CN111647591A

CN111647591A - Method for preparing statin intermediate by using immobilized enzyme

Info

Publication number: CN111647591A
Application number: CN202010588965.2A
Authority: CN
Inventors: 陶荣盛; 郑云; 朱傅赟; 沈青; 沈正权; 孙梁栋; 潘震华; 原犇犇; 胡海亮; 刘萍
Original assignee: Huzhou Yisheng Biotechnology Co Ltd
Current assignee: Huzhou Yisheng Biotechnology Co Ltd
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2020-09-11

Abstract

The invention provides a method for preparing a statin intermediate by using immobilized enzyme, which comprises the following steps: immobilizing carbonyl reductase by using immobilized carriers Seplite LX-1000NH and LX-G20 to obtain immobilized enzyme; immobilized enzyme is used for catalyzing the tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate to carry out reduction reaction to obtain a statin intermediate tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, and the immobilized enzyme is used for catalyzing the tert-butyl (S) -6-chloro-5-hydroxy-3-carbonyl hexanoate to carry out reduction reaction to obtain the statin intermediate tert-butyl 6-chloro- (3R,5S) -dihydroxyhexanoate. The method has good stability, can generate products with high optical purity, effectively reduces the production cost, and is suitable for industrial application.

Description

Method for preparing statin intermediate by using immobilized enzyme

Technical Field

The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing a statin intermediate by using immobilized enzyme.

Background

Statins are the most common lipid regulators in clinic and are important basic drugs for preventing and treating cardiovascular and cerebrovascular diseases at home and abroad. Atorvastatin intermediate 6-cyano- (3R,5R) -tert-butyl dihydroxyhexanoate shown as the following formula A7 (CAS number: 125971-93-9), formula A8 (4R,6R) -6-cyanomethyl-2, 2-dimethyl-1, 3-dioxolane-tert-butyl acetate (CAS number: 125971-94-0, atorvastatin calcium side chain ATS-8) and formula D3, rosuvastatin intermediate 6-chloro- (3R,5S) -tert-butyl dihydroxyhexanoate (CAS number: 154026-93-4) are all important chiral compound intermediates of statins.

The inventor reports that three carbonyl reductases SEQ ID NO 2-4 can catalyze the asymmetric reduction reaction of the formula A6 to obtain a compound A7 in a patent document CN 110387359A; and catalyzing the asymmetric reduction of formula D2 to give compound D3. The method can solve the problem of serious pollution caused by chemical production.

However, in the production by this enzyme catalysis method, free carbonyl reductase is used, and after one reaction, expensive enzyme is discarded and cannot be reused, which leads to high raw material cost. In addition, as a protein, the mechanical stability and thermostability of free enzyme are weak in stirring reaction, and the application of the protein in the industrial production of statin intermediates is also influenced.

Compared with the free enzyme method, the application of the immobilized enzyme technology has the advantages of simplified production process, improved production efficiency and the like. Meanwhile, the immobilized enzyme can be used for multiple times, and the stability is improved, so that the productivity of unit enzyme is effectively improved; and secondly, the immobilized enzyme is easily separated from the substrate and the product, so that the product purification process is simplified, and the product quality is stable.

Disclosure of Invention

In order to overcome the above-mentioned problems of the free carbonyl reductase, the inventors have conducted studies and extensive experimental studies on the technique of enzyme immobilization. Surprisingly, it was found that the carbonyl reductase reported in patent document CN110387359A can maintain higher enzyme activity and significantly improved stability after being immobilized on a separate carrier by adsorption method, and the chiral purity of the product does not decrease or increase inversely, which is very beneficial for the pharmaceutical field in which chiral purity of optically active products is sought. Specifically, the present invention includes the following technical solutions.

A method for preparing a statin intermediate by using immobilized enzyme comprises the following steps:

A. immobilizing carbonyl reductase by using immobilized carriers Seplite LX-1000NH and LX-G20 to obtain immobilized enzyme; B. and B, catalyzing the tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate by using the immobilized enzyme obtained in the step A to perform a reduction reaction to obtain a statin intermediate tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, or catalyzing the tert-butyl (S) -6-chloro-5-hydroxy-3-carbonyl hexanoate to perform a reduction reaction to obtain a statin intermediate tert-butyl 6-chloro- (3R,5S) -dihydroxyhexanoate.

