CN106754787A - A kind of oxidizing ferment and its application - Google Patents

Abstract

The invention relates to the acquisition and clone expression of an L-alpha-hydroxyacid oxidase gene derived from Carnimonas nigrificans, which belongs to the field of bioengineering. Its substrate specificity is disclosed, and at the same time, the L-α-hydroxy acid oxidase can oxidize (S)-α-hydroxy acid ester, and can be applied to the preparation of optically pure (R)-α-hydroxy acid ester.

Description

A kind of oxidase and its application

technical field

The invention clones and expresses an L-α-hydroxyacid oxidase, discloses its nucleotide sequence, amino acid sequence, enzymatic properties and application, and belongs to the field of industrial microorganisms.

Background technique

L-α-hydroxyacid oxidase (L-α-hydroxyacid oxidase) is a dehydrogenase with FMN (FAD) as a coenzyme (Aliphatic l-α-hydroxyacid oxidase from rat livers purification and properties. Biochimica et Biophysica Acta (BBA )-Enzymology 1968,167:9-22), usually contains glycolate oxidase (glycolate oxidase) (Preparation and some properties ofcrystalline glycolic acid oxidase of spinach.J.Biol.Chem.1958,231(1):135– 57), L-lactate oxidase (Conversion of L-lactate oxidase to a longchain alpha-hydroxyacid oxidase by site-directed mutagenesis of alanine 95toglycine.J Biol Chem.1996 8;271(45):28300 -28305). It can be used in biosensors to determine the content of lactic acid, or to oxidize L-lactic acid to produce pyruvate. It is also used in the preparation of optically pure α-hydroxy acids (Chinese patent 201210109290.4)

So far, it has been tested in Pseudomonas putida, Aerococcus viridians, Streptococcus sp., Pediococcus sp., Lactococcus lactis, Edwardsimus spp. L-lactate oxidase was cloned and expressed in bacteria such as Edwardsiella tarda, Mycobacterium smegmatis, Zymomonas mobilis and Acetobacter peroxidans. L-lactate oxidase has also been detected in some fungi such as Geotrichum candidum and Yarrowia lipolytica. (Search for Lactate Oxidase Producer Microorganisms, Applied Biochemistry and Microbiology, 2007, 43(2)178–181)

The present invention clones and expresses a novel L-alpha-hydroxyacid oxidase from Carnimonas nigrificans for the first time, which can not only oxidize (S)-alpha-hydroxyacids, but also oxidize (S)-alpha-hydroxyacids, It can be applied to the preparation of optically pure (R)-α-hydroxy acid ester and (R)-α-hydroxy acid.

Contents of the invention

The present invention clones a gene of L-alpha-hydroxyacid oxidase from Carnimonas nigrificans, utilizes Escherichia coli engineering bacteria to express heterologously, discloses its related enzymatic characteristics, and conducts applied research.

Technical scheme of the present invention is as follows:

1. Strains

The source strain of the L-α-hydroxyacid oxidase gene of the present invention is: Carnimonas nigrificans ATCC BAA-78, purchased from the American ATCC strain bank.

2. Cloning of L-α-hydroxyacid oxidase gene

The total genomic DNA of Carnimonas nigrificans ATCC BAA-78 was extracted. Design specific primers and use PCR method to amplify the full-length coding frame sequence of L-α-hydroxyacid oxidase gene. and construct recombinant plasmids.

3. Expression and purification of L-α-hydroxyacid oxidase

The recombinant plasmid was introduced into E.coli BL21(DE3) to induce expression. The crude enzyme solution was obtained after the bacterial cells were crushed, which was purified and freeze-dried for later use.

4. Analysis of enzymatic properties of L-α-hydroxyacid oxidase

Using L-lactic acid as a substrate, the effect of pH on the enzyme activity of L-α-hydroxyacid oxidase described in the present invention was studied.

The effect of temperature on the enzyme activity of L-α-hydroxyacid oxidase of the present invention was studied by using L-lactic acid as a substrate.

Substrate specificity analysis of L-α-hydroxyacid oxidase: the substrates used are L-lactic acid, glycolic acid, L-phenyllactic acid, L-tartaric acid, L-malic acid, L-p-hydroxyphenyllactic acid, L- Danshensu, L-mandelic acid.

