CN106701705A - Oxidase and application thereof - Google Patents

Oxidase and application thereof Download PDF

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CN106701705A
CN106701705A CN201710006611.0A CN201710006611A CN106701705A CN 106701705 A CN106701705 A CN 106701705A CN 201710006611 A CN201710006611 A CN 201710006611A CN 106701705 A CN106701705 A CN 106701705A
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alpha
acid
ester
ala
oxidizing ferment
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CN106701705B (en
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蔡宇杰
卢欢
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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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • 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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    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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    • 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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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
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    • 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)

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Abstract

The invention relates to obtaining and cloning and expression of a D-lactate oxidase gene derived from extended Brevibacterium linens, and belongs to the field of biological engineering. The invention discloses substrate specificity of the D-lactate oxidase gene. D-lactate oxidase can oxidize (R)-alpha-hydroxy esters and can be used for preparing optical pure (S)-alpha- hydroxy esters.

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, belongs to industrial microorganism field.
Background technology
D-ALPHA-Hydroxypropionic acid oxidizing ferment (D-lactate oxidase) is a kind of alpha-hydroxy acid oxidizing ferment with FAD (FMN) as coenzyme (being traditionally referred to as D-ALPHA-Hydroxypropionic acid oxidizing ferment).D-ALPHA-Hydroxypropionic acid oxidizing ferment can be used in biology sensor determine the content of lactic acid, or Oxidation D-ALPHA-Hydroxypropionic acid production pyruvic acid.Also have and be used for the preparation (Chinese patent 201210109290.4) of optical voidness alpha-hydroxy acid
So far, in edwardsiella tarda (Edwardsiella tarda) and zymomonas mobilis D-ALPHA-Hydroxypropionic acid oxidizing ferment is found that 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)
Clonal expression goes out a kind of new D- to the present invention from extension brevibacterium (Brevibacterium linens) first LO, the enzyme can not only aoxidize (R)-alpha-hydroxy acid, and can aoxidize (R)-alpha-hydroxy acid ester, the reaction and NAD (NADP) for the reaction of 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.
The content of the invention
Present invention clone from extension brevibacterium (Brevibacterium linens) has obtained one kind with FAD as coenzyme D-ALPHA-Hydroxypropionic acid oxidizing ferment gene, using colibacillus engineering heterogenous expression, disclose its related enzymatic property, and carry out Application study.
Technical scheme is as follows：
1st, bacterial strain
The source bacterial strain of D-ALPHA-Hydroxypropionic acid oxidase gene of the present invention is：Brevibacterium linens ATCC 8377, purchase From U.S.'s ATCC strain libraries.
2nd, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract the phage gene group STb genes of Brevibacterium linens ATCC 8377.Design specific primer, should With PCR method, D-ALPHA-Hydroxypropionic acid oxidase gene total length encoder block sequence is amplified.And construction recombination plasmid.
3rd, D-ALPHA-Hydroxypropionic acid Oxidase Expression and purifying
Recombinant plasmid is imported in E.coli BL21 (DE3), induced expression.Crude enzyme liquid is obtained after bacterial cell disruption, after purification Freeze-drying is standby.
4th, the characterization analysis of D-ALPHA-Hydroxypropionic acid oxidizing ferment
Influence with D-ALPHA-Hydroxypropionic acid as substrate research pH to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention.
Influence with D-ALPHA-Hydroxypropionic acid as substrate research temperature to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention.
The substrate specificity analysis of D-ALPHA-Hydroxypropionic acid oxidizing ferment：Substrate used have D-ALPHA-Hydroxypropionic acid, glycolic, D- phenyllactic acids, D- pairs Hydroxyphenyl lactic acid, D- tartaric acid, D-malic acid, D- mandelic acids, D- danshensus.
Enzyme activity determination method is：According to Characterization of a Lactate Oxidase from a Strain of Gram Negative Bacterium from Soil, Applied Biochemistry and Biotechnology,56,1996,278-288.Methods described is carried out.
5th, D-ALPHA-Hydroxypropionic acid oxidizing ferment splits the alpha-hydroxy acid ester of DL
The method of resolution of alpha-carboxylic esters (alpha-hydroxy esters) is：0.1 gram of the enzyme for having purified is taken in 50mL tri- In the bottle of angle, in adding dissolved with the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, in 30 DEG C, converted in 150rpm shaking baths 16h, liquid-phase chromatographic analysis supernatant after conversion.(R) Alpha-hydroxy in-alpha-hydroxy acid ester is dehydrogenated and is oxidized to corresponding 2-ketoacid Ester, (S)-alpha-hydroxy acid ester is not oxidized.
