CN108949720B

CN108949720B - Oxidized polysucrose modified epoxide hydrolase and application thereof

Info

Publication number: CN108949720B
Application number: CN201810839572.7A
Authority: CN
Inventors: 邹树平; 郑裕国; 轩秀玲; 王之见
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-07-27
Filing date: 2018-07-27
Publication date: 2021-03-19
Anticipated expiration: 2038-07-27
Also published as: CN108949720A

Abstract

The invention discloses an oxidized ficoll modified epoxide hydrolase and application thereof in resolution of epichlorohydrin, wherein the enzyme is prepared by dissolving oxidized ficoll and epoxide hydrolase in 0.2M, pH8.0, phosphate buffer solution, uniformly mixing, reacting at 0-15 ℃ and 600rpm for 3-30h, dialyzing with distilled water, taking trapped fluid, precipitating with ethanol aqueous solution with volume concentration of 50%, taking precipitate, and obtaining the ficoll modified epoxide hydrolase; the invention carries out chemical modification on epoxide hydrolase through polysucrose, greatly improves the specific activity and the thermal stability of the epoxide hydrolase, and has wide prospect in the enzymatic preparation and application of optical pure epoxide. The enzyme has simple production method, low cost and wide industrial application prospect.

Description

Oxidized polysucrose modified epoxide hydrolase and application thereof

(I) technical field

The invention relates to a method for simultaneously improving the specific activity and the thermal stability of epoxide hydrolase, in particular to a chemical modification method for modifying epoxide hydrolase by utilizing polysucrose.

(II) background of the invention

The chiral epoxide is an important chiral synthetic building block and is widely applied in the fields of medicine, chemical industry, materials, pesticides and the like. The chiral epoxide produced by the chemical method has the problems of high cost of metal catalyst, more byproducts, serious environmental pollution and the like. The biocatalysis method has the advantages of high selectivity, low cost, few byproducts, environmental protection and the like, and has good development prospect and application value.

Epoxide hydrolases (EH, EC 3.3.2.3) are capable of stereoselectively hydrolyzing and cleaving racemic epoxides to obtain optically active epoxides and the corresponding 1, 2-diols. The enzyme has wide sources, is commonly found in nature, and can be found in various organisms such as plants, insects, mammals, bacteria and the like. However, epoxide hydrolases generally have the problems of low activity, easy variability, poor stability and the like, and directly affect the production efficiency and production cost of the industrial application of the epoxide hydrolases.

Chemical modification is a very effective way to increase the stability of enzymes, and the presence of sugar groups increases the rigidity of the protein and affects the correct folding of the protein and its conformation. The carbohydrate group may also have pharmacological properties on the protein, such as biological activity, immunogenicity, receptor binding capacity, etc. Since the saccharides are highly hydrophilic, the hydration layer around the protein can be stabilized, promoting the formation of new hydrogen bonds. In addition, by binding carbohydrates, potential cleavage sites are hidden inside the structure, avoiding proteolytic attack. It follows that the glycosyl groups also play an important role in improving the stability of the protein itself. In recent years, although researchers have developed a variety of microorganisms capable of producing epoxide hydrolase such as Pseudomonas, Rhodococcus, Agrobacterium, Xanthobacter, Chryseomonas, Chaetomium globosum, Cunning-hamella, Alenteraria, Pleurotus, Rhodotorula, etc., epoxide hydrolase is also used for asymmetric resolution of racemic epichlorohydrin to produce chiral epichlorohydrin.

Disclosure of the invention

The invention aims to provide an oxidized ficoll modified epoxide hydrolase and application thereof for simultaneously improving relative enzyme activity and thermal stability of the epoxide hydrolase.

The technical scheme adopted by the invention is as follows:

the inventionThere is provided an oxidized ficoll-modified epoxide hydrolase prepared by the following method: (1) oxidized polysucrose: dissolving Polysucrose in 0.25M NaIO₄Reacting in water solution at 37 deg.C and 150rpm in dark for 6h, adding ethylene glycol to terminate the reaction, dialyzing the reaction solution in distilled water, and freeze-drying the dialyzed retentate to obtain oxidized polysucrose; the NaIO₄The volume dosage of the aqueous solution is 5-50mL/g (preferably 40mL/g) based on the mass of the polysucrose; (2) dissolving oxidized polysucrose and epoxide hydrolase in 0.2M, pH8.0 and Phosphate (PBS) buffer solution, uniformly mixing, reacting at 0-15 ℃ and 600rpm for 3-30h (preferably 4 ℃ and 600rpm for 16h), dialyzing with distilled water (the cut-off molecular weight of a dialysis bag is 14kDa, dialyzing for 24h, and exchanging dialysate once for 12 h), taking the cut-off solution, precipitating with 50% ethanol aqueous solution with volume concentration, taking the precipitate, and obtaining the epoxide hydrolase modified by polysucrose; the mass ratio of the oxidized ficoll to the epoxide hydrolase is 0.5-500:1, preferably 15: 1.

further, the enzyme activity of the epoxide hydrolase in the step (2) is 10-1000U/mg, preferably 200U/mg.

Further, the molecular weight of the ficoll in the step (1) is 70-700kDa, preferably 400 kDa.

Further, in the step (1), the dialysis time is 24h, the dialysis solution is replaced once at intervals of 12h, and the molecular weight cut-off of the dialysis bag is 14000 Da.

Further, the epoxide hydrolase in the step (2) is an enzyme solution obtained by separating and extracting wet bacteria after fermentation culture of a strain containing the epoxide hydrolase, wherein the strain containing the epoxide hydrolase comprises Aspergillus niger, radioactive Agrobacterium radiobacter, Rhodotorula glutinis and recombinant Escherichia coli thereof.

Further, the nucleotide sequence of the epoxide hydrolase gene is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 1.

Further, the volume dosage of the phosphate buffer solution in the step (2) is 0.1-10, preferably 0.3ml/mg based on the mass of the epoxide hydrolase.

Further, the epoxide hydrolase is prepared as follows:

(1) wet thalli:

inoculating a strain containing epoxide hydrolase gene (preferably Aspergillus niger CCTCC M2010275) to a slant culture medium, and culturing at 37 ℃ for 12h to obtain slant thallus; the final concentration of the slant culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 20g/L of agar and deionized water as a solvent, wherein the pH value is natural;

inoculating the slant thallus to a seed culture medium, and culturing at 37 ℃ for 10h to obtain a seed solution; the final concentration composition of the seed culture medium is 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, deionized water as a solvent, and the pH value is natural;

the seed solution was inoculated into a fermentation medium containing 50. mu.g/mL kanamycin at a final concentration of 2% by volume, incubated at 37 ℃ until OD600 became equal to 0.6-0.8, then IPTG at a final concentration of 0.1mM was added, and the resulting mixture was incubated at 28 ℃ for 12 hours, and the broth was centrifuged to collect wet cells. The final concentration composition of the fermentation medium is as follows: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast extract, deionized water as a solvent and natural pH value;

(2) separation and purification of epoxide hydrolase

Suspending the wet thallus prepared in the step (1) in PBS buffer solution with pH of 8.0 and 200mM, shaking uniformly, and then crushing under ultrasonic waves (power of 300W), wherein the ultrasonic frequency is 1s and the interval is 1s, and the crushing time is 20 min; centrifuging the crushed solution at 10000r/min at 4 ℃ for 10min to remove cell fragments, and obtaining supernatant which is crude enzyme solution; the obtained crude enzyme solution was purified by Ni-NTA, the Ni-NTA column was equilibrated with a loading buffer (20mM PBS buffer, 500mM NaCl and 20mM imidazole, pH8.0) for 30min, followed by loading the crude enzyme solution, followed by elution with the loading buffer at a rate of 1mL/min to remove non-adsorbed proteins, and finally elution with an elution buffer (20mM PBS buffer, 500mM NaCl and 500mM imidazole, pH8.0) at a rate of 1mL/min to collect the target protein. The flow rate was maintained at 1mL/min throughout the process. The enzyme solution was dialyzed overnight at 20mM, pH8.0, 0 ℃ in PBS buffer, and the retentate was lyophilized to obtain purified epoxide hydrolase.

The invention also provides an application of the oxidized ficoll modified epoxide hydrolase in the resolution of epichlorohydrin, and the application method comprises the following steps: using PBS buffer solution with pH of 8.0 and 0.2M as a reaction medium, using oxidized polysucrose modified epoxide hydrolase as a catalyst and racemic epichlorohydrin as a substrate to form a reaction system, reacting at the temperature of 35 ℃ and the rotation speed of 100rpm, and separating and purifying reaction liquid after the reaction is completed to obtain R-epichlorohydrin; in the reaction system, the final concentration of a substrate is 800mM, and the dosage of the catalyst is 100-1000U/ml, preferably 350U/ml.

Compared with the prior art, the invention has the following beneficial effects: the invention carries out chemical modification on epoxide hydrolase through polysucrose, greatly improves the specific activity and the thermal stability of the epoxide hydrolase, and has wide prospect in the enzymatic preparation and application of optical pure epoxide. The enzyme has simple production method, low cost and wide industrial application prospect. When the concentration of substrate epichlorohydrin is 800mM, the epoxide hydrolase modified by polysucrose (400kDa) can completely split the epichlorohydrin, and after the reaction is carried out for 30 minutes, the enantiomeric excess value (ee value) of (R) -epichlorohydrin can reach more than 99 percent; the unmodified original enzyme is completely inactivated after reacting for 10 minutes under the high substrate concentration, and the final (R) -epichlorohydrin ee value is less than 10%. This indicates that the polysucrose modification increases the tolerance and catalytic activity of epoxide hydrolase to high concentrations of substrate.

(IV) description of the drawings

FIG. 1 bar graph of specific activity of different species or molecular weight of polysaccharide-modified epoxide hydrolases.

FIG. 2 is a bar graph of the thermostability of different species or molecular weight of polysaccharide-modified epoxide hydrolases.

FIG. 3 temperature optimum curves for the polysucrose (400kDa) modified epoxide hydrolase and the proenzyme; tangle-solidup, a modifying enzyme; ● proenzyme.

FIG. 4 pH optima curves for the polysucrose (400kDa) modified epoxide hydrolase and the proenzyme; tangle-solidup, a modifying enzyme; ● proenzyme.

FIG. 5 shows the comparison of the effect of resolving racemic epichlorohydrin with modified enzyme to prepare R-epichlorohydrin; ● proenzyme.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

EXAMPLE 1 preparation of epoxide hydrolase

(1) Enzyme-containing bacteria

Epoxide hydrolase (shown in SEQ ID NO.1 and SEQ ID NO.2 in the sequence) derived from Aspergillus niger CCTCC M2010275 (disclosed in patent application 201110350712.2) is transferred into Escherichia coli BL21(DE3)/pET-28b, and positive clones are screened to obtain recombinant Escherichia coli.

Inoculating recombinant escherichia coli expressing an aspergillus niger CCTCC M2010275 epoxide hydrolase gene to a slant culture medium, and culturing at 37 ℃ for 12h to obtain slant thalli; the final concentration of the slant culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 20g/L of agar and deionized water as a solvent, wherein the pH value is natural;

inoculating the seed solution into a fermentation culture medium containing kanamycin with the final concentration of 50 mu g/mL by an inoculation amount of 2% of volume concentration, culturing at the constant temperature of 37 ℃ until OD600 is equal to 0.6-0.8, adding IPTG with the final concentration of 0.1mM, culturing at the temperature of 28 ℃ for 12h, centrifuging the fermentation liquor, and collecting wet thalli; the final concentration composition of the fermentation medium is as follows: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast extract and deionized water as a solvent, and the pH value is natural.

(2) Separation and purification of epoxide hydrolase

And (2) suspending the wet thalli prepared in the step (1) in 20mL of PBS (phosphate buffer solution) with the pH value of 8.0 and the concentration of 200mM, shaking uniformly, and then crushing under ultrasonic waves (the power is 300W), wherein the ultrasonic frequency is 1s, the interval is 1s, and the crushing time is 20 min. Centrifuging the crushed solution at 10000r/min at 4 ℃ for 10min to remove cell fragments, and obtaining supernatant which is crude enzyme solution. The obtained crude enzyme solution was purified by Ni-NTA, the Ni-NTA column was equilibrated with a loading buffer (20mM PBS buffer, 500mM NaCl and 20mM imidazole, pH8.0) for 30min, followed by loading of the crude enzyme solution, followed by elution with the loading buffer to remove non-adsorbed proteins, and finally elution with an elution buffer (20mM PBS buffer, 500mM NaCl and 500mM imidazole, pH8.0) to collect the target protein. The flow rate was maintained at 1mL/min throughout the process. The enzyme solution was dialyzed overnight at 20mM, pH8.0, 0 ℃ in phosphate buffer, and the retentate was lyophilized to obtain purified epoxide hydrolase which was stored at 20 ℃ for further use.