The 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. The method is carried out.

5. L-α-hydroxyacid oxidase resolves swirling α-hydroxyesters

The method for splitting α-hydroxy esters (alpha-hydroxy esters) is: take 0.1 g of the purified enzyme in a 50 mL Erlenmeyer flask, add 5 mM of α-hydroxy esters into a pH 7 phosphate buffer, and 30° C., 150 rpm in a water-bath shaker for 16 h, and the supernatant was analyzed by liquid chromatography after conversion. The α-hydroxyl group in the (S)-α-hydroxy ester is dehydrogenated to the corresponding α-ketoester, and the (R)-α-hydroxy ester is not oxidized.

The optical purity of the product (R)-α-hydroxyester was assessed by enantiomeric excess (%e.e):

Enantiomeric excess value %ee=[(S _R -S _S )/(S _R +S _S )]×100%

(R)-α-hydroxy acid ester yield (%)=(S _R /S ₀ )×100%

In the formula, S _R is the peak area of the (R)-enantiomer after the reaction, S _S is the liquid chromatography peak area of the (S ₎ -enantiomer after the reaction, and S is the front of the reaction (R)- and (S) - The sum of the liquid chromatography peak areas of the enantiomers.

The liquid chromatography conditions for product determination are: Chiralcel OD-H chiral column (4.6×250mm), the mobile phase volume ratio is n-hexane:isopropanol:trifluoroacetic acid=80:20:0.1, and the flow rate is 0.5mL/min, The column temperature is 25°C, the detection wavelength is 210nm, and the injection volume is 20μL.

The α-hydroxy acid ester is one of the following: bornyl danshensu, isopropyl danshensu, bornyl phenyl lactate, isopropyl phenyl lactate, bornyl p-hydroxybenzoate, isopropyl p-hydroxyphenyl lactate, Bornyl Mandelate, Isopropyl Mandelate, Danshensu Asaryl Octyl Ester, Bornyl Lactate, Asaryl Octyl Phenyl Lactate, Asaryl Octyl P-Hydroxyphenyl Lactate.

The α-hydroxy acid ester is synthesized according to the methods published in Chinese patents 200610042787.3, 201410180490.8, 201410175950.8 and 20140699506.6.

Advantages of the present invention: An L-alpha-hydroxyacid oxidase was cloned from Carnimonas nigrificans ATCC BAA-78, which can oxidize (S)-alpha-hydroxyacids and (S)-alpha-hydroxyacids The acid ester can be used for large-scale preparation of chiral pure (R)-α-hydroxy acid ester, and has important industrial application value.

detailed description

Example 1

This example is the cloning of the L-α-hydroxyacid oxidase gene of the present invention and the construction of Escherichia coli engineering bacteria.

1. Extraction of Carnimonas nigrificans ATCC BAA-78DNA

Cultivate the Carnimonas nigrificans ATCC BAA-78 strain in LB medium for 12h, centrifuge at 12,000rmp/min for 10min to obtain the bacterial cells, use the Bacterial Genomic DNA Extraction Kit (TaKaRa Company) to extract the total genomic DNA of the bacterial cells according to its operation, and put them in the refrigerator spare.

2. Competent preparation of Escherichia coli

(1) Inoculate E. coli DH5α and BL21(DE3) into 250 mL shake flasks containing 20 mL LB medium respectively, and culture overnight at 37° C. and 200 rpm/min.

(2) Inoculate in 50 mL LB medium according to 1% inoculum amount, and culture at 37°C until OD ₆₀₀ is about 0.6 (about 2-3 hours).

(3) Transfer the bacterial solution to a 50 mL pre-cooled centrifuge tube, place on ice for 30 min, and centrifuge at 8000 rpm/min at 4°C for 5 min.

(4) Discard the supernatant, add 5 mL of pre-cooled 0.1 mol/L CaCl ₂ solution to suspend the cells, place on ice for 20 min, and centrifuge at 8000 rpm/min at 4°C for 5 min. Repeat 2 times.

(5) Discard the supernatant, add 1.5 mL of pre-cooled 0.1 mol/L CaCl ₂ solution (containing 15% glycerol), gently suspend the bacteria, and then add 100 μL of the bacteria solution to each centrifuge tube (1.5 mL). Store in -70°C refrigerator for later use.