Preferably, the immobilized carrier is Seplite LX-1000 NH.

The amino acid sequence of the carbonyl reductase is preferably SEQ ID NO: 1:

MSTPLNALVTGASRGIGAATAIKLAENGYSVTLAARNVAKLNEVKEKLPVVKDGQKHHIWELDLASVEAASSFKGAPLPASDYDLFVSNAGIAQITPTADQTDKDFLNILTVNLSSPIALTKALLKGVRERSNEKPFHIIFLSSVAALHGVPQAAVYSASKAGLDGFVRSLAREVGPKGIHVNVIHPGWTKTDMTDGIDDPNDTPIKGWIQPEAIADAVVFLAKSKNITGTNIVVDNGLLA (SEQ ID NO: 1). It is carbonyl reductase mutant SEQ ID NO. 4 reported in patent document CN 110387359A.

In a preferred embodiment, the carbonyl reductase SEQ ID NO 1 described above can be expressed from Escherichia coli, thereby enabling mass production and direct use for the preparation of immobilized enzymes without purification after fermentation.

For example, the step a is: and (3) centrifuging the escherichia coli fermentation liquor to obtain bacterial sludge, breaking the cell wall, centrifuging, taking the supernatant to obtain a crude enzyme solution, mixing the crude enzyme solution with an immobilized carrier in a phosphate buffer solution, and adsorbing to obtain the immobilized enzyme.

Preferably, the pH of the reaction system in the step B is 7.5 to 8.5, preferably 7.8 to 8.2, more preferably about 8.0. For example, the reaction system may be a phosphate buffer solution of pH7.5 to 8.5.

The reaction temperature in the step B is 30-45 ℃. Preferably 35-42 deg.C, more preferably 37-40 deg.C.

Preferably, isopropanol and coenzyme NADP + may be added to the reaction system. NADP + (nicotinamide adenine dinucleotide phosphate, coenzyme II) functions as an oxidant to scavenge electrons, carbonyl reductase reduces NADP + to NADPH using isopropanol, producing sufficient NADPH as a biosynthetic reductant, thereby facilitating the reduction reaction.

The immobilized carbonyl reductase provided by the invention can catalyze the compound A6 to carry out asymmetric reduction reaction with high activity to generate a statin intermediate A7 with high optical purity; and the compound D2 is catalyzed to carry out asymmetric reduction reaction to generate the statin intermediate D3 with high optical purity, so that the method has the advantages of good stability and effectively reduced production cost, and is beneficial to realizing the industrial production of the statin intermediate.

Detailed Description

The enzyme is immobilized by blocking free enzyme on solid material or limiting in a certain area by physical or chemical means, and the enzyme can still play a catalytic role and can be recycled. Compared with free enzyme, the immobilized enzyme has the advantages of high stability, convenient recovery, easy control, repeated use, low cost and the like, and plays an important role in the aspects of biological industry, medical and clinical diagnosis, chemical analysis, environmental protection, energy development, basic research and the like.

As is well known in the field of biological catalysis, compared with a free enzyme method, the application of an immobilized enzyme technology has the advantages of simplified production process, improved production efficiency and the like. Meanwhile, the enzyme can be used for multiple times, and the stability of the enzyme is improved, so that the productivity of unit enzyme is effectively improved; and secondly, the immobilized enzyme is easily separated from the substrate and the product, the purification process is simplified, the yield is high, and the product quality is good. However, enzyme immobilization also has a number of disadvantages, enzyme activity is usually lost during immobilization, and the chiral purity of the catalytic reaction product sometimes changes, which often makes it difficult to ensure high chiral purity.

