CN106701703A - Oxidase and application thereof - Google Patents

Abstract

The invention relates to the acquisition and clone expression of an L-alpha-hydroxyacid oxidase gene derived from Proteus mirabilis, belonging 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-α-hydroxyacid oxidase from Kerstersia gyiorum DSM 16618 for the first time, which can not only oxidize (S)-α-hydroxyacid, but also oxidize (S)-α-hydroxyacid Esters, which can be applied to the preparation of optically pure (R)-α-hydroxy acid esters and (R)-α-hydroxy acids.

Contents of the invention

The present invention clones a gene of L-alpha-hydroxyacid oxidase with FMN as a coenzyme from Proteus mirabilis, utilizes Escherichia coli engineering bacteria for heterologous expression, and discloses its related enzymatic characteristics, and 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: Proteus mirabilis ATCC 25933, purchased from the American ATCC strain bank.

2. Cloning of L-α-hydroxyacid oxidase gene

The total genomic DNA of Proteus mirabilis ATCC 25933 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 _S is the peak area of the (S)-enantiomer after the reaction, S _R is the liquid chromatography peak area of the ₍ R)-enantiomer after the reaction, and S is the front of the reaction (S)- and (R) - 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: Cloning and expressing a novel L-α-hydroxyacid oxidase from Proteus mirabilis, which can oxidize (S)-α-hydroxyacid and (S)-α-hydroxyacid The 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 Proteus mirabilis ATCC 25933 DNA

The Proteus mirabilis ATCC 25933 strain was cultured in LB medium for 12 hours, centrifuged at 12,000rmp/min for 10 minutes to obtain the bacteria, and the bacterial genome DNA extraction kit (TaKaRa Company) was used to extract the total genomic DNA of the bacteria according to its operation, and stored in the refrigerator for later use.

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) to pack, 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'GCCGGGATCCATGAAACATACATTGTTAAAAAC 3'

Primer 2: 5'GCCGTCTAGATCGTTTTTAACAATGTATGTTTCAT 3'

(2) PCR amplification

Using the two primers synthesized above, the genomic DNA of Proteus mirabilis ATCC 25933 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 L-α-hydroxyacid oxidase, 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 ten minutes of equilibration , put B1 into the eluent, exhaust once more, and equilibrate for 20 minutes, then load the crude enzyme solution, and use 500mM high-concentration imidazole buffer solution (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 8.0 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 enzyme activity of the substrate was measured 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 8.0.

