CN106754794B - A kind of oxidizing ferment and its application - Google Patents
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- 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)
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Abstract
The present invention relates to a kind of acquisition of L- alpha-hydroxy acid oxidase gene from Carnimonas nigrificans and its clonal expressions, belong to bioengineering field.Its substrate specificity is disclosed, while the L- alpha-hydroxy acid oxidizing ferment can aoxidize (S)-alpha-hydroxy acid ester, can be applied to the preparation of optical voidness (R)-alpha-hydroxy acid ester.
Description
Technical field
A kind of L- alpha-hydroxy acid 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 technique
L- alpha-hydroxy acid oxidizing ferment (L- α-hydroxyacid oxidase) is a kind of dehydrogenase with FMN (FAD) for coenzyme
(Aliphatic l-α-hydroxyacid oxidase from rat livers purification and
Properties. Biochimica et Biophysica Acta (BBA)-Enzymology 1968,167:9-22), usually
It 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).It can be used for measuring lactic acid in biosensor
Content, or oxidation Pfansteihl produce pyruvic acid.Also there is the preparation (Chinese patent for being used for 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), shame dirt branch
Bacillus (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
It is 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 novel L- alpha-hydroxy acid oxidation to the present invention for the first time
Enzyme, the enzyme can not only aoxidize (S)-alpha-hydroxy acid, but also 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.
Summary of the invention
The present invention is cloned from Carnimonas nigrificans has obtained a kind of gene of L- alpha-hydroxy acid oxidizing ferment, benefit
With colibacillus engineering heterogenous expression, its relevant enzymatic property is disclosed, and has carried out application study.
Technical scheme is as follows:
1, bacterial strain
The source bacterial strain of L- alpha-hydroxy acid oxidase gene of the present invention are as follows: Carnimonas nigrificans ATCC BAA-
78, it is purchased from U.S. ATCC strain library.
2, the clone of L- alpha-hydroxy acid oxidase gene
Extract Carnimonas nigrificans ATCC BAA-78 phage gene group total DNA.Specific primer is designed,
Using PCR method, L- alpha-hydroxy acid oxidase gene overall length encoder block sequence is amplified.And construction recombination plasmid.
3, L- alpha-hydroxy acid Oxidase Expression and purifying
Recombinant plasmid is imported in E.coli BL21 (DE3), inducing expression.Crude enzyme liquid is obtained after bacterial cell disruption, after purification
It is freeze-dried spare.
4, the characterization analysis of L- alpha-hydroxy acid oxidizing ferment
Influence of the pH to L- alpha-hydroxy acid oxidizing ferment enzyme activity of the present invention is studied by substrate of Pfansteihl.
Influence of the temperature to L- alpha-hydroxy acid oxidizing ferment enzyme activity of the present invention is studied by substrate of Pfansteihl.
The substrate specificity of L- alpha-hydroxy acid oxidizing ferment is analyzed: substrate used has Pfansteihl, glycolic, L- phenyllactic acid, L-
Tartaric acid, L MALIC ACID, L- para hydroxybenzene lactic acid, L- danshensu, L- mandelic acid.
Enzyme activity determination method are as follows: according to Characterization of a Lactate Oxidase from a
Strain of Gram Negative Bacterium from Soil, Applied Biochemistry and
Biotechnology,56, 1996,278-288.The method carries out.
5, L- alpha-hydroxy acid oxidizing ferment splits the alpha-hydroxy acid ester of mixed
The method of resolution of alpha-carboxylic esters (alpha-hydroxy esters) are as follows: take 0.1 gram of purified enzyme in 50 mL tri-
In the bottle of angle, it is added dissolved in the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, is converted in 30 DEG C, 150rpm shaking bath
16h, liquid-phase chromatographic analysis supernatant after conversion.(S) Alpha-hydroxy in-alpha-hydroxy acid ester, which is dehydrogenated, is oxidized to corresponding 2-ketoacid
Ester, (R)-alpha-hydroxy acid ester are not oxidized.
Product (R)-alpha-hydroxy acid ester optical purity 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 formulaRFor the peak area of (R)-enantiomer after reaction, SSFor reaction after (S)-enantiomer liquid chromatogram peak area,
S0For the sum of the liquid chromatogram peak area of (R)-and (S)-enantiomer before reaction.
Product measures liquid phase chromatogram condition are as follows: Chiralcel OD-H chiral column (4.6 × 250mm), mobile phase volume ratio
For n-hexane: isopropanol: trifluoroacetic acid=80:20:0.1, flow velocity 0.5mL/min, 25 DEG C of column temperature, Detection wavelength 210nm,
20 μ L of sample volume.
