CN106754788A - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754788A
CN106754788A CN201710006572.4A CN201710006572A CN106754788A CN 106754788 A CN106754788 A CN 106754788A CN 201710006572 A CN201710006572 A CN 201710006572A CN 106754788 A CN106754788 A CN 106754788A
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acid
alpha
ester
leu
oxidizing ferment
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CN106754788B (en
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蔡宇杰
王亚红
曹憬
白亚军
郑晓晖
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Zhuohong Chaoyuan Biotechnology Zhengzhou Co ltd
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Jiangnan University
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    • 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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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
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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 present invention relates to one kind from morganella morganii rub root subspecies (Morganella morganii subsp.morganii) D Lactate oxidase genes acquisition and its clonal expression, belong to bioengineering field.Its substrate specificity is disclosed, while the D LOs can aoxidize (R) alpha hydroxy acid ester, the preparation of optical voidness (S) alpha hydroxy acid ester is can be applied to.

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)
During the present invention rubs root subspecies (Morganella morganii subsp.morganii) from morganella morganii first Clonal expression goes out a kind of new D-ALPHA-Hydroxypropionic acid oxidizing ferment, and the enzyme can not only aoxidize (R)-alpha-hydroxy acid, and can aoxidize (R)- Alpha-hydroxy acid ester, reaction back reaction compared with the reaction that NAD (NADP) is participated in for the lactic dehydrogenase of coenzyme is atomic weak, can apply In the preparation of optical voidness (S)-alpha-hydroxy acid ester and (S)-alpha-hydroxy acid.
The content of the invention
The present invention is cloned in rubbing root subspecies (Morganella morganii subsp.morganii) from morganella morganii A kind of gene of the D-ALPHA-Hydroxypropionic acid oxidizing ferment with FAD as coenzyme has been obtained, using colibacillus engineering heterogenous expression, it has been disclosed Related enzymatic property, and carried 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：Morganella morganii subsp.morganii ATCC 258297, purchased 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 Morganella morganii subsp.morganii ATCC 258297.If Meter specific primer, using PCR method, amplifies D-ALPHA-Hydroxypropionic acid oxidase gene total length encoder block sequence.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：From Morganella morganii subsp.morganii ATCC258297 Clonal expression goes out a kind of new D-ALPHA-Hydroxypropionic acid oxidizing ferment, and the enzyme 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, 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 Morganella morganii subsp.morganii ATCC 258297DNA
The bacterial strains of Morganella morganii subsp.morganii ATCC 258297 are cultivated in LB culture mediums 12h, 12,000rmp/min centrifugation 10min obtain thalline, are pressed using bacterial genomes DNA extraction agents box (TaKaRa companies) Phage gene group STb gene is extracted according to its operation, refrigerator is put 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'GCCGGGATCCATGATCAACAATCAACAACCTGACG 3'
Primer 2：5'GCCGTCTAGATTACTGATTATACTTTCCGCAGCCA 3'
(2) PCR amplifications
Two primers of synthesis more than, with Morganella morganii subsp.morganii ATCC258297 Genomic DNA for template enter 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 6.0 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 45 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 3-9,45 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 6.0.
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 table 2D- LOs 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> 1746
<212> DNA
<213> Morganella morganii subsp. morganii ATCC 258297
<400> 1
atgatcaaca atcaacaacc tgacggcgca cgtcttattc aggcactgac agatattgtc 60
agtcacaaac acattttaac agagccgcgt aagacggagc gataccgcaa aggcttccgt 120
tccggagaag gcagcgcgct ggcggtggtg ttcccgggca cgctgtatga actgtggcag 180
gtgtttaaag ccgctgttga agctgacaga attgtgatca tgcaggcggc caataccggt 240
ctgaccgaag gctccacccc gagcggggat gattatgacc gcgaaattat tattatcagc 300
acactgcgcc tggataaaat tcaggtactg acaaaacagc agcaggtagt ggcactgccg 360
ggcagcaccc tgtggcacct ggaaaaagtc ctgaaaccgc tgggtcgtga gccgcattca 420
gtgatcggtt cttcctgtat cggggcttcc gtggtcggcg ggatttgtaa taactccggc 480
ggttcactgg tgcagcgcgg tcccgcttat accgaactgg cgctgtacgg ccgcgttaat 540
