CN106754781B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754781B
CN106754781B CN201710006545.7A CN201710006545A CN106754781B CN 106754781 B CN106754781 B CN 106754781B CN 201710006545 A CN201710006545 A CN 201710006545A CN 106754781 B CN106754781 B CN 106754781B
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CN106754781A (en
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蔡宇杰
于凤川
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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Abstract

The present invention relates to a kind of acquisition of D-ALPHA-Hydroxypropionic acid oxidase gene from Hao Shi proteus (Proteus hauseri) 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 Hao Shi proteus (Proteus hauseri) goes out a kind of novel D- cream to the present invention for the first time Acid oxidase, the enzyme can not only aoxidize (R)-alpha-hydroxy acid, but also can aoxidize (R)-alpha-hydroxy acid ester, the reaction and NAD (NADP) reaction for the lactic dehydrogenase participation of coenzyme is atomic weak compared to back reaction, can be applied to optical voidness (S)-alpha-hydroxy acid ester The preparation of (S)-alpha-hydroxy acid.
Summary of the invention
Present invention clone from Hao Shi proteus (Proteus hauseri) has obtained a kind of using FAD as the D- of coenzyme The gene of lactate oxidase discloses its relevant enzymatic property, and answered using colibacillus engineering heterogenous expression With research.
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: Proteus hauseri ATCC 13315, purchased from beauty State's ATCC strain library.
2, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract 13315 phage gene group total DNA of Proteus hauseri ATCC.Specific primer is designed, using PCR Method amplifies D-ALPHA-Hydroxypropionic acid oxidase gene overall length encoder block sequence.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 a kind of novel from Proteus hauseri ATCC 13315 D-ALPHA-Hydroxypropionic acid oxidizing ferment, the enzyme can aoxidize (R)-alpha-hydroxy acid and (R)-alpha-hydroxy acid ester, can be used for prepare with scale chiral purity (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 Proteus hauseri ATCC 13315DNA
13315 bacterial strain of Proteus hauseri ATCC is cultivated into 12h in LB culture medium, 12,000 rmp/min from Heart 10min obtains thallus, operates using bacterial genomes DNA extraction agent box (TaKaRa company) according to it and extracts 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 D-ALPHA-Hydroxypropionic acid oxidase gene
(1) design of primers
Design primer sequence are as follows:
Primer 1:5'GCCGGGATCCATGAATGAGAGTAACAGCTCCG 3'
Primer 2: 5'GCCGTCTAGATTATTTTTCTATTTCGTTATCAGCG 3'
(2) PCR amplification
With two primers synthesized above, using the genomic DNA of Proteus hauseri ATCC 13315 as template into Row 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, DNA4 μ Each 0.5 μ l of 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 5.0 phosphate buffer, measures the enzyme activity of D-ALPHA-Hydroxypropionic acid oxidizing ferment, The optimal reactive temperature for determining enzyme is 45 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 45 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 5.0 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 1 D of the table-lactate oxidase 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> 1755
<212> DNA
<213> Proteus hauseri ATCC13315
<400> 1
atgaatgaga gtaacagctc cgctaaccaa cattttattc gtaagttaga aggcatcgtc 60
gggaaaaaac aagtactaac acaagcacat aaaaccgagc gatatcgcaa aggctttcgt 120
tcagggcagg gtaaggcatt agctgttgta tttccaagtt ctttactaga acagtggcgc 180
gtatttaaag cctgtgttga agccgataag attattatta tgcaagcggc aaatactggg 240
ttaacagaag gttcaacacc taatggtgat gattatgacc gagacattgt tattatcagc 300
actttgcgcc aagataaaat tcaagtgctc tcagaacata accaagttat tgcttttcct 360
ggtagtacat tatggcattt agagaaagtc ttaaagccac ttggccgtga gcctcattcc 420
gttattggtt cttcttgtat tggtgcttct gttattggtg gcatttgtaa taactcaggt 480
ggcgctttgg ttcgtcgtgg tcctgcatat actgaattat cgctttatgc tcgtgtaaat 540
gaaaaaggtg aagctgaatt agttaatcat ttaggaatag atttaggtga aacacctgaa 600
gagattttaa ctaatcttga taatcgccat tatcatgcaa aacaaattca tgctacagaa 660
aaactggctt cagatcatga ataccatgag cgagtgcgtg atgttgatgc agatacacct 720
tctcgtttta ataacgatga gcgacgccta tatgatgccg caggaagcgc aggaaaactg 780
tctgtttttg ctgtgcgatt agatacattc ccagctgatc atcgtactca agtattctat 840