In order to study the immobilization of the carbonyl reductase mutant SEQ ID NO:1 (i.e., the carbonyl reductase mutant SEQ ID NO:4 reported in patent document CN 110387359A), the inventors have tried various types of immobilization methods, including adsorption, cross-linking, entrapment, carrier coupling (also called covalent binding), and the like. In the selection of carriers for adsorption/carrier coupling, the ion exchange resins are considered with emphasis, including commercial amino-immobilized carriers such as

Series EC-HA, EC-EA; seplite LX-1000HA and Seplite LX-1000NH of Xian blue Xiao Tech Co., Ltd; relizyme^TMThe series HA403, EA 403; commercial epoxy-based immobilization supports such as

The series EC-EP, EC-HFA; seplite LX-1000EP, Seplite LX-1000HFA, LX-1000ODS, LXES-J420, LX-Q650C, LX-T300, LX-G20 from Xian blue-Xiao science and technology Co., Ltd; relizyme^TMSeries EP403, HFA403, etc. The types of these ion exchange resins are listed in table 1 below.

TABLE 1 partial ion exchange resin Classification

It was found experimentally that the carbonyl reductase SEQ ID NO 1 is able to form the desired combination with two immobilization supports Seplite LX-1000NH and LX-G20, especially LX-1000 NH. After the immobilization is carried out by an adsorption method, chiral reduction can be efficiently and stably carried out on substrates A6 and D2 to respectively obtain compounds A7 and D3 with high optical activity, so that the bottleneck problem existing in the conventional free enzyme catalysis process is solved, the production cost is reduced, and a foundation is laid for realizing industrialization of chiral intermediates of statins produced by an enzyme method.

Surprisingly, the optical purity (ee value) of the product of the reaction catalyzed by the supported LX-1000NH immobilized carbonyl reductase SEQ ID NO:1 exceeded that of the reaction catalyzed by the free enzyme, suggesting that the stereoselectivity of the LX-1000NH immobilized carbonyl reductase SEQ ID NO:1 is enhanced for reasons to be further investigated. Probably because the carbonyl reductase SEQ ID NO 1 is immobilized by the carrier LX-1000NH, the three-dimensional conformation change or the space change of the enzyme active site occurs, the correct folding of enzyme protein is promoted, the enzyme activity is enhanced, and the selectivity of a substrate is improved.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

In the examples, the addition, content and concentration of various substances are mentioned, wherein the percentages refer to mass percentages unless otherwise indicated.

Example 1 immobilization of enzymes

1.1 preparation of crude enzyme solution

The bacterial species and culture methods for expressing carbonyl reductase SEQ ID NO 1 are provided by CN110387359A, Inc., Yihui Biotech, Huzhou.

The preparation of the culture medium and the method of shake flask fermentation are as follows:

TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K₂HPO₄.3H₂O、2.31g/LKH₂PO₄5g/L of glycerol, pH7.0-7.5, and sterilizing at 121 deg.C for 20 min;

slant culture medium: mixing 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 20g/L agar powder, subpackaging the mixture into eggplant bottles according to the liquid loading amount of 30-40mL, vertically placing the eggplant bottles at 121 ℃ for high-temperature high-pressure sterilization for 20min, cooling, adding 100 mu g/mL kanamycin sulfate, placing the eggplant bottles into an inclined plane, and condensing the eggplant bottles into a solid.

Seed activation: taking 100 mu L of seed preservation solution from a seed glycerol preservation tube, uniformly coating an inoculating loop on the inclined plane of an eggplant bottle, and then placing the eggplant bottle in a 37 ℃ incubator for culturing for 18 h;

seed culture: introducing 100mL of sterile water into an eggplant bottle to prepare a bacterial suspension, taking 50 mu l of the bacterial suspension, inoculating into a 250mL shake flask filled with 50mL of LTB culture medium, and culturing at 30 ℃ and 220rpm for 16 h;

fermentation: inoculating the primary seed culture solution into 5L shake flask containing 1L TB medium, culturing at 37 deg.C and 220rpm for 4-6 hr, adding 0.3mM IPTG, cooling to 28 deg.C, and inducing at 220rpm for 12 hr.

And (3) collecting thalli: collecting fermentation liquor, centrifuging at 4000rpm for 30min, removing supernatant, collecting bacterial sludge, and freezing at-20 deg.C for storage.

Weighing thallus or cryopreserved bacterial sludge, suspending with 0.02M potassium phosphate buffer (pH 7.5 +/-0.2) to prepare liquid with bacterial content of 100 g/L. And then pressing and breaking cells by using a homogenizer, and centrifuging to obtain a crude enzyme solution for later use.

