30results about How to "Increased substrate tolerance" patented technology

The invention relates to application of novel recombinant R-Omega-transaminase and a high-activity mutant of the recombinant R-Omega-transaminase in asymmetrically synthesizing sitagliptin by catalyzing sitagliptin precursor ketone. The amino acid sequence of the recombinant R-Omega-transaminase is shown by SEQ ID:1, and the mutant of the recombinant R-Omega-transaminase is obtained by performingsingle-site mutation or multisite mutation on one or more of a 60th site, 113th site, 178th site, 233th site, 146th site, 214th site and 186th site of the amino acid sequence shown by SEQ ID:1. The mutant of the novel R-Omega-transaminase with high activity and high stereoselectivity is provided; the mutant has milestone significance in realizing autonomization and localization of a biological catalysis preparation technology for the sitagliptin; and the technology monopoly situation that an enzyme source is single in the process of asymmetrically synthesizing the sitagliptin at present can bechanged.

The invention relates to a preparation method of 2,6-difluorobenzamide by utilizing rhodococcus ruber. The method comprises the following steps: (1) fermentation and culture of seeds; (2) preparation of rhodococcus ruber suspension fluid; (3) conversion of 2,6-difluorobenzonitrile; (4) preparation of finished products. The invention provides a preparation method of 2,6-difluorobenzamide by utilizing rhodococcus ruber to convert 2,6-difluorobenzonitrile into 2,6-difluorobenzamide. The microorganism conversion method has a very high catalytic efficiency, increases the substrate conversion rate, under a material concentration of 3.5 mol / L, the 2,6-difluorobentrinile conversion rate is as high as 100%, the 2,6-difluorobenamide selectivity is 100%, and no by product 2,6- difluorobenzoic acid is generated. The separation and purification technology of target product is simplified, and the method has the advantages of mild reaction conditions, low requirements on equipment, and easy application to production.

The invention discloses a recombinant carbonyl reductase mutant, genes, a carrier, engineering bacteria, and applications thereof. The recombinant carbonyl reductase mutant is obtained through combination mutation of the 145th and the 152th sites of an amino acid sequence represented by SEQ ID NO.2, the 145th site phenylalanine is changed into methionine, and the 152th site threonine is changed into serine; the 145th site phenylalanine is changed into tyrosine, and the 152th site threonine is changed into serine. The invention also provided a recombinant carbonyl reductase mutant capable of reducing (S)-6-chloro-5-hydroxy-3-tert-butyl oxohexanoate, the catalytic activity and the substrate survivability of the recombinant carbonyl reductase mutant are much better than those of a wild type enzyme in the above conversion, and reaction process is shortened obviously.

The invention discloses an immobilized recombinant penicillin G acylase and an application thereof to preparation of (S)-2-aryl-amino acid and cephalosporin antibiotics. The immobilized recombinant penicillin G acylase disclosed by the invention is capable of converting an S-type substrate into an S-type product within shorter time, high in substrate tolerance and strict in S selectivity for the substrate and structural analogues thereof; due to the addition of cobalt ions as well as a protective agent with phenylacetic acid and glycerin as enzyme active centers, the immobilized recombinant penicillin G acylase disclosed by the invention is prevented from being successfully subjected to multipoint covalent immobilization on the surface of an epoxy resin carrier under the condition that enzyme molecules are seriously inactivated in an immobilization process, and the immobilization method is simple and feasible, cheap in raw materials and suitable for large-scale operation; the immobilized recombinant penicillin G acylase can be used for synthesizing cephalosporin antibiotics and splitting the prepared (S)-2-aryl-amino acid, and can be continuously used for more than 50 batches before the activity of the immobilized enzyme is not remarkably reduced.

The invention discloses an acylase for producing 7-ACA (Aminocephalosporanic acid) by splitting CPC (Cephalosporin C) by utilizing a one-step method, polynucleotide encoding the same, a recombinant expression vector containing a nucleotide sequence, a recombinant expression transformant containing the nucleotide sequence, a preparation method of the acylase and an application of a recombinase in synthesis of the 7-ACA. A CPC acylase is a protein of (a) or (b) as follows: (a) a protein which has CPC acylase activity and an amino acid sequence represented by SEQ ID NO.3, wherein 180-site valine is substituted; and (b) a protein which has the CPC acylase activity and an amino acid sequence formed by adding one or multiple amino acids to an N terminal or a C terminal of the amino acid sequence in the (a) and is derivative from (a). According to the CPC acylase disclosed by the invention, both the enzyme activity and the substrate tolerance are improved, so that a strong precondition is provided for manual determinated evolution transformation of the CPC acylase.

The invention discloses a method for preparing magnetic nano carrier immobilized aldolase with high substrate tolerance. The method comprises the following steps of: 1) preparing super-paramagnetic Fe3O4 nano particles by co-precipitation of a mixture of ferrous and ferric iron salts, modifying the particles by using a silane coupling agent, and activating the outer surfaces of the modified particles by using glutaraldehyde to obtain surface activated magnetic nano particles; 2) stirring a purified and desalted aldolase buffer solution and a magnetic carrier at a low temperature, washing, performing freeze drying, and thus obtaining the magnetic nano carrier immobilized aldolase; and 3) catalyzing 2-deoxy-D-ribose-5-phosphoric acid by using the magnetic nano carrier immobilized aldolase as a catalyst to obtain 3-glyceraldehyde phosphate and acetaldehyde. In the reaction of catalyzing the 2-deoxy-D-ribose-5-phosphoric acid by using the immobilized aldolase, the tolerance of the acetaldehyde substrate is remarkably improved, and the recycling operation of enzyme is greatly simplified.

The invention relates to the application of a novel recombinant R-ω-transaminase and its high-activity mutant in catalyzing the asymmetric synthesis of sitagliptin precursor ketone. The amino acid sequence of the recombinant R-ω-transaminase is shown in SEQ ID: 1, and its mutant is the 60th, 113, 178, 233, 146, 214 or 186 of the amino acid sequence shown in SEQ ID: 1 One or more single-site mutations or multi-site mutations are obtained. The present invention provides a novel R-omega-TA mutant with high activity and high stereoselectivity, which is a milestone in realizing the self-management and localization of sitagliptin biocatalytic preparation technology, and can change the current asymmetric synthesis of sitagliptin The technical monopoly situation of a single enzyme source in the process of Gliptin.

The invention provides a carbonyl reductase mutant mut-AcCR (I147V / G152L) and its application and coding gene. Carbonyl reductase AcCR can catalyze the asymmetric reduction of various latent chiral carbonyl compounds, but its activity and substrate tolerance to aromatic compounds are low. The present invention adopts enzyme molecular transformation method to mutate glycosylation reductase AcCR , the mutant mut‑AcCR (I147V / G152L) was obtained. The specific enzyme activity of the mutant for 2‑hydroxyacetophenone can reach 6.4 U / mg, which is 17.4 times higher than that of the unmutated carbonyl reductase. The substrate tolerance concentration was increased from 50 mmol / L to 200 mmol / L. The carbonyl reductase mutants of the present invention are widely used in the asymmetric reduction of carbonyl compounds.

The invention belongs to the technical field of enzyme engineering and relates to a tyrosinase mutant and its application. The tyrosinase mutant has multiple amino acid positions mutated in the wild-type tyrosinase in the amino acid sequence shown in SEQ ID NO.1. Utilizing the tyrosinase mutant and the method for preparing theaflavin of the present invention can make the mutant have higher specific activity than wild-type tyrosinase, and have higher yield when catalyzing the preparation of theaflavin.

The invention relates to the technical field of biotransformation, in particular to a method for preparing cycloastragalol by compound transformation of astragaloside IV with double enzymes. The method of the present invention takes astragaloside IV as a substrate, utilizes xylosidase and glucosidase double-enzyme complex, and then undergoes one-step hydrolysis to cleave the substrate C. 3 position of the xylosidic bond and C 6 The method for obtaining cycloastragalus by the position of the glucosidic bond. The purity of cycloastragalol obtained by the invention can reach more than 98%, and the method has simple operation, no pollution, milder reaction temperature, clear enzymatic transformation mechanism and wider enzyme substrate adaptability, and is suitable for industrial production.

The invention discloses a carbonyl reductase mutant mut-AcCR (E144A / G152L) and its application and encoding gene. Carbonyl reductase AcCR can catalyze the asymmetric reduction of various latent chiral carbonyl compounds, but its activity and substrate tolerance to aromatic compounds are low. The present invention adopts enzyme molecular transformation method to mutate glycosylation reductase AcCR , the mutant mut‑AcCR (E144A / G152L) was obtained, the specific activity of the mutant for 4'‑chloroacetophenone could reach 92.7 U / mg, which was 17.93 times higher than that of the unmutated carbonyl reductase. The substrate tolerance concentration was increased from 50 mmol / L to 200 mmol / L. The carbonyl reductase mutants of the present invention are widely used in the asymmetric reduction of carbonyl compounds.

The invention provides an AMA synthetase and its application in synthesizing AMA or its derivatives, belonging to the technical field of genetic engineering. The AMA synthetase gene sequence of the present invention is shown in SEQ ID No.2, and its encoded protein sequence is shown in SEQ ID No.1. The present invention finds that the AMA synthetase derived from fungi can react O-acetylserine or O-phosphoserine as a substrate with natural amino acids at low cost and efficiently to generate Toxin A or its derivatives and further catalyze Toxin A or its derivatives Any of AMA and / or AMA derivatives are produced. The AMA synthetase provided by the invention has broad application prospects in the field of antibiotic adjuvant preparation.

The invention discloses a recombinant carbonyl reductase mutant, a gene, a vector, an engineering bacterium and application of the recombinant carbonyl reductase mutant. The mutant is obtained by performing single-point mutation on the 95th locus, the 144th locus or the 156th locus of an amino acid sequence shown as SEQ ID NO.2. The recombinant carbonyl reductase mutant has reduced (S)-6-chloro-5-hydroxy-3-oxohexanoate, and compared with a wild type enzyme, has the advantages that the catalytic activity and the substrate tolerance are greatly improved when such reaction is converted, and the time consumption of a reaction process is obviously reduced. Compared with a chemical method for preparing (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, such a technology has the advantages that an obtained product is high in stereoselectivity, a tedious chemical catalysis step is simplified, the reaction condition is milder, the requirement on equipment is low, the reaction cost is reduced, and the technology is environment-friendly.

The present invention discloses a (R)-ω-transaminase mutant and its application in the preparation of sitagliptin intermediate, the mutant consists of arginine at position 77 of the amino acid sequence shown in SEQ ID NO.1 , leucine at position 181, arginine at position 130, tyrosine at position 139 and threonine at position 273 were obtained by multiple point mutations. The present invention screens novel (R)‑ω‑TA recombinases through gene mining technology, and carries out molecular transformation through protein engineering technology to obtain (R)‑ω‑TA recombinase with high enzyme activity, high substrate tolerance and high stereoselectivity. Omega-TA mutant catalyst, this mutant can be precursor ketone analogue 1-(pyrrolidin-1-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione as Synthesis of sitagliptin intermediate (R)-3-amino-1-(pyrrolidin-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one by substrate asymmetric catalysis , and the conversion rate is high, up to 94.6%.

The invention relates to a recombinant R-ω-transaminase and a mutant, as well as its application in the asymmetric synthesis of sitagliptin. The present invention screens novel R-ω-transaminase, and recombines with existing high-efficiency transaminase, the amino acid sequence of the obtained recombinant is shown in SEQ ID: 15, and its mutant is the amino acid sequence shown in SEQ ID: 15. One or more of 71, 135, or 292 obtained by single point mutation or multi-site mutation, the amino acid sequence of the best R-ω-transaminase mutant strain is shown in SEQ ID: 17, the corresponding nucleotide The sequence is shown as SEQ ID:18. The present invention provides R-ω-TA mutants with higher activity (582.4U / g) and stereoselectivity (e.e. value 99.9%), which can catalyze the conversion of 500mM substrate into sitagliptin, and the conversion rate is as high as 99% , which is of great significance for improving the biocatalytic preparation technology of sitagliptin.

The invention discloses a carbonyl reductase mutant mut-AcCR (G152L / Y189N) and its application and coding gene. Carbonyl reductase AcCR can catalyze the asymmetric reduction of various latent chiral carbonyl compounds, but its activity and substrate tolerance to aromatic compounds are low. The present invention adopts enzyme molecular transformation method to mutate glycosylation reductase AcCR , to obtain the mutant mut-AcCR (G152L / Y189N), the specific enzyme activity of the mutant for 2-oxo-4-phenylbutyrate ethyl ester can reach 88.9U / mg, which is higher than that of the carbonyl reductase before the mutation The specific vitality is increased by 61.3 times. The substrate tolerance concentration was increased from 50mmol / L to 200mmol / L. The mutant catalyzed ethyl 2‑oxo‑4‑phenyl‑butyrate with absolute selectivity, and the enantiomeric excess value of the product increased from 82.9% to >99%. The carbonyl reductase mutant of the present invention plays an important role in catalyzing the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate.

The present invention discloses a mutant of (R)-ω-transaminase and its application. The mutant is composed of leucine at position 182, arginine at position 79, arginine at position 51 in the amino acid sequence shown in SEQ ID NO.1. Glutamine at position 1, valine at position 149, leucine at position 235 and glycine at position 216 were obtained through multiple point mutations. The present invention screens novel (R)-ω-TA enzymes through gene mining technology, and carries out molecular transformation through protein engineering technology to obtain (R)-ω-TA enzymes with high enzyme activity, high substrate tolerance and high stereoselectivity ‑TA mutant catalyst capable of biocatalyzing the precursor ketone analog 1‑(3‑oxypyrrolidin‑1‑yl)‑4‑(2,4,5‑trifluorophenyl)‑1,3‑ Synthesis of sitagliptin intermediate (R)-1-[3-amino-4-(2,4,5-trifluorophenyl) butyryl]pyrrole-3-one from diacetyl, with high conversion rate, It can reach up to 95.4%, which is a milestone for breaking through the biocatalytic preparation technology of sitagliptin.

The invention discloses a lactamase, its application and a method for enzymatic resolution and preparation of (1R, 4S)-vins lactone. The amino acid sequence of the lactamase is shown in SEQ ID NO.1. The nucleotide sequence of the nucleic acid molecule encoding the above-mentioned lactamase is shown in SEQ ID NO.2. The method for enzymatic resolution and preparation of (1R, 4S)-wens lactone comprises: in a buffer system, the substrate and the above-mentioned lactamase are mixed and reacted, and the substrate is 2-azabicyclo[2.2.1]heptane ‑5‑en‑3‑one. The lactamase has the characteristics of high substrate tolerance and high activity, and it is used in the method of enzymatic resolution and preparation of (1R, 4S)-Wens lactone, which can make a higher concentration of the substrate Fully reacting under the action of the lactamase, the (1R, 4S)-wens lactone with higher yield and chiral purity is obtained, which improves production efficiency and reduces production cost.