CN106754787A - A kind of oxidizing ferment and its application - Google Patents
A kind of oxidizing ferment and its application Download PDFInfo
- Publication number
- CN106754787A CN106754787A CN201710006567.3A CN201710006567A CN106754787A CN 106754787 A CN106754787 A CN 106754787A CN 201710006567 A CN201710006567 A CN 201710006567A CN 106754787 A CN106754787 A CN 106754787A
- Authority
- CN
- China
- Prior art keywords
- alpha
- acid
- ester
- hydroxy
- oxidizing ferment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Acquisition and its clonal expression the present invention relates to a kind of L alpha hydroxy acid oxidase genes from Carnimonas nigrificans, belong to bioengineering field.Its substrate specificity is disclosed, while the L alpha hydroxy acids oxidizing ferment can aoxidize (S) alpha hydroxy acid ester, the preparation of optical voidness (R) alpha hydroxy acid ester is can be applied to.
Description
Technical field
A kind of L- alpha-hydroxy acids 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 technology
L- alpha-hydroxy acids oxidizing ferment (L- α-hydroxyacid oxidase) is a kind of dehydrogenase with FMN (FAD) as coenzyme
(Aliphatic l-α-hydroxyacid oxidase from rat livers purification and
properties.Biochimica et Biophysica Acta(BBA)-Enzymology 1968,167:9-22), generally
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).Can be used to determine lactic acid in biology sensor
Content, or oxidation Pfansteihl production pyruvic acid.Also have and be used for the preparation (Chinese patent of 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), mycobacterium smegmatis
(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
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 Carnimonas nigrificans goes out a kind of new L- alpha-hydroxy acids oxidation to the present invention first
Enzyme, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, and can aoxidize (S)-alpha-hydroxy acid ester, can be applied to optical voidness (R)-α-hydroxyl
The preparation of acid esters and (R)-alpha-hydroxy acid.
The content of the invention
Present invention clone from Carnimonas nigrificans has obtained a kind of gene of L- alpha-hydroxy acids oxidizing ferment, profit
Colibacillus engineering heterogenous expression is used, its related enzymatic property is disclosed, and carried out application study.
Technical scheme is as follows：
1st, bacterial strain
The source bacterial strain of L- alpha-hydroxy acids oxidase gene of the present invention is：Carnimonas nigrificans ATCC BAA-
78, purchased from U.S.'s ATCC strain libraries.
2nd, the clone of L- alpha-hydroxy acids oxidase gene
Extract Carnimonas nigrificans ATCC BAA-78 phage gene group STb genes.Design specific primer,
Using PCR method, L- alpha-hydroxy acid oxidase gene total length encoder block sequences are amplified.And construction recombination plasmid.
3rd, L- alpha-hydroxy acids 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 L- alpha-hydroxy acids oxidizing ferment
Influence with Pfansteihl as substrate research pH to L- alpha-hydroxy acids oxidizing ferment enzyme activity of the present invention.
Influence with Pfansteihl as substrate research temperature to L- alpha-hydroxy acids oxidizing ferment enzyme activity of the present invention.
The substrate specificity analysis of L- alpha-hydroxy acid oxidizing ferment：Substrate used has Pfansteihl, glycolic, L- phenyllactic acids, L-
Tartaric acid, L MALIC ACID, L- para hydroxybenzenes lactic acid, L- danshensus, L- mandelic acids.
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, L- alpha-hydroxy acids 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.(S) Alpha-hydroxy in-alpha-hydroxy acid ester is dehydrogenated and is oxidized to corresponding 2-ketoacid
Ester, (R)-alpha-hydroxy acid ester is not oxidized.
The optical purity of product (R)-alpha-hydroxy acid ester 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 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：Clone has obtained one from Carnimonas nigrificans ATCC BAA-78
L- alpha-hydroxy acid oxidizing ferment is planted, 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, with important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of L- alpha-hydroxy acids oxidase gene of the present invention and colibacillus engineering build.