The optical purity of product (S)-alpha-hydroxy acid ester 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 formulaRIt is the peak area of (R)-enantiomer after reaction, SSIt is the liquid chromatogram peak area of (S)-enantiomer after reaction, S0It is the liquid chromatogram peak area sum of (R)-and (S)-enantiomer before reaction.
Product determines liquid phase chromatogram condition：Chiralcel OD-H chiral columns (4.6 × 250mm), mobile phase volume ratio It is n-hexane：Isopropanol：Trifluoroacetic acid=80:20:0.1, flow velocity is 0.5mL/min, and 25 DEG C of column temperature, Detection wavelength 210nm enters The μ L of sample amount 20.
Described alpha-hydroxy acid ester is one of following：Tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, benzene breast Isopropyl propionate, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene isopropyl lactate, mandelic acid norbornene ester, almond isopropyl propionate, the red sage root Plain asarum alcohol ester, lactic acid norbornene ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
Described alpha-hydroxy acid ester, according to Chinese patent 200610042787.3,201410180490.8, The 201410175950.8 and 20140699506.6 method synthesis announced.
Originally bright usefulness is delivered：Clonal expression goes out one kind from Brevibacterium linens ATCC 8377 New D-ALPHA-Hydroxypropionic acid oxidizing ferment, the enzyme can aoxidize (R)-alpha-hydroxy acid and (R)-alpha-hydroxy acid ester, can be used for prepare with scale chiral Pure (S)-alpha-hydroxy acid ester, with 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 build.
1st, the extraction of Brevibacterium linens ATCC 8377DNA
The bacterial strains of Brevibacterium linens ATCC 8377 are cultivated into 12h, 12,000rmp/ in LB culture mediums Min centrifugations 10min obtains thalline, and bacterium is extracted according to its operation using bacterial genomes DNA extraction agents box (TaKaRa companies) Body genome DNA, puts refrigerator standby.
2nd, prepared by E. coli competent
(1) inoculation E.coli DH5 α and BL21 (DE3) is respectively in the 250mL shaking flasks containing 20mL LB culture mediums, and 37 DEG C, 200rpm/min overnight incubations.
(2) it is inoculated in 50mL LB culture mediums 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 precoolings, 30min, 8000rpm/min, 4 DEG C of centrifugations is placed on ice 5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl of 5mL precoolings are added2Solution, makes thalline suspend, and 20min is placed 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 precoolings are added2Solution (contains 15% glycerine), 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 standby.
3rd, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
(1) design of primers
Designing primer sequence is：
Primer 1：5'GCCGGGATCC ATGTTCGGACGACAGCGTCAGCACC 3'
Primer 2：5'GCCGTCTAGACAGGCCCAGTTCTTGTGCGGTGAG 3'
(2) PCR amplifications
Two primers of synthesis more than, the genomic DNA with Brevibacterium linens ATCC 8377 is as mould Plate enters performing PCR amplification.
Amplification system is in this step：
Amplification program is：
98 DEG C, 10min
98 DEG C, 10sec；55 DEG C, 15sec；72 DEG C, 2min reacts 30 circulations
72 DEG C, 10min
PCR primer send the gene order that the enzyme is obtained after Hua Da gene sequencing, such as SEQ ID NO:Shown in 1.According to the base Because of the amino acid sequence such as SEQ ID NO that sequence is obtained:Shown in 2.
(3) double digestion and connection
The plasmids of pCold II and PCR primer are carried out into double digestion, digestion system is：10×cut buffer 3μl,DNA 4μ Each 0.5 μ l of l, enzyme BamHI and XbaI, the μ l of sterilized water 2 totally 30 μ l.Double digestion 1h under 37 DEG C of water-baths.DNA fragmentation is cloned into On the carriers of pCold II, and it is transformed into E.coli DH5 α competent cells.Linked system：10×DNA ligase buffer 2.5 μ l, the μ l of DNA fragmentation 8,2 μ l, T4DNA ligase of carrier DNA 1 μ l, the μ l of sterilized water 11.5 totally 25 μ l.Connect under 16 DEG C of water-baths Meet 12h-16h.
