CN106754791B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754791B
CN106754791B CN201710006591.7A CN201710006591A CN106754791B CN 106754791 B CN106754791 B CN 106754791B CN 201710006591 A CN201710006591 A CN 201710006591A CN 106754791 B CN106754791 B CN 106754791B
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蔡宇杰
武旭攀
曹憬
白亚军
郑晓晖
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Zhuohong Chaoyuan Biotechnology Zhengzhou Co ltd
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Jiangnan University
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
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Abstract

The present invention relates to a kind of acquisition of D-ALPHA-Hydroxypropionic acid oxidase gene from providencia rettgeri (Providencia rettgeri) and its clonal expressions, belong to bioengineering field.Its substrate specificity is disclosed, while the D-ALPHA-Hydroxypropionic acid oxidizing ferment can aoxidize (R)-alpha-hydroxy acid ester, can be applied to the preparation of optical voidness (S)-alpha-hydroxy acid ester.

Description

A kind of oxidizing ferment and its application
Technical field
A kind of D-ALPHA-Hydroxypropionic acid oxidizing ferment of clonal expression of the present invention, and disclose its nucleotide sequence and amino acid sequence and enzyme Property and application are learned, industrial microorganism field is belonged to.
Background technique
D-ALPHA-Hydroxypropionic acid oxidizing ferment (D-lactate oxidase) is a kind of alpha-hydroxy acid oxidizing ferment with FAD (FMN) for coenzyme (being traditionally referred to as D-ALPHA-Hydroxypropionic acid oxidizing ferment).D-ALPHA-Hydroxypropionic acid oxidizing ferment can be used in biosensor measuring the content of lactic acid, or It aoxidizes D-ALPHA-Hydroxypropionic acid and produces pyruvic acid.Also there is the preparation (Chinese patent 201210109290.4) for being used for optical voidness alpha-hydroxy acid
So far, in edwardsiella tarda (Edwardsiella tarda) and zymomonas mobilis D-ALPHA-Hydroxypropionic acid oxidizing ferment is had found 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 clonal expression from providencia rettgeri (Providencia rettgeri) goes out one kind newly to the present invention for the first time The D-ALPHA-Hydroxypropionic acid oxidizing ferment of type, the enzyme can not only aoxidize (R)-alpha-hydroxy acid, but also can aoxidize (R)-alpha-hydroxy acid ester, the reaction with NAD (NADP) is that the reaction that the lactic dehydrogenase of coenzyme participates in is atomic weak compared to back reaction, can be applied to optical voidness (S)-α-hydroxyl The preparation of acid esters and (S)-alpha-hydroxy acid.
Summary of the invention
Present invention clone from providencia rettgeri (Providencia rettgeri) has obtained one kind with FAD Its relevant enzymatic property is disclosed using colibacillus engineering heterogenous expression for the gene of the D-ALPHA-Hydroxypropionic acid oxidizing ferment of coenzyme, And application study is carried out.
Technical scheme is as follows:
1, bacterial strain
The source bacterial strain of D-ALPHA-Hydroxypropionic acid oxidase gene of the present invention are as follows: Providencia rettgeri ATCC 29944, purchase From U.S.'s ATCC strain library.
2, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract 29944 phage gene group total DNA of Providencia rettgeri ATCC.Specific primer is designed, is answered With PCR method, D-ALPHA-Hydroxypropionic acid oxidase gene overall length encoder block sequence is amplified.And construction recombination plasmid.
3, D-ALPHA-Hydroxypropionic 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment
Influence of the pH to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention is studied by substrate of D-ALPHA-Hydroxypropionic acid.
Influence of the temperature to D-ALPHA-Hydroxypropionic acid oxidizing ferment enzyme activity of the present invention is studied by substrate of D-ALPHA-Hydroxypropionic acid.
The substrate specificity of D-ALPHA-Hydroxypropionic acid oxidizing ferment is analyzed: substrate used has D-ALPHA-Hydroxypropionic acid, glycolic, D- phenyllactic acid, D- pairs Hydroxyphenyl lactic acid, D- tartaric acid, D-malic acid, D- mandelic acid, D- danshensu.
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, D-ALPHA-Hydroxypropionic 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.(R) Alpha-hydroxy in-alpha-hydroxy acid ester, which is dehydrogenated, is oxidized to corresponding 2-ketoacid Ester, (S)-alpha-hydroxy acid ester are not oxidized.
Product (S)-alpha-hydroxy acid ester optical purity 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 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: clonal expression goes out one kind from Providencia rettgeri ATCC 29944 Novel 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 chirality Pure (S)-alpha-hydroxy acid ester has 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 construct.
1, the extraction of 29944 DNA of Providencia rettgeri ATCC
29944 bacterial strain of Providencia rettgeri ATCC is cultivated into 12h, 12,000 rmp/ in LB culture medium Min centrifugation 10min obtains thallus, operates using bacterial genomes DNA extraction agent box (TaKaRa company) according to it and extracts bacterium Body genome 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 D-ALPHA-Hydroxypropionic acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCATGAATGATGCAAAGAAACCTGAGA 3'
Primer 2: 5'GCCGTCTAGATTAATGATCATGAGTATGAGCGCAG 3'
(2) PCR amplification
With two primers synthesized above, the genomic DNA with Providencia rettgeri ATCC 29944 is Template carries out PCR amplification.
