CN106754785B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754785B
CN106754785B CN201710006561.6A CN201710006561A CN106754785B CN 106754785 B CN106754785 B CN 106754785B CN 201710006561 A CN201710006561 A CN 201710006561A CN 106754785 B CN106754785 B CN 106754785B
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alpha
ester
acid
hydroxy acid
ala
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CN106754785A (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 Hao Shi proteus (Proteus hauseri) 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 Kerstersia gyiorum DSM 16618 goes out a kind of novel L- α-hydroxyl to the present invention for the first time Acid oxidase, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, but also can aoxidize (S)-alpha-hydroxy acid ester, can be applied to optical voidness (R) preparation of-alpha-hydroxy acid ester and (R)-alpha-hydroxy acid.
Summary of the invention
Present invention clone from the Hao Shi proteus (Proteus hauseri) has obtained a kind of L- alpha-hydroxy acid oxidizing ferment Gene 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: Proteus hauseri ATCC 13315 is purchased from U.S.'s ATCC strain library.
2, the clone of L- alpha-hydroxy acid oxidase gene
Extract 13315 phage gene group total DNA of Proteus hauseri ATCC.Specific primer is designed, using PCR Method amplifies L- alpha-hydroxy acid oxidase gene overall length encoder block sequence.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 a kind of L- α-from Proteus hauseri ATCC 13315 Hydroxy acid oxidase, the enzyme can aoxidize (S)-alpha-hydroxy acid and (S)-alpha-hydroxy acid ester, can be used for prepare with scale chiral purity (R)-α- Carboxylic esters have 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 Proteus hauseri ATCC 13315DNA
13315 bacterial strain of Proteus hauseri ATCC is cultivated into 12h in LB culture medium, 12,000 rmp/min from Heart 10min obtains thallus, operates using bacterial genomes DNA extraction agent box (TaKaRa company) 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, 8000 rpm/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'AGCCGGGATCCATGAAACGTCAAATATTAAAAGCTA 3'
Primer 2: 5'GCCGTCTAGATTATTGATTAAAACTTGCATCAGTA 3'
(2) PCR amplification
With two primers synthesized above, using the genomic DNA of Proteus hauseri ATCC 13315 as template into Row 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 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 5.0 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 50 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 50 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 5.0 pH It is high.
Embodiment 5
The present embodiment is listed in table 1 from the response characteristic of different substrates for L- alpha-hydroxy acid oxidizing ferment of the present invention.
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> 1197
<212> DNA
<213> Proteus hauseri ATCC13315
<400> 1
atgaaacgtc aaatattaaa agctaccgct attgctatgg cattaagtgt cggtgttgca 60
caggcggctg aatataaagc aagtactgct gaaggtccaa ttaaaattgt aaacttaaaa 120
gcaatggaag ctcaagttca agcaaatatg gaaaaaggtg cctttggtta tattcgtggt 180
ggtgctgaag acgaaaataa tttacgtgcg aatacacgcg catttgataa aaaatatatt 240
atgccacgtt cattacaagg catcgaattt tctgacataa acctaaaaac agaattccta 300
gggattaaat tagatacgcc tattattcag gcgccgatgg cagcccaagg gcttgctcat 360
caacaaggcg aagttgccac agcaaaaggt atggcgaaag cgggctctat tttttcatta 420
agcacttatg gtaataaaac cattaaagaa gtggcggatg cccaacccgg ttaccccttc 480
ttctttcagt tatacatgag taaaaatgat gcctttaatg agtatatttt atctcaagca 540
aaacagtatg gcgctaaagg tatcattatg actatcgact cttctgttgg tggttatcgt 600
gaagatgatg tgaaaaataa tttccaattt ccactcggct ttgccaactt agaagcgttt 660