3. Cloning of L-α-hydroxyacid oxidase gene

(1) Primer design

The primer sequences were designed as:

Primer 1: 5'GCCGGGATCCATGAGTACTCCGCTAACTCCACGTC 3'

Primer 2: 5'GCCGTCTAGACGTCCCGCGCCGATAG 3'

(2) PCR amplification

Using the two primers synthesized above, the genomic DNA of Carnimonas nigrificans ATCC BAA-78 was used as a template for PCR amplification.

In this step, the amplification system is:

The amplification procedure is:

98℃, 10min

98°C, 10sec; 55°C, 15sec; 72°C, 2min for 30 cycles

72℃, 10min

The PCR product was sent to BGI for sequencing to obtain the gene sequence of the enzyme, as shown in SEQ ID NO:1. The amino acid sequence obtained according to the gene sequence is shown in SEQ ID NO:2.

(3) Double digestion and ligation

The pColdⅡ plasmid and PCR product were subjected to double enzyme digestion. The enzyme digestion system was: 10×cut buffer 3 μl, DNA 4 μl, enzymes BamHI and XbaI 0.5 μl each, sterile water 2 μl, a total of 30 μl. Double enzyme digestion was carried out in a water bath at 37°C for 1 hour. The DNA fragment was cloned into pColdⅡ vector and transformed into E.coli DH5α competent cells. Ligation system: 10×DNA ligase buffer 2.5 μl, DNA fragment 8 μl, carrier DNA 2 μl, T4 DNA ligase 1 μl, sterile water 11.5 μl, a total of 25 μl. Connect in a water bath at 16°C for 12h-16h.

(4) Conversion

step:

1 Add 100 μl DH5α competent bacteria to the connection system, mix gently, and ice-bath for 30 minutes.

2 Place in a preheated 42°C water bath for 90 seconds for heat shock treatment.

3 immediately ice bath 2min.

4 Add 1ml of LB culture medium without antibiotics, incubate at 37°C for 1 hour to recover the bacteria.

5 Spread the bacteria evenly on the LB plate containing antibiotics.

6 After 24 hours of cultivation, it grows well. Pick a single colony for colony PCR, nucleic acid electrophoresis verification, and extract recombinant plasmids. The recombinant plasmid was introduced into BL21 Escherichia coli competent and stored for future use.

Example 2

This example is the induced expression and isolation and purification of the L-α-hydroxyacid oxidase of the present invention.

1. Add 500μl of recombinant bacteria solution to 50ml of LB culture solution. Cultivate at 37°C for 2.5h, and stand at 15°C for 0.5h. Then add 20 μl of 0.5M IPTG, and incubate at 15°C for 24 hours. Centrifuge the fermentation broth (8000rmp/min, 10min) to obtain the thallus, redissolve the thallus with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (20mmol/L, pH 7.0), break it with an ultrasonic breaker, and centrifuge (8000rmp/min , 10 min) to collect the supernatant to obtain the crude enzyme solution.

2. Use the AKTA avant 150 protein purification system to purify the crude enzyme liquid obtained in step 1 with a nickel column. The elution method is: put the four pipelines A1, A2, B1, and B2 into water, and set the system flow to 20ml/ min flow rate, exhaust. Then set system flow 1ml/min, flow path (column position 3), delta pressure 0.3, pre-pressure 0.5, Gradient 0, inset A1, install the column after the water droplets flow out evenly, and put A1 into the binding solution after equilibrating for ten minutes In the process, put B1 into the eluent, exhaust once again, and equilibrate for 20 minutes, then load the crude enzyme solution, and use 500mM high-concentration imidazole buffer (the solution where B1 is located) to gradiently elute the target protein, and the adsorbed The protein is eluted on the ion column to obtain the purified enzyme. The purified enzyme was freeze-dried for further use.

Example 3

This example is the optimal temperature of the L-α-hydroxyacid oxidase of the present invention. Using L-lactic acid as the substrate, put the substrate and phosphate buffer solution with a pH of 5.0 in a water bath for 15 minutes under different temperature conditions of 30-60°C, measure the enzyme activity of L-α-hydroxyacid oxidase, and determine the optimal activity of the enzyme. The reaction temperature was 35°C.