1.2 immobilization of enzymes Using ion exchange resins shown in Table 1 as immobilization carriers, respectively

The amino immobilized carrier needs to be activated before use, and the method comprises the following steps: immersing 10g of the vector in 40ml of 0.1M potassium phosphate buffer (pH4.2-4.5), and shaking at 150rpm at 20-25 deg.C for 15 min; maintaining the pH within 8.0 + -0.2, and adjusting if necessary; standing, and removing a supernatant; the carrier was re-immersed in 40ml of 0.02M potassium phosphate buffer (pH 8.0. + -. 0.2), shaken at 150rpm and 20-25 ℃ for 5 min; removing supernatant, immersing the carrier in 40ml of 0.02M potassium phosphate buffer (pH8.0 + -0.2) containing 2% glutaraldehyde, shaking at 150rpm at 20-25 deg.C for 60 min; standing, and removing a supernatant; washing the carrier with 0.02M potassium phosphate buffer (pH8.0 + -0.2) twice and draining.

Epoxy and adsorption immobilized carriers can be directly used for enzyme immobilization without activation.

Immobilization of an amino-based immobilization support and carbonyl reductase, taking LX-1000NH as an example, the immobilized enzyme is prepared by the following method: according to the protein carrier ratio of 1: 10, at 20-25 ℃ and 150 rpm. Stopping shaking after 1min, detecting and adjusting pH to 8.0 + -0.1, shaking for 18 hr, removing supernatant, and shaking with 40ml 0.02M potassium phosphate buffer (pH8.0 + -0.2) at 25 deg.C for 1-2 min; removing supernatant, shaking with 0.02M potassium phosphate buffer (pH8.0 + -0.2) containing 0.5M NaCl at 20-25 deg.C and 150rpm for 45 min; the supernatant was removed, washed again with 0.02M potassium phosphate buffer (pH 8.0. + -. 0.2), and dried with a vacuum pump to obtain the immobilized enzyme for transformation test.

The immobilization of other amino immobilized carriers and carbonyl reductase is the same as the preparation of LX-1000 NH.

The immobilization of the epoxy and adsorption-type immobilized carrier and the carbonyl reductase, taking the adsorption-type carrier LX-G20 as an example, the preparation method of the immobilized enzyme is as follows: according to the protein carrier ratio of 1: 10, placing in 0.1M potassium phosphate buffer (pH8.0 +/-0.2), and shaking at 150rpm at 20-25 ℃; stopping shaking after 1min, detecting and adjusting pH to 8.0 + -0.1, and continuing shaking for 18 h; stopping shaking, and standing at the same temperature for 20-24 h; removing supernatant, and shaking with 40ml 0.02M potassium phosphate buffer (pH8.0 + -0.2) at 20-25 deg.C for 1-2 min; removing supernatant, and washing with the same buffer solution for 45 min; the supernatant was removed, washed again with 40ml of 0.02M potassium phosphate buffer (pH 8.0. + -. 0.2), and dried by vacuum pump to obtain the immobilized enzyme for transformation test.

The immobilization of other epoxy and adsorption immobilized carriers and carbonyl reductase is the same as the preparation of LX-G20.

Example 2 enzyme-catalyzed assay of immobilized enzymes for Compound A6

2.1 enzymatic method (150 ml system as an example):

the substrate A6, namely 30g (200g/L) of tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate, 22.5ml of isopropanol and 127.5ml of water are accurately weighed and mixed in a triangular flask, the pH value is adjusted to about 8.0 by 2N sodium hydroxide solution, and the temperature is increased to 37 ℃.

Then, 9g of the immobilized enzyme obtained in example 1 was added, followed by addition of NADP + at a final concentration of 0.04g/L, sealing, and reacting at 37 ℃ at 180 rpm.

Samples were taken after 2h and 6h of reaction to check the content of the reduced reaction product A7 by HPLC and the optical purity of A7 was checked by checking the optical purity of the product A8 of the further reaction of A7.