The specific activity of the purified epoxide hydrolase was found to be 419U/mg.

(3) Determination of enzymatic and specific Activity of epoxide hydrolase

Enzyme activity determination reaction system: adding 25 μ L of purified epoxide hydrolase enzyme solution (1.0mg/mL, PBS buffer solution with pH8.0) obtained in step (2) into 955 μ L of 0.2M PBS buffer solution with pH8.0, keeping the temperature of the mixed solution at 40 deg.C for 10min, adding 20 μ L of epichlorohydrin, reacting for 4min, adding 100 μ L of 2M H₂SO₄The reaction was terminated. Taking a proper amount of sample from the reaction liquid, extracting with four times of volume of ethyl acetate, rotating at 12000rpm, centrifuging for 2min, then taking the ethyl acetate phase obtained after centrifugation to dilute by a proper amount, adding anhydrous sodium sulfate to absorb water, drying, and carrying out gas chromatography detection.

The gas chromatography detection conditions are as follows: keeping the column temperature at 60 ℃ for 4min, heating to 160 ℃ at 20 ℃/min, and keeping the column temperature for 1 min; the temperature of a front sample inlet is 230 ℃; the FID temperature of the detector is 250 ℃; nitrogen (N)₂) The flow rate is 54.0mL/min, the split ratio is 1:40, the retention time: (S) -ECH 5.985min, (R) -ECH 6.095 min.

The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze hydrolysis of 1 mu mol of Epichlorohydrin (ECH) per minute at 40 ℃ and pH 8.0.

Protein concentration determination: diluting the collected enzyme solution by 10 times with PBS (0.02M and pH8.0) as blank control, mixing 100 μ L with 5mL Coomassie brilliant blue reagent, standing for 2min, and measuring absorbance at 595nm with phosphate buffer (0.02M and pH8.0) as blank control. Protein concentration was calculated from the measured absorbance and the standard curve.

Specific activity (U/mg) ═ volume enzyme activity (U/mL)/concentration of enzyme protein (mg/mL)

Example 2 preparation of a Polysucrose-modified epoxide hydrolase

1. Preparation of oxidized polysaccharides

1g of polysaccharides of different species or molecular weights (dextran (70kDa, 250kDa), polysucrose (70kDa,400kDa and 700kDa), pectin (360kDa) and amylose (300kDa)) were weighed out separately and dissolved in 40mL of 0.25M NaIO₄In an aqueous solution, the reaction was carried out at 37 ℃ and 150rpm in the absence of light for 6 hours, and then 1.2mL of ethylene glycol was added to terminate the reaction. Dialyzing the reaction solution in distilled water for 24h (the cut-off molecular weight of the dialysis bag is 14000Da), replacing the dialyzate once after 12h, and finally freeze-drying the dialyzed cut-off solution to respectively obtain 450mg of various oxidized polysaccharides which are stored at 4 ℃ for later use.

2. Polysaccharide modification of epoxide hydrolases

Dissolving 150mg of the oxidized polysaccharide prepared in the step 1 and 10mg of the epoxide hydrolase prepared in the example 1 in 3mL of PBS buffer solution with the pH value of 8.0 and the pH value of 0.2M, uniformly mixing, reacting for 16h at 4 ℃ and 600rpm, dialyzing the reaction solution in distilled water for 24h (the cut-off molecular weight of a dialysis bag is 14kDa), replacing the dialyzate once after 12h, precipitating the dialyzed cut-off solution by using ethanol aqueous solution with the volume concentration of 50%, and taking the precipitate to obtain the epoxide hydrolase modified by the polysaccharide. The 1mg precipitate was dissolved in 3mL PBS buffer (0.2M, pH8.0), which was the polysaccharide-modified epoxide hydrolase enzyme solution, and the specific activity was determined (the same procedure as in example 1), and the results are shown in FIG. 1.