gcaaaaatca gtgatgataa atctaaaaca ggtaaaggtg caggtattag cgaaatttat 720
gcacaagcta aacaagcctt cacaccagca gatattcagt atgtgaaaaa aatgtctggt 780
ttaccggtca ttgtaaaagg tatcgaatca cctgaagacg cagatactgc tattaaagcc 840
ggtgcagatg caatttgggt ttctaaccat ggtggtcgtc aattagatag cgcacccgca 900
acgattgatg tattacctgc gattgcaaaa gtggtgaata aacgtgttcc tatcgtcttt 960
gatagtggcg tacgtcgtgg ctcacacgta tttaaagccc tcgcaagtgg tgcggatgtg 1020
gttgcggtag gtcgcccgat tctttatggc ttaaatttag gtggatctga aggtgtgaat 1080
tcagttattc aacatttaaa taaagagctg agaattaata tgatgttggg tggtgcaaaa 1140
acagtgaaag atattcaagc tactcctctt tatactgatg caagttttaa tcaataa 1197
<210> 2
<211> 398
<212> PRT
<213> Proteus hauseri ATCC13315
<400> 2
Met Lys Arg Gln Ile Leu Lys Ala Thr Ala Ile Ala Met Ala Leu Ser
1 5 10 15
Val Gly Val Ala Gln Ala Ala Glu Tyr Lys Ala Ser Thr Ala Glu Gly
20 25 30
Pro Ile Lys Ile Val Asn Leu Lys Ala Met Glu Ala Gln Val Gln Ala
35 40 45
Asn Met Glu Lys Gly Ala Phe Gly Tyr Ile Arg Gly Gly Ala Glu Asp
50 55 60
Glu Asn Asn Leu Arg Ala Asn Thr Arg Ala Phe Asp Lys Lys Tyr Ile
65 70 75 80
Met Pro Arg Ser Leu Gln Gly Ile Glu Phe Ser Asp Ile Asn Leu Lys
85 90 95
Thr Glu Phe Leu Gly Ile Lys Leu Asp Thr Pro Ile Ile Gln Ala Pro
100 105 110
Met Ala Ala Gln Gly Leu Ala His Gln Gln Gly Glu Val Ala Thr Ala
115 120 125
Lys Gly Met Ala Lys Ala Gly Ser Ile Phe Ser Leu Ser Thr Tyr Gly
130 135 140
Asn Lys Thr Ile Lys Glu Val Ala Asp Ala Gln Pro Gly Tyr Pro Phe
145 150 155 160
Phe Phe Gln Leu Tyr Met Ser Lys Asn Asp Ala Phe Asn Glu Tyr Ile
165 170 175
Leu Ser Gln Ala Lys Gln Tyr Gly Ala Lys Gly Ile Ile Met Thr Ile
180 185 190
Asp Ser Ser Val Gly Gly Tyr Arg Glu Asp Asp Val Lys Asn Asn Phe
195 200 205
Gln Phe Pro Leu Gly Phe Ala Asn Leu Glu Ala Phe Ala Lys Ile Ser
210 215 220
Asp Asp Lys Ser Lys Thr Gly Lys Gly Ala Gly Ile Ser Glu Ile Tyr
225 230 235 240
Ala Gln Ala Lys Gln Ala Phe Thr Pro Ala Asp Ile Gln Tyr Val Lys
245 250 255
Lys Met Ser Gly Leu Pro Val Ile Val Lys Gly Ile Glu Ser Pro Glu
260 265 270
Asp Ala Asp Thr Ala Ile Lys Ala Gly Ala Asp Ala Ile Trp Val Ser
275 280 285
Asn His Gly Gly Arg Gln Leu Asp Ser Ala Pro Ala Thr Ile Asp Val
290 295 300
Leu Pro Ala Ile Ala Lys Val Val Asn Lys Arg Val Pro Ile Val Phe
305 310 315 320
Asp Ser Gly Val Arg Arg Gly Ser His Val Phe Lys Ala Leu Ala Ser
325 330 335
Gly Ala Asp Val Val Ala Val Gly Arg Pro Ile Leu Tyr Gly Leu Asn
340 345 350
Leu Gly Gly Ser Glu Gly Val Asn Ser Val Ile Gln His Leu Asn Lys
355 360 365
Glu Leu Arg Ile Asn Met Met Leu Gly Gly Ala Lys Thr Val Lys Asp
370 375 380
Ile Gln Ala Thr Pro Leu Tyr Thr Asp Ala Ser Phe Asn Gln
385 390 395

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 Hao Shi proteus The L- alpha-hydroxy acid oxidizing ferment of (Proteus hauseri), 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 L- alpha-hydroxy acid oxidizing ferment is classified as SEQ Shown in ID NO:1.
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CN102660470A (en) * 2012-04-13 2012-09-12 浙江工业大学 Sinorhizobium fredii and its application in producing chiral alpha-hydroxy acid by biologically splitting alpha-hydroxy acid raceme

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CA2165940C (en) * 1993-06-25 2005-09-13 David Leroy Anton Process for the preparation of pyruvic acid

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Publication number Priority date Publication date Assignee Title
CN102660470A (en) * 2012-04-13 2012-09-12 浙江工业大学 Sinorhizobium fredii and its application in producing chiral alpha-hydroxy acid by biologically splitting alpha-hydroxy acid raceme

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WP_023583683.1:oxidase[Proteushauseri];NCBI;《NCBI》;20131125;ORIGIN部分 *

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