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CN-122012429-A - Imine reductase mutant and application thereof in vitamin Bei Gelong synthesis

CN122012429ACN 122012429 ACN122012429 ACN 122012429ACN-122012429-A

Abstract

The invention discloses an imine reductase mutant and application thereof in the synthesis of vitamin Bei Gelong. The mutant is based on imine reductase mutant IR-13-M5 with the following mutation：M203P-F269W-I149G-S241Q-L200T-G268Q,M203P-F269W-I149G-L200T, M203P-F269W-I149G-S241Q, M203P-F269W-I149G-S241Q-L200T or M203P-F269W-I149G-L200T-G268Q. The mutant can be used for asymmetrically catalyzing and synthesizing S-selective chiral amine product vitamin Bei Gelong, the conversion rate reaches 78%, the stereoselectivity reaches 99%, and the mutant has good industrial application prospect.

Inventors

  • GAO SHUSHAN
  • CUI CHENGSEN
  • BAO JINPING
  • Ju Xiaozhi
  • Tang dongyu
  • ZHANG HAN
  • Yan Juzhang

Assignees

  • 中国科学院天津工业生物技术研究所

Dates

Publication Date
20260512
Application Date
20260127

Claims (10)

  1. 1. An imine reductase mutant, characterized in that the mutation ： M203P-F269W-I149G-S241Q-L200T-G268Q,M203P-F269W-I149G-L200T、M203P-F269W-I149G-S241Q、M203P-F269W-I149G-S241Q-L200T or M203P-F269W-I149G-L200T-G268Q is present on the basis of the imine reductase mutant IR-13-M5, wherein the amino acid sequence of the imine reductase mutant IR-13-M5 is shown in SEQ ID NO. 2.
  2. 2. The mutant nucleic acid of claim 1 which encodes.
  3. 3. A recombinant vector, such as a pET28a plasmid, comprising the coding nucleic acid of claim 2.
  4. 4. Recombinant host bacteria comprising the coding nucleic acid according to claim 2 or the recombinant vector according to claim 3, preferably E.coli, such as E.coli BL21 (DE 3).
  5. 5. The recombinant host bacterium of claim 4, wherein the nitroreductase gene is knocked out or attenuated in E.coli.
  6. 6. Use of a mutant according to claim 1 or a nucleic acid encoding the same, or a recombinant host bacterium according to claim 4 or 5, for the synthesis of vitamin Bei Gelong, in particular by the reduction of vitamin Bei Gelong imine to give a vitamin Bei Gelong chiral amine precursor.
  7. 7. A method for preparing a key chiral amine intermediate of vitamin Bei Gelong is characterized in that, Asymmetrically catalyzing imine reduction reaction of substrate vitamin Bei Gelong imine, namely (R) - ((R) -5- (4-nitrobenzyl) -3, 4-dihydro-2H-pyrrol-2-yl) (phenyl) methanol to obtain a vitamin E precursor compound by using the mutant as a catalyst under the condition of a coenzyme NADPH (NADPH) circulating system; further, the coenzyme NADPH circulatory system comprises coenzyme NADP + salt, glucose and glucose dehydrogenase; Specifically, a chiral amine intermediate of vitamin Bei Gelong is obtained by reacting a reaction system comprising a substrate vitamin Bei Gelong imine, the mutant of claim 1, D-glucose, NADP + , glucose dehydrogenase, and phosphate.
  8. 8. The method according to claim 7, wherein the reaction system uses phosphate buffer as a solvent and DMSO as a cosolvent, the glucose dehydrogenase is a glucose dehydrogenase extract prepared by knocking out a nitroreductase gene from Escherichia coli, and the mutant according to claim 1 is added into the reaction system in the form of pure enzyme or a bacterial lysate of the recombinant host bacterium according to claim 4 or a wet cell of the recombinant host bacterium according to claim 4.
  9. 9. The method of claim 8, wherein the substrate concentration in the reaction system is 20-80mM, the mass-to-volume ratio of the mutant reaction mixture is 8-16 g/L, D-the mass ratio of glucose to substrate is 1-2:1, the amount of NADP+ is 0.6-1.5 mmol/L, the amount of glucose dehydrogenase extract is 0.2-0.6 g/L, the amount of phosphate is 60-150 mmol/L, and the volume amount of DMSO is 15-50% of the total volume of the reaction mixture; The pH of the reacted solution is 6.5-7.5, the reaction temperature is 25-35 ℃ and the reaction time is 10-30 hours.
  10. 10. The method according to claim 8, wherein the reaction comprises the steps of adding buffer solution of DMSO and sodium phosphate salt into a reactor according to the volume ratio of the reaction, adding substrate vitamin Bei Gelong imine, the mutant or the mixed solution of the wet cells, D-glucose and nicotinamide adenine dinucleotide phosphate disodium salt into the reactor for reaction to obtain a product; In gram scale reaction, the reaction is carried out for 20-28 hours and then comprises the following steps of quenching the reaction with sodium carbonate solution to obtain solution with pH of 8.0-9.0, adding ethyl acetate for extraction for two-four times, filtering the mixture remained after extraction on a diatomite layer, flushing the mixture with ethyl acetate to obtain filtrate, extracting the mixture with ethyl acetate for two-four times again, merging the organic phase extracts, washing the organic phase with water, drying the organic phase with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain the product.