2.2 the detection method of the A7 content of the product is as follows:

mobile phase A: 2.72g of potassium dihydrogen phosphate are weighed into 1L of water and adjusted to ph 4.0 with phosphoric acid

Mobile phase B: acetonitrile

Sample volume 5 μ l flow rate: 1.0ml/min column: SB-C18 (250X 4.6X 5)

Detection wavelength: column temperature of 215 nm: 40-degree running time: 40min

Gradient elution procedure

2.3 chirality of Compound A7 was determined by measuring the chirality of A8 as follows:

2.3.1 Synthesis of Compound A8: adding 250mL of ethyl acetate and 25g of diatomite into the feed liquid after the enzyme catalysis reaction, heating to 50-60 ℃, filtering, demixing, extracting the water layer twice with 150mL of ethyl acetate, combining the organic layers, and concentrating to obtain a crude product of the compound A7, which is an oily substance. The obtained oily substance, 25g of 2, 2-dimethoxypropane, 50mL of toluene and 0.2g of methanesulfonic acid were charged into a flask, and the mixture was heated to 25 ℃ and reacted for 3 hours while maintaining the temperature. After completion of the reaction, 100mL of toluene was added, and a saturated aqueous solution of sodium hydrogencarbonate was added to neutralize the pH to 7.5, and the aqueous layer was separated and the dry organic layer was concentrated. Adding 75mL of hexane, heating to dissolve, cooling to 0 ℃, crystallizing, and filtering to obtain a crude compound A8. Then recrystallizing with mixed solvent of n-hexane (50mL) and ethanol (2.5mL), filtering, and drying to obtain compound A8.

2.3.2 chiral analysis method of Compound A8: a detection instrument: agilent 1200 type high performance liquid chromatograph, liquid phase mobile phase: n-hexane: isopropyl alcohol 98: 2, wavelength λ 215nm, flow rate 0.6mL/min, solvent: a mobile phase; a chromatographic column: chiralcel OD-H250 is multiplied by 4.6mm and 5 mu m; sample introduction amount: 20 μ l, column temperature: 30 ℃, run time: and (6) taking 28 min. The retention time of the compound A8 is about 19.8 min; the retention time of the 4R, 6S-isomer is about 14.1 min; the retention time of the 4S, 6S-isomer is about 15.5 min; the retention time of the 4S, 6R-isomer is about 16.9 min. And (3) sample analysis: 200mg of A8 sample obtained by catalysis and treatment is weighed and placed in a 10ml volumetric flask, dissolved by a solvent, subjected to constant volume and shaken up to be used as a sample solution. A sample solution (20. mu.L) was taken and injected into a liquid chromatograph for detection.

2.4 conversion (product peak area/(product peak area + substrate peak area)) and ee-value comparison results when the immobilized enzyme catalyzes A6 for 2h are shown in Table 2 below.

Table 2 comparison of the conversion of product and ee value in the reaction of A6 catalyzed by partially immobilized enzyme for 2 hours

As can be seen from Table 2, the enzyme activities of the immobilized carbonyl reductases of vectors LX-1000NH and LX-G20 are more prominent, and the conversion rates respectively reach 58.53% and 50.07%. In particular, vector LX-1000NH, whose product ee at 58.53% conversion was 99.99%, gave statin intermediate a7 as an almost homochiral compound.

Compared with the catalytic reaction of the crude enzyme liquid, although the catalytic activity of the crude enzyme liquid is obviously higher and the reaction speed is obviously higher, the optical purity of the product A7 with the content of about 55 percent is 99.75 percent and is lower than the stereospecificity of the catalytic product of the LX-1000NH immobilized enzyme.

The LX-1000NH immobilized enzyme is intensively studied in the following experiments.

Example 3 stability examination of reaction of substrate A6 catalyzed by Carrier LX-1000NH immobilized enzyme

The stability of different batches of LX-1000NH immobilized enzyme catalyzing the reduction reaction of A6 was examined according to the method in example 2, and the conversion rate and ee value of the product A7 were measured at 2h and 6h of reaction, respectively.

3.1 extraction of Compound A7

And (3) repeatedly using LX-1000NH immobilized enzyme fed at the same time to catalyze the substrate A6 of different batches, wherein when each batch is reacted for 6 hours, the liquid phase detection is complete (the conversion rate is more than 99%).