Example 4

This example is the optimum pH value of the L-α-hydroxyacid oxidase of the present invention. Using L-lactic acid as the substrate, the enzyme activity of the substrate was measured at pH 3-9 in a 35°C water bath for 15 minutes. It was found that the enzyme activity of L-α-hydroxyacid oxidase was the highest at pH 5.0.

Example 5

In this example, the reaction characteristics of the L-α-hydroxyacid oxidase of the present invention with different substrates are listed in Table 2.

Table 2 Activities of L-α-hydroxyacid oxidase on different substrates

Example 6

According to the method in the summary of the invention, various racemic α-hydroxy acid esters are resolved, and the results are shown in the table below:

Table 3 The effect of resolving various racemic α-hydroxy esters

It can be seen from the above table that when the reaction time is sufficient, various types of highly optically pure (R)-α-hydroxyesters can be obtained, and the optical specificity of the enzyme is very good.

SEQUENCE LISTING

<110> Jiangnan University

<120> A kind of oxidase and its application

<130> No

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1188

<212>DNA

<213> Carnimonas nigrificans ATCC BAA-78

<400> 1

atgagtactc cgctaactcc acgtcgcagc gatcaaaagc gttatctgaa tctggacgac 60

tacagcgcgg ctgcacagcg cctgttgcca cgggcgctgt tcgcctacgt ttcaggctcg 120

gcaggcgacg gtgaaacact ggctgcgaac cgcgatgcct tcaagcagtg ggcgctaata 180

ccgcagcagc tgcgcggcgt cgcttcacga agtgccgcca tcgagctact cggccagcgt 240

tttgccatgc cgattggcgt gtcggcattc ggcgcggcgg cggttttggg ctataacgct 300

gatatcgcga tggcggcagc ggcatataac gcaaatgtgc cttatcagct gagtgccaac 360

tccattacgc ccatggaaga ggtaatcaag gtgaatcccg aggcgtggtt cgccgcctat 420

ctgccagacg atgcagcgct gatcagaggc gttgtcgagc gcgttagcaa cgcaggcttc 480

aagactctgg taattacggt cgatgtgccg gtggctgccc gacgcgagcc ggaaacgcgg 540

gctggctacg ccatgccgtt caaggcatcg gcgcgcttgg ggtttgatat gctccgccat 600

ccgcgctggg tgctgagccg cttcactcgt accctgctgc ggcgcgggat tccgcacatt 660

gaaaacgtta ccccccagcg tggccccagt atctttgctt ccaacacagg gtcgatcggt 720

ggagcggcca acttcaactg ggacaatatt cgtgctattc gcgcacagtg gcccggcaag 780

ctgctgctga agggtattct ttccgaacgc gatgcgctaa cagcacagga gattggcgtc 840

gatggcgtga ttgtctcgaa tcatggcgcc cgtttaagcg attcatccat cacaccgctg 900

gaagccctgc caaacatccg cgcagcctgc cctgagctga cggttctgct cgatggcggc 960

gtgcggcgtg ccggggatgc gttaaaggcg atcgcgctgg gggcagatgg cgtgatgatt 1020

ggccgtccac tgttttacgc caccatcctt ggtggccggc gcggcctcgt tcatgcgctg 1080

catttgctga gcgcagaact ggatcgggag atgggctttc atggcttgct aaacgttaac 1140

gaagtacgtg aggaagatcg gctctatcgg cgcgggacgc ttgcctaa 1188

<210> 2

<211> 395

<212> PRT

<213> Carnimonas nigrificans ATCC BAA-78

<400> 2

Met Ser Thr Pro Leu Thr Pro Arg Arg Ser Asp Gln Lys Arg Tyr Leu

1 5 10 15

Asn Leu Asp Asp Tyr Ser Ala Ala Ala Gln Arg Leu Leu Pro Arg Ala

20 25 30

Leu Phe Ala Tyr Val Ser Gly Ser Ala Gly Asp Gly Glu Thr Leu Ala