1st, the extraction of Carnimonas nigrificans ATCC BAA-78DNA
Carnimonas nigrificans ATCC BAA-78 bacterial strains are cultivated into 12h in LB culture mediums, 12,
000rmp/min centrifugations 10min obtains thalline, is operated according to it using bacterial genomes DNA extraction agents box (TaKaRa companies)
Phage gene group STb gene is extracted, 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 L- alpha-hydroxy acids oxidase gene
(1) design of primers
Designing primer sequence is：
Primer 1：5'GCCGGGATCCATGAGTACTCCGCTAACTCCACGTC 3'
Primer 2：5'GCCGTCTAGACGTCCCGCGCCGATAG 3'
(2) PCR amplifications
Two primers of synthesis more than, with the genomic DNA of Carnimonas nigrificans ATCC BAA-78
For template 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 L- alpha-hydroxy acids 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, systemflow 20ml/min flow velocitys are set, arranged
Gas.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 L- alpha-hydroxy acids oxidizing ferment of the present invention.With Pfansteihl as substrate, by substrate with
PH be 5.0 phosphate buffer under 30-60 DEG C of different temperature conditionss water-bath 15min, determine L- alpha-hydroxy acid oxidizing ferment enzyme
Living, the optimal reactive temperature for determining enzyme is 35 DEG C.
Embodiment 4
The present embodiment is the optimum pH of L- alpha-hydroxy acids oxidizing ferment of the present invention.With Pfansteihl as substrate, substrate is existed
As a result pH 3-9,35 DEG C of enzyme activity of water-bath 15min measure enzymes find that L- alpha-hydroxy acids oxidizing ferment enzyme activity is most under the conditions of pH 5.0
It is high.
Embodiment 5
The present embodiment is that L- alpha-hydroxy acids oxidizing ferment of the present invention is listed in Table 2 below from the response characteristic of different substrates.
Activity of the L- alpha-hydroxy acids 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 R)-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> 1188
<212> DNA
<213> Carnimonas nigrificans ATCC BAA-78
<400> 1
atgagtactc cgctaactcc acgtcgcagc gatcaaaagc gttatctgaa tctggacgac 60
tacagcgcgg ctgcacagcg cctgttgcca cgggcgctgt tcgcctacgt ttcaggctcg 120
gcaggcgacg gtgaaacact ggctgcgaac cgcgatgcct tcaagcagtg ggcgctaata 180
ccgcagcagc tgcgcggcgt cgcttcacga agtgccgcca tcgagctact cggccagcgt 240
tttgccatgc cgattggcgt gtcggcattc ggcgcggcgg cggttttggg ctataacgct 300
gatatcgcga tggcggcagc ggcatataac gcaaatgtgc cttatcagct gagtgccaac 360
tccattacgc ccatggaaga ggtaatcaag gtgaatcccg aggcgtggtt cgccgcctat 420
ctgccagacg atgcagcgct gatcagaggc gttgtcgagc gcgttagcaa cgcaggcttc 480
aagactctgg taattacggt cgatgtgccg gtggctgccc gacgcgagcc ggaaacgcgg 540
gctggctacg ccatgccgtt caaggcatcg gcgcgcttgg ggtttgatat gctccgccat 600
ccgcgctggg tgctgagccg cttcactcgt accctgctgc ggcgcgggat tccgcacatt 660