Adding 250mL of ethyl acetate into the reacted feed liquid, heating to 50-60 ℃, filtering, layering, extracting the water layer twice with 150mL of ethyl acetate, combining the organic layers, concentrating to obtain a compound A7, weighing, and calculating the yield.

3.2 chiral determination of product A7

The chiral detection method of the product A7 is shown in step 2.3, and the result is shown in Table 3.

Table 3 shows that the same LX-1000NH immobilized enzyme catalyzes the product A7 yield and ee value of different batches of substrate A6 to react for 2h and 6h

As can be seen from Table 3, after 10 batches of catalytic reactions of the immobilized carbonyl reductase carried by the carrier LX-1000NH, the yield of the product A7 is quite stable, and the ee value of the product A7 is always over 99.98% (the content of 4S, 6S-enantiomer is not more than 0.02%, and other isomers are not detected), which indicates that the immobilized enzyme is repeatedly used for 10 times, the enzyme activity and the stereoselectivity of the immobilized enzyme are kept quite stable, and the immobilized enzyme has very high stability.

Example 4 catalytic test of Compound D2 with Carrier LX-1000NH immobilized enzyme

4.1 enzymatic method (150 ml system as an example):

30g (200g/L) of D2 substrate tert-butyl 6-chloro-5-hydroxy-3-carbonylhexanoate, 22.5ml of isopropanol and 127.5ml of water are accurately weighed and uniformly mixed in a triangular flask, the pH is adjusted to about 8.0 by 2N sodium hydroxide, and the temperature is increased to 37 ℃.

Then, 9g of immobilized enzyme was added, followed by addition of NADP + at a final concentration of 0.04g/L, sealing, and reacting at 37 ℃ at 180 rpm. Samples were taken after 2h and 6h of reaction and checked by HPLC.

4.2 determination method of substrate D2 and product D3:

a detection instrument: agilent model 1200 hplc. The detection method comprises the following steps: HPLC column: ZORBAXSB-C8, 4.6X 150mm, 5-Micron, mobile phase: 30% acetonitrile, flow rate: 1.0mL/min, column temperature: 40 ℃, detection wavelength: 210 nm. Product D3 retention time: about 7.1 min; substrate D2 retention time: about 12.0 min. And calculating the content by an external standard method.

4.3 product D3 extraction

And (3) continuing to react for 6 hours by the LX-1000NH immobilized enzyme, completely reacting liquid phase detection, adding 250mL of ethyl acetate into the reacted feed liquid, heating to 40-50 ℃, filtering, layering, extracting the water layer twice by using 150mL of ethyl acetate, washing with saline water once, combining the organic layers, concentrating to obtain a compound D3, weighing, and calculating the yield.

4.4 chiral determination of product D3

The detection method comprises the following steps: using a chiral chromatography column OD-H column (250 × 4.6mm, 5 μm), mobile phase: n-hexane: isopropanol 85: 15, flow rate 1mL/min, detection wavelength 215 nm. The retention times of compound D3 and the (3S,5S) -isomer were 5.1min and 4.9min, respectively. The results show that under the liquid phase chiral analysis conditions, the content of the enantiomer in the sample is lower than 0.03%, and other isomers are not detected. The carbonyl reductase constructed by the invention is fixed on the LX-1000NH carrier and has high stereospecificity to the substrate D2, and the result data are shown in the following table 4.

Table 4 shows the yield and ee value of the product D3 in different batches of LX-1000NH immobilized enzyme catalyzing D2 to react for 2h and 6h

As can be seen from Table 4, the yield of the product D3 is quite stable after the immobilized carbonyl reductase of the carrier LX-1000NH is subjected to catalytic reaction of 10 batches, and the ee value of the product D3 is always over 99.93 percent, which shows that the enzyme activity and the stereoselectivity of the immobilized enzyme are kept quite stable and have high stability after the immobilized enzyme is repeatedly used for 10 times.

The experiments show that carbonyl reductase SEQ ID NO 1 can be ideally matched with two immobilized carriers Seplite LX-1000NH and LX-G20, especially the combination of LX-1000NH and carbonyl reductase SEQ ID NO 1 can keep the high catalytic activity and stability of carbonyl reductase, can also improve the stereospecificity of carbonyl reductase, and has important significance for the application of industrial production of statin intermediates A7, A8 and D3.

Sequence listing

<110> Yisheng Biotechnology Ltd of Huzhou

<120> method for preparing statin intermediate using immobilized enzyme

<130>SHPI2010276

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<170>SIPOSequenceListing 1.0

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<213> Artificial sequence ()

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Met Ser Thr Pro Leu Asn Ala Leu Val Thr Gly Ala Ser Arg Gly Ile

1 5 10 15

Gly Ala Ala Thr Ala Ile Lys Leu Ala Glu Asn Gly Tyr Ser Val Thr

20 25 30

Leu Ala Ala Arg Asn Val Ala Lys Leu Asn Glu Val Lys Glu Lys Leu

35 40 45

Pro Val Val Lys Asp Gly Gln Lys His His Ile Trp Glu Leu Asp Leu

50 5560

Ala Ser Val Glu Ala Ala Ser Ser Phe Lys Gly Ala Pro Leu Pro Ala

65 70 75 80

Ser Asp Tyr Asp Leu Phe Val Ser Asn Ala Gly Ile Ala Gln Ile Thr

85 90 95

Pro Thr Ala Asp Gln Thr Asp Lys Asp Phe Leu Asn Ile Leu Thr Val

100 105 110

Asn Leu Ser Ser Pro Ile Ala Leu Thr Lys Ala Leu Leu Lys Gly Val

115 120 125

Arg Glu Arg Ser Asn Glu Lys Pro Phe His Ile Ile Phe Leu Ser Ser

130 135 140

Val Ala Ala Leu His Gly Val Pro Gln Ala Ala Val Tyr Ser Ala Ser

145 150 155 160

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

165 170 175

Pro Lys Gly Ile His Val Asn Val Ile His Pro Gly Trp Thr Lys Thr

180 185 190

Asp Met Thr Asp Gly Ile Asp Asp Pro Asn Asp Thr Pro Ile Lys Gly

195 200 205

Trp Ile Gln Pro Glu Ala Ile Ala Asp Ala Val Val Phe Leu Ala Lys

210 215220

Ser Lys Asn Ile Thr Gly Thr Asn Ile Val Val Asp Asn Gly Leu Leu

225 230 235 240

Ala

Claims

1. A method for preparing a statin intermediate by using immobilized enzyme comprises the following steps:

A. immobilizing carbonyl reductase by using immobilized carriers Seplite LX-1000NH and LX-G20 to obtain immobilized enzyme;

B. and B, catalyzing the tert-butyl 6-cyano- (5R) -hydroxy-3-carbonyl hexanoate by using the immobilized enzyme obtained in the step A to perform a reduction reaction to obtain a statin intermediate tert-butyl 6-cyano- (3R,5R) -dihydroxyhexanoate, or catalyzing the tert-butyl (S) -6-chloro-5-hydroxy-3-carbonyl hexanoate to perform a reduction reaction to obtain a statin intermediate tert-butyl 6-chloro- (3R,5S) -dihydroxyhexanoate.

2. The method of claim 1, wherein the immobilized support is Seplite LX-1000 NH.

3. The method of claim 2, wherein the carbonyl reductase has the amino acid sequence of SEQ ID NO 1.

4. The method of claim 3, wherein SEQ ID NO 1 is expressed in E.coli.

5. The method of claim 4, wherein step A is: and (3) centrifuging the fermented escherichia coli thallus, breaking the wall, centrifuging, taking the supernatant to obtain a crude enzyme solution, mixing the crude enzyme solution with an immobilized carrier in a phosphate buffer solution, and adsorbing to obtain the immobilized enzyme.

6. The method of claim 1, wherein the reaction system of step B has a pH of 7.5 to 8.5.

7. The method according to claim 6, wherein isopropanol and coenzyme NADP + are added to the reaction system.

8. The process of claim 7, wherein the reaction temperature is from 30 to 45 ℃.