The invention relates to a biological preparation method of an intermediate of atazanavir. (S)-tertiary butyl (4-chloro-3-carbonyl-1-benzene butyl-2-yl) carbamic acid ester is taken as a substrate. The preparation method comprises the following steps of adding the substrate, a cosolvent and glucose into a reactor, stirring uniformly, weighing ketoreductase powder, glucose dehydrogenase and cofactor, dissolving in a water phase buffered solution with pH of 8-9, adding the obtained mixed solution into the reactor, stirring, controlling the temperature to be at 32DEG C-34 DEG C, and beginning reacting, wherein at the beginning of the reaction, the mass volume ratio concentration of the substrate is 0.05-0.2g/ml, and the mass ratio of the added ketoreductase to cofactor to glucose to the substrate is (0.02-0.03):(0.001-0.005):(0.8-1.2):1. Compared with the prior art, the biological preparation method realizes more environment-friendly and mild biological conversion process with high reaction efficiency, and has important application value.

Biological preparation method of intermediate of atazanavir

The invention relates to recombinant ketoreductase, nucleotide and amino acid sequences for coding the recombinant ketoreductase and application of the recombinant ketoreductase in production of an oxiracetam intermediate compound through catalytic reduction. The invention also particularly provides a method for preparing ethyl 4-chloro-3-hydroxybutyrate through a biological catalysis method by using the recombinant ketoreductase. The method is mild in reaction conditions and low in solvent consumption, has less pollution and energy consumption, greatly lowers the cost, and is greener and more environment-friendly, thus being more suitable for industrial production.

Recombinant ketoreductase and method for preparing oxiracetam intermediate compound by using same

A biological preparation method of ethyl (R)-4-cyano-hydroxybutanoate. The reaction system of enzymatic synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate is properly treated, and recombinant halohydrin dehalogenase is subsequently added. Under the effect of recombinant halohydrin dehalogenase, ethyl (S)-4-chloro-3-hydroxybutanoate reacts with sodium cyanide to give a target product ethyl (R)-4-cyano-hydroxybutanoate, and the reaction takes place in an aqueous phase of pH 6-8. Ethyl (S)-4-chloro-3-hydroxybutanoate is prepared by asymmetric reduction reaction of ethyl 4-chloro-3-oxobutanoate under the presence of recombinant ketoreductase enzyme, a cofactor and a hydrogen donor, the hydrogen donor is isopropanol, and the asymmetric reduction reaction takes place in a mixed system of an aqueous phase buffer solution of pH 7.0-9.0 and toluene. In the present invention, the two reactions can share one reaction system, which can reduce the material and energy consumption, effectively reduce the cost, solve the problem of high material and energy consumption of a single-step enzyme method, and solve the problem of a coupled enzyme method that the product cannot be produced effectively due to mutual interference.

Biological preparation method of ethyl (r)-4-cyano-hydroxybutanoate

The invention provides a ketoreductase with high catalytic activity, strong enantioselectivity and good substrate tolerance and an enzyme chemical synthesis method for catalytic synthesis of (R)-HPBE through the ketoreductase and further synthesis of a pril drug. The invention also provides a nucleic acid sequence for coding the ketoreductase, a recombinant expression vector containing the nucleic acid sequence, a recombinant expression transformant, a preparation method of the ketoreductase and a use of the ketoreductase in catalysis of carbonyl substrate asymmetric reduction. Compared with other asymmetric reduction preparation methods, the preparation method utilizing the ketoreductase has the advantages of high product concentration, high product optical purity, mild reaction conditions, good environmental friendliness, operation simpleness and industry expanding easiness.

Ketoreductase and its use in asymmetric synthesis of chiral hydroxyl compound

The invention discloses a method for preparing a statin side chain intermediate by means of whole-cell catalysis The method is characterized by comprising the following steps: adding a prepared water-phase buffering solution into a reaction container; then sequentially adding substrates (5S/R)-6-X-3-hydroxyl-5-carbonylcaproic tert-butyl ester 1(X=Cl, CN or OR), isopropanol, and whole cells of colon bacillus of recombined ketoreductase; stirring and reacting at the temperature of 25-40 DEG C; detecting the reaction progress by using a gas chromatography (GC) method; ending the reaction until the reaction conversion rate is as high as 95% to 100%; adding ethyl acetate for a plurality of times of extraction; and combining organic phases and removing reagents by evaporation to obtain (3R, 5S/R)-6-X-3,5-dihydroxyl caproic tert-butyl ester 2(X=Cl, CN or OR) The method disclosed by the invention is simple in steps, low in cost and high in product purity, and the method is a green biological preparation method applicable to large-scale production of the (3R, 5S/R)-6-X-3,5-dihydroxyl caproic tert-butyl ester 2(X=Cl, CN or OR)

Method for preparing statin side chain intermediate by means of whole-cell catalysis

The invention relates to a biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl, which uses 4-chloro-3-carbonyl ethyl butyrate as a substrate and enables the substrate to be subjected to asymmetric reduction reaction in the presence of a biological catalyst, a cofactor and a hydrogen donor to produce the (S)-4-chloro-3-hydroxybutyrate ethyl, wherein the biological catalyst is recombinant ketone reductase and the hydrogen donor is isopropyl alcohol; the asymmetric reduction reaction is carried out in a hybrid system of aqueous buffer solution and toluene with pH of 7.0-9.0, and the volume ratio of the aqueous buffer solution to the toluene is 4-20:1. The method provided by the invention solves the problems of requirement of additional coenzyme system, too much added organic solvent, low efficiency of catalyst and high cost in the traditional biological preparation method. The method provided by the invention has the advantages of mild reaction condition, high reaction efficiency and simple operation, and the concentration of the substrate can be increased to 40%, thus improving the preparation efficiency of (S)-4-chloro-3-hydroxybutyrate ethyl and reducing the reaction cost greatly.

Tang Yuanyuan

Papers

Biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl