CN106754794B - A kind of oxidase 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 alpha-hydroxy esters (alpha-hydroxy esters) is: take 0.1 g of the purified enzyme in a 50 mL Erlenmeyer flask, add 5 mM of alpha-hydroxy esters to the phosphate buffer solution of pH 7, Transform at 30° C., 150 rpm in a water-bath shaker for 16 hours, and analyze the supernatant by liquid chromatography after transformation. 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 ways

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 cell, use the bacterial genome DNA extraction kit (TaKaRa company) to extract the total genomic DNA of the bacterial cell according to its operation, and put it in the refrigerator spare.

2. Competent preparation of Escherichia coli

(1) Inoculate E.coli DH5α and BL21(DE3) into 250mL shake flasks containing 20mL LB medium, culture overnight at 37°C and 200rpm/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 it 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'GCCGGGATCCATGATTATTTCCAGCCCAACTGATT 3'

Primer 2: 5'GCCGTCTAGACGAACCACTTGATCCAGACGG 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 6.0 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 50°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 substrate was tested for enzyme activity at pH 3-9 in a 50°C water bath for 15 minutes. It was found that the enzyme activity of L-α-hydroxyacid oxidase was the highest at pH 6.0.

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 1.

Table 1 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 2 The effect of resolution of 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-hydroxy acid esters 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> 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 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.