gaaaacgtta ccccccagcg tggccccagt atctttgctt ccaacacagg gtcgatcggt 720
ggagcggcca acttcaactg ggacaatatt cgtgctattc gcgcacagtg gcccggcaag 780
ctgctgctga agggtattct ttccgaacgc gatgcgctaa cagcacagga gattggcgtc 840
gatggcgtga ttgtctcgaa tcatggcgcc cgtttaagcg attcatccat cacaccgctg 900
gaagccctgc caaacatccg cgcagcctgc cctgagctga cggttctgct cgatggcggc 960
gtgcggcgtg ccggggatgc gttaaaggcg atcgcgctgg gggcagatgg cgtgatgatt 1020
ggccgtccac tgttttacgc caccatcctt ggtggccggc gcggcctcgt tcatgcgctg 1080
catttgctga gcgcagaact ggatcgggag atgggctttc atggcttgct aaacgttaac 1140
gaagtacgtg aggaagatcg gctctatcgg cgcgggacgc ttgcctaa 1188
<210> 2
<211> 395
<212> PRT
<213> Carnimonas nigrificans ATCC BAA-78
<400> 2
Met Ser Thr Pro Leu Thr Pro Arg Arg Ser Asp Gln Lys Arg Tyr Leu
1 5 10 15
Asn Leu Asp Asp Tyr Ser Ala Ala Ala Gln Arg Leu Leu Pro Arg Ala
20 25 30
Leu Phe Ala Tyr Val Ser Gly Ser Ala Gly Asp Gly Glu Thr Leu Ala
35 40 45
Ala Asn Arg Asp Ala Phe Lys Gln Trp Ala Leu Ile Pro Gln Gln Leu
50 55 60
Arg Gly Val Ala Ser Arg Ser Ala Ala Ile Glu Leu Leu Gly Gln Arg
65 70 75 80
Phe Ala Met Pro Ile Gly Val Ser Ala Phe Gly Ala Ala Ala Val Leu
85 90 95
Gly Tyr Asn Ala Asp Ile Ala Met Ala Ala Ala Ala Tyr Asn Ala Asn
100 105 110
Val Pro Tyr Gln Leu Ser Ala Asn Ser Ile Thr Pro Met Glu Glu Val
115 120 125
Ile Lys Val Asn Pro Glu Ala Trp Phe Ala Ala Tyr Leu Pro Asp Asp
130 135 140
Ala Ala Leu Ile Arg Gly Val Val Glu Arg Val Ser Asn Ala Gly Phe
145 150 155 160
Lys Thr Leu Val Ile Thr Val Asp Val Pro Val Ala Ala Arg Arg Glu
165 170 175
Pro Glu Thr Arg Ala Gly Tyr Ala Met Pro Phe Lys Ala Ser Ala Arg
180 185 190
Leu Gly Phe Asp Met Leu Arg His Pro Arg Trp Val Leu Ser Arg Phe
195 200 205
Thr Arg Thr Leu Leu Arg Arg Gly Ile Pro His Ile Glu Asn Val Thr
210 215 220
Pro Gln Arg Gly Pro Ser Ile Phe Ala Ser Asn Thr Gly Ser Ile Gly
225 230 235 240
Gly Ala Ala Asn Phe Asn Trp Asp Asn Ile Arg Ala Ile Arg Ala Gln
245 250 255
Trp Pro Gly Lys Leu Leu Leu Lys Gly Ile Leu Ser Glu Arg Asp Ala
260 265 270
Leu Thr Ala Gln Glu Ile Gly Val Asp Gly Val Ile Val Ser Asn His
275 280 285
Gly Ala Arg Leu Ser Asp Ser Ser Ile Thr Pro Leu Glu Ala Leu Pro
290 295 300
Asn Ile Arg Ala Ala Cys Pro Glu Leu Thr Val Leu Leu Asp Gly Gly
305 310 315 320
Val Arg Arg Ala Gly Asp Ala Leu Lys Ala Ile Ala Leu Gly Ala Asp
325 330 335
Gly Val Met Ile Gly Arg Pro Leu Phe Tyr Ala Thr Ile Leu Gly Gly
340 345 350
Arg Arg Gly Leu Val His Ala Leu His Leu Leu Ser Ala Glu Leu Asp
355 360 365
Arg Glu Met Gly Phe His Gly Leu Leu Asn Val Asn Glu Val Arg Glu
370 375 380
Glu Asp Arg Leu Tyr Arg Arg Gly Thr Leu Ala
385 390 395
Claims (5)
1. a kind of L- alpha-hydroxy acid oxidizing ferment from Carnimonas nigrificans, its amino acid sequence is SEQ ID
NO:Shown in 2.
2. L- alpha-hydroxy acids oxidizing ferment according to claim 1, its nucleotides sequence is classified as SEQ ID NO:Shown in 1.