As can be seen from FIG. 1, the epoxide hydrolase specific activities modified by three polysucroses (70kDa,400kDa and 700kDa) are all improved remarkably, wherein the modification effect of the polysucrose with the molecular weight of 400kDa is the best, the specific activity of the epoxide hydrolase is improved from 419U/mg to 694U/mg before modification, and the specific activity is improved by 66%. The activities of glucan (250kDa), pectin (360kDa) and amylose (300kDa) modified epoxide hydrolases were all slightly reduced.

3. Stability of polysaccharide-modified epoxy hydrolase

Prepared in step (2)0.2mL of the enzyme solution of the polysaccharide-modified epoxide hydrolase is respectively kept in water baths of 50 ℃, 60 ℃ and 70 ℃ for 2h, samples are taken every 20min, and the samples are immediately subjected to ice bath for 10min to measure the enzyme activity (the method is the same as the example 1). The initial enzyme activity of the enzyme solution is 100%, the logarithm of the percentage of the corresponding residual enzyme activity at different time is used as the ordinate, the time is used as the abscissa for plotting, and the half-life period of the polysucrose modified epoxide hydrolase at different temperatures is calculated. The half-life is defined as the time when the enzyme loses half of its activity at a specific temperature, and is denoted as t_1/2The calculation formula is as follows:

ln2 in formula (2) is 0.693, t_1/2Is half-life, K_dIs the inactivation rate constant of the enzyme. The results are shown in FIG. 2, using the epoxide hydrolase enzyme solution prepared in step (2) of example 1 as a control.

As can be seen from FIG. 2, the stability of the epoxide hydrolase is significantly improved after the modification of the polysucrose, wherein the stability of the epoxide hydrolase modified by the polysucrose (400kDa) is the best, and the half-life is improved by 268% compared with that of the original enzyme.

Example 3 Effect of reaction temperature and reaction pH on enzymatic Activity of Polysucrose modified epoxide hydrolase

A. Measurement of optimum reaction temperature:

taking 25 μ L of the polysucrose (400kDa) modified epoxide hydrolase enzyme solution prepared in step 2 of example 2, adjusting pH to 8.0, respectively placing at 30, 35, 40, 45, 50, 55, and 60 deg.C for 10min, adding 100 μ L of 2M H₂SO₄The reaction was terminated. The enzyme activities at different temperatures were measured separately (same method as in example 1). The highest enzyme activity was taken as 100%, then the relative enzyme activities at other temperatures were calculated, and 1mg of the pure enzyme prepared in step (2) of example 1 was dissolved in 3ml of 0.2M PBS buffer pH8.0 to prepare an epoxide hydrolase enzyme solution as a control, and the results are shown in FIG. 3.

B. Determination of optimum pH:

prepared as in step 2 of example 2Adding polysucrose (400kDa) modified epoxide hydrolase enzyme solution 25 μ L, adjusting pH to 5, 6, 7, 8, 9, 10, 11, reacting at 40 deg.C for 4min, adding 100 μ L2M H₂SO₄The reaction was terminated, the enzyme activities at different pH were measured (same as example 1), the highest enzyme activity was taken as 100%, then the relative enzyme activities at other pH were calculated, and simultaneously, 1mg of the pure enzyme prepared in step (2) of example 1 was dissolved in 3ml of 0.2M PBS buffer solution (pH8.0) to prepare epoxide hydrolase enzyme solution as a control, and the results are shown in FIG. 4.

As can be seen from FIG. 3, the optimum temperatures for the reactions of epoxide hydrolase proenzymes were 40 ℃ and the optimum temperature range for the modified enzyme was broadened to 40-45 ℃. As can be seen from FIG. 4, the optimum reaction pH of the modified enzyme and the proenzyme are both 8.0, but the enzyme activities of the modified enzyme are substantially the same between pH 7.0 and 9.0, and the optimum pH range is wider than that of the proenzyme.

