179 results about "Carbonyl Reductase" patented technology

The invention belongs to the field of biotechnology, and in particular relates to a mutant of diketoreductase (DKR) that can be used as a catalyst for chiral alcohol synthesis; the invention discloses a mutant of diketoreductase, the diketoreductase The amino acid sequence of the mutant has more than 80% homology with any amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.20. Compared with the wild-type double carbonyl reductase, the double carbonyl reductase mutant of the present invention has higher transformation efficiency and higher stereoselectivity.

The invention provides a Pichia guilliermondii X25 which is enriched and screened from soil, has high diastereoselectivity and is a carbonyl reductase active microbial new strain, and application of the Pichia guilliermondii X25 in preparation of 6-cyano-(3R, 5R)-dyhydroxyl hexanoic acid tert-butyl ester by biological asymmetric reduction (R)-6-cyano-5-hydroxyl-3-carbonyl hexanoic acid tert-butylester. The strain is preserved in China Center for Type Culture Collection (CCTCC), the address is Wuhan university, Wuhan, China, the post code is 430072, the CCTCC No. is M 2011386, and the preservation date is November11th,2011. The optical pure 6-cyano-(3R, 5R)-dyhydroxyl hexanoic acid tert-butyl ester prepared by using carbonyl reductase generated by the Pichia guilliermondii X25 to convert (R)-6-cyano-5-hydroxyl-3-carbonyl hexanoic acid tert-butyl ester is high in stereoselectivity, mild in reaction condition and environment-friendly.

The invention discloses an application of carbonyl reductase with the amino acid sequence as shown in SEQ ID NO:2 to prepare (S)-4-chloro-3-hydroxybutanoic ethyl ester by asymmetric reduction of 4-chloracetyl ethyl acetate. The carbonyl reductase with the amino acid sequence as shown in SEQ ID NO:2 is taken as a catalyst, the ethyl 4-chloracetyl ethyl acetate is taken as a substrate, and NADPH is taken as a cofactor, and the (S)-4-chloro-3-hydroxybutanoic ethyl ester is prepared by the asymmetric reduction. The carbonyl reductase with the amino acid sequence as shown in SEQ ID NO:2 is applied to preparing the (S)-4-chloro-3-hydroxybutanoic ethyl ester by the asymmertric reduction of the 4-chloroacetyl ethyl acetate for the first time, which produces good effect; the yield of the substrate is up to 95%, and the enantiomer excess value of the product is 100%, the yield is high, and the production cost is greatly reduced.

The invention discloses a simple and convenient method for recombinase immobilization. According to the method, the characteristic that a histidine label of recombinant protein and metal ion affinity chromatography resin are combined specifically is utilized, and therefore recombinase is immobilized on the resin, and the immobilized recombinase is prepared. Immobilized carbonyl reductase is used for continuous production of chiral alcohol, and the space time yield is greatly increased.

The invention discloses a carbonyl reductase expressed recombination engineering bacterium and application thereof. The engineering bacterium comprises a host cell and a recombinant vector transferred into the host cell, wherein the recombinant vector consists of an original vector and a target gene connected into the original vector, the host cell is Escherichiacoli Rosetta (DE3), the target gene is a carbonyl reductase gene, and the base sequence is shown in SEQ ID NO.1. According to the recombination engineering bacterium, the expression level of a carbonyl reductase is high, bacterial cells which are prepared through induced culture are used for converting N,N-dimethyl-3-oxy-3-(2-thienyl)-l-propylamine hydrochloride into (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-l-propylamine or converting 4-chloroethyl acetoacetate into (S)-4-chloro-3-hydroxyethyl butyrate, and the two products are higher in both optical purity and conversion rate.

The invention discloses a method for preparing pro-xylane from a biological enzyme by a one-pot method. The method is characterized by comprising the following steps of taking xylose and isopropanol as substrates, generating the pro-xylane under the catalytic action of isopropanol dehydrogenase, pro-xylane synzyme, carbonyl reductase and coenzyme nicotinamide adenine dinucleotide. The method is simple in process, free of chemical reagents, low in cost, high in yield and suitable for industrial production.

The invention belongs to the technical field of gene engineering and particularly relates to carbonyl reductase AcCR and an encoding gene and application thereof. The amino acid sequence of the carbonyl reductase AcCR is shown as SEQ ID NO.1. The nucleotide sequence of the gene for encoding the carbonyl reductase AcCR is shown as SEQ ID NO.2. The carbonyl reductase AcCR comes from bacillus aceticus XZY003 and can catalyze 13 carbonyl compounds to generate corresponding chiral alcohol of single enantiomers. The carbonyl reductase AcCR and glucose dehydrogenase GDH are co-expressed in a prokaryotic expression system or a eukaryotic expression system, the biological reaction process of biological catalytic conversion is conducted through the carbonyl reductase, in-situ regeneration of coenzyme is achieved, production cost is greatly reduced, the optical purity of an obtained product is high, the reaction process is efficient, and conditions are moderate.

The invention discloses a method for asymmetrically reducing a carbonyl compound by biocatalysis in a water/ion liquid biphasic system, which belongs to the biochemical engineering technical field. The method takes a selective strain producing carbonyl reductase as a starting strain, and prochiral ketone as a substrate to perform reducing preparation of corresponding chiral alcohol in the water/ion liquid biphasic system; the method comprises the following steps that: Aureobasidium pullulans CGMCC No.1244 is used to catalyze 4-chloro-acetoacetic acid ethyl ester to perform the asymmetrical reduction preparation of (S)-4-chloro-3-hydroxybutyric acid ethyl ester, and Saccharoinyces uvarum ATCC 26602 is used to catalyze 4,4,4-trifluoroacetoacetate to perform the asymmetrical reduction preparation of (R)-4, 4, 4-trilfluoro-3-hydroxybutyrate. The method shows the advantages of no toxicity, no smell, difficult volatilization, good biocompatibility, no environmental pollution, simple product separation, easy recovery, repeated use and so on, of ion liquid. At the same time, the method improves the transformation rate, the concentration and an enantiomeric excess value of a reaction product, and quickens the process of the reaction.

The invention discloses a carbonyl reductase (i)Ch (/i) KRED15 derived from Chryseobacterium ((i) Chryseobacterium (/i) (i) (/i) sp. CA49), and a coding gene thereof; the invention also discloses application of the carbonyl reductase as a biocatalyst in preparing a Duloxetine chiral intermediate ((i) S (/i))-N-methyl-3-hydroxy-3-(2-thienyl) propanamide. The enantiomer excess of products is greater than 99.9%. 20 g/L of substrate can be catalyzed by Ch(/i) KRED15 crude enzyme (2U/mL), and the conversion rate of over 99% can be obtained in 2 hours; a coenzyme circulation system is simple, and has large industrial application prospect.

The invention discloses a carbonyl reductase mutant as well as a coding gene and application thereof. A carbonyl reductase mutant gene has a sequence shown as SEQ ID NO. 1; an amino acid sequence of acarbonyl reductase mutant coded by the carbonyl reductase mutant gene is shown as SEQ ID NO. 2. A genetically engineered bacterium for producing carbonyl reductase contains the carbonyl reductase gene provided by the invention. A preparation method of the carbonyl reductase comprises the following step: culturing the genetically engineered bacterium for producing the carbonyl reductase to obtainthe carbonyl reductase through recombinant expression. The carbonyl reductase prepared by the recombinant expression is used as catalyst, and optical active chiral alcohol is prepared through an asymmetric reducing carbonyl compound; preparation of the recombinant carbonyl reductase is realized, and the recombinant carbonyl reductase is used for asymmetrically synthesizing and catalyzing (R)-2-hydroxyl-4-ethyl phenylbutyrate. The substrate conversion rate reaches 96.8 percent and the ee of a reduction product is greater than 99 percent.

It is an object of the present invention to provide an inexpensive and efficient industrial method for obtaining (S)-2-pentanol, (S)-2-hexanol, 1-methylalkyl malonic acid and 3-methyl carboxylic acid at a high optical purity. The present invention provides a method of producing (S)-2-pentanol or (S)-2-hexanol which comprises allowing certain types of microorganisms or transformed cells, a product obtained by treating said microorganisms or cells, a culture solution of said microorganisms or cells, and/or a crude purified product or purified product of a carbonyl reductase fraction obtained from said microorganisms or cells, to act on 2-pentanone or 2-hexanone.

The invention relates to a method for improving the transformation efficiency of (S)-phenyl glycol by coupling glucose-6-phosphate dehydrogenase and (S)-carbonyl reductase, belonging to the technical field of biological catalytic asymmetric transformation. The invention provides the method for preparing the (S)-phenyl glycol by recombinant Pichia pastoris with CGMCCNo.4198 and 2-hydroxyacetophenone catalyzed by the recombinant Pichia pastoris with CGMCCNo.4198 in multiple batches. In the invention, the Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase gene and the Candida parapsilosis (S)-carbonyl reductase gene are simultaneously integrated into the Pichia pastoris genome, under the participation of 5% (w/v) glucose, the whole cells are reused for 10 batches, and the optical purity and yield of the (S)-phenyl glycol prepared by asymmetric transformation are maintained at 100% and higher than 85% all the time. The recombinant strain improves the batch stability of full cell transformation of the (S)-phenyl glycol by polygene coexpression, which well relieves the limit of coenzyme regenerative cycle in a biological transformation reaction; and simultaneously an efficient approach for preparing the (S)-phenyl glycol industrially in multiple batches at low cost is provided.

The invention discloses a method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase and belongs to the technical field of biological catalytic asymmetric transformation. The invention provides recombinant escherichia coli E. coli RIL/pET-R-SD-AS-G. The recombinant escherichia coli E. coli RIL/pET-R-SD-AS-G is preserved in the China center for type culture collection (CCTCC) and has a preservation number of CCTCC NO: M2015170. R-carbonyl reductase and glucose dehydrogenase are coupled by a coexpression way, and the coupled enzyme, a cultured recombinant strain and 2-hydroxyacetophenone as a substrate undergo a catalytic asymmetric transformation reaction under optimized biotransformation reaction conditions to produce (R)-phenylglycol with optical purity of 100% and a yield of 99.9%. The method utilizes a SD-AS sequence high efficiency coupling double-enzyme technology, solves the problem of limitation of chiral catalytic reaction coenzyme regeneration cycle and provides an effective approach for high efficiency bio-preparation of the (R)-phenylglycol.

The present invention is to provide a process for efficiently producing an optically active alcohol including (R)-3-hydroxy-3-phenylpropanenitrile. One of the features of the present invention is a polypeptide having an activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile isolated from a microorganism belonging to the genus Candida to produce (R)-3-hydroxy-3-phenylpropanenitrile, DNA encoding the polypeptide and a transformant of producing the polypeptide. Another feature of the present invention is a process for producing an optically active alcohol such as (R)-3-hydroxy-3-phenylpropanenitrile by reducing a carbonyl compound such as 3-oxo-3-phenylpropanenitrile by use of the polypeptide or the transformant.

The present invention provides methods for producing (S)-1,1,1-trifluoro-2-propanol, which include the step of reacting an enzyme of any one of alcohol dehydrogenase CpSADH, alcohol dehydrogenase ReSADH, carbonyl reductase ScoPAR, (2S,3S)-butanediol dehydrogenase ZraSBDH, carbonyl reductase ScGCY1, tropinone reductase HnTR1, tropinone reductase DsTR1, or alcohol dehydrogenase BstADHT, a microorganism or a transformant strain that functionally expresses the enzyme, or a processed material thereof, with 1,1,1-trifluoroacetone. The present invention also provides methods for producing (R)-1,1,1-trifluoro-2-propanol, which include the step of reacting alcohol dehydrogenase PfODH, a microorganism or a transformant strain that functionally expresses the enzyme, or a processed material thereof, with 1,1,1-trifluoroacetone.

An object of the present invention is to modify a wild-type enzyme that is less reactive in the presence of an organic solvent to provide altered carbonyl reductases having better reactivity in the presence of the organic solvent than the wild-type enzyme, and/or to provide transformants producing such reductases. The present inventors have found altered carbonyl reductases having better reactivity in the presence of an organic solvent than the wild-type enzyme, from among a mutant enzyme library prepared by randomly mutating the wild-type enzyme gene, thereby arriving at completion of the present invention.

The invention discloses a carbonyl reductase EbSDR8 mutant as well as a construction method and application thereof, wherein the amino acid sequence of carbonyl reductase EbSDR8, as shown in SEQ ID NO.2, has single-point mutation or two-point combination mutation in 97th-site alanine or 160th-site lysine, wherein the 97th-site alanine is mutated to leucine and the amino acid sequence of the leucine is as shown in SEQ ID NO.4; the 160th-site lysine is mutated to glutamic acid and the amino acid sequence of the glutamic acid is as shown in SEQ ID NO.6. The invention aims at providing the carbonyl reductase EbSDR8 mutant as well as the construction method and application thereof, and the catalytic activity of the carbonyl reductase EbSDR8 is improved.

The invention belongs to the technical fields of biological pharmacy and biological engineering, and particularly relates to a carbonyl reductase mutant and application thereof in preparation of (R)-4-chloro-3-hydroxy-butyrate. The carbonyl reductase mutant disclosed by the invention is obtained by replacing phenylalanine at the 85th site of a sequence shown as SEQ ID NO.2 with methionine, or is obtained by replacing one or more sites except the 85th site with amino acid residues on the basis that phenylalanine at the 85th site of the sequence shown as SEQ ID NO.2 is replaced with methionine.The invention also relates to a recombinant expression vector containing the carbonyl reductase mutant gene, an engineering bacterium containing the carbonyl reductase mutant gene and a glucose dehydrogenase gene, and application of the genetic engineering bacterium in preparation of (R)-4-chloro-3-hydroxy-butyrate by asymmetric reduction of chloroacetoacetate. Compared with a wild enzyme, the carbonyl reductase mutant disclosed by the invention has the advantages that the reducing capability on chloroacetoacetate is obviously improved and the carbonyl reductase mutant has good industrial application prospect.

The invention discloses a fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH, and belongs to the technical field of genetic engineering and biological catalysis. Fusion expression is carried out on carbonyl reductase and glucose dehydrogenase to achieve coupling between the two enzymes, and a separated and purified fusion protein in a successfully constructed recombinant bacteria package is utilized to carry out biotransformation COBE to obtain S-CHBE. By optimizing reaction conditions, a substrate of the fusion protein can be totally converted, the optical purity of a product is above 99%, and the TTN reaches up to 1200. The final concentration of the substrate is 305 mmol/L through a substrate batch replenishing strategy, the yield of the product reaches up to 53%, the product of 162 mmol/L is obtained, and the e.e. value remains above 99%. The fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH provide a novel research thought for coupling a coenzyme regeneration cycle approach into a biotransformation approach of the S-CHBE and have great significance for simplifying the a chiral transformed way.

The invention discloses a biscarbonyl reductase, and a coding gene and application thereof. The biscarbonyl reductase has one of the following amino acid sequences: 1) amino acid sequence disclosed as SEQ NO.1; or 2) SEQ NO.1-derived amino acid sequence subjected to substitution, and/or deletion and/or addition of one or more amino acids with the function of stereoselectively reducing the formula disclosed in the specification into the formula disclosed in the specification, wherein the SEQ NO.1-derived amino acid sequence has more than 80% of homology with SEQ NO.1.R1 is selected from aryl group, alkyl group, cycloalkyl group, alkyl-substituted aryl group, halogen-substituted aryl group, aryl alkyl heterocyclic group, and cycloheteroalkyl or cyclohetero alkylated alkyl group; and R2 is selected from alkyl group, cycloalkyl group, and haloalkyl group or halocycloalkyl group. The biscarbonyl reductase can reduce a diketone substrate in one step to obtain 3R,5S-dihydroxy compounds with single optical purity.

A method for preparing (S)-phenylethylene glycol by coupling of carbonyl reductase and pyrimidine nucleotide transhydrogenase, which belongs to the technical field of biocatalysis asymmetric transformation. In the invention, (R)-carbonyl reductase, (S)-carbonyl reductase and pyrimidine nucleotide transhydrogenaseA, B are respectively constructed to two co-expression vectors, and the constructed recombinant plasmid pETDuet-rcr-scr and pACYCDuet-pnta-pntb are co-transformed into Escherichia coli to obtain recombinant strain E. Coli BL21(pETDuet-rcr-scr/pACYCDuet-pnta-pntb). The accession number is CCTCC NO: M208126. The recombinant strain not only realizes one-step transformation from substrate (R)-phenylethylene glycol to product (S)-phenylethylene glycol, but also removes a restriction of coenzyme regeneration cycle in a transformation reaction. The method provides an effective way for producing chiral alcohol through catalytic reaction of enzyme and regeneration of in-situ coenzyme under high substrate concentration.

The invention provides a novel carbonyl reductase and a method for asymmetric reduction by using recombinant carbonyl reductase. Compared with asymmetric ketone reduction catalyzed other enzymes, the carbonyl reductase provided by the invention has the following advantages in the asymmetric reduction: the carbonyl reductase has wide substrate selectivity, can catalyze reduction of a plurality of substituted aromatic ketones like a benzene ring, a naphthalene ring, pyridine and benzopyridine and enables the optical purity of a product to be as high as more than 99% under the condition of catalysis of a substrate with a concentration as high as 100 g/L. A preparation method for the carbonyl reductase in the invention has the advantages of high concentration and high optical purity of the produced carbonyl reductase, mild reaction conditions, environment friendliness, simple operation and easy industrial scaling-up.