CN106754784A - A kind of oxidizing ferment and its application - Google Patents
A kind of oxidizing ferment and its application Download PDFInfo
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Abstract
The present invention relates to one kind, the acquisition from the D Lactate oxidase genes of Ke Shi citric acid bacillus (Citrobacter koseri) 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
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)
The present invention first from Ke Shi citric acid bacillus (Citrobacter koseri) clonal expression go out it is a kind of new
D-ALPHA-Hydroxypropionic acid oxidizing ferment, 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 Ke Shi citric acid bacillus (Citrobacter koseri) 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：Citrobacter koseri ATCC BAA-895, purchase
From U.S.'s ATCC strain libraries.
2nd, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract Citrobacter koseri ATCC BAA-895 phage gene group STb genes.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 Citrobacter koseri ATCC BAA-895
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 Citrobacter koseri ATCC BAA-895 DNA
Citrobacter koseri ATCC BAA-895 bacterial strains 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'GCCGGGATCCATGAGAAAGCGTATTGTCATACAAA 3'
Primer 2：5'GCCGTCTAGATCGTCGGACGCCGCT 3'
(2) PCR amplifications
Two primers of synthesis more than, the genomic DNA with Citrobacter koseri ATCC BAA-895 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, T4 DNA ligase of carrier DNA 1 μ l, the μ l of sterilized water 11.5 totally 25 μ l.Under 16 DEG C of water-baths
Connection 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 9.0 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 9.0.
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> 1803
<212> DNA
<213> Citrobacter koseri ATCC BAA-895
<400> 1
atgagaaagc gtattgtcat acaaagcgct atgcttagtt ccgatagttt gtcccaccac 60
aaggagtgga gaatgtcttc cattacaacg acagataata aagcctttct taatgagctc 120
acccgtctgg tgggtcattc gcacctgctc accgatcctg caaaaaccgc ccgctaccgc 180
aaggggttcc gttccggtca gggcgaagcg ctggccgtgg tcttccccgg ctcgctgctg 240
gaattgtggc gcgtactgaa cgcctgcgta aacgccgata aaatcattct gatgcaggcc 300
gccaataccg gcctgactga aggttccacg ccgaacggca acgattacga tcgcgaaatt 360
gtcatcatca gcacgctacg cctcgacaag ctgcacgtcc tcagtaaagg cgaacaggtg 420
ctggcatacc caggcaccac gctgtactca ctggaaaaag cgcttaaacc ttttggtcgc 480
gaaccgcact cggttatcgg ttcctcctgt atcggcgcgt cagttattgg cggcatttgc 540
aataactccg gcgggtcgct ggtacaacgc ggcccggcgt ataccgaaat gtcgctgttc 600
gcgcgcattg atgaacaggg caaattacag ctggtgaacc atctgggcat cgaactgggc 660
cagaccccgg aacagatcct cagcacactc gatgatgaac gcatcaaaga tgaagatgtg 720
cgccacgatg gccgccacgc ccacgattac gattacgtga ctcgcgtcag ggacattgag 780
gccgataccc ctgcccgcta taacgctgac ccggatcgcc tgttcgaatc ctccggctgc 840
gcgggtaagc tggcggtgtt cgccgtgcgg ctggacactt tcgaggcgga aaaaaaccag 900
caggtctttt acatcggcac caatcagccc gacgtgctga ccgagattcg tcgtcatatc 960
ctggcgaagt tcgacaacct gccggttgcc ggtgaatata tgcaccgcga tatttacgac 1020
atcgcggaga aatatggcaa agatacgttc ctgatgatcg acaagctcgg caccgacaaa 1080
atgccgttct tctttacgct caaaggccgt accgacgcga tgctggaaaa ggtaaaaatc 1140
ttccgcccgc atttcactga ccgcgcgatg cagaagttcg gccatttatt ccctaaccat 1200
ctgccgccgc gcatgaaaag ctggcgcgac aaatatgagc atcatctgct gttaaaaatg 1260
gcgggagacg gggtggcgga agcacaaagc tggctgaccg agtttttcaa aaccgccgag 1320
ggcgatttct ttgcctgtac cccggaggaa ggcagcaaag cgttcctgca ccgttttgcc 1380
gcggcaggcg cggcgatccg ttatcaggcg gtgcatgccg atgaggtgga ggatattctc 1440
gcactggata tcgccttacg gcgtaacgac accgaatggt atgagcatct gccgccggaa 1500
attgacagcc agctggtgca taaactctat tacggtcatt tcatgtgcta tgtcttccat 1560
caggattaca tcgtgaagaa aggcgtcgat gcccatgcgc tgaaagagca aatgctggaa 1620
ctgctgcgcc agcgcggcgc gcaatatccg gcagagcata acgttggtca tttgtacaaa 1680