The alpha-hydroxy acid ester is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, benzene cream
Isopropyl propionate, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene isopropyl lactate, mandelic acid norbornene ester, almond isopropyl propionate, Radix Salviae Miltiorrhizae
Plain asarum alcohol ester, lactic acid norbornene ester, phenyllactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
The alpha-hydroxy acid ester, according to Chinese patent 200610042787.3,201410180490.8,
201410175950.8 the method synthesis announced with 20140699506.6.
Originally deliver bright usefulness: clone has obtained one from Carnimonas nigrificans ATCC BAA-78
Kind 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 has important industrial application value.
Specific embodiment
Embodiment 1
The present embodiment is that the clone of L- alpha-hydroxy acid oxidase gene of the present invention and colibacillus engineering construct.
1, the extraction of Carnimonas nigrificans ATCC BAA-78DNA
Carnimonas nigrificans ATCC BAA-78 bacterial strain is cultivated into 12h in LB culture medium, 12,
000rmp/min centrifugation 10min obtains thallus, using bacterial genomes DNA extraction agent box (TaKaRa company) according to its behaviour
Make to extract phage gene group total DNA, it is spare to put refrigerator.
2, prepared by E. coli competent
(1) inoculation E.coli DH5 α and BL21 (DE3) is respectively in the 250mL shaking flask containing 20mL LB culture medium, and 37
DEG C, 200rpm/min overnight incubation.
(2) it is inoculated in 50mL LB culture medium 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 pre-cooling, places 30min, 8000rpm/min, 4 DEG C of centrifugations on ice
5min。
(4) supernatant is abandoned, the 0.1mol/L CaCl of 5mL pre-cooling is added2Solution makes thallus suspend, and places 20min 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 pre-cooling is added2Solution (contains 15% glycerol), 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 spare.
3, the clone of L- alpha-hydroxy acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCATGATTATTTCCAGCCCAACTGATT 3'
Primer 2: 5'GCCGTCTAGACGAACCACTTGATCCAGACGG 3'
(2) PCR amplification
With two primers synthesized above, with the genomic DNA of Carnimonas nigrificans ATCC BAA-78
PCR amplification is carried out for template.
Amplification system in this step are as follows:
Amplification program are as follows:
98 DEG C, 10min
98 DEG C, 10sec；55 DEG C, 15sec；72 DEG C, 2min reacts 30 circulations
72 DEG C, 10min
PCR product obtains the gene order of the enzyme after sending Hua Da gene sequencing, as shown in SEQ ID NO:1.According to the base
The amino acid sequence obtained by sequence is as shown in SEQ ID NO:2.
(3) double digestion and connection
II plasmid of pCold and PCR product are subjected to double digestion, digestion system are as follows: 10 × cut buffer, 3 μ l, DNA 4
Each 0.5 μ l of μ l, enzyme BamHI and XbaI, 2 μ l of sterile water totally 30 μ l.Double digestion 1h under 37 DEG C of water-baths.DNA fragmentation is cloned into
On II carrier of pCold, and it is transformed into E.coli DH5 α competent cell.Linked system: 10 × DNA ligase buffer
2.5 μ l, 8 μ l of DNA fragmentation, 2 μ l, T4 DNA ligase of carrier DNA 1 μ l, 11.5 μ l of sterile water totally 25 μ l.Under 16 DEG C of water-baths
Connect 12h-16h.
(4) it converts
Step:
1 is added 100 μ l DH5 α competent bacterias in linked system, light to mix, ice bath 30min.
2 are put into 42 DEG C of water-baths of preheating, place 90s and carry out heat shock processing.
3 ice bath 2min immediately.
4 are added the not antibiotic LB culture solution of 1ml, and 37 DEG C of culture 1h make thallus recover.
5 are uniformly coated on thallus on antibiotic LB plate.
6 cultures are grown fine for 24 hours.It chooses single colonie and carries out bacterium colony PCR, recombinant plasmid is extracted in nucleic acid electrophoresis verifying.It will recombination
Plasmid imports in BL21 E. coli competent, saves backup.
Embodiment 2
The present embodiment is the inducing expression of L- alpha-hydroxy acid oxidizing ferment of the present invention and isolates and purifies.
1, plus 500 μ l recombination bacterium solution is into 50ml LB culture solution.37 DEG C of culture 2.5h stand 0.5h at 15 DEG C.Again plus 20
The IPTG of μ l 0.5M, cold-induction culture is for 24 hours at 15 DEG C.Fermentation liquid is centrifuged (8000rmp/min, 10min) and obtains bacterium
Body redissolves thallus with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (20mmol/L, pH 7.0), and Ultrasonic Cell Disruptor is broken,
Centrifugation (8000rmp/min, 10 min) collects supernatant and obtains crude enzyme liquid.
