 B CN106754778 B CN 106754778B CN 201710006540 A CN201710006540 A CN 201710006540A CN 106754778 B CN106754778 B CN 106754778B
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ester
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CN106754778A (en
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蔡宇杰
武旭攀
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 a kind of acquisition of D-ALPHA-Hydroxypropionic acid oxidase gene from phlegm Ta Temu Salmonella (Tatumella ptyseos) and its clonal expressions, 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 clonal expression from phlegm Ta Temu Salmonella (Tatumella ptyseos) goes out a kind of novel D- to the present invention for the first time Lactate oxidase, the enzyme can not only aoxidize (R)-alpha-hydroxy acid, but also can aoxidize (R)-alpha-hydroxy acid ester, the reaction and NAD (NADP) reaction for the lactic dehydrogenase participation of coenzyme is atomic weak compared to back reaction, can be applied to optical voidness (S)-alpha-hydroxy acid ester The preparation of (S)-alpha-hydroxy acid.
Summary of the invention
Present invention clone from phlegm Ta Temu Salmonella (Tatumella ptyseos) has obtained a kind of using FAD as coenzyme The gene of D-ALPHA-Hydroxypropionic acid oxidizing ferment discloses its relevant enzymatic property, and carry out using colibacillus engineering heterogenous expression 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: Tatumella ptyseos ATCC 33301 is purchased from U.S.'s ATCC strain library.
2, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract 33301 phage gene group total DNA of Tatumella ptyseos ATCC.Design specific primer, application PCR method amplifies D-ALPHA-Hydroxypropionic acid oxidase gene overall length encoder block sequence.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 deliver bright usefulness: clonal expression goes out a kind of novel from Tatumella ptyseos ATCC 33301 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 Tatumella ptyseos ATCC 33301DNA
33301 bacterial strain of Tatumella ptyseos ATCC is cultivated into 12h, 12,000 rmp/min in LB culture medium Centrifugation 10min obtains thallus, operates using bacterial genomes DNA extraction agent box (TaKaRa company) according to it and extracts thallus base Because of a 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'GCCGGGATCCATGAATAACCGTCAGGAGCTGACC 3'
Primer 2: 5'GCCGTCTAGATTACTTGCCCCAGAATTTTTTACGG 3'
With two primers synthesized above, using the genomic DNA of Tatumella ptyseos ATCC 33301 as 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> 1698
<212> DNA
<213> Tatumella ptyseos ATCC 33301
<400> 1
atgaataacc gtcaggagct gacccgcaag ctgcacgaaa tcgcgggaga aaaacaggtt 60
ttactgagtg aagacgataa ggccttttac accaaaggat tccgcgtcgg tcagggtgaa 120
gcactggccg tggtactgcc acagacgctg ttacagctct ggcaattgtt agaagtttgt 180
gttgccgaag atgttatcat tctgatgcag gcctccaata ccggcgtcac cggaggttca 240
accccggacg gtaatgatta cgaccgcgat attgtcatta tcagcacccg tttactgacc 300
ggggttcagg tcattgatga tcaccaacag gttatcgctt ttccgggcac gaccctcacc 360
gagcttgaag acaccctgaa gcctctgcag cgtgaacccc attcggtgat cggttcctcc 420
tgtatcggcg cgtctgtcat tggcgggtta tgtaataact ccgggggctc gctgatccgt 480
cgtggtccgg catttacaga aaaatcgctg tttgcccgca ttactgatga cggacatctt 540
gaactggtta accatctggg tattgagctg ggcgatactc cggagaggat cattaatgcg 600
ctggagacac gcgaatacac gaccggcgat gctgccgact ggcagggtaa aatctgggca 660
gatgattatt ctgaacaact gcgcgatact catgcggcat caccggccag attcaacggc 720
gataaacgtt atctgtatga cagtgcggga tgtgccggaa aagtggtcgt ctttgccgca 780
cgtttgccga cgtttgccgc cagcgaagct acccgtacct tctatatcgg cagcaatgat 840
gaaagcgaac tgatcggttt acgccgtttt ctgctggaaa aaatggatga actgccctta 900
caggcggaat atattcacgc cggtgcgttt gacctgacat tgcgttacgc aaaacatatg 960
tatctggcca tccgtaagct ggggccgcag gccattcccg gtttaatggc caacaaagcg 1020
cgctgggatg tgcggatcag acatgcccgt tttctgccgc ataattttac cgataagctg 1080
atccagggct ttaatcagat aaccccctcg tttatcgcac cgcggatcat ggattaccgc 1140