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, T4DNA 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 D-ALPHA-Hydroxypropionic 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 D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.Using D-ALPHA-Hydroxypropionic acid as substrate, by substrate and pH It is lauched bath 15min in 30-60 DEG C of different temperature condition for 6.0 phosphate buffer, measures the enzyme activity of D-ALPHA-Hydroxypropionic acid oxidizing ferment, The optimal reactive temperature for determining enzyme is 55 DEG C.
Embodiment 4
The present embodiment is the optimum pH of D-ALPHA-Hydroxypropionic acid oxidizing ferment of the present invention.Using D-ALPHA-Hydroxypropionic acid as substrate, by substrate in pH 3-9, the enzyme activity of 55 DEG C of water-bath 15min measurement enzymes, as a result, it has been found that D- lactate oxidase enzyme activity highest under the conditions of 6.0 pH.
Embodiment 5
The present embodiment is the response characteristic of D-ALPHA-Hydroxypropionic acid oxidizing ferment and different substrates of the present invention, is listed in table 1.
Activity of the 1 D-ALPHA-Hydroxypropionic 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 (S)-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> 1740
<212> DNA
<213> Providencia rettgeri ATCC 29944
<400> 1
atgaatgatg caaagaaacc tgagaatcaa cggttattac tgacgcttag gaatattgtt 60
ggcaaaaaaa atattttaac agagccacat aaaacagaac gttatcgtaa aggcttccgc 120
tctggtatgg gggatgcaat cgcggttgta tttccaacaa cattgctcga acagtggtat 180
gttttcaagg cgtgtgttga agcagacaaa attgtcctga tgcaagcggc taatacaggg 240
ttaaccgaag ggtcgacacc gagtggctat gactatgatc gcgatatcgt cattattagt 300
acgttgaaat taaaccaaat tcaagtttta ccggaattca accaagtcgt tgcgatgccg 360
gggagtactt tgtacgactt ggagaaaatc ttaaaaccat tagggcgtga accgcactct 420
ttgattggct cttcttgtat tggagcttca gttgtgggtg gtatttgtaa tagttctggc 480
ggttcattag tgcgtagagg ccccgcctat actgagcttg ctctttatgg ccgagtcagt 540
gacgatggaa aagtggagct tgtcaatcac ctaggtattg atttaggtaa cacaccagaa 600
gaaatcctga cgaatttaca aaatcgtggc tatagtagtg aagacgtaca agttagcgat 660
aaactcgctt ctgaccatga gtatgcagag cgcgttcgtg atgtcgatgc agatacacca 720
tcacgcttta acgcggatga acgtcgtcta tttgaagcat cagggtgcgc aggaaaatta 780
gccgtgtttg cggttcgttt agataccttc ccagccgata aaaaaacaca ggtattttat 840
atcggttcaa ataaccctga cgttttggaa gatatacgcc gttatatttt aagtgaattt 900
aaaaacctgc cagttgcagg tgaatatatg caccgtggtt gcttcgatat agcagaagtt 960
tatggtaaag ataccttttt aatgattgat aaatttggta ctgacaaaat gccaatgttt 1020
ttcactatta aagggcgtat tgatgcagta ttaaataaag tcccattcct gccatcaaat 1080
ctgatcgacc gtactatgca ggttctcagt aaattatggc cttcccattt accaccacgt 1140
atgaaagagt ttcgtgatcg ttatgaacac catttaatgt taaaaatggc cggagatggc 1200
attgatgagg ctaaagtttg gctaaaatct tattttgcaa gcgccgatgg cggctatttt 1260
gaatgtacac cagaagaggg ggcaaaagca tttttacacc gctttgccgc tgctggctcc 1320
gctgttcgtt atcacgctgt tcataataaa gatgtggaag atgtattacc cctcgacatt 1380
gctttacgtc gcaatgatag aaattggttt gaaacattac caccagaaat tgaaaagcta 1440
ttagttcata aattatactg cggtcatttt atgtgccatg ttatgcacca agactatgtc 1500
ttgaaaaaag gcgttgaccc gaaagaacta aaagaaaaaa tgttagcttt attagatgaa 1560
aggggggctc aatatcctgc ggaacataac gtagggcatt tatataaagc cccagaacaa 1620
ttaaaatcat tttataaaca atctgaccca acaaatacaa tgaaccccgg tttaggtaaa 1680
acaactaaac gcaaaaattg ggaaggggat tgtggctgcg ctcatactca tgatcattaa 1740
<210> 2
<211> 579
<212> PRT
<213> Providencia rettgeri ATCC 29944
<400> 2
Met Asn Asp Ala Lys Lys Pro Glu Asn Gln Arg Leu Leu Leu Thr Leu
1 5 10 15