(4) convert
Step：
1 adds 100 μ l DH5 α competence bacteriums in linked system, light to mix, ice bath 30min.
2 are put into 42 DEG C of water-baths of preheating, and placing 90s carries out heat shock treatment.
3 ice bath 2min immediately.
4 add LB nutrient solutions of the 1ml without antibiotic, and cultivating 1h for 37 DEG C makes thalline recover.
5 are uniformly coated on the LB flat boards containing antibiotic thalline.
6 culture 24h grow fine.Choosing single bacterium colony carries out bacterium colony PCR, and recombinant plasmid is extracted in nucleic acid electrophoresis checking.Will restructuring Plasmid is imported in BL21 E. coli competents, is saved backup.
Embodiment 2
The present embodiment is the induced expression of D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention and isolates and purifies.
1st, plus 500 μ l recombinate bacterium solution in 50ml LB nutrient solutions.37 DEG C of culture 2.5h, 0.5h is stood at 15 DEG C.Plus 20 again The IPTG of μ l 0.5M, cold-induced culture 24h at 15 DEG C.Zymotic fluid is centrifuged (8000rmp/min, 10min) and is obtained bacterium Body, thalline is redissolved with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (20mmol/L, pH 7.0), and Ultrasonic Cell Disruptor is crushed, from The heart (8000rmp/min, 10min) collects supernatant and obtains crude enzyme liquid.
2nd, the crude enzyme liquid for obtaining step 1 carries out ni-sepharose purification using the operation of the protein purification systems of AKTA avant 150, Elution process is：All put tetra- pipelines of A1, A2, B1, B2 into water, system flow 20ml/min flow velocitys are set, carry out Exhaust.Then system flow 1ml/min, flow path (column position 3), delta pressure are set 0.3rd, pre-pressure 0.5, Gradient 0, inset A1, after filling pillar after water droplet uniformly outflow, balance ten minutes it A1 is put into reference in liquid afterwards, B1 is put into eluent, then is exhausted once, balance 20 minutes, then loading crude enzyme liquid, With high concentration imidazole buffer (solution residing for B1) gradient elution destination protein of 500mM, the albumen on ion column will be adsorbed Elute the enzyme for being purified.Enzyme after purification is freeze-dried standby.
Embodiment 3
The present embodiment is the optimum temperature of D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.With D-ALPHA-Hydroxypropionic acid as substrate, by substrate and pH For 8.0 phosphate buffer under 30-60 DEG C of different temperature conditionss water-bath 15min, determine D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity, really The optimal reactive temperature for determining enzyme is 40 DEG C.
Embodiment 4
The present embodiment is the optimum pH of D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.With D-ALPHA-Hydroxypropionic acid as substrate, by substrate in pH 3-9,40 DEG C of water-bath 15min determine the enzyme activity of enzyme, as a result find the D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity highest under the conditions of pH 8.0.
Embodiment 5
The present embodiment is D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention and the response characteristic of different substrates, is listed in Table 2 below.
Activity of the D-ALPHA-Hydroxypropionic acid oxidizing ferment of table 2 to different substrates
Embodiment 6
Method in the content of the invention splits various racemic ' alpha '-carboxylic esters, as a result as shown in the table：