Example 4 comparison of Effect of Primary enzyme and Polysucrose-modified epoxide hydrolase on catalytic resolution of epichlorohydrin

The polysucrose (400kDa) modified epoxide hydrolase enzyme liquid prepared in the step 2 of the embodiment 2 is used as an enzyme for conversion, and racemic epichlorohydrin is used as a substrate to carry out resolution reaction to produce R-epichlorohydrin. The specific operation is as follows: the reaction was carried out in 150mL standard shake flasks, 20mL reaction system: 15mL of PBS buffer solution with the pH value of 8.0 and the enzyme activity of the reaction system of 350U/mL and the final concentration of 800mM of racemic epichlorohydrin, the temperature of 35 ℃, the revolution of 100rpm, extracting 200 mul of reaction solution sample in 800 mul of ethyl acetate after every 5 minutes, adding 10 mul of 2M H₂SO₄The reaction was terminated. Centrifuging at 8000rpm for 5min, collecting upper layer extract 600 μ L, adding small amount of anhydrous sodium sulfate to remove water, and detecting the concentration of R-epichlorohydrin by gas chromatography (same as example 1). And calculating the enantiomeric excess value (ee value) of the R-epichlorohydrin at different reaction times. Meanwhile, the pure enzyme prepared in step (2) of example 1 was dissolved in 0.2M, pH8.0PBS buffer to prepare an epoxide hydrolase solution (enzyme activity: 350U/ml) as a control, and the results are shown in FIG. 5.

As can be seen from FIG. 5, when the concentration of epichlorohydrin as a substrate is 800mM, the epoxide hydrolase modified by polysucrose (400kDa) can completely split the epichlorohydrin, and the ee value of (R) -epichlorohydrin can reach more than 99% after the reaction is carried out for 30 minutes; the unmodified original enzyme is completely inactivated after reacting for 10 minutes under the high substrate concentration, and finally the (R) -epichlorohydrin ee is less than 10 percent. This indicates that the polysucrose modification increases the tolerance and catalytic activity of epoxide hydrolase to high concentrations of substrate.

Sequence listing

<110> Zhejiang industrial university

<120> oxidized ficoll modified epoxide hydrolase and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 398

