CN106754800B - A kind of oxidizing ferment and its application - Google Patents
A kind of oxidizing ferment and its application Download PDFInfo
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- 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)
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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 Kerstersia gyiorums 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
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.19968；271(45):28300-28305).It can be used for measuring containing for lactic acid in biosensor
Amount, 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
The present invention is cloned from Kerstersia gyiorum DSM 16618 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: Kerstersia gyiorum DSM 16618, purchase
From DSMZ- Germany Microbiological Culture Collection Center.
2, the clone of L- alpha-hydroxy acid oxidase gene
Extract 16618 phage gene group total DNA of Kerstersia gyiorum DSM.Design specific primer, application
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- from Kerstersia gyiorum DSM 16618
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 Kerstersia gyiorum DSM 16618DNA
16618 bacterial strain of Kerstersia gyiorum DSM 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 L- alpha-hydroxy acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCATGTCTGTTTTTCAACCGAACGCAA 3'
Primer 2: 5'GCCGTCTAGAGAAATAGTGCGCGGCACCAGG 3'
(2) PCR amplification
With two primers synthesized above, using the genomic DNA of Kerstersia gyiorum DSM 16618 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 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 7.0 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 7.0 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 1 L of the table-alpha-hydroxy acid oxidizing ferment 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> 1203
<212> DNA
<213> Kerstersia gyiorum DSM16618
<400> 1
atgtctgttt ttcaaccgaa cgcaaaatca aaggcaagcg agccccgcag actgcgcggc 60
gtgctgaact tgcaggattt cgagcgccag gcccggcggc acttacccag gccgctgttc 120
gagtatgtcg ccggtaccgt ggaggatggc cgcgccggac gagacaacca gcatgccttg 180
ctggaatacg gcctgctgcc acgcattctg acggacgtgt ccgggcgcag ccagaaagtc 240
gaactgttcg gccgcaccta tgacagcccc ttcggtattg cccccatggg cctggccgcg 300
ctgaccgcct accgtggcga cgtcgtgctg gcccgcgctg cgcgtgctgc gaatattcct 360
gcagtcctga gcggcacttc cgtcattccg ctggaaactg tcatggaaga tgtgccgggg 420
acctggttcc aggcctatct gcccggggat tccgaacgca ttgccgcctt gctggaccgt 480
gtggcgcgta ccggatgcga tacgctggtc gtgacagtgg atatccccgt caatgccaat 540
cgcgaaaata atatccgcgc cggtttctcc acgccgctca agcccagctt gcggctggcc 600
tgggatggcc tgacgcggcc ttcctggctg ttcggcacct ttctgcgtac gctggcgcgt 660
cacggcatgc cgcattttga gaactctttc gcgacgcgcg gcgcgcccat cctgtcggcc 720
acggtgctgc gggatttcac tgcccgggac catctcaact ggcgccattt cgctgaaatc 780
cgccgtcaat ggcacggtaa cctggtgatc aagggcatct tgcatccggc cgacgcgaag 840
ctggcccgcg agcatggtgc ggatgccgtc atcgtctcca accacggggg ccgccagctc 900
gacggtgcca tctcgccgtt gcgctccctg caggcggtcg tggctgctgt gccggacctg 960
cccgttctgg ttgacagtgg ttttcggcgc ggttcggacg tgatggttgc gctggcgatg 1020
ggggcacgca tggtctttgt cggacgcccc ttcaattacg ccgccgccat tgccggcgaa 1080
gcgggcgttg cccatggcat ccggctattg cgcgaggaaa tcgaccgcaa catggggatg 1140
ctgggcatta accgttgcga tgaactgact cccgcgtgcc tggtgccgcg cactatttcc 1200
tga 1203
<210> 2
<211> 400
<212> PRT
<213> Kerstersia gyiorum DSM16618
<400> 2
Met Ser Val Phe Gln Pro Asn Ala Lys Ser Lys Ala Ser Glu Pro Arg
1 5 10 15
Arg Leu Arg Gly Val Leu Asn Leu Gln Asp Phe Glu Arg Gln Ala Arg
20 25 30
Arg His Leu Pro Arg Pro Leu Phe Glu Tyr Val Ala Gly Thr Val Glu
35 40 45
Asp Gly Arg Ala Gly Arg Asp Asn Gln His Ala Leu Leu Glu Tyr Gly
50 55 60
Leu Leu Pro Arg Ile Leu Thr Asp Val Ser Gly Arg Ser Gln Lys Val
65 70 75 80
Glu Leu Phe Gly Arg Thr Tyr Asp Ser Pro Phe Gly Ile Ala Pro Met
85 90 95
Gly Leu Ala Ala Leu Thr Ala Tyr Arg Gly Asp Val Val Leu Ala Arg
100 105 110
Ala Ala Arg Ala Ala Asn Ile Pro Ala Val Leu Ser Gly Thr Ser Val
115 120 125
Ile Pro Leu Glu Thr Val Met Glu Asp Val Pro Gly Thr Trp Phe Gln
130 135 140
Ala Tyr Leu Pro Gly Asp Ser Glu Arg Ile Ala Ala Leu Leu Asp Arg
145 150 155 160
Val Ala Arg Thr Gly Cys Asp Thr Leu Val Val Thr Val Asp Ile Pro
165 170 175
Val Asn Ala Asn Arg Glu Asn Asn Ile Arg Ala Gly Phe Ser Thr Pro
180 185 190
Leu Lys Pro Ser Leu Arg Leu Ala Trp Asp Gly Leu Thr Arg Pro Ser
195 200 205
Trp Leu Phe Gly Thr Phe Leu Arg Thr Leu Ala Arg His Gly Met Pro
210 215 220
His Phe Glu Asn Ser Phe Ala Thr Arg Gly Ala Pro Ile Leu Ser Ala
225 230 235 240
Thr Val Leu Arg Asp Phe Thr Ala Arg Asp His Leu Asn Trp Arg His
245 250 255
Phe Ala Glu Ile Arg Arg Gln Trp His Gly Asn Leu Val Ile Lys Gly
260 265 270
Ile Leu His Pro Ala Asp Ala Lys Leu Ala Arg Glu His Gly Ala Asp
275 280 285
Ala Val Ile Val Ser Asn His Gly Gly Arg Gln Leu Asp Gly Ala Ile
290 295 300
Ser Pro Leu Arg Ser Leu Gln Ala Val Val Ala Ala Val Pro Asp Leu
305 310 315 320
Pro Val Leu Val Asp Ser Gly Phe Arg Arg Gly Ser Asp Val Met Val
325 330 335
Ala Leu Ala Met Gly Ala Arg Met Val Phe Val Gly Arg Pro Phe Asn
340 345 350
Tyr Ala Ala Ala Ile Ala Gly Glu Ala Gly Val Ala His Gly Ile Arg
355 360 365
Leu Leu Arg Glu Glu Ile Asp Arg Asn Met Gly Met Leu Gly Ile Asn
370 375 380
Arg Cys Asp Glu Leu Thr Pro Ala Cys Leu Val Pro Arg Thr Ile Ser
385 390 395 400
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 Kerstersia
The L- alpha-hydroxy acid oxidizing ferment of gyiorum DSM 16618, 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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