Table 3 splits the effect of various racemic ' alpha '-carboxylic esters
As can be seen from the above table, when abundant in the reaction time, optically pure (the S)-alpha-hydroxy acid ester of all kinds of height can be obtained, The optics selectivity of 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> 1710
<212> DNA
<213> Brevibacterium linens ATCC 8377
<400> 1
atgttcggac gacagcgtca gcaccaatca caggcggtca gcgccttcat cgacatcatg 60
ggcagtcgtc atgtgctcac ctccgcccgg gcgacggagc ccttcgccaa aggcaatcgc 120
ttcggcggcg gcagtgtgct cgcggtcctc aagccgggca gccttgtcga tatgtggcgg 180
gctctgcagg tctgcgtcga tcacgatctc atcgtcatcc cacagtcggc gaacaccggc 240
ctgaccggag gttcgggtcc cggcgatcag gactacgatc gtgagatcgt catcatctcc 300
accctgcgca tcaaccagat ccatctcatc aacgatgctc gtgaggccgt gtgtctggcc 360
ggatccacgc tgtacgaact cgatgacaag ctggcacctc acgggcgtga acctcattcg 420
gtgatcggtt cgtcgtcgat cggtgcttcg gtcgtcggcg gcatcgcgaa caactccggc 480
ggcagccaga ttcgcaaggg cccggccttc acgaagcacg cgatcttcgc ccgggtcgat 540
gacgagggca aggtcgaact cgtcaatcac ctgggcatca gcctcggcga tgatccggcc 600
catatcctcg acagactcca acgcggcgaa tgggacgaag ccgatgtcac gccgcccccg 660
gaagacagtc tcgataccga atacagcgag cacgtccgcg aaatcaccga ttctcctgcc 720
cgctacaacg ccaacccgaa gttcctccac gaggcgtcag ggtcggccgg caagctcatg 780
gtcttcgccg tgcgcacgcg gaccttcccc aagcagaagg attcgacgac cttctacgtg 840
ggcaccaaca gccccgccga gctcgaggcc ctgcgccggg cgatcctgac ttcggacatg 900
ccgctgccga tctccggtga gtacatggga cggccctcct tcgaccttgc cgagaagtac 960
ggcaaggaca cgttcgtctc cgtcaagcac gccggcagcc gcgaacagat caagctcttc 1020
gcgttgaaga actgggcgaa cggggtgttc gcgaagctgc ccttcttcgg acagaccgtt 1080
gccgattcca ttgcgcaggg tgccttcagc ttgctgccgc agcagctgcc ggaccgcatg 1140
cttgagtacc gcgatcgctt tgaacaccat ctgctcctcg tcgtcagcgg cgaccagaag 1200
cagaggaccg aggagttcct cacgaccttc ttcgccgagg tggggcatga cggtgacttc 1260
ttcatctgca ctgccgatga agctcagtcc gccatgctcc accgcttcgg tgccgccagt 1320
gccgccacac gctacttcaa tctccaccgt gacgagtcct cggagatgat caccttcgac 1380
gttgcgctga cacgcgatga cgaggattgg ctcgaggtgc tgccaacgga gatctcagac 1440
cagctgcaca tcagttccta ttacggccac ttcttctgcc acgtcctcca tcaggaccat 1500
gtggcgaaga agggtgttga cccggtggcg ttgaaaaagc agatgatggc gctgctcgag 1560
gaacgtggag ccgccgtgcc cgccgagcac aactatggtc gcctctacca ggtgcctccg 1620
gagatggagg ctcacttcaa ggagctcgac ccgtgcaacg tcttcaaccc gggggtcggc 1680
gagacctcac cgcacaagaa ctgggcctga 1710
<210> 2
<211> 569
<212> PRT
<213> Brevibacterium linens ATCC 8377
<400> 2
Met Phe Gly Arg Gln Arg Gln His Gln Ser Gln Ala Val Ser Ala Phe
1 5 10 15
Ile Asp Ile Met Gly Ser Arg His Val Leu Thr Ser Ala Arg Ala Thr
20 25 30
Glu Pro Phe Ala Lys Gly Asn Arg Phe Gly Gly Gly Ser Val Leu Ala
35 40 45
Val Leu Lys Pro Gly Ser Leu Val Asp Met Trp Arg Ala Leu Gln Val
50 55 60
Cys Val Asp His Asp Leu Ile Val Ile Pro Gln Ser Ala Asn Thr Gly
65 70 75 80
Leu Thr Gly Gly Ser Gly Pro Gly Asp Gln Asp Tyr Asp Arg Glu Ile
85 90 95
Val Ile Ile Ser Thr Leu Arg Ile Asn Gln Ile His Leu Ile Asn Asp
100 105 110
Ala Arg Glu Ala Val Cys Leu Ala Gly Ser Thr Leu Tyr Glu Leu Asp
115 120 125
Asp Lys Leu Ala Pro His Gly Arg Glu Pro His Ser Val Ile Gly Ser
130 135 140
Ser Ser Ile Gly Ala Ser Val Val Gly Gly Ile Ala Asn Asn Ser Gly
145 150 155 160
Gly Ser Gln Ile Arg Lys Gly Pro Ala Phe Thr Lys His Ala Ile Phe
165 170 175
Ala Arg Val Asp Asp Glu Gly Lys Val Glu Leu Val Asn His Leu Gly
180 185 190
Ile Ser Leu Gly Asp Asp Pro Ala His Ile Leu Asp Arg Leu Gln Arg