gcggacggca gcgcagaact ggttaaccat ctggggattg atttaggatc cacgccggaa 600
gaaattctga attgtctgga ataccgtcag tatcaggatg cggatattga ggacacggat 660
aaaccggcct ctgaccatga ttatcatcag cgtatccgtg atgtggatgc ggacacccca 720
tcccgtttca acgccgacga acgccgcctg tttgaagcct ccggctgtgc cggtaagctg 780
gtggtgtttg cagtgcgcct tgataccttc ccgtctcagg gagagagcaa agttttctat 840
atcggcacca atacgccttc tgtgctggaa gatatccgcc gtcatattct tgcggagttt 900
gagcatctgc cggtggccgg tgaatatatg caccgcgaat gttatgacat cgcaaaggtc 960
tacggcaaag acagcttcct gatgattgat aaactcggca ctgagaaaat gccgaagctg 1020
ttcactatca aagggcggat ggatgctgtt ttcaataaaa tcccgctgct gccgaaaaat 1080
ctgattgacc gcacaatgca gttattcagt catctgtggc cgaaccatct gccgcgccgg 1140
atggaagagt accgcgataa atatgagcat cacctgatgc tgaaaatggc cggagagggg 1200
attgatgaag cccgtaactg gctgagcact ttctttgaca atgcagaggg tgctttcttt 1260
gagtgtgatg ccaaagaggg ggcggatgct ttcctgcacc gctttgtggc cgccggtgcc 1320
gccatccgct atcacgcggt caacagcagc aaagtggaag atatcctcgc actggatatt 1380
gcactgcgcc gtaatgacca ggcgtggctg gaagtcctgc cgccggaaat tgagagcaaa 1440
ctggtgcata aactctacta cggacatttt ttatgccacg ttttccatca ggactatatt 1500
gtgaagaaag gcgtggatcc gcatgcgctg aaagaggaaa tgctggaaat cctgaacacg 1560
cgcggggcgg aatatccggc agagcataat gtcggccatc tgtataaagc gaaacctcag 1620
ctgaaagcct tctataaagc caccgatccg accaacaccc tgaacccggg actgggcaaa 1680
accagtaaat taaaatattg gggggaaggg cacgaaggat gtggctgcgg aaagtataat 1740
cagtaa 1746
<210> 2
<211> 581
<212> PRT
<213> Morganella morganii subsp. morganii ATCC 258297
<400> 2
Met Ile Asn Asn Gln Gln Pro Asp Gly Ala Arg Leu Ile Gln Ala Leu
1 5 10 15
Thr Asp Ile Val Ser His Lys His Ile Leu Thr Glu Pro Arg Lys Thr
20 25 30
Glu Arg Tyr Arg Lys Gly Phe Arg Ser Gly Glu Gly Ser Ala Leu Ala
35 40 45
Val Val Phe Pro Gly Thr Leu Tyr Glu Leu Trp Gln Val Phe Lys Ala
50 55 60
Ala Val Glu Ala Asp Arg Ile Val Ile Met Gln Ala Ala Asn Thr Gly
65 70 75 80
Leu Thr Glu Gly Ser Thr Pro Ser Gly Asp Asp Tyr Asp Arg Glu Ile
85 90 95
Ile Ile Ile Ser Thr Leu Arg Leu Asp Lys Ile Gln Val Leu Thr Lys
100 105 110
Gln Gln Gln Val Val Ala Leu Pro Gly Ser Thr Leu Trp His Leu Glu
115 120 125
Lys Val Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser
130 135 140
Ser Cys Ile Gly Ala Ser Val Val Gly Gly Ile Cys Asn Asn Ser Gly
145 150 155 160
Gly Ser Leu Val Gln Arg Gly Pro Ala Tyr Thr Glu Leu Ala Leu Tyr
165 170 175
Gly Arg Val Asn Ala Asp Gly Ser Ala Glu Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly Ser Thr Pro Glu Glu Ile Leu Asn Cys Leu Glu Tyr
195 200 205
Arg Gln Tyr Gln Asp Ala Asp Ile Glu Asp Thr Asp Lys Pro Ala Ser
210 215 220
Asp His Asp Tyr His Gln Arg Ile Arg Asp Val Asp Ala Asp Thr Pro
225 230 235 240
Ser Arg Phe Asn Ala Asp Glu Arg Arg Leu Phe Glu Ala Ser Gly Cys
245 250 255
Ala Gly Lys Leu Val Val Phe Ala Val Arg Leu Asp Thr Phe Pro Ser
260 265 270
Gln Gly Glu Ser Lys Val Phe Tyr Ile Gly Thr Asn Thr Pro Ser Val
275 280 285
Leu Glu Asp Ile Arg Arg His Ile Leu Ala Glu Phe Glu His Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Glu Cys Tyr Asp Ile Ala Lys Val
305 310 315 320
Tyr Gly Lys Asp Ser Phe Leu Met Ile Asp Lys Leu Gly Thr Glu Lys
325 330 335
Met Pro Lys Leu Phe Thr Ile Lys Gly Arg Met Asp Ala Val Phe Asn
340 345 350
Lys Ile Pro Leu Leu Pro Lys Asn Leu Ile Asp Arg Thr Met Gln Leu
355 360 365
Phe Ser His Leu Trp Pro Asn His Leu Pro Arg Arg Met Glu Glu Tyr
370 375 380
Arg Asp Lys Tyr Glu His His Leu Met Leu Lys Met Ala Gly Glu Gly
385 390 395 400
Ile Asp Glu Ala Arg Asn Trp Leu Ser Thr Phe Phe Asp Asn Ala Glu
405 410 415
Gly Ala Phe Phe Glu Cys Asp Ala Lys Glu Gly Ala Asp Ala Phe Leu
420 425 430
His Arg Phe Val Ala Ala Gly Ala Ala Ile Arg Tyr His Ala Val Asn
435 440 445
Ser Ser Lys Val Glu Asp Ile Leu Ala Leu Asp Ile Ala Leu Arg Arg
450 455 460
Asn Asp Gln Ala Trp Leu Glu Val Leu Pro Pro Glu Ile Glu Ser Lys
465 470 475 480
Leu Val His Lys Leu Tyr Tyr Gly His Phe Leu Cys His Val Phe His
485 490 495
Gln Asp Tyr Ile Val Lys Lys Gly Val Asp Pro His Ala Leu Lys Glu
500 505 510
Glu Met Leu Glu Ile Leu Asn Thr Arg Gly Ala Glu Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Leu Tyr Lys Ala Lys Pro Gln Leu Lys Ala Phe
530 535 540
Tyr Lys Ala Thr Asp Pro Thr Asn Thr Leu Asn Pro Gly Leu Gly Lys
545 550 555 560
Thr Ser Lys Leu Lys Tyr Trp Gly Glu Gly His Glu Gly Cys Gly Cys
565 570 575
Gly Lys Tyr Asn Gln
580

Claims (5)

1. one kind from morganella morganii rub root subspecies (Morganella morganii subsp.morganii) D- breast Acid oxidase, its amino acid sequence 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 45 DEG C, and optimal reaction pH is 6.
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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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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