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

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 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 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 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 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.