195 200 205
Gly Glu Trp Asp Glu Ala Asp Val Thr Pro Pro Pro Glu Asp Ser Leu
210 215 220
Asp Thr Glu Tyr Ser Glu His Val Arg Glu Ile Thr Asp Ser Pro Ala
225 230 235 240
Arg Tyr Asn Ala Asn Pro Lys Phe Leu His Glu Ala Ser Gly Ser Ala
245 250 255
Gly Lys Leu Met Val Phe Ala Val Arg Thr Arg Thr Phe Pro Lys Gln
260 265 270
Lys Asp Ser Thr Thr Phe Tyr Val Gly Thr Asn Ser Pro Ala Glu Leu
275 280 285
Glu Ala Leu Arg Arg Ala Ile Leu Thr Ser Asp Met Pro Leu Pro Ile
290 295 300
Ser Gly Glu Tyr Met Gly Arg Pro Ser Phe Asp Leu Ala Glu Lys Tyr
305 310 315 320
Gly Lys Asp Thr Phe Val Ser Val Lys His Ala Gly Ser Arg Glu Gln
325 330 335
Ile Lys Leu Phe Ala Leu Lys Asn Trp Ala Asn Gly Val Phe Ala Lys
340 345 350
Leu Pro Phe Phe Gly Gln Thr Val Ala Asp Ser Ile Ala Gln Gly Ala
355 360 365
Phe Ser Leu Leu Pro Gln Gln Leu Pro Asp Arg Met Leu Glu Tyr Arg
370 375 380
Asp Arg Phe Glu His His Leu Leu Leu Val Val Ser Gly Asp Gln Lys
385 390 395 400
Gln Arg Thr Glu Glu Phe Leu Thr Thr Phe Phe Ala Glu Val Gly His
405 410 415
Asp Gly Asp Phe Phe Ile Cys Thr Ala Asp Glu Ala Gln Ser Ala Met
420 425 430
Leu His Arg Phe Gly Ala Ala Ser Ala Ala Thr Arg Tyr Phe Asn Leu
435 440 445
His Arg Asp Glu Ser Ser Glu Met Ile Thr Phe Asp Val Ala Leu Thr
450 455 460
Arg Asp Asp Glu Asp Trp Leu Glu Val Leu Pro Thr Glu Ile Ser Asp
465 470 475 480
Gln Leu His Ile Ser Ser Tyr Tyr Gly His Phe Phe Cys His Val Leu
485 490 495
His Gln Asp His Val Ala Lys Lys Gly Val Asp Pro Val Ala Leu Lys
500 505 510
Lys Gln Met Met Ala Leu Leu Glu Glu Arg Gly Ala Ala Val Pro Ala
515 520 525
Glu His Asn Tyr Gly Arg Leu Tyr Gln Val Pro Pro Glu Met Glu Ala
530 535 540
His Phe Lys Glu Leu Asp Pro Cys Asn Val Phe Asn Pro Gly Val Gly
545 550 555 560
Glu Thr Ser Pro His Lys Asn Trp Ala
565

Claims (5)

1. one kind derives from the D-ALPHA-Hydroxypropionic acid oxidizing ferment of extension brevibacterium (Brevibacterium linens), its amino acid sequence It is SEQ ID NO:Shown in 2.
2. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, its nucleotides sequence is classified as SEQ ID NO:Shown in 1.
3. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, its optimal reactive temperature is 40 DEG C, and optimal reaction pH is 8.
4. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, oxidable D-ALPHA-Hydroxypropionic acid, glycolic, D- phenyllactic acids, D- para hydroxybenzenes Lactic acid, D- tartaric acid, D-malic acid, D- mandelic acids, D- danshensus, generate corresponding ketone acid.
5. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, (the R)-alpha-hydroxy acid ester in oxidable racemic ' alpha '-carboxylic esters, tear open Divide and prepare corresponding optical voidness (S)-alpha-hydroxy acid ester and alpha-keto ester, described alpha-hydroxy acid ester is one of following：Danshensu Norbornene ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene lactic acid It is isopropyl ester, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu asarum alcohol ester, phenyllactic acid asarum alcohol ester, right Hydroxyphenyl lactic acid asarum alcohol ester.
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Citations (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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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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无: "《NCBI：WP_009884587.1》", 《NCBI》 *

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