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

<400> 1

Met Ser Ala Pro Phe Ala Lys Leu Pro Ser Ser Ala Ser Ile Ser Pro

1 5 10 15

Thr Pro Phe Thr Val Ser Ile Pro Asp Glu Gln Leu Asn Asp Leu Lys

20 25 30

Thr Leu Ile Arg Leu Ser Lys Ile Ala Pro Pro Thr Tyr Glu Asn Leu

35 40 45

Gln Ser Asp Gly Arg Phe Gly Val Thr Ser Glu Trp Leu Ser Ser Met

50 55 60

Arg Glu Lys Trp Val Ser Glu Phe Asp Trp Arg Thr Phe Glu Ala Arg

65 70 75 80

Met Asn Ser Phe Pro Gln Phe Thr Thr Glu Ile Glu Gly Leu Thr Val

85 90 95

His Phe Ala Ala Leu Phe Ser Gln Arg Glu Asp Ala Val Pro Ile Ala

100 105 110

Leu Leu His Gly Trp Pro Gly Asn Phe Val Glu Phe Tyr Pro Ile Leu

115 120 125

Gln Leu Phe Ser Glu Glu Tyr Ser Pro Glu Thr Leu Pro Phe His Leu

130 135 140

Ile Val Pro Ser Leu Pro Gly Tyr Thr Phe Ser Ser Gly Pro Pro Leu

145 150 155 160

Asp Arg Asp Phe Gly Leu Val Asp Ile Ala Arg Val Val Asp Gln Leu

165 170 175

Met Lys Asp Leu Gly Phe Gly Ser Gly Tyr Val Ile Gln Gly Gly Asp

180 185 190

Ile Gly Ser Phe Val Gly Arg Val Leu Gly Val Ser Phe Asp Ala Cys

195 200 205

Lys Ala Val His Leu Asn Leu Cys Ala Met Arg Ala Pro Pro Glu Gly

210 215 220

Leu Ser Thr Glu Ser Leu Thr Ala Ala Glu Lys Glu Gly Val Ala Arg

225 230 235 240

Met Glu Lys Phe Met Thr Asn Gly Leu Ala Tyr Ala Leu Glu His Ser

245 250 255

Thr Arg Pro Ser Thr Ile Gly His Val Leu Ser Ser Ser Pro Ile Ala

260 265 270

Leu Leu Ala Trp Val Gly Glu Lys Tyr Leu Gln Trp Val Asp Glu Pro

275 280 285

Leu Pro Ser Thr Thr Ile Leu Glu Met Val Ser Leu Tyr Trp Leu Thr

290 295 300

Glu Ser Phe Pro Arg Ala Ile Tyr Ser Tyr Arg Glu Thr Thr Pro Thr

305 310 315 320

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

325 330 335

His Lys Pro Phe Gly Phe Ser Phe Phe Pro Lys Asp Leu Cys Pro Val

340 345 350

Pro Arg Ser Trp Ile Ala Thr Thr Gly Asp Leu Val Phe Phe Gln Asp

355 360 365

His Ser Glu Gly Gly His Phe Ala Ala Leu Glu Arg Pro Arg Glu Leu

370 375 380

Lys Ala Asp Leu Thr Ala Phe Val Glu Gln Val Trp Gln Lys

385 390 395

<210> 2

<211> 1197

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<400> 2

atgtccgctc cgttcgccaa gcttccctcc tcggccagca tttctcctac tcccttcact 60

gtctccatcc ccgatgagca gctgaatgac ctgaaaaccc tcatccgact atccaaaatc 120

gcccctccca cctatgagaa cctgcaatca gatggccggt ttggcgtcac ttccgaatgg 180

ctgtcatcca tgcgggagaa atgggtctcg gaatttgact ggcgaacatt tgaagctcga 240

atgaactctt tcccccagtt tactacggag attgagggtc tcacagtgca ctttgctgcc 300

ttattttctc agagggagga tgctgtgccc atcgcattgc tccatggttg gcccggcaac 360

ttcgttgaat tctacccaat cctccagcta ttcagcgagg agtactctcc tgaaacctta 420

cctttccatc taattgttcc atcccttcct gggtacacct tctcgtctgg tcccccactg 480

gacagggatt tcggcttggt tgacatcgcc cgggtcgtag accagttgat gaaggacctc 540

gggttcggca gtggctatgt tatccaggga ggtgatattg gtagttttgt agggcgggtt 600

ctgggcgtaa gcttcgacgc ctgcaaagcg gtacatttga acctatgcgc aatgagagct 660

ccccctgaag gcctgtcaac ggagagcttg actgcggcgg agaaagaggg agtcgcgcga 720

atggagaaat tcatgaccaa tggcctagct tatgccctgg agcacagtac tcggcccagt 780

acaatcggcc atgtactgtc cagcagtccg atcgctttac ttgcatgggt tggtgagaaa 840

tacctccaat gggtggatga acccctccct tctacgacca ttcttgagat ggtaagcctg 900

tattggctca ctgagagttt tccacgggct atttattcct accgtgagac tacccccacc 960

gcctctgtgc ccaacggagc gacgatgctg cagaacgaat tatatattca caaaccattc 1020

ggattctcgt tcttccccaa ggacctttgc cccgtgcctc gaagctggat tgctaccaca 1080

ggagatctag tcttcttcca ggatcattca gagggaggac actttgccgc attggagcgt 1140

ccacgcgagc tcaaggccga tctaacggca tttgtcgagc aggtgtggca gaagtag 1197

Claims

1. An oxidized ficoll-modified epoxide hydrolase, characterized in that said enzyme is prepared by the following method: (1) oxidized polysucrose: dissolving Polysucrose in 0.25M NaIO₄Reacting in water solution at 37 ℃ and 150rpm in a dark place for 6h, adding ethylene glycol to terminate the reaction, dialyzing the reaction solution in distilled water for 24h, replacing primary dialyzate at intervals of 12h, wherein the cut-off molecular weight of the dialysis bag is 14000Da, and freeze-drying the dialyzed cut-off solution to obtain oxidized ficoll; the NaIO₄The volume dosage of the aqueous solution is 40mL/g based on the mass of the polysucrose; the molecular weight of the polysucrose is 70-700 kDa; (2) dissolving oxidized polysucrose and epoxide hydrolase in 0.2M phosphate buffer solution with the pH value of 8.0, uniformly mixing, reacting at the temperature of 4 ℃ and the rpm of 600 for 16h, dialyzing with distilled water for 24h, changing dialysate once for 12h, taking the retentate with the molecular weight of 14kDa from a dialysis bag, precipitating the retentate with ethanol aqueous solution with the volume concentration of 50%, and taking the precipitate to obtain the polysucrose modified epoxide hydrolase; the mass ratio of the oxidized ficoll to the epoxide hydrolase is 15: 1.

