CN106754789A - A kind of oxidizing ferment and its application - Google Patents
A kind of oxidizing ferment and its application Download PDFInfo
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- CN106754789A CN106754789A CN201710006577.7A CN201710006577A CN106754789A CN 106754789 A CN106754789 A CN 106754789A CN 201710006577 A CN201710006577 A CN 201710006577A CN 106754789 A CN106754789 A CN 106754789A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- 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
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/50—Polycarboxylic acids having keto groups, e.g. 2-ketoglutaric acid
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- 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
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- 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)
Abstract
The present invention relates to one kind, the acquisition from the L alpha hydroxy acid oxidase genes of extension brevibacterium (Brevibacterium linens) and its clonal expression, 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)
Clonal expression goes out a kind of new L- to the present invention from extension brevibacterium (Brevibacterium linens) first
Alpha-hydroxy acid oxidizing ferment, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, and can aoxidize (S)-alpha-hydroxy acid ester, can be applied to optics
The preparation of pure (R)-alpha-hydroxy acid ester and (R)-alpha-hydroxy acid.
The content of the invention
Present invention clone from extension brevibacterium (Brevibacterium linens) has obtained a kind of L- alpha-hydroxy acids oxidation
The gene of enzyme, using colibacillus engineering heterogenous expression, discloses its related enzymatic property, 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：Brevibacterium linens ATCC 8377,
Purchased from U.S.'s ATCC strain libraries.
2nd, the clone of L- alpha-hydroxy acids oxidase gene
Extract the phage gene group STb genes of Brevibacterium linens ATCC 8377.Design specific primer, should
With PCR method, alpha-hydroxy acid oxidase gene total length encoder block sequence is 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 kind from Brevibacterium linens ATCC 8377
L- alpha-hydroxy acid oxidizing ferment, the enzyme can aoxidize (S)-alpha-hydroxy acid and (S)-alpha-hydroxy acid ester, can be used for prepare with scale chiral purity
(R)-alpha-hydroxy acid ester, 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 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 L- alpha-hydroxy acids oxidase gene
(1) design of primers
Designing primer sequence is：
Primer 1：5'GCCGGGATCCATGAAACGTCGACTGCCTGATCTC 3'
Primer 2：5'GCCGTCTAGAGAGTCGGCCGGCAGCTCC 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 obtains this gene order, such as SEQ ID NO after sending Hua Da gene sequencing:Shown in 1.According to the gene
The amino acid sequence that sequence is obtained such as SEQ ID NO: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 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, 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 L- alpha-hydroxy acids oxidizing ferment of the present invention.With Pfansteihl as substrate, by substrate with
PH be 6.0 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 40 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 3-9,40 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 6.0
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> 1254
<212> DNA
<213> Brevibacterium linens ATCC 8377
<400> 1
atgaaacgtc gactgcctga tctcaaagag cttgccccct tgctgcagtt cgacctgcct 60
cgcctcgacc gggtcgccgc caggctggaa gcggccgcag acatctggga cctccgccgc 120
atcgccaagc gcgtgactcc tacagccccg ttcgactatg tcgacggtgc cgcactggac 180
gagcggacat tggcgaagaa ccgggacgct ctgggcaacg tcgagcttct tcctcgcatc 240
ctccacggag tcgacgcccc ggatacgtcg acgacgatcg ccggcgcccg tgttcgactg 300
cccttcggca tcgctccgac cggttacaca cgcatgatgc actccgaagg cgaagtcgcc 360
ggagtgcgtg cggccgcgaa ggccggaatc ccgttctcgc tgtcgacgat gggaacgacc 420
tcggtcgaag acgtcgcgca ggcggcaccg gattccaccc ggtggttcca gctctacctg 480
tggaaggacc gacagcgcag cctcgacctg atccagcgag ccgccgccag cggatacaag 540
actctgctgg tcaccgtcga caccccgatc actggccagc gtctgcgcga tcaccgcaac 600
ggtctgacga tcccgccccg gctgacgctg ggaacgattc tcgacgcctc ctaccggccc 660
ggctggtggt tcaacttcct caccaccgaa ccgccgaagt acgcgtcact gtcgaacacc 720
tctcagtccc tggcggagat gacgcagacg atgttcgacc cgacccttga cctcgaggac 780
ctgcggtgga tccgcgaaca gtggaacgga cgactgttcg tcaagggcat tctcaccgcc 840
gacgatgccc gacgtgccca atccgtcgga gccgatgggc tcgtcgtgtc caaccacggc 900
ggtcgccaac tcgatcgggc cccggactcg ctgacctcac tggccgaagt ccgcgcggag 960
gtcgggccgg agatggagct gatcttcgac tccgggatca tgtccggcac cgatgtcgtc 1020
gccgcgctgt gtgccggagc ggacttcgtg ctcatcggtc gggcgtatct atacgggctc 1080
atggccggcg gtcagcgagg cgtcgagagg gccatcgcac tcatccaaca ggagatcctc 1140
accgcgatgg ggctcatggg tgcccgcagc atctctgatc tcggccccga actcgttcgt 1200
ggcctgcccc aggcaccgaa cacagatcag gagctgccgg ccgactccat gtga 1254
<210> 2
<211> 417
<212> PRT
<213> Brevibacterium linens ATCC 8377
<400> 2
Met Lys Arg Arg Leu Pro Asp Leu Lys Glu Leu Ala Pro Leu Leu Gln
1 5 10 15
Phe Asp Leu Pro Arg Leu Asp Arg Val Ala Ala Arg Leu Glu Ala Ala
20 25 30
Ala Asp Ile Trp Asp Leu Arg Arg Ile Ala Lys Arg Val Thr Pro Thr
35 40 45
Ala Pro Phe Asp Tyr Val Asp Gly Ala Ala Leu Asp Glu Arg Thr Leu
50 55 60
Ala Lys Asn Arg Asp Ala Leu Gly Asn Val Glu Leu Leu Pro Arg Ile
65 70 75 80
Leu His Gly Val Asp Ala Pro Asp Thr Ser Thr Thr Ile Ala Gly Ala
85 90 95
Arg Val Arg Leu Pro Phe Gly Ile Ala Pro Thr Gly Tyr Thr Arg Met
100 105 110
Met His Ser Glu Gly Glu Val Ala Gly Val Arg Ala Ala Ala Lys Ala
115 120 125
Gly Ile Pro Phe Ser Leu Ser Thr Met Gly Thr Thr Ser Val Glu Asp
130 135 140
Val Ala Gln Ala Ala Pro Asp Ser Thr Arg Trp Phe Gln Leu Tyr Leu
145 150 155 160
Trp Lys Asp Arg Gln Arg Ser Leu Asp Leu Ile Gln Arg Ala Ala Ala
165 170 175
Ser Gly Tyr Lys Thr Leu Leu Val Thr Val Asp Thr Pro Ile Thr Gly
180 185 190
Gln Arg Leu Arg Asp His Arg Asn Gly Leu Thr Ile Pro Pro Arg Leu
195 200 205
Thr Leu Gly Thr Ile Leu Asp Ala Ser Tyr Arg Pro Gly Trp Trp Phe
210 215 220
Asn Phe Leu Thr Thr Glu Pro Pro Lys Tyr Ala Ser Leu Ser Asn Thr
225 230 235 240
Ser Gln Ser Leu Ala Glu Met Thr Gln Thr Met Phe Asp Pro Thr Leu
245 250 255
Asp Leu Glu Asp Leu Arg Trp Ile Arg Glu Gln Trp Asn Gly Arg Leu
260 265 270
Phe Val Lys Gly Ile Leu Thr Ala Asp Asp Ala Arg Arg Ala Gln Ser
275 280 285
Val Gly Ala Asp Gly Leu Val Val Ser Asn His Gly Gly Arg Gln Leu
290 295 300
Asp Arg Ala Pro Asp Ser Leu Thr Ser Leu Ala Glu Val Arg Ala Glu
305 310 315 320
Val Gly Pro Glu Met Glu Leu Ile Phe Asp Ser Gly Ile Met Ser Gly
325 330 335
Thr Asp Val Val Ala Ala Leu Cys Ala Gly Ala Asp Phe Val Leu Ile
340 345 350
Gly Arg Ala Tyr Leu Tyr Gly Leu Met Ala Gly Gly Gln Arg Gly Val
355 360 365
Glu Arg Ala Ile Ala Leu Ile Gln Gln Glu Ile Leu Thr Ala Met Gly
370 375 380
Leu Met Gly Ala Arg Ser Ile Ser Asp Leu Gly Pro Glu Leu Val Arg
385 390 395 400
Gly Leu Pro Gln Ala Pro Asn Thr Asp Gln Glu Leu Pro Ala Asp Ser
405 410 415
Met
Claims (5)
1. one kind derives from the L- alpha-hydroxy acid oxidizing ferment of extension brevibacterium (Brevibacterium linens), its amino acid sequence
It is classified as 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 40 DEG C, and optimal reaction pH is 6.
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.
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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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2017
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Patent Citations (1)
| 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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| Title |
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| 无: "WP_062861473.1", 《NCBI》 * |
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