145 results about "Carbonyl Reductase" patented technology

The invention discloses a carbonyl reductase mutant and a method for catalytically synthesizing high-concentration glassine by using the carbonyl reductase mutant, and belongs to the field of biological catalysis. According to the invention, a one-pot method is adopted to catalyze beta-acetone xyloside to synthesize high-concentration boine, carbonyl reductase CnMDR coded enzyme and rational design of a substrate pocket key are combined with multiple sites, beneficial mutations are combined, dominant mutant enzyme recombinant bacteria are used as a catalyst, beta-acetone xyloside is used as a substrate, and formate dehydrogenase variants and NAD + kinase are introduced at the same time, so that the high-concentration boine is synthesized. The NADP < + >-NADPH coenzyme is driven to circulate, the yield of the synthesized boine is 200g / L, and the conversion rate is gt; the ee value of the product is gt; the invention provides a one-pot efficient low-cost method for synthesizing the boine, only a trace amount (0.3 mM) of coenzyme needs to be added, byproducts in a reaction system are easy to separate, and great convenience is provided for industrial large-scale production.

The invention provides a carbonyl reductase mutant. The carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation on the 17th site, the 40th site and the 64th site of an amino acid sequence as shown in SEQ ID NO.1. The invention also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and an application of the co-expression engineering bacterium. Compared with a wild type enzyme, the carbonyl reductase mutant has high activity and high stereoselectivity, the enzyme activity can reach more than two times that of the wild type enzyme, the catalytic efficiency on a substrate precursor ketone is remarkably improved, the yield of a statin drug intermediate and chiral alcohol synthesized by an enzyme method of the carbonyl reductase mutant is remarkably improved, and the yield of a rosuvastatin intermediate (3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R) is remarkably improved. The highest yields of the (3R, 5R)-6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester and the atorvastatin intermediate (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoic acid tert-butyl ester reach 99% and 97.6% respectively, e.e. Is larger than 99%, and the method has high industrial application value.

The invention discloses a carbonyl reductase, a mutant and an application of the carbonyl reductase in synthesis of a chiral alcohol compound. The carbonyl reductase mutant is obtained by performing single-site or multi-site mutation on the 96th site, the 145th site, the 202nd site or the 206th site of the amino acid sequence of the carbonyl reductase LbCR derived from lactobacillus brevis as shown in SEQ ID No.2. The activity and stability of the carbonyl reductase LbCR mutant are obviously improved, and the carbonyl reductase LbCR mutant has a wide substrate spectrum. Compared with a wild type, the mutant LbCRM4 has the advantages that the enzyme activity is improved by 86.3 times, the substrate conversion rate is greater than 99%, the e.e.p value of the product is always kept at 99.5% or above, the space time yield reaches 1288.9 g / L / d, and the half-life period at 40 DEG C is 101.4 h and is improved by 83.5 times. The LbCR mutant has an industrial application prospect.

The invention discloses a high-performance carbonyl reductase mutant and application thereof in synthesis of a series of chiral alcohols. The high-performance carbonyl reductase mutant is obtained by performing the following mutations on an amino acid sequence as shown in SEQ ID NO.2: the 69th alanine mutates into any one of lysine, leucine, aspartic acid or asparagine, and the 69th alanine mutates into any one of lysine, leucine, aspartic acid or asparagine; and / or the 100th glycine is mutated into any one of glutamic acid, serine, lysine or asparagine. Compared with a wild type enzyme, the thermal stability and the catalytic activity of the obtained mutant are remarkably improved, particularly, single point mutants Mut-A69N, Mut-G100E and Mut-G100N and a combined mutant Mut-A69N-G100N show excellent relative enzyme activity, thermal stability and substrate tolerance. The mutant has high catalytic activity and stability to various chiral alcohols, and the contradiction that the traditional carbonyl reductase is easy to inactivate at high temperature and the catalytic efficiency is difficult to achieve at the same time is solved. The invention provides an efficient and stable enzyme catalysis tool for green biological preparation of chiral drug intermediates.