3. L- alpha-hydroxy acids oxidizing ferment according to claim 1, its optimal reactive temperature is 35 DEG C, and optimal reaction pH is 5.
4. L- alpha-hydroxy acids oxidizing ferment according to claim 1, oxidable Pfansteihl, glycolic, L- phenyllactic acids, L- are to hydroxyl
Phenyllactic acid, L-TARTARIC ACID, L MALIC ACID, L- mandelic acids, L- danshensus, generate corresponding ketone acid.
5. L- alpha-hydroxy acids oxidizing ferment according to claim 1, (the S)-alpha-hydroxy acid ester in oxidable racemic ' alpha '-carboxylic esters,
Fractionation prepares corresponding optical voidness (R)-alpha-hydroxy acid ester and alpha-keto ester, and described alpha-hydroxy acid ester is one of following：The red sage root
Plain norbornene ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene breast
Isopropyl propionate, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu asarum alcohol ester, phenyllactic acid asarum alcohol ester,
Para hydroxybenzene lactic acid asarum alcohol ester.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710006567.3A CN106754787B (en) | 2017-01-05 | 2017-01-05 | A kind of oxidizing ferment and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710006567.3A CN106754787B (en) | 2017-01-05 | 2017-01-05 | A kind of oxidizing ferment and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754787A true CN106754787A (en) | 2017-05-31 |
| CN106754787B CN106754787B (en) | 2019-11-15 |
Family
ID=58949569
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710006567.3A Active CN106754787B (en) | 2017-01-05 | 2017-01-05 | A kind of oxidizing ferment and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754787B (en) |
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 |
-
2017
- 2017-01-05 CN CN201710006567.3A patent/CN106754787B/en active Active
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 |
|---|
| ETHAN S.SIMON ET AL.: "D-Lactate Dehydrogenase", 《APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》 * |
| NCBI: "WP_025733596.1", 《GENBANK》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754787B (en) | 2019-11-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106591250B (en) | A kind of oxidizing ferment and its application | |
| CN106754785B (en) | A kind of oxidizing ferment and its application | |
| CN106754801B (en) | A kind of oxidizing ferment and its application | |
| CN106701700A (en) | Oxidase and application thereof | |
| CN106754778A (en) | A kind of oxidizing ferment and its application | |
| CN106754790B (en) | A kind of oxidizing ferment and its application | |
| CN106754800B (en) | A kind of oxidizing ferment and its application | |
| CN106754799B (en) | A kind of oxidizing ferment and its application | |
| CN106754797B (en) | A kind of oxidizing ferment and its application | |
| CN106754794B (en) | A kind of oxidizing ferment and its application | |
| CN106754787B (en) | A kind of oxidizing ferment and its application | |
| CN106754789B (en) | A kind of oxidizing ferment and its application | |
| CN106701706B (en) | A kind of oxidizing ferment and its application | |
| CN106754796B (en) | A kind of oxidizing ferment and its application | |
| CN106754798B (en) | A kind of oxidizing ferment and its application | |
| CN106701703B (en) | A kind of oxidizing ferment and its application | |
| CN106754786B (en) | A kind of oxidizing ferment and its application | |
| CN106754795B (en) | A kind of oxidizing ferment and its application | |
| CN106701704A (en) | Oxidase and application thereof | |
| CN106701702B (en) | A kind of oxidizing ferment and its application | |
| CN106591251A (en) | Oxidase and application thereof | |
| CN106754784A (en) | A kind of oxidizing ferment and its application | |
| CN106754793A (en) | A kind of oxidizing ferment and its application | |
| CN106754791A (en) | A kind of oxidizing ferment and its application | |
| CN106754782A (en) | A kind of oxidizing ferment and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230216 Address after: Floor 20, Unit 2, Building 1, Jinlan West Jingyuan, No. 56, Shinan Road, Science Avenue, High-tech Industrial Development Zone, Zhengzhou City, Henan Province, 450000 Patentee after: Zhuohong Chaoyuan Biotechnology (Zhengzhou) Co.,Ltd. Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |
|
| TR01 | Transfer of patent right |