CN106754779B - A kind of oxidizing ferment and its application - Google Patents

A kind of oxidizing ferment and its application Download PDF

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CN106754779B
CN106754779B CN201710006541.9A CN201710006541A CN106754779B CN 106754779 B CN106754779 B CN 106754779B CN 201710006541 A CN201710006541 A CN 201710006541A CN 106754779 B CN106754779 B CN 106754779B
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ester
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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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    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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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)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)

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Abstract

The present invention relates to a kind of acquisition from the D-ALPHA-Hydroxypropionic acid oxidase gene for dispersing general bacterium (Pantoea dispersa) 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)
Clonal expression goes out a kind of novel D-ALPHA-Hydroxypropionic acid oxygen to the present invention from dispersion general bacterium (Pantoea dispersa) for the first time Change enzyme, which can not only aoxidize (R)-alpha-hydroxy acid, but also can aoxidize (R)-alpha-hydroxy acid ester, which is with NAD (NADP) The reaction that the lactic dehydrogenase of coenzyme participates in is atomic weak compared to back reaction, can be applied to optical voidness (S)-alpha-hydroxy acid ester and (S)-α- The preparation of hydroxy acid.
Summary of the invention
Present invention clone from the general bacterium (Pantoea dispersa) of dispersion has obtained a kind of using FAD as the D- of coenzyme cream The gene of acid oxidase discloses its relevant enzymatic property, and applied using colibacillus engineering heterogenous expression 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: Pantoea dispersa ATCC 14593, purchased from beauty State's ATCC strain library.
2, the clone of D-ALPHA-Hydroxypropionic acid oxidase gene
Extract 14593 phage gene group total DNA of Pantoea dispersa 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 Pantoea dispersa ATCC 14593 D-ALPHA-Hydroxypropionic acid oxidizing ferment, which 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 Pantoea dispersa ATCC 14593DNA
14593 bacterial strain of Pantoea dispersa 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'GCCGGGATCCATGAATAACAACACCTCACACCCTG 3'
Primer 2: 5'GCCGTCTAGATTTCACCGCCCAGCCTTTG 3'
(2) PCR amplification
With two primers synthesized above, using the genomic DNA of Pantoea dispersa ATCC 14593 as template Carry 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, 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 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 4.0 phosphate buffer, measures the enzyme activity of D-ALPHA-Hydroxypropionic acid oxidizing ferment, The optimal reactive temperature for determining enzyme is 40 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 40 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 4.0 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> 1710
<212> DNA
<213> Pantoea dispersa ATCC14593
<400> 1
atgaataaca acacctcaca ccctgccctg ctgtccgagc tgcgccgtct ggtcggcgcg 60
tcacagctgc tgaccgacgc agagcaaacc gcgcgctatc gtaagggttt ccgttccggc 120
gagggcgatg cgctggcggt ggttttcccc ggctcgctgc tggagctatg gcgcgtactg 180
caggcgctgg tacaggccga tgccatcgtg ctgatgcagg ccgccaatac cggcctgacc 240
gaaggttcaa cgcccaacgg caacgattac gatcgcccgg tggtgatcat cagtaccctg 300
cgtctcgaca aactgcagct gatcgacggc ggcaggcaga ttctcgcctt ccccggcagc 360
acgctgtatc agctggagaa aacccttaaa ccgctcgggc gcgagccgca ctcggtgatt 420
ggctcctcgt gcatcggcgc ctcggtgctg ggcggcatct gcaataactc cggcggctcg 480
ctgatcaaac gcggcccggc ctacagcgaa atggcgctgt ttgcacaggt ggacgccgac 540
ggcaagctcc ggctggtcaa ccatctgggc attgacctcg gtcattcgcc ggaagagatt 600
ctcggccggc tggacgatga gcgctggcag ccggatgatg tacgctggga tgcgcgccac 660
gcgtccgacc acgaatacgc cgagcgggtg cgggacattc atgccgatac gccggcgcgt 720
ttcaacgccg accagcggcg cctgtttgag gcgtcaggct gcgccggcaa actggcggtg 780
tttgccgtgc ggctcgacac cttccccacc gagccgaagc agcaggtgtt ttacattggc 840
accaacgacc cggcgacgct cggcgagctg cgtcgccaca tactggcgga gtttacccat 900
ttaccggtcg ccggtgagta catgcaccgc gatattttcg acatcgccga ggtctacggc 960
aaagacacct ttgtgatgat tgataaactc ggcaccgaca agatgccact gtttttcacc 1020
ctcaaaggcc gggtggatgc ctggctcagc aagctgagct tcgttaagcc gcacctgacc 1080
gatcgcctgc tgcaacgcat cagccgctgg ttcccggcgc acctgccgaa gcgcctgaag 1140
cagtatcgtg acaaatacga gcaccacctg atgctgaaga tgtctggcga cggcgtggcc 1200
gaagcgcagg cgtatctgca ggagtatttc cgcgaaggcg gtggtgagtt ctttgcctgc 1260
gacgccaaag agggcgcgca cgcctttttg catcgttttg ccgcagccgg tgccgccgtg 1320
cgctatcacg cggtgcacgc tgacgaagtg gaggatattc tggcgctgga cattgcgctg 1380
cgccgcaacg atcaggagtg gtttgagcgc ctgccgccgg agtttgataa cgatctgatt 1440
catcgccttt actacggcca cttcttctgc catgtgttcc atcaggatta cattgtgaag 1500
aaaggcgtag atgcccatgc gctgaaggag aagatgctgg cgatcctcgc ggcgcgcggc 1560
gcggagtatc cggcagagca taacgttggc catctgtatg cggccaaacc gcagctgcag 1620
gcgttttacc gccagctcga tccgaccaat acctttaacc cgggcatcgg gaaaaccagt 1680
aagcacaaag gctgggcggt gaaagcctga 1710
<210> 2
<211> 569
<212> PRT
<213> Pantoea dispersa ATCC14593
<400> 2
Met Asn Asn Asn Thr Ser His Pro Ala Leu Leu Ser Glu Leu Arg Arg
1 5 10 15
Leu Val Gly Ala Ser Gln Leu Leu Thr Asp Ala Glu Gln Thr Ala Arg
20 25 30
Tyr Arg Lys Gly Phe Arg Ser Gly Glu Gly Asp Ala Leu Ala Val Val
35 40 45
Phe Pro Gly Ser Leu Leu Glu Leu Trp Arg Val Leu Gln Ala Leu Val
50 55 60
Gln Ala Asp Ala Ile Val Leu Met Gln Ala Ala Asn Thr Gly Leu Thr
65 70 75 80
Glu Gly Ser Thr Pro Asn Gly Asn Asp Tyr Asp Arg Pro Val Val Ile
85 90 95
Ile Ser Thr Leu Arg Leu Asp Lys Leu Gln Leu Ile Asp Gly Gly Arg
100 105 110
Gln Ile Leu Ala Phe Pro Gly Ser Thr Leu Tyr Gln Leu Glu Lys Thr
115 120 125
Leu Lys Pro Leu Gly Arg Glu Pro His Ser Val Ile Gly Ser Ser Cys
130 135 140
Ile Gly Ala Ser Val Leu Gly Gly Ile Cys Asn Asn Ser Gly Gly Ser
145 150 155 160
Leu Ile Lys Arg Gly Pro Ala Tyr Ser Glu Met Ala Leu Phe Ala Gln
165 170 175
Val Asp Ala Asp Gly Lys Leu Arg Leu Val Asn His Leu Gly Ile Asp
180 185 190
Leu Gly His Ser Pro Glu Glu Ile Leu Gly Arg Leu Asp Asp Glu Arg
195 200 205
Trp Gln Pro Asp Asp Val Arg Trp Asp Ala Arg His Ala Ser Asp His
210 215 220
Glu Tyr Ala Glu Arg Val Arg Asp Ile His Ala Asp Thr Pro Ala Arg
225 230 235 240
Phe Asn Ala Asp Gln Arg Arg Leu Phe Glu Ala Ser Gly Cys Ala Gly
245 250 255
Lys Leu Ala Val Phe Ala Val Arg Leu Asp Thr Phe Pro Thr Glu Pro
260 265 270
Lys Gln Gln Val Phe Tyr Ile Gly Thr Asn Asp Pro Ala Thr Leu Gly
275 280 285
Glu Leu Arg Arg His Ile Leu Ala Glu Phe Thr His Leu Pro Val Ala
290 295 300
Gly Glu Tyr Met His Arg Asp Ile Phe Asp Ile Ala Glu Val Tyr Gly
305 310 315 320
Lys Asp Thr Phe Val Met Ile Asp Lys Leu Gly Thr Asp Lys Met Pro
325 330 335
Leu Phe Phe Thr Leu Lys Gly Arg Val Asp Ala Trp Leu Ser Lys Leu
340 345 350
Ser Phe Val Lys Pro His Leu Thr Asp Arg Leu Leu Gln Arg Ile Ser
355 360 365
Arg Trp Phe Pro Ala His Leu Pro Lys Arg Leu Lys Gln Tyr Arg Asp
370 375 380
Lys Tyr Glu His His Leu Met Leu Lys Met Ser Gly Asp Gly Val Ala
385 390 395 400
Glu Ala Gln Ala Tyr Leu Gln Glu Tyr Phe Arg Glu Gly Gly Gly Glu
405 410 415
Phe Phe Ala Cys Asp Ala Lys Glu Gly Ala His Ala Phe Leu His Arg
420 425 430
Phe Ala Ala Ala Gly Ala Ala Val Arg Tyr His Ala Val His Ala Asp
435 440 445
Glu Val Glu Asp Ile Leu Ala Leu Asp Ile Ala Leu Arg Arg Asn Asp
450 455 460
Gln Glu Trp Phe Glu Arg Leu Pro Pro Glu Phe Asp Asn Asp Leu Ile
465 470 475 480
His Arg Leu Tyr Tyr Gly His Phe Phe Cys His Val Phe His Gln Asp
485 490 495
Tyr Ile Val Lys Lys Gly Val Asp Ala His Ala Leu Lys Glu Lys Met
500 505 510
Leu Ala Ile Leu Ala Ala Arg Gly Ala Glu Tyr Pro Ala Glu His Asn
515 520 525
Val Gly His Leu Tyr Ala Ala Lys Pro Gln Leu Gln Ala Phe Tyr Arg
530 535 540
Gln Leu Asp Pro Thr Asn Thr Phe Asn Pro Gly Ile Gly Lys Thr Ser
545 550 555 560
Lys His Lys Gly Trp Ala Val Lys Ala
565

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 source is in the general bacterium (Pantoea of dispersion Dispersa D-ALPHA-Hydroxypropionic acid oxidizing ferment), amino acid sequence are shown in SEQ ID NO:2；The alpha-hydroxy acid ester be it is following it One: tanshinol borneol ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid norbornene ester, right Hydroxyphenyl lactic acid isopropyl ester, lactic acid norbornene ester, mandelic acid norbornene ester, almond isopropyl propionate, danshensu asarum alcohol ester, phenyllactic acid are thin Octanol 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.
CN201710006541.9A 2017-01-05 2017-01-05 A kind of oxidizing ferment and its application Active CN106754779B (en)

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