The invention discloses a carbonyl reductase mutant as well as a preparation method and application thereof, and belongs to the technical field of enzyme engineering. According to the invention, on the basis of carbonyl reductase BcKRED from Bacillus cereus, a series of dominant mutants are obtained through a method of directed evolution in the field of enzyme engineering. The carbonyl reductase dominant mutant obtained by the invention has high catalytic activity, thermal stability and chiral selectivity reversed with wild-type carbonyl reductase. A green and efficient way is provided for industrial production of chiral alcohol by taking carbonyl reductase as a biocatalyst, and the method has a wide application prospect.

The invention relates to the technical field of biology, and discloses a carbonyl reductase mutant and application thereof in enzymatic synthesis of chiral alcohol. The amino acid sequence as shown in SEQ ID NO.2 is subjected to mutation that 36th lysine is mutated into any one of aspartic acid, threonine and valine, or / and 125th histidine is mutated into any one of lysine, serine and leucine, or 170th histidine is mutated into any one of lysine, cysteine and leucine. The carbonyl reductase mutants, especially single-site mutants Mut-T75K, Mut-T75D and Mut-E216M and a combined mutant Mut-T75K-E216M, with activity improved to different extents compared with that of a wild type are obtained, have excellent relative enzyme activity, thermal stability and substrate tolerance, have relatively high activity and stability for various chiral alcohols, and can be used for preparing the carbonyl reductase mutants. The catalyst has the advantages of high catalytic activity, high thermal stability, strong substrate tolerance, easiness in fermentation and the like.

The invention provides a carbonyl reductase mutant and application thereof in preparation of chiral alcohol compounds, and belongs to the technical field of gene engineering and enzyme catalysis. According to the invention, on the basis of the wild carbonyl reductase, the mutant with activity obviously superior to that of the wild carbonyl reductase is obtained through specific mutation, the selectivity and stability of the enzyme are improved, and the mutant has a wide application prospect in catalytic synthesis of chiral alcohol compounds.

The invention belongs to the technical field of perfume synthesis, and particularly relates to a method for efficiently preparing chiral oxo-ionol by an enzymic method, which comprises the following steps: putting a substrate containing oxo-ionone and an auxiliary substrate into a buffer solution, and stirring at room temperature until complete dissolution to obtain a substrate solution; the method comprises the following steps: dispersing carbonyl reductase ChKRED20 or a mutant thereof and coenzyme into a substrate solution, and carrying out heat preservation and stirring; and after the reaction is finished, adding water to dilute, extracting with ethyl acetate, washing with water, drying, and concentrating the ethyl acetate to obtain the chiral oxo-ionol. The enzyme catalysis method disclosed by the invention is mild in reaction condition and high in selectivity, and a target product can be obtained at high yield at room temperature and normal pressure. The coenzyme is regenerated by adopting a substrate coupling method, the use amount of the coenzyme is reduced, and a substrate coupling strategy that an auxiliary substrate is used as a substrate is adopted, so that a reaction system is simple, products are easy to separate, and the cost is reduced. According to the technical scheme, the conversion rate is greater than 99%, the ee value of the product reaches 99% or above, and the industrial application value is achieved.

The invention provides a carbonyl reductase mutant and an application of the carbonyl reductase mutant in synthesis of S-glassine. The mutant protein is a non-natural protein, and the mutant protein can significantly improve the activity of catalyzing the reduction of carbonyl in 1-C-(beta-D-xylopyranosyl)-acetone to generate S-configuration alcohol. When the activity-improved carbonyl reductase mutant strain is applied to the production process of S-configuration glassine, the carbonyl reduction capacity of the S-configuration glassine can be improved, 100-240 g / L of substrate can be completely converted, and the S-configuration glassine is generated.