gcgccggaga cgctggcgcg tttttatcgt gaaaatgacc ccaccaacag catgaatccg 1740
ggtattggta aaacaagtaa gctgaaattc tggaaagaag cggcgtccga cgagacgcat 1800
tga 1803
<210> 2
<211> 600
<212> PRT
<213> Citrobacter koseri ATCC BAA-895
<400> 2
Met Arg Lys Arg Ile Val Ile Gln Ser Ala Met Leu Ser Ser Asp Ser
1 5 10 15
Leu Ser His His Lys Glu Trp Arg Met Ser Ser Ile Thr Thr Thr Asp
20 25 30
Asn Lys Ala Phe Leu Asn Glu Leu Thr Arg Leu Val Gly His Ser His
35 40 45
Leu Leu Thr Asp Pro Ala Lys Thr Ala Arg Tyr Arg Lys Gly Phe Arg
50 55 60
Ser Gly Gln Gly Glu Ala Leu Ala Val Val Phe Pro Gly Ser Leu Leu
65 70 75 80
Glu Leu Trp Arg Val Leu Asn Ala Cys Val Asn Ala Asp Lys Ile Ile
85 90 95
Leu Met Gln Ala Ala Asn Thr Gly Leu Thr Glu Gly Ser Thr Pro Asn
100 105 110
Gly Asn Asp Tyr Asp Arg Glu Ile Val Ile Ile Ser Thr Leu Arg Leu
115 120 125
Asp Lys Leu His Val Leu Ser Lys Gly Glu Gln Val Leu Ala Tyr Pro
130 135 140
Gly Thr Thr Leu Tyr Ser Leu Glu Lys Ala Leu Lys Pro Phe Gly Arg
145 150 155 160
Glu Pro His Ser Val Ile Gly Ser Ser Cys Ile Gly Ala Ser Val Ile
165 170 175
Gly Gly Ile Cys Asn Asn Ser Gly Gly Ser Leu Val Gln Arg Gly Pro
180 185 190
Ala Tyr Thr Glu Met Ser Leu Phe Ala Arg Ile Asp Glu Gln Gly Lys
195 200 205
Leu Gln Leu Val Asn His Leu Gly Ile Glu Leu Gly Gln Thr Pro Glu
210 215 220
Gln Ile Leu Ser Thr Leu Asp Asp Glu Arg Ile Lys Asp Glu Asp Val
225 230 235 240
Arg His Asp Gly Arg His Ala His Asp Tyr Asp Tyr Val Thr Arg Val
245 250 255
Arg Asp Ile Glu Ala Asp Thr Pro Ala Arg Tyr Asn Ala Asp Pro Asp
260 265 270
Arg Leu Phe Glu Ser Ser Gly Cys Ala Gly Lys Leu Ala Val Phe Ala
275 280 285
Val Arg Leu Asp Thr Phe Glu Ala Glu Lys Asn Gln Gln Val Phe Tyr
290 295 300
Ile Gly Thr Asn Gln Pro Asp Val Leu Thr Glu Ile Arg Arg His Ile
305 310 315 320
Leu Ala Lys Phe Asp Asn Leu Pro Val Ala Gly Glu Tyr Met His Arg
325 330 335
Asp Ile Tyr Asp Ile Ala Glu Lys Tyr Gly Lys Asp Thr Phe Leu Met
340 345 350
Ile Asp Lys Leu Gly Thr Asp Lys Met Pro Phe Phe Phe Thr Leu Lys
355 360 365
Gly Arg Thr Asp Ala Met Leu Glu Lys Val Lys Ile Phe Arg Pro His
370 375 380
Phe Thr Asp Arg Ala Met Gln Lys Phe Gly His Leu Phe Pro Asn His
385 390 395 400
Leu Pro Pro Arg Met Lys Ser Trp Arg Asp Lys Tyr Glu His His Leu
405 410 415
Leu Leu Lys Met Ala Gly Asp Gly Val Ala Glu Ala Gln Ser Trp Leu
420 425 430
Thr Glu Phe Phe Lys Thr Ala Glu Gly Asp Phe Phe Ala Cys Thr Pro
435 440 445
Glu Glu Gly Ser Lys Ala Phe Leu His Arg Phe Ala Ala Ala Gly Ala
450 455 460
Ala Ile Arg Tyr Gln Ala Val His Ala Asp Glu Val Glu Asp Ile Leu
465 470 475 480
Ala Leu Asp Ile Ala Leu Arg Arg Asn Asp Thr Glu Trp Tyr Glu His
485 490 495
Leu Pro Pro Glu Ile Asp Ser Gln Leu Val His Lys Leu Tyr Tyr Gly
500 505 510
His Phe Met Cys Tyr Val Phe His Gln Asp Tyr Ile Val Lys Lys Gly
515 520 525
Val Asp Ala His Ala Leu Lys Glu Gln Met Leu Glu Leu Leu Arg Gln
530 535 540
Arg Gly Ala Gln Tyr Pro Ala Glu His Asn Val Gly His Leu Tyr Lys
545 550 555 560
Ala Pro Glu Thr Leu Ala Arg Phe Tyr Arg Glu Asn Asp Pro Thr Asn
565 570 575
Ser Met Asn Pro Gly Ile Gly Lys Thr Ser Lys Leu Lys Phe Trp Lys
580 585 590
Glu Ala Ala Ser Asp Glu Thr His
595 600
Claims (5)
1. one kind derives from the D-ALPHA-Hydroxypropionic acid oxidizing ferment of Ke Shi citric acid bacillus (Citrobacter koseri), 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 9.
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1125961A (en) * | 1993-06-25 | 1996-07-03 | 纳幕尔杜邦公司 | Process for the preparation of pyruvic acid |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1125961A (en) * | 1993-06-25 | 1996-07-03 | 纳幕尔杜邦公司 | Process for the preparation of pyruvic acid |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| NCBI: "ABV11815.1：hypothetical protein CKO_00661 [Citrobacter koseri ATCC BAA-895]", 《NCBI》 * |
| UNIPROT: "A8AEA2", 《UNIPROT》 * |
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