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CN-121406597-B - Immobilized ketoreductase mutant and application thereof in synthesis of benananate intermediate

CN121406597BCN 121406597 BCN121406597 BCN 121406597BCN-121406597-B

Abstract

The invention provides an immobilized ketoreductase, an immobilization method and application thereof in preparation of a key intermediate of benazelate, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO.2, preferably a ketoreductase mutant SEQ ID NO.16, and the immobilized ketoreductase comprises resin and corresponding ketoreductase. The ketoreductase mutant and the immobilized ketoreductase prepared by the ketoreductase mutant have high enzyme activity when subjected to ketoreduction reaction, and the solvent is pure water and is environment-friendly when the ketoreductase mutant is used for preparing a key intermediate of the benazelate. The immobilized transaminase has high conversion rate, good stereoselectivity, good stability, repeated use and simpler operation, thereby reducing the production cost and being beneficial to industrial production.

Inventors

  • ZHANG XIN
  • LUO ZHONGHUA
  • XUE LIAN
  • YAO LIN
  • PAN XUE
  • DONG JIAZHENG
  • YU NA
  • LI CHENLU
  • YANG HUIZHAO
  • WANG GUOWEI

Assignees

  • 宜昌东阳光制药有限公司

Dates

Publication Date
20260508
Application Date
20251230

Claims (7)

  1. 1. A mutant of ketoreductase is characterized in that the mutant of ketoreductase is obtained by three site mutation of 190 site, 193 site and 201 site of ketoreductase in SEQ ID NO. 2, wherein the mutation is specifically that alanine residue at 190 site is replaced by methionine, serine residue at 193 site is replaced by cysteine, tyrosine residue at 201 site is replaced by tryptophan, and the amino acid sequence after mutation is SEQ ID NO. 16; the mutant of the ketoreductase is used for synthesizing a benananate intermediate shown as a formula B: Formula B; The synthesis process of the phenylbanacol comprises the steps of reacting a substrate shown in a formula A in a buffer solution in the presence of an enzyme catalytic system by utilizing ketoreductase, obtaining a compound shown in the formula B by using water as a solvent of the buffer solution, preparing the phenylbanacol on the basis of the compound shown in the formula B, 。
  2. 2. An isolated nucleotide encoding a mutant of the ketoreductase enzyme of claim 1.
  3. 3. A recombinant plasmid is characterized in that, the recombinant plasmid carries the nucleotide of claim 2.
  4. 4. A genetically engineered bacterium comprising a mutant of the ketoreductase of claim 1 or the nucleotide of claim 2 or the recombinant plasmid of claim 3.
  5. 5. An immobilized ketoreductase, which is characterized by comprising resin and the mutant of the ketoreductase as claimed in claim 1, wherein the ketoreductase and the resin are connected through covalent bonds.
  6. 6. The immobilized ketoreductase according to claim 5, wherein the resin is an amino or epoxy resin.
  7. 7. The mutant ketoreductase according to claim 1, wherein the concentration of the compound of formula a is from 10g/L to 200g/L.

Description

Immobilized ketoreductase mutant and application thereof in synthesis of benananate intermediate Technical Field The invention belongs to the field of molecular sieve synthesis, and particularly relates to an immobilized ketoreductase mutant and application thereof in synthesis of a benananate intermediate. Background Benbazaar is a medicament for treating epilepsy developed by Korean Aikekai biopharmaceutical company, is approved by the FDA in U.S. for marketing at 2019.11.21, is approved by the EMA in Europe at 2021.06.16, and clinical test results show that the proportion of patients without seizures in the Benbazaar treatment group exceeds 20 percent, and has obvious clinical advantages compared with other similar products. According to IMS data statistics, the sales of the benananate in 2024 reaches 4.5 hundred million dollars, the raw material medicine demand reaches 4 tons, the market expansion speed is doubled, and the raw material medicine in China only becomes Du Obang at 2024.10.10 to declare CDE at present and cannot meet the market demands of the raw material medicine at home and abroad. The chemical structural formula is as follows: C (C) Wherein (R) -1- (2-chlorophenyl) -2- (2H tetrazol-2-yl) ethanol is a key chiral intermediate thereof. The chemical structural formula is as follows: B (B) Korean Aisi patent CN 101228138B discloses a method for preparing (R) -1- (2-chlorophenyl) -2- (2H-tetrazol-2-yl) ethanol (5), wherein 2-bromo-2' -chloroacetophenone (compound 2) is used as a raw material, and ketone reductase is used for catalyzing and preparing compound 3, but substitution reaction can be carried out at the 1 position and the 2 position of 1H-tetrazole in the process of synthesizing 5 from 4 to respectively generate (R) 1- (2-chlorophenyl) -2- (1H-tetrazol-1-yl) ethane-1-ol and compound 5, and the (R) -1- (2H-tetrazol-1-yl) ethane-1-ol is required to be separated by column chromatography, has high cost and is not suitable for large-scale production. And the final optical purity is more than 98 percent, which does not meet the quality requirement of the bulk drug. Subsequently, the route was improved in korean patent. The technological route is redesigned in the patent CN102803233B, CN02574821B, and asymmetric metal catalysis and biological enzyme catalysis are respectively adopted for the compound. The ruthenium catalyst is high in price, so that the cost of raw materials is high, and the risk of exceeding the standard of heavy metals in finished products is increased. The main disadvantages of the biological enzyme catalysis, which adopts crude enzyme liquid to carry out catalytic reaction and uses crude enzyme liquid or wet thalli and other free enzyme forms, are that the enzyme is difficult to recycle, thus leading to higher cost, the stability is poor, the enzyme is easy to inactivate due to environmental sensitivity and difficult to store and transport, the separation between the enzyme and the product is difficult after the reaction, the downstream purification process is complex, the purity of the product is affected, and the continuous industrial production is difficult to realize. In 2024, yu Zenghui et al, published new technology literature on cilazalide, modified in route 2, used commercially available ketoreductase, but still used free enzyme solution in catalytic form and substrate concentration of only 110g/L. Based on the above-mentioned status quo. Aiming at a series of problems of industry of adopting metal catalysts or free enzymes in biological enzyme catalysis in the construction of key chiral centers in the synthesis process of the benananate and the intermediate thereof, the patent specifically screens and verifies the ketone reduction of 3 different strains, further develops corresponding immobilized enzyme ketone reductase, and reacts as pure water reaction. The method simplifies the process flow, effectively reduces the production cost, and has obvious industrial advantages. Disclosure of Invention Aiming at the technical problems, the invention aims to solve the problems that the enzyme is difficult to recycle, the cost is high, the wastewater treatment pressure is high, the environmental pollution is serious, the stability and the storage are poor, the downstream purification process is complex, the product purity is influenced, the continuous industrial production is difficult to realize due to the difficulty in separating the enzyme from the product after the reaction, and the like when the free enzyme is adopted for catalysis in the prior art. Therefore, three ketoreductase enzymes from different strains are screened through sequence comparison and analysis, the conversion of the compound A can be efficiently and highly stereoselectively catalyzed, key active sites are identified through design, one ketoreductase enzyme is subjected to directed evolution through a site-specific saturation mutation technology, and the enzyme activity is further improved, so t