The invention belongs to the technical field of biocatalysts, and discloses an alkene reductase mutant and application of the alkene reductase mutant in catalytic preparation of a brivaracetam synthesis intermediate. The amino acid sequence of the alkene reductase mutant is as shown in SEQ ID NO.1, a compound enzyme composed of the mutant, carbonyl reductase K1 and glucose dehydrogenase can take a compound 5-hydroxy-4-n-propyl-2-furanone as a substrate, double bonds are reduced, hydroxyl is removed, a compound (R)-4-propyl dihydrofuran-2 (3H)-ketone is obtained, and the compound (R)-4-propyl dihydrofuran-2 (3H)-ketone can be used for preparing the alkene reductase. The ee value of the (R)-4-propyl dihydrofuran-2 (3H)-ketone obtained through catalysis reaches 93.0%, the (R)-4-propyl dihydrofuran-2 (3H)-ketone with the ee value larger than 99.8% can be further obtained through diastereoisomer crystallization, and the method can be used for efficient and green preparation of the brivaracetam key intermediate (R)-4-propyl dihydrofuran-2 (3H)-ketone.

The invention discloses a carbonyl reductase mutant as well as a preparation method and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, wild-type carbonyl reductase of Stachybotrys chlorohallonata is taken as an evolution template, and a plurality of mutants with catalytic activity, thermal stability and substrate spectrum obviously superior to those of the wild-type carbonyl reductase are obtained through an enzyme engineering technology. The carbonyl reductase mutant can catalyze various prochiral carbonyl compounds to obtain chiral alcohol with high stereoselectivity (ee is greater than 99%). The carbonyl reductase mutant provides a novel, efficient and environment-friendly technical route for industrial production of chiral alcohol products, and has very high industrial application potential.

The invention discloses a high-stability carbonyl reductase mutant and application of the high-stability carbonyl reductase mutant in chiral alcohol synthesis. An amino acid sequence as shown in SEQ ID NO.2 is mutated as follows: 20th leucine is replaced by any one of lysine, threonine, aspartic acid or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine. Compared with a wild type enzyme, the stability of the obtained mutant is remarkably improved, particularly, single-point mutants Mut-L20I and Mut-L55I and a combined mutant Mut-L20I-L55I show excellent relative enzyme activity, thermal stability and substrate tolerance. The mutant has high catalytic activity and stability to various chiral alcohols, has the advantages of high catalytic efficiency, high thermal stability, strong substrate tolerance, easiness in fermentation production and the like, and is suitable for industrial application of enzymatic synthesis of chiral alcohols.

The invention relates to the technical field of NADPH regeneration and the technical field of antidepressant drug synthesis, and discloses a method for catalytically synthesizing (S)-3-chloro-1-phenyl-1-propanol by driving a WO3-based photoenzyme catalyst through infrared light, and the method comprises the following steps: step S1, preparing an oxygen-deficient WO3 nanosheet; s2, preparing a NaCBR carbonyl reductase (NaCBR carbonyl reductase); s3, mixing the oxygen-deficient WO3 nanosheet and NaCBR carbonyl reductase according to the mass ratio of (1: 0.5)-(1: 2), and stirring for 3-6 hours to obtain the photo-enzyme catalyst (WO3 / NaCBR). According to the method, artificial photosynthesis, namely a method for driving NADPH regeneration, is utilized, other materials are not additionally used as hydrogen transfer and electron transfer media, and the (S)-3-chloro-1-phenylpropanol is catalytically synthesized by utilizing infrared light to drive a photo-enzyme catalyst (WO3 / NaCBR). The method can greatly reduce the cost, is green and environment-friendly, has high catalytic efficiency and mild reaction conditions, is simple to operate, is easy to recover and recycle, and is suitable for large-scale industrial production and application.

The invention relates to the technical field of biology, and discloses a recombinant carbonyl reductase mutant and application thereof in synthesis of chiral alcohol. According to the invention, the 36th-site lysine is mutated into any one of aspartic acid, threonine and valine, or / and the 125th-site histidine is mutated into any one of lysine, serine and leucine, or / and the 170th-site histidine is mutated into any one of lysine, cysteine and leucine; the carbonyl reductase mutants, especially single-site mutants Mut-K36D and Mut-H125K and a combined mutant Mut-K36D-H125K, with activity improved to different extents compared with that of a wild type are obtained, have excellent thermal stability and relative enzyme activity, have relatively high activity and stability aiming at various chiral alcohols, and can be used for preparing the carbonyl reductase. The method has the advantages of high catalytic activity, high enzyme expression quantity, easiness in fermentation and the like.

The invention relates to a preparation method of D-panthenol. The preparation method comprises the following steps: (1) carrying out mixed reaction on DL-pantoic acid lactone and D-pantoic acid lactone hydrolase, extracting, and separating a water phase to obtain D-pantoic acid; (2) mixing the D-pantoic acid with a compound enzyme auxiliary agent for reaction to obtain D-pantoic acid lactone; (3) mixing the D-pantoic acid lactone with beta aminopropanol for reaction to obtain D-panthenol crude product slurry; (4) purifying the D-panthenol crude product slurry to obtain D-panthenol; the compound enzyme auxiliary agent comprises esterase, carbonyl reductase, alcohol dehydrogenase and alcohol nitrile enzyme. According to the method disclosed by the invention, the esterase, the carbonyl reductase, the alcohol dehydrogenase and the alcohol nitrile enzyme act synergistically to catalyze an esterification reaction in D-pantoic acid molecules and promote the D-pantoic acid molecules to form a lactone structure, so that side reactions are reduced, the efficiency of the esterification reaction of the D-pantoic acid is improved, and finally, the yield and the purity of the D-panthenol are improved.

The invention relates to the technical field of medical intermediates, in particular to a biosynthesis method of carbonyl reductase and a remegapam intermediate. The amino acid sequence of the carbonyl reductase is shown as SEQ ID NO: 1, the carbonyl reductase can be used for synthesizing a remegapam intermediate, and the specific method is as follows: a compound II is taken as a substrate, and in the presence of the carbonyl reductase, a coenzyme, an enzyme circulating hydrogen donor, a coenzyme circulating enzyme, a cosolvent and a buffer solution, a biological catalytic reaction is performed to generate the remegapam intermediate, namely a compound I; the synthesis method disclosed by the invention is mild in reaction condition, simple and convenient to operate, environment-friendly, low in cost, few in by-products and high in ee value of the product, and efficient synthesis of the remegapam intermediate is realized.

The invention relates to a carbonyl reductase mutant based on cosubstrate catalytic ability optimization and application thereof, the mutant is constructed on the basis of a carbonyl reductase BaSDRX amino acid sequence as shown in SEQ ID No.2, the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.1 in a sequence table, and the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.2 in the sequence table. The mutant is single-point mutation or multi-point combined mutation on the basis of an amino acid sequence of carbonyl reductase BaSDRX as shown in SEQ ID No.2: threonine Thr133 at the 133 site, arginine Arg137 at the 137 site and glutamic acid Glu142 at the 142 site; when the mutant enzyme or a recombinant cell containing the mutant enzyme is applied to synthesis of (S)-1-(2, 6-difluorophenyl) ethanol through asymmetric reduction of 2, 6-difluoroacetophenone, high catalytic activity and high stereoselectivity are achieved, and high-optical-purity (S)-1-(2, 6-difluorophenyl) ethanol (eet; therefore, the method has a good industrial application and development prospect.

The invention relates to the technical field of biology, in particular to a carbonyl reductase mutant, a gene, an engineering bacterium and application. The amino acid sequence of the carbonyl reductase mutant is shown as SEQ ID NO: 1, the catalytic efficiency of asymmetric reductive hydrogenation of prochiral ketone compounds is improved, the stereoselectivity of chiral hydroxyl compounds formed through catalysis is improved, and the synthesis efficiency of (S)-1-(5-fluoro-2-iodophenyl) ethanol is improved.