2, the crude enzyme liquid for obtaining step 1 carries out ni-sepharose purification using the operation of 150 protein purification system of AKTA avant,
Elution process are as follows: all put the tetra- root canal road A1, A2, B1, B2 into water, system flow 20ml/min flow velocity is set, carry out
Exhaust.Then system flow 1ml/min, flow path (column position 3), delta pressure are set
0.3, pre-pressure 0.5, Gradient 0, inset A1, fill pillar after water droplet uniformly flows out, balance ten minutes it
A1 is put into conjunction in liquid afterwards, B1 is put into eluent, then primary, balance 20 minutes is exhausted, then loading crude enzyme liquid,
With high concentration imidazole buffer (solution locating for B1) gradient elution destination protein of 500mM, the albumen that will be adsorbed on ion column
Elute the enzyme purified.Enzyme after purification is freeze-dried spare.
Embodiment 3
The present embodiment is the optimum temperature of L- alpha-hydroxy acid oxidizing ferment of the present invention.Using Pfansteihl as substrate, by substrate with
The phosphate buffer that pH is 6.0 is lauched bath 15min in 30-60 DEG C of different temperature condition, measures the enzyme of L- alpha-hydroxy acid oxidizing ferment
It is living, determine that the optimal reactive temperature of enzyme is 50 DEG C.
Embodiment 4
The present embodiment is the optimum pH of L- alpha-hydroxy acid oxidizing ferment of the present invention.Using Pfansteihl as substrate, substrate is existed
PH 3-9, the enzyme activity of 50 DEG C of water-bath 15min measurement enzymes, as a result, it has been found that L- alpha-hydroxy acid oxidizing ferment enzyme activity is most under the conditions of 6.0 pH
It is high.
Embodiment 5
The present embodiment is that L- alpha-hydroxy acid oxidizing ferment of the present invention is listed in table 1 from the response characteristic of different substrates.
Activity of the 1 L- alpha-hydroxy acid oxidizing ferment of table to different substrates
Embodiment 6
Various racemic ' alpha '-carboxylic esters are split according to the method in summary of the invention, as a result as shown in the table:
Table 2 splits the effect of various racemic ' alpha '-carboxylic esters
As can be seen from the above table, when the reaction time is abundant, available all kinds of optically pure (R)-α-hydroxy acids of height
The optics specificity of ester, 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> 1143
<212> DNA
<213> Carnimonas nigrificans ATCC BAA-78
<400> 1
atgattattt ccagcccaac tgattaccgg ttagcggcca aaaaacgcct tccccgcttc 60
ttgtttgatt acatcgacgg cggtgccgtc accgaaggta ccatgcgcag caacactgaa 120
gcgttgcagg cgattgctct gcgtcagcgt gtcttgcagg gaggcggcaa tcccaacctg 180
gaatcagaga ttctcggcag ccaatggaag ctccccattg cgcttgctcc ggtaggcgcc 240
accggtatgt acgcacgccg tggagaagta caggccgcga aagccgccga acaggcaggt 300
attccttata cgctctcaac cgtaggggtc tgcccgatcg aggaagtggc cgccggcgcg 360
aacggtgagc agtggtcgca gctttatgtt ctcaaggacc gtggctatat gaagaactcg 420
ctggagcgcg cgtgggcaaa cggcatgaag acgctggtct tcaccgtcga tatgccgctg 480
cccggctcac gctatcgcga tgcccattca ggcatgtcag gccccaacgc gaccatgcgc 540
caatacctgc aagcctgcat gcacccgcac tgggccatcg acgtaggttt gcagggccgc 600
ccactgtcgt ttggcaacat cgagaagtac accggtcatc gcatgaccat gaacgactac 660
atgggctttc ttagcaagaa cttcgatcca tccatcgcct ggagcgagct tgaatggatt 720
cgcgatagct ggaaaggcaa tctgatcatt aaaggaattc tcgataaaga ggatgcccgc 780
