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

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 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 lactamase, an application of the lactamase and a method for preparing (1R,4S)-gamma-lactam through enzymatic resolution. An amino acid sequence of the lactamase is as shown inSEQ ID NO.1. A nucleotide sequence of a nucleic acid molecule for encoding the lactamase is as shown in SEQ ID NO. 2. The method for preparing the (1R,4S)-gamma-lactam through enzymatic resolution comprises the following step of in a buffer solution system, mixing a substrate and the lactamase to react, wherein the substrate is 2-azabicyclo[2.2.1]hept-5-ene-3-one. The lactamase has the characteristics of high substrate tolerance and high activity, and when the lactamase is applied to the method for preparing the (1R,4S)-gamma-lactam through enzymatic resolution, the substrate with relatively high concentration can fully react under the action of the lactamase to obtain the (1R,4S)-gamma-lactam with relatively high yield and chiral purity, so that the production efficiency is improved, andthe production cost is reduced.

The invention relates to the technical field of biotransformation, in particular to a method for preparing cycloastragenol through double-enzyme compounding transformation of astragaloside. Accordingto the method, the astragaloside is used as a substrate, xylosidase and glucosidase are subjected to double-enzyme compounding, then xyloside bonds at the C3 position of the substrate and glucoside bonds at the C6 position of the substrate are broken through one-step hydrolysis, and the cycloastragenol is obtained. The purity of the obtained cycloastragenol can reach 98% or above, and the method is easy to operate, free of pollution, milder in reaction temperature, clear in enzyme conversion mechanism, and wider in enzyme substrate adaptability and is suitable for industrial production.

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 application thereof. According to the tyrosinase mutant, a plurality of amino acid sites are mutated in wild type tyrosinase with an amino acid sequence as shown in SEQ ID NO.1. According to the tyrosinase mutant and the method for preparing theaflavin by using the tyrosinase mutant, the mutant has higher specific activity than wild type tyrosinase, and has higher yield during catalytic preparation of theaflavin.

The invention discloses a multicellular system for regulating products by changing the ventilating condition and a fermenting method. The multicellular system comprises anaerobic clostridium strains,facultative klebsiella pneumonia strains and escherich's bacillus strains, the total number of effective living bacteria is 97 percent or above that in the multicellular system, wherein the ratio of effective living bacteria of the anaerobic clostridium strains, facultative klebsiella pneumonia strains and escherich's bacillus strains is (60-90):(5-40):(1-10). The multicellular system has stable fermenting performance and strong tolerance under micro-aerobic/anaerobic condition, and can be used for efficiently transforming crude glycerine into 1,3-propylene glycol, lactic acid and butyric acid, different multicellular factories can be constructed from cell sizes by flexible combination of different cells of microorganisms, and the material flow and energy flow of cellular metabolism of microorganisms can be re-distributed by manually controlled recombination and optimization, so that platform compounds can be efficiently prepared.

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 an application of oxidoreductase and a mutant thereof in biosynthesis of noottanone, and belongs to the technical field of bioengineering. According to the present invention, the oxidation-reduction enzyme having high nodulariol conversion ability is obtained from the marine killer yeast (Wickhamomyces anomalusM15) for the first time, and the oxidation-reduction enzyme mutant having high ability is obtained through site-specific mutagenesis; compared with the existing oxidoreductase capable of catalyzing nodulariol, the oxidoreductase and the mutant thereof have good substrate tolerance, high conversion rate and high salt tolerance. According to the oxidoreductase and the mutant thereof provided by the invention, conditions are provided for synthesizing noottanone in an in-vitro enzyme catalysis manner, and noottanone products are synthesized by catalyzing noottanol in a green and efficient manner. According to the invention, an important tool enzyme is provided for the synthesis of noottanone, and huge economic benefits are brought to the synthesis industry of noottanone.

The invention discloses a preparation method of chitosan microspheres for cell immobilization and drug delivery, and relates to the field of preparation of chitosan microspheres. According to the method, chitin is deacetylated by combining a compound enzyme with a microwave induction process technology, the chitosan is subjected to heavy metal green chelation by adopting tetrasodium glutamate diacetate or citric acid, and the extraction and removal capabilities of a chelating agent on residual metal ions are further improved by further utilizing a carbonate dissolution promoting technology; and trace proteins and lipids in crude chitosan products are hydrolyzed and separated by selecting a proper biological enzyme, refined chitosan is obtained, enzymatic molecular modification is performed on the refined chitosan by using transglycosides or oxidase, and finally the chitosan microspheres are prepared by using a green and environment-friendly spray drying method. The prepared chitosan microspheres are smooth in shape and uniform in size, the particle size is 1-250 microns, the chitosan microspheres are of a core-shell structure, and the application range of chitosan in the fields of medicine, food and other industries is widened by utilizing chitosan immobilized enzyme.

The invention provides an AMA synthetase and its application in synthesis of AMA or its derivatives, and belongs to the technical field of genetic engineering. The gene sequence of the AMA synthetaseis shown as SEQ ID No. 2, and the encoded protein sequence is shown as SEQ ID No. 1. It is found that the AMA synthetase derived from fungi can effectively make O-acetylserine or O-phosphoserine usedas a substrate react with natural amino acids to generate Toxin A or derivatives thereof and further catalyze Toxin A or derivatives thereof to generate AMA and/or any one of AMA derivatives. The AMAsynthetase provided by the invention has broad application prospects in the field of antibiotic adjuvant preparation.

The present invention discloses an application of a bacillus DL-1 whole cell in catalytic resolution of styralyl acetate. The bacillus DL-1 whole cell can be used as a catalyst to prepare chiral (R)-1-phenylethanol and (S)-styralyl acetate with high optical purity. Under optimal reaction conditions, an enantiomeric excess value of the (R)-1-phenylethanol obtained by the bacillus DL-1 whole cell catalytic resolution of (+ or -)-styralyl acetate is 96.6%, a conversion rate is 24.5% and yield is 48.1%; the enantiomeric excess value of the obtained (S)-styralyl acetate by the catalytic resolutionof the (+ or -)-styralyl acetate is 98.4%, the conversion rate is 56.6% and the yield is 86.2%. The bacillus DL-1 whole cell as the biocatalyst has relatively high substrate tolerance and optical selectivity in the chiral resolution of the (+ or -)-styralyl acetate, and has relatively great application value in the field of biochemical engineering.