69 results about "Penicillin G Acylase" patented technology
Filter
Efficacy Topic
Property
Owner
Technical Advancement
Application Domain
Technology Topic
Technology Field Word
Patent Country/Region
Patent Type
Patent Status
Application Year
Inventor
Penicillin G acylase (PDB ID: 1AJQ) is found in the organism Escherichia coli. Penicillin G acylase has a molecular weight of 94,642.59 Da, and its isoelectric point (pI) is 6.17. It is part of the larger family of penicillin acylases that come from such microorganisms as bacteria, yeast, and fungi.
The invention relates to a gene, mutantplasmid and engineeringbacteria which have improved synthesis performance to penicillin G acylase and are obtained by a gene site-directed mutagenesis method, and mutantenzyme can also be obtained with improved synthesis performance to penicillin G acylase by fermenting and purifying the engineeringbacteria. Two enzymes Kpn I and Pst I are firstly used for cutting pUC18 by the invention, then T4 polymerase is adopted to make the ends blunt, and pZ01 is obtained through self-linkage; the enzyme of EcoR I is used for cutting pZ01, and then connected with pEES102 that is also cut by the enzyme of EcoR I, thereby obtaining the recombinant plasmid pY020; the pY020 is adopted as a template plasmid, and TaKaRa MuTanBEST Kit is utilized for conducting the site-directed mutagenesis to B.megaterium PGA, thereby obtaining the mutant plasmid with improved synthesis performance to the penicillin G acylase. The mutant plasmid is transformed to bacillus subtilