Method for preparing statin intermediate by using immobilized enzyme
阅读说明：本技术 利用固定化酶制备他汀中间体的方法 (Method for preparing statin intermediate by using immobilized enzyme ) 是由 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 于 2020-06-24 设计创作，主要内容包括：本发明提供了一种利用固定化酶制备他汀中间体的方法,包括如下步骤：使用固定化载体Seplite LX-1000NH和LX-G20将羰基还原酶固定化,得到固定化酶；使用固定化酶催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯进行还原反应得到他汀中间体6-氰基-(3R,5R)-二羟基己酸叔丁酯,催化(S)-6-氯-5-羟基-3-羰基己酸叔丁酯进行还原反应得到他汀中间体6-氯-(3R,5S)-二羟基己酸叔丁酯。本发明的方法稳定性好,能够生成高光学纯度的产物,有效降低生产成本,适合于工业化应用。(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.)
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 ℃.
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 asSeries EC-HA, EC-EA; seplite LX-1000HA and Seplite LX-1000NH of Xian blue Xiao Tech Co., Ltd; relizymeTMThe series HA403, EA 403; commercial epoxy-based immobilization supports such as
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.
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