attggtacta ataatcctga tgagttagaa gatattcgtc gtcatatttt gagtaacttt 900
aaaacactcc ctgtagccgg tgagtatatg cacagaagtt attatgaaat ggcagaagtt 960
tatggaaaag acacttttct tgttatcgat aaattaggta ctgataaaat gccaaccctt 1020
tttgcaatta aagggcgatt tgatgctgta ttaaataaag tgccattttt acctaaaaat 1080
atggttgata gaacaatgca gttaatgagt aaattatggc cctcgcattt acctgcacga 1140
atgactgatt ttcgcgataa atatgagcat cacttgatgt taagaatggc agatggtggt 1200
attgatgagg catcaactta tttaaaagaa tattttaaac aagcttctgg cgattatttt 1260
gaatgcacag aagaagaggg aaataaagcc tttttacatc gctttgctgc tgccggcgct 1320
gctgttcgtt atcatgcggt tcatgtcaat gaggtagagg atgttttacc tttagatatt 1380
gcattgcgtc gcaatgatag agagtggttt gaaaaattac caccagaaat agagagtaaa 1440
ttattgttta aactctattg cggacatttt atgtgtcatg ttatgcatca agattatatt 1500
attaaaaaag gtgttgatgc caaggcacta aaagcccaaa tgctagaatt attggataaa 1560
cgtggtgctg aatatcccgc agagcataac gtaggacata tgtatcatgc taaaccacaa 1620
ttaaaggcgt tttatcagca taatgatcca accaatagta tgaatccggg aattggtaaa 1680
acatcaaaat taaaatattg gggtggtgag tgtggttgtg agcaaactca cgctgataac 1740
gaaatagaaa aataa 1755
<210> 2
<211> 584
<212> PRT
<213> Proteus hauseri ATCC13315
<400> 2
Met Asn Glu Ser Asn Ser Ser Ala Asn Gln His Phe Ile Arg Lys Leu
1 5 10 15
Glu Gly Ile Val Gly Lys Lys Gln Val Leu Thr Gln Ala His Lys Thr
20 25 30
Glu Arg Tyr Arg Lys Gly Phe Arg Ser Gly Gln Gly Lys Ala Leu Ala
35 40 45
Val Val Phe Pro Ser Ser Leu Leu Glu Gln Trp Arg Val Phe Lys Ala
50 55 60
Cys Val Glu Ala Asp Lys Ile Ile Ile Met Gln Ala Ala Asn Thr Gly
65 70 75 80
Leu Thr Glu Gly Ser Thr Pro Asn Gly Asp Asp Tyr Asp Arg Asp Ile
85 90 95
Val Ile Ile Ser Thr Leu Arg Gln Asp Lys Ile Gln Val Leu Ser Glu
100 105 110
His Asn Gln Val Ile Ala Phe Pro Gly Ser Thr Leu Trp His Leu Glu
115 120 125
Lys Val Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser
130 135 140
Ser Cys Ile Gly Ala Ser Val Ile Gly Gly Ile Cys Asn Asn Ser Gly
145 150 155 160
Gly Ala Leu Val Arg Arg Gly Pro Ala Tyr Thr Glu Leu Ser Leu Tyr
165 170 175
Ala Arg Val Asn Glu Lys Gly Glu Ala Glu Leu Val Asn His Leu Gly
180 185 190
Ile Asp Leu Gly Glu Thr Pro Glu Glu Ile Leu Thr Asn Leu Asp Asn
195 200 205
Arg His Tyr His Ala Lys Gln Ile His Ala Thr Glu Lys Leu Ala Ser
210 215 220
Asp His Glu Tyr His Glu Arg Val Arg Asp Val Asp Ala Asp Thr Pro
225 230 235 240
Ser Arg Phe Asn Asn Asp Glu Arg Arg Leu Tyr Asp Ala Ala Gly Ser
245 250 255
Ala Gly Lys Leu Ser Val Phe Ala Val Arg Leu Asp Thr Phe Pro Ala
260 265 270
Asp His Arg Thr Gln Val Phe Tyr Ile Gly Thr Asn Asn Pro Asp Glu
275 280 285
Leu Glu Asp Ile Arg Arg His Ile Leu Ser Asn Phe Lys Thr Leu Pro
290 295 300
Val Ala Gly Glu Tyr Met His Arg Ser Tyr Tyr Glu Met Ala Glu Val
305 310 315 320
Tyr Gly Lys Asp Thr Phe Leu Val Ile Asp Lys Leu Gly Thr Asp Lys
325 330 335
Met Pro Thr Leu Phe Ala Ile Lys Gly Arg Phe Asp Ala Val Leu Asn
340 345 350
Lys Val Pro Phe Leu Pro Lys Asn Met Val Asp Arg Thr Met Gln Leu
355 360 365
Met Ser Lys Leu Trp Pro Ser His Leu Pro Ala Arg Met Thr Asp Phe
370 375 380
Arg Asp Lys Tyr Glu His His Leu Met Leu Arg Met Ala Asp Gly Gly
385 390 395 400
Ile Asp Glu Ala Ser Thr Tyr Leu Lys Glu Tyr Phe Lys Gln Ala Ser
405 410 415
Gly Asp Tyr Phe Glu Cys Thr Glu Glu Glu Gly Asn Lys Ala Phe Leu
420 425 430
His Arg Phe Ala Ala Ala Gly Ala Ala Val Arg Tyr His Ala Val His
435 440 445
Val Asn Glu Val Glu Asp Val Leu Pro Leu Asp Ile Ala Leu Arg Arg
450 455 460
Asn Asp Arg Glu Trp Phe Glu Lys Leu Pro Pro Glu Ile Glu Ser Lys
465 470 475 480
Leu Leu Phe Lys Leu Tyr Cys Gly His Phe Met Cys His Val Met His
485 490 495
Gln Asp Tyr Ile Ile Lys Lys Gly Val Asp Ala Lys Ala Leu Lys Ala
500 505 510
Gln Met Leu Glu Leu Leu Asp Lys Arg Gly Ala Glu Tyr Pro Ala Glu
515 520 525
His Asn Val Gly His Met Tyr His Ala Lys Pro Gln Leu Lys Ala Phe
530 535 540
Tyr Gln His Asn Asp Pro Thr Asn Ser Met Asn Pro Gly Ile Gly Lys
545 550 555 560
Thr Ser Lys Leu Lys Tyr Trp Gly Gly Glu Cys Gly Cys Glu Gln Thr
565 570 575
His Ala Asp Asn Glu Ile Glu Lys
580

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 Hao Shi proteus The D-ALPHA-Hydroxypropionic acid oxidizing ferment of (Proteus hauseri), amino acid sequence are shown in SEQ ID NO:2；The alpha-hydroxy acid ester It is one of following: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid ice Piece 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. D-ALPHA-Hydroxypropionic acid oxidizing ferment according to claim 1, nucleotides sequence is classified as shown in SEQ ID NO:1.
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