gaagcagtac gcatgggcgc cgacggcatc atcgtctcca accacggagg ccgccagctc 840
gacggtgcca aacccaccgc ccacgcactg cccgagattg ccgcaacggt tggcgatgac 900
ctgaccgtgc tcgccgattc aggcatccgc tctggccttg acgtagtgcg catgatggcg 960
ctgggcgcca aaggcgtact gctgggacgc tcctatatct acgcactcgc cgccggtggg 1020
cagcaaggcg ttagcaatct gctcagattg atagaagccg acatgaaggt gaccatgtgc 1080
ttgattggtg ccaactcccc tgcagaaatt accgctgacc gtctggatca agtggttcgg 1140
tag 1143
<210> 2
<211> 380
<212> PRT
<213> Carnimonas nigrificans ATCC BAA-78
<400> 2
Met Ile Ile Ser Ser Pro Thr Asp Tyr Arg Leu Ala Ala Lys Lys Arg
1 5 10 15
Leu Pro Arg Phe Leu Phe Asp Tyr Ile Asp Gly Gly Ala Val Thr Glu
20 25 30
Gly Thr Met Arg Ser Asn Thr Glu Ala Leu Gln Ala Ile Ala Leu Arg
35 40 45
Gln Arg Val Leu Gln Gly Gly Gly Asn Pro Asn Leu Glu Ser Glu Ile
50 55 60
Leu Gly Ser Gln Trp Lys Leu Pro Ile Ala Leu Ala Pro Val Gly Ala
65 70 75 80
Thr Gly Met Tyr Ala Arg Arg Gly Glu Val Gln Ala Ala Lys Ala Ala
85 90 95
Glu Gln Ala Gly Ile Pro Tyr Thr Leu Ser Thr Val Gly Val Cys Pro
100 105 110
Ile Glu Glu Val Ala Ala Gly Ala Asn Gly Glu Gln Trp Ser Gln Leu
115 120 125
Tyr Val Leu Lys Asp Arg Gly Tyr Met Lys Asn Ser Leu Glu Arg Ala
130 135 140
Trp Ala Asn Gly Met Lys Thr Leu Val Phe Thr Val Asp Met Pro Leu
145 150 155 160
Pro Gly Ser Arg Tyr Arg Asp Ala His Ser Gly Met Ser Gly Pro Asn
165 170 175
Ala Thr Met Arg Gln Tyr Leu Gln Ala Cys Met His Pro His Trp Ala
180 185 190
Ile Asp Val Gly Leu Gln Gly Arg Pro Leu Ser Phe Gly Asn Ile Glu
195 200 205
Lys Tyr Thr Gly His Arg Met Thr Met Asn Asp Tyr Met Gly Phe Leu
210 215 220
Ser Lys Asn Phe Asp Pro Ser Ile Ala Trp Ser Glu Leu Glu Trp Ile
225 230 235 240
Arg Asp Ser Trp Lys Gly Asn Leu Ile Ile Lys Gly Ile Leu Asp Lys
245 250 255
Glu Asp Ala Arg Glu Ala Val Arg Met Gly Ala Asp Gly Ile Ile Val
260 265 270
Ser Asn His Gly Gly Arg Gln Leu Asp Gly Ala Lys Pro Thr Ala His
275 280 285
Ala Leu Pro Glu Ile Ala Ala Thr Val Gly Asp Asp Leu Thr Val Leu
290 295 300
Ala Asp Ser Gly Ile Arg Ser Gly Leu Asp Val Val Arg Met Met Ala
305 310 315 320
Leu Gly Ala Lys Gly Val Leu Leu Gly Arg Ser Tyr Ile Tyr Ala Leu
325 330 335
Ala Ala Gly Gly Gln Gln Gly Val Ser Asn Leu Leu Arg Leu Ile Glu
340 345 350
Ala Asp Met Lys Val Thr Met Cys Leu Ile Gly Ala Asn Ser Pro Ala
355 360 365
Glu Ile Thr Ala Asp Arg Leu Asp Gln Val Val Arg
370 375 380
Claims (2)
1. a kind of method of resolution of alpha-carboxylic esters (alpha-hydroxy esters), which is characterized in that the method are as follows: take pure
0.1 gram of the enzyme changed is added in 50mL triangular flask dissolved in the phosphate buffer of the pH 7 of alpha-hydroxy acid ester 5mM, in 30 DEG C,
16h is converted in 150rpm shaking bath, liquid-phase chromatographic analysis supernatant after conversion；The enzyme is from Carnimonas
The L- alpha-hydroxy acid oxidizing ferment of nigrificans, amino acid sequence are shown in SEQ ID NO:2；Under the alpha-hydroxy acid ester is
One of column: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid borneol
Ester, para hydroxybenzene isopropyl lactate, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu asarum alcohol ester, benzene
Lactic acid asarum alcohol ester, para hydroxybenzene lactic acid asarum alcohol ester.
2. the method according to claim 1, wherein the nucleotides sequence of the L- alpha-hydroxy acid oxidizing ferment is classified as SEQ
Shown in ID NO:1.
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