2. The oxidized ficoll-modified epoxide hydrolase according to claim 1, wherein the epoxide hydrolase activity in step (2) is 10 to 1000U/mg.

3. The oxidized ficoll-modified epoxide hydrolase according to claim 1, wherein the epoxide hydrolase in the step (2) is a pure enzyme isolated from a wet cell obtained by fermentation culture of a strain containing an epoxide hydrolase gene; the strain includes Aspergillus niger, Agrobacterium radiobacter or Rhodotorula glutinis.

4. The oxidized ficoll-modified epoxide hydrolase according to claim 3, wherein the nucleotide sequence of the epoxide hydrolase gene is represented by SEQ ID No. 2.

5. The oxidized ficoll-modified epoxide hydrolase according to claim 1, wherein the phosphate buffer in step (2) is used in a volume of 0.1 to 10mL/mg based on the mass of the epoxide hydrolase.

6. The oxidized ficoll-modified epoxide hydrolase according to claim 3, wherein said epoxide hydrolase is prepared by the following method:

(1) wet thalli:

inoculating a strain containing epoxide hydrolase genes to a slant culture medium, and culturing at 37 ℃ for 12h to obtain slant thalli; the final concentration of the slant culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 20g/L of agar and deionized water as a solvent, wherein the pH value is natural;

inoculating the seed solution into a fermentation medium containing kanamycin to a final concentration of 50 μ g/mL at a constant temperature of 37 deg.C to OD₆₀₀Adding IPTG with final concentration of 0.1mM after the concentration is equal to 0.6-0.8, culturing at 28 ℃ for 12h, centrifuging fermentation liquor, and collecting wet thalli; the final concentration composition of the fermentation medium is as follows: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast extract, deionized water as a solvent and natural pH value;

(2) separation and purification of epoxide hydrolase

Suspending the wet thallus prepared in the step (1) in PBS buffer solution with pH of 8.0 and 200mM, shaking uniformly, crushing under the ultrasonic power of 300W, wherein the ultrasonic frequency is 1s and the interval is 1s, the crushing time is 20min, centrifuging the crushed liquid for 10min at the temperature of 4 ℃ at 10000r/min to remove cell fragments, and obtaining supernatant which is crude enzyme liquid; purifying the crude enzyme solution by using Ni-NTA, balancing a Ni-NTA column by using a loading buffer solution for 30min, loading the crude enzyme solution, eluting by using the loading buffer solution at the speed of 1mL/min to remove unadsorbed protein, eluting by using an elution buffer solution at the speed of 1mL/min, and collecting target protein; dialyzing the target protein in 20mM PBS buffer solution with pH of 8.0 and 0 ℃ overnight, taking the retentate, and freeze-drying to obtain purified epoxide hydrolase; the loading buffer solution is 20mM PBS buffer solution containing 500mM NaCl and 20mM imidazole, and the pH value is 8.0; the elution buffer was 20mM PBS buffer containing 500mM NaCl and 500mM imidazole, pH 8.0.

7. Use of the oxidized ficoll-modified epoxide hydrolase of claim 1 for the resolution of epichlorohydrin.

8. The use according to claim 7, characterized in that the method of application is: using PBS buffer solution with pH of 8.0 and 0.2M as a reaction medium, using oxidized polysucrose modified epoxide hydrolase as a catalyst and racemic epichlorohydrin as a substrate to form a reaction system, reacting at the temperature of 35 ℃ and the rotation speed of 100rpm, and separating and purifying reaction liquid after the reaction is completed to obtain R-epichlorohydrin; in the reaction system, the final concentration of a substrate is 800mM, and the dosage of a catalyst is 100-1000U/ml.