Description

Imine reductase mutant and application thereof in vitamin Bei Gelong synthesis Technical Field The invention belongs to the technical field of enzyme engineering, and particularly relates to an imine reductase mutant with high stereoselectivity and high catalytic activity and application of the mutant in asymmetric catalytic synthesis of a key chiral amine intermediate of vitamin Bei Gelong. Background Vitamin Bei Gelong (Vibegron) is a β 3 -adrenergic receptor agonist for use in the treatment of overactive bladder (OAB). The molecular structure of the chiral amine contains a key chiral amine fragment, and the efficient and high-stereoselective synthesis of the fragment is a core challenge of industrial production. At present, the chemical catalytic synthesis of chiral amine mainly depends on noble metal chiral catalysts, high-pressure catalytic environment or multi-step continuous reaction, and has the problems of poor atomic economy, serious environmental pollution, insufficient optical purity and the like. The biocatalysis method, in particular to the imine reductase catalytic imine direct asymmetric reduction synthesis chiral amine, has the advantages of mild reaction condition, high selectivity, environmental friendliness and the like. In the previous study, the inventor clones an imine reductase IR-13 from Actinoalloteichus hymeniacidonis, and uses the mutant IR-13-M5 obtained by engineering the active center for asymmetric catalytic reduction of imine to synthesize a vitamin Bei Gelong (Vibegron) drug intermediate (hereinafter referred to as IR-M5). The IR-M5 has good catalytic activity on a five-membered ring imine structure of a vitamin Bei Gelong (Vibegron) drug intermediate, but has poor stereoselectivity (ee value is 36% R) and cannot meet the requirement of industrial production. Therefore, the mutant is further engineered to obtain the enzyme capable of efficiently and selectively catalyzing the vitamin Bei Gelong (Vibegron) drug intermediate, and the mutant has important industrial value and practical significance. Disclosure of Invention The invention aims to provide an engineered imine reductase mutant to solve the technical bottlenecks that the reaction conditions are harsh and the stereoselectivity (S configuration) is not up to standard when a chemical method or the existing imine reductase catalyzes key imine intermediates of a vitamin Bei Gelong medicine. The present invention provides mutants obtained by further mutation based on the imine reductase mutant IR-13-M5, said further mutation being selected from ： M203P-F269W-I149G-S241Q-L200T-G268Q,M203P-F269W-I149G-L200T、M203P-F269W-I149G-S241Q、M203P-F269W-I149G-S241Q-L200T and M203P-F269W-I149G-L200T-G268Q. The amino acid sequence of the imine reductase mutant IR-13-M5 is shown as SEQ ID NO. 2. The invention also provides a nucleic acid encoding the mutant. The invention further provides recombinant vectors containing the encoding nucleic acids. The invention also provides recombinant host bacteria containing the coding nucleic acid, or the recombinant vector. The invention provides, inter alia, the use of said mutants, encoding nucleic acids, in the synthesis of vitamin Bei Gelong, in particular by the imine reduction of vitamin Bei Gelong to obtain a key chiral amine intermediate of vitamin Bei Gelong. The invention provides a method for preparing vitamin Bei Gelong, which takes the mutant of claim 1 as a catalyst to asymmetrically catalyze the imine reduction reaction of (R) - ((R) -5- (4-nitrobenzyl) -3, 4-dihydro-2H-pyrrol-2-yl) (phenyl) methanol under the condition of a coenzyme NADPH circulating system to obtain a vitamin Bei Gelong chiral amine precursor compound; further, the coenzyme NADPH circulatory system comprises coenzyme NADP + salt, glucose and glucose dehydrogenase; specifically, a reaction system containing substrate vitamin Bei Gelong imine, the mutant, D-glucose, NADP +, glucose dehydrogenase and phosphate is reacted to obtain the vitamin Bei Gelong chiral amine compound. Furthermore, the reaction system takes DMSO as a solvent, the glucose dehydrogenase is a glucose dehydrogenase extract prepared by escherichia coli from which nitroreductase genes are knocked out, and the mutant is added into the reaction system in the form of pure enzyme or bacterial lysate of the recombinant host bacteria or wet cells of the recombinant host bacteria. In specific embodiments, in the reaction system, the substrate concentration is 20-80mM, the mass-volume ratio of the mutant reaction mixture is 8-16 g/L, D-the mass ratio of glucose to substrate is 1-2:1, the amount of NADP + is 0.6-1.5 mmol/L, the amount of glucose dehydrogenase extract is 0.2-0.6 g/L, the amount of phosphate is 60-150 mmol/L, and the volume amount of DMSO is 15-50% of the total volume of the reaction solution; The pH of the reacted solution is 6.5-7.5, the reaction temperature is 25-35 ℃ and the reaction time is 10-30 hours. More specifically, the reaction compri