cggcgctttg tgcaccatct gatgattaaa attgaagccc gtcaggctga acaactgacg 1200
gcattgcttg aggagtattt cagccagcgt ccgggggaat atttcctgtg cgatgaccgg 1260
gaagctgccg atgcctttct gatccgtttc gcggtgggcg gctgtaccat ctcctactgt 1320
gactacctcg gctatgatac caaccagcga ttaattgctt ttgacgtggc tctgcgccgt 1380
aatgatgacc agtggcggat ccatctgcca cctcatcttg cggcgcaggt ccaggccgat 1440
tcctgttgcg ggcatttctt ttgctttgtg aaccaccagg attacgtcct taaagccggg 1500
gtggatgcga agaaattcaa acaggaggtg atggagtatc tggaacagcg tggtgcccgc 1560
tatcctgcgg agcacaatgt cgggcacctg tatcaggcgt cctgtgaaca tgaacaacac 1620
tggcgtgaac tggatccgac caatacctgc aacccgggga ttgggaaaac cagccgtaaa 1680
aaattctggg gcaagtaa 1698
<210> 2
<211> 565
<212> PRT
<213> Tatumella ptyseos ATCC 33301
<400> 2
Met Asn Asn Arg Gln Glu Leu Thr Arg Lys Leu His Glu Ile Ala Gly
1 5 10 15
Glu Lys Gln Val Leu Leu Ser Glu Asp Asp Lys Ala Phe Tyr Thr Lys
20 25 30
Gly Phe Arg Val Gly Gln Gly Glu Ala Leu Ala Val Val Leu Pro Gln
35 40 45
Thr Leu Leu Gln Leu Trp Gln Leu Leu Glu Val Cys Val Ala Glu Asp
50 55 60
Val Ile Ile Leu Met Gln Ala Ser Asn Thr Gly Val Thr Gly Gly Ser
65 70 75 80
Thr Pro Asp Gly Asn Asp Tyr Asp Arg Asp Ile Val Ile Ile Ser Thr
85 90 95
Arg Leu Leu Thr Gly Val Gln Val Ile Asp Asp His Gln Gln Val Ile
100 105 110
Ala Phe Pro Gly Thr Thr Leu Thr Glu Leu Glu Asp Thr Leu Lys Pro
115 120 125
Leu Gln Arg Glu Pro His Ser Val Ile Gly Ser Ser Cys Ile Gly Ala
130 135 140
Ser Val Ile Gly Gly Leu Cys Asn Asn Ser Gly Gly Ser Leu Ile Arg
145 150 155 160
Arg Gly Pro Ala Phe Thr Glu Lys Ser Leu Phe Ala Arg Ile Thr Asp
165 170 175
Asp Gly His Leu Glu Leu Val Asn His Leu Gly Ile Glu Leu Gly Asp
180 185 190
Thr Pro Glu Arg Ile Ile Asn Ala Leu Glu Thr Arg Glu Tyr Thr Thr
195 200 205
Gly Asp Ala Ala Asp Trp Gln Gly Lys Ile Trp Ala Asp Asp Tyr Ser
210 215 220
Glu Gln Leu Arg Asp Thr His Ala Ala Ser Pro Ala Arg Phe Asn Gly
225 230 235 240
Asp Lys Arg Tyr Leu Tyr Asp Ser Ala Gly Cys Ala Gly Lys Val Val
245 250 255
Val Phe Ala Ala Arg Leu Pro Thr Phe Ala Ala Ser Glu Ala Thr Arg
260 265 270
Thr Phe Tyr Ile Gly Ser Asn Asp Glu Ser Glu Leu Ile Gly Leu Arg
275 280 285
Arg Phe Leu Leu Glu Lys Met Asp Glu Leu Pro Leu Gln Ala Glu Tyr
290 295 300
Ile His Ala Gly Ala Phe Asp Leu Thr Leu Arg Tyr Ala Lys His Met
305 310 315 320
Tyr Leu Ala Ile Arg Lys Leu Gly Pro Gln Ala Ile Pro Gly Leu Met
325 330 335
Ala Asn Lys Ala Arg Trp Asp Val Arg Ile Arg His Ala Arg Phe Leu
340 345 350
Pro His Asn Phe Thr Asp Lys Leu Ile Gln Gly Phe Asn Gln Ile Thr
355 360 365
Pro Ser Phe Ile Ala Pro Arg Ile Met Asp Tyr Arg Arg Arg Phe Val
370 375 380
His His Leu Met Ile Lys Ile Glu Ala Arg Gln Ala Glu Gln Leu Thr
385 390 395 400
Ala Leu Leu Glu Glu Tyr Phe Ser Gln Arg Pro Gly Glu Tyr Phe Leu
405 410 415
Cys Asp Asp Arg Glu Ala Ala Asp Ala Phe Leu Ile Arg Phe Ala Val
420 425 430
Gly Gly Cys Thr Ile Ser Tyr Cys Asp Tyr Leu Gly Tyr Asp Thr Asn
435 440 445
Gln Arg Leu Ile Ala Phe Asp Val Ala Leu Arg Arg Asn Asp Asp Gln
450 455 460
Trp Arg Ile His Leu Pro Pro His Leu Ala Ala Gln Val Gln Ala Asp
465 470 475 480
Ser Cys Cys Gly His Phe Phe Cys Phe Val Asn His Gln Asp Tyr Val
485 490 495
Leu Lys Ala Gly Val Asp Ala Lys Lys Phe Lys Gln Glu Val Met Glu
500 505 510
Tyr Leu Glu Gln Arg Gly Ala Arg Tyr Pro Ala Glu His Asn Val Gly
515 520 525
His Leu Tyr Gln Ala Ser Cys Glu His Glu Gln His Trp Arg Glu Leu
530 535 540
Asp Pro Thr Asn Thr Cys Asn Pro Gly Ile Gly Lys Thr Ser Arg Lys
545 550 555 560
Lys Phe Trp Gly Lys
565

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 phlegm Ta Temu Salmonella The D-ALPHA-Hydroxypropionic acid oxidizing ferment of (Tatumella ptyseos), amino acid sequence are shown in SEQ ID NO:2；The alpha-hydroxy acid Ester is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid Norbornene ester, para hydroxybenzene isopropyl lactate, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu kakuol 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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