Arg Asn Ile Val Gly Lys Lys Asn Ile Leu Thr Glu Pro His Lys Thr
20 25 30
Glu Arg Tyr Arg Lys Gly Phe Arg Ser Gly Met Gly Asp Ala Ile Ala
35 40 45
Val Val Phe Pro Thr Thr Leu Leu Glu Gln Trp Tyr Val Phe Lys Ala
50 55 60
Cys Val Glu Ala Asp Lys Ile Val Leu Met Gln Ala Ala Asn Thr Gly
65 70 75 80
Leu Thr Glu Gly Ser Thr Pro Ser Gly Tyr Asp Tyr Asp Arg Asp Ile
85 90 95
Val Ile Ile Ser Thr Leu Lys Leu Asn Gln Ile Gln Val Leu Pro Glu
100 105 110
Phe Asn Gln Val Val Ala Met Pro Gly Ser Thr Leu Tyr Asp Leu Glu
115 120 125
Lys Ile Leu Lys Pro Leu Gly Arg Glu Pro His Ser Leu Ile Gly Ser
130 135 140
Ser Cys Ile Gly Ala Ser Val Val Gly Gly Ile Cys Asn Ser Ser Gly
145 150 155 160
Gly Ser Leu Val Arg Arg Gly Pro Ala Tyr Thr Glu Leu Ala Leu Tyr
165 170 175
Gly Arg Val Ser Asp Asp Gly Lys Val Glu Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly Asn Thr Pro Glu Glu Ile Leu Thr Asn Leu Gln Asn
195 200 205
Arg Gly Tyr Ser Ser Glu Asp Val Gln Val Ser Asp Lys Leu Ala Ser
210 215 220
Asp His Glu Tyr Ala Glu Arg Val Arg Asp Val Asp Ala Asp Thr Pro
225 230 235 240
Ser Arg Phe Asn Ala Asp Glu Arg Arg Leu Phe Glu Ala Ser Gly Cys
245 250 255
Ala Gly Lys Leu Ala Val Phe Ala Val Arg Leu Asp Thr Phe Pro Ala
260 265 270
Asp Lys Lys Thr Gln Val Phe Tyr Ile Gly Ser Asn Asn Pro Asp Val
275 280 285
Leu Glu Asp Ile Arg Arg Tyr Ile Leu Ser Glu Phe Lys Asn Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Gly Cys Phe Asp Ile Ala Glu Val
305 310 315 320
Tyr Gly Lys Asp Thr Phe Leu Met Ile Asp Lys Phe Gly Thr Asp Lys
325 330 335
Met Pro Met Phe Phe Thr Ile Lys Gly Arg Ile Asp Ala Val Leu Asn
340 345 350
Lys Val Pro Phe Leu Pro Ser Asn Leu Ile Asp Arg Thr Met Gln Val
355 360 365
Leu Ser Lys Leu Trp Pro Ser His Leu Pro Pro Arg Met Lys Glu Phe
370 375 380
Arg Asp Arg Tyr Glu His His Leu Met Leu Lys Met Ala Gly Asp Gly
385 390 395 400
Ile Asp Glu Ala Lys Val Trp Leu Lys Ser Tyr Phe Ala Ser Ala Asp
405 410 415
Gly Gly Tyr Phe Glu Cys Thr Pro Glu Glu Gly Ala Lys Ala Phe Leu
420 425 430
His Arg Phe Ala Ala Ala Gly Ser Ala Val Arg Tyr His Ala Val His
435 440 445
Asn Lys Asp Val Glu Asp Val Leu Pro Leu Asp Ile Ala Leu Arg Arg
450 455 460
Asn Asp Arg Asn Trp Phe Glu Thr Leu Pro Pro Glu Ile Glu Lys Leu
465 470 475 480
Leu Val His Lys Leu Tyr Cys Gly His Phe Met Cys His Val Met His
485 490 495
Gln Asp Tyr Val Leu Lys Lys Gly Val Asp Pro Lys Glu Leu Lys Glu
500 505 510
Lys Met Leu Ala Leu Leu Asp Glu Arg Gly Ala Gln Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Leu Tyr Lys Ala Pro Glu Gln Leu Lys Ser Phe
530 535 540
Tyr Lys Gln Ser Asp Pro Thr Asn Thr Met Asn Pro Gly Leu Gly Lys
545 550 555 560
Thr Thr Lys Arg Lys Asn Trp Glu Gly Asp Cys Gly Cys Ala His Thr
565 570 575
His Asp His

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 be from Lei Shi Providian this The D-ALPHA-Hydroxypropionic acid oxidizing ferment of bacterium (Providencia rettgeri), amino acid sequence are shown in SEQ ID NO:2；The α- Carboxylic esters are one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene Lactic acid norbornene ester, para hydroxybenzene isopropyl lactate, 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.
2. the method according to claim 1, wherein the nucleotides sequence of the D-ALPHA-Hydroxypropionic acid oxidizing ferment is classified as SEQ ID Shown in NO:1.
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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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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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