35 40 45

Ala Asn Arg Asp Ala Phe Lys Gln Trp Ala Leu Ile Pro Gln Gln Leu

50 55 60

Arg Gly Val Ala Ser Arg Ser Ala Ala Ile Glu Leu Leu Gly Gln Arg

65 70 75 80

Phe Ala Met Pro Ile Gly Val Ser Ala Phe Gly Ala Ala Ala Val Leu

85 90 95

Gly Tyr Asn Ala Asp Ile Ala Met Ala Ala Ala Ala Tyr Asn Ala Asn

100 105 110

Val Pro Tyr Gln Leu Ser Ala Asn Ser Ile Thr Pro Met Glu Glu Val

115 120 125

Ile Lys Val Asn Pro Glu Ala Trp Phe Ala Ala Tyr Leu Pro Asp Asp

130 135 140

Ala Ala Leu Ile Arg Gly Val Val Glu Arg Val Ser Asn Ala Gly Phe

145 150 155 160

Lys Thr Leu Val Ile Thr Val Asp Val Pro Val Ala Ala Arg Arg Glu

165 170 175

Pro Glu Thr Arg Ala Gly Tyr Ala Met Pro Phe Lys Ala Ser Ala Arg

180 185 190

Leu Gly Phe Asp Met Leu Arg His Pro Arg Trp Val Leu Ser Arg Phe

195 200 205

Thr Arg Thr Leu Leu Arg Arg Gly Ile Pro His Ile Glu Asn Val Thr

210 215 220

Pro Gln Arg Gly Pro Ser Ile Phe Ala Ser Asn Thr Gly Ser Ile Gly

225 230 235 240

Gly Ala Ala Asn Phe Asn Trp Asp Asn Ile Arg Ala Ile Arg Ala Gln

245 250 255

Trp Pro Gly Lys Leu Leu Leu Lys Gly Ile Leu Ser Glu Arg Asp Ala

260 265 270

Leu Thr Ala Gln Glu Ile Gly Val Asp Gly Val Ile Val Ser Asn His

275 280 285

Gly Ala Arg Leu Ser Asp Ser Ser Ile Thr Pro Leu Glu Ala Leu Pro

290 295 300

Asn Ile Arg Ala Ala Cys Pro Glu Leu Thr Val Leu Leu Asp Gly Gly

305 310 315 320

Val Arg Arg Ala Gly Asp Ala Leu Lys Ala Ile Ala Leu Gly Ala Asp

325 330 335

Gly Val Met Ile Gly Arg Pro Leu Phe Tyr Ala Thr Ile Leu Gly Gly

340 345 350

Arg Arg Gly Leu Val His Ala Leu His Leu Leu Ser Ala Glu Leu Asp

355 360 365

Arg Glu Met Gly Phe His Gly Leu Leu Asn Val Asn Glu Val Arg Glu

370 375 380

Glu Asp Arg Leu Tyr Arg Arg Gly Thr Leu Ala

385 390 395

Claims (5)

1. A L-alpha-hydroxyacid oxidase derived from Carnimonas nigrificans, the amino acid sequence of which is shown in SEQ ID NO:2. 2. The L-alpha-hydroxyacid oxidase according to claim 1, whose nucleotide sequence is shown in SEQ ID NO:1. 3. The L-alpha-hydroxyacid oxidase according to claim 1, whose optimum reaction temperature is 35° C., and the optimum reaction pH is 5. 4. L-alpha-hydroxyacid oxidase according to claim 1, can oxidize L-lactic acid, glycolic acid, L-phenyllactic acid, L-p-hydroxyphenyllactic acid, L-tartaric acid, L-malic acid, L- Mandelic acid and L-danshensu generate corresponding keto acids. 5. L-alpha-hydroxy acid oxidase according to claim 1, (S)-alpha-hydroxy acid ester in the oxidizable racemic alpha-hydroxy acid ester, splits and prepares to obtain corresponding optically pure (R )-α-hydroxyester and α-ketoester, the α-hydroxyester is one of the following: borneyl danshensu, isopropyl danshensu, bornyl phenyl lactate, isopropyl phenyl lactate, p- Bornyl Hydroxyphenyl Lactate, Isopropyl p-Hydroxyphenyl Lactate, Bornyl Lactate, Bornyl Mandelate, Isopropyl Mandelate, Danshensu Asaryl Octyl Lactate, Asaryl Octyl Phenyl Lactate, Asaryl Octyl P-Hydroxyphenyl Lactate ester.