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> 1194

<212>DNA

<213> Proteus mirabilis ATCC 25933

<400> 1

atgaaacata cattgttaaa aacgaccgca attgctatgg cattaagtgt gggagttgta 60

caagcagctg aatataaagc aagtaccgca gaaggtccaa tcaaaatagt taatttaaaa 120

gccatggaag cacaggtaga agcaaatatg gataaaggtg ctttcggtta tattcgtggc 180

ggggctgagg atgaaaataa tttaagagca aatactcgag catttgataa aaaatatatt 240

atgccccgtt cattacaagg tattgagttt tcagattatta atctaaaaac agaattttta 300

ggtattaaat tagatacgcc tattattcag gcaccgatgg cagcacaagg gcttgctcat 360

caacaaggcg aagttgccac agcaaaaggt atggcgaaag cgggttctat tttctcatta 420

agtacttatg gtaataaaac cattaaagaa gttgcgcaag cacaaccggg ttatccgttc 480

ttttttcagc tatatatgag taaaaacgac gcgtttaatc agtatatttt atcgcaagca 540

aaacagtatg gcgcgaaagg cattattatg actatcgact cctctgttgg tggttatcgt 600

gaagatgatg tcaaaaacaa ctttcaattt ccgttaggtt ttgccaattt ggaagcattc 660

gccaaaatca gtgatgataa atcaaaaaca ggtaaaggtt cgggtattag tgaaatctat 720

gctcaagcta aacaagcatt tactcctgct gatattcaat atgtgaaaaa gatgtcaggt 780

ttaccagtca ttgtgaaagg tattgaatca cctgaagatg ccgatactgc aattaaagcg 840

ggtgctgatg cgatttgggt gtctaatcat ggtggtcgtc agttagatag tgcaccagca 900

accattgatg tattgccagc tattgctaaa gtcgtgaaca aacgtgttcc tatcgttttc 960

gtagtggtg ttcgtcgtgg ctcacatgtc tttaaagcgt tagcgagtgg tgcagatgtt 1020

gttgctgtgg ggcgtccaat tctttatgga ttaaatttag gcggtgctga aggtgttaat 1080

tcagttatcc agcaattaaa taaagagtta agaattaata tgatgttagg tggtgcaaga 1140

aacgtcaaag aaattcaagc aacgcacttg tatacagatg ctgactttaa ataa 1194

<210> 2

<211> 397

<212> PRT

<213> Proteus mirabilis ATCC 25933

<400> 2

Met Lys His Thr Leu Leu Lys Thr Thr Ala Ile Ala Met Ala Leu Ser

1 5 10 15

Val Gly Val Val Gln Ala Ala Glu Tyr Lys Ala Ser Thr Ala Glu Gly

20 25 30

Pro Ile Lys Ile Val Asn Leu Lys Ala Met Glu Ala Gln Val Glu Ala

35 40 45

Asn Met Asp Lys Gly Ala Phe Gly Tyr Ile Arg Gly Gly Ala Glu Asp

50 55 60

Glu Asn Asn Leu Arg Ala Asn Thr Arg Ala Phe Asp Lys Lys Tyr Ile

65 70 75 80

Met Pro Arg Ser Leu Gln Gly Ile Glu Phe Ser Asp Ile Asn Leu Lys

85 90 95

Thr Glu Phe Leu Gly Ile Lys Leu Asp Thr Pro Ile Ile Gln Ala Pro

100 105 110

Met Ala Ala Gln Gly Leu Ala His Gln Gln Gly Glu Val Ala Thr Ala

115 120 125

Lys Gly Met Ala Lys Ala Gly Ser Ile Phe Ser Leu Ser Thr Tyr Gly

130 135 140

Asn Lys Thr Ile Lys Glu Val Ala Gln Ala Gln Pro Gly Tyr Pro Phe

145 150 155 160

Phe Phe Gln Leu Tyr Met Ser Lys Asn Asp Ala Phe Asn Gln Tyr Ile

165 170 175

Leu Ser Gln Ala Lys Gln Tyr Gly Ala Lys Gly Ile Ile Met Thr Ile

180 185 190

Asp Ser Ser Val Gly Gly Tyr Arg Glu Asp Asp Val Lys Asn Asn Phe

195 200 205

Gln Phe Pro Leu Gly Phe Ala Asn Leu Glu Ala Phe Ala Lys Ile Ser

210 215 220

Asp Asp Lys Ser Lys Thr Gly Lys Gly Ser Gly Ile Ser Glu Ile Tyr

225 230 235 240

Ala Gln Ala Lys Gln Ala Phe Thr Pro Ala Asp Ile Gln Tyr Val Lys

245 250 255

Lys Met Ser Gly Leu Pro Val Ile Val Lys Gly Ile Glu Ser Pro Glu

260 265 270

Asp Ala Asp Thr Ala Ile Lys Ala Gly Ala Asp Ala Ile Trp Val Ser

275 280 285

Asn His Gly Gly Arg Gln Leu Asp Ser Ala Pro Ala Thr Ile Asp Val

290 295 300

Leu Pro Ala Ile Ala Lys Val Val Asn Lys Arg Val Pro Ile Val Phe

305 310 315 320

Asp Ser Gly Val Arg Arg Gly Ser His Val Phe Lys Ala Leu Ala Ser

325 330 335

Gly Ala Asp Val Val Ala Val Gly Arg Pro Ile Leu Tyr Gly Leu Asn

340 345 350

Leu Gly Gly Ala Glu Gly Val Asn Ser Val Ile Gln Gln Leu Asn Lys

355 360 365

Glu Leu Arg Ile Asn Met Met Leu Gly Gly Ala Arg Asn Val Lys Glu

370 375 380

Ile Gln Ala Thr His Leu Tyr Thr Asp Ala Asp Phe Lys

385 390 395

Claims (5)

1. one kind derives from the L- alpha-hydroxy acid oxidizing ferment of proteus mirabilis (Proteus mirabilis), its amino acid sequence It is SEQ ID NO:Shown in 2.

2. L- alpha-hydroxy acids oxidizing ferment according to claim 1, its nucleotides sequence is classified as SEQ ID NO:Shown in 1.

3. L- alpha-hydroxy acids oxidizing ferment according to claim 1, its optimal reactive temperature is 50 DEG C, and optimal reaction pH is 8.

4. L- alpha-hydroxy acids oxidizing ferment according to claim 1, oxidable Pfansteihl, glycolic, L- phenyllactic acids, L- are to hydroxyl Phenyllactic acid, L-TARTARIC ACID, L MALIC ACID, L- mandelic acids, L- danshensus, generate corresponding ketone acid.

5. L- alpha-hydroxy acids oxidizing ferment according to claim 1, (the S)-alpha-hydroxy acid ester in oxidable racemic ' alpha '-carboxylic esters, Fractionation prepares corresponding optical voidness (R)-alpha-hydroxy acid ester and alpha-keto ester, and described alpha-hydroxy acid ester is one of following：The red sage root Plain norbornene ester, danshensu isopropyl ester, phenyllactic acid norbornene ester, phenyllactic acid isopropyl ester, para hydroxybenzene lactic acid norbornene ester, para hydroxybenzene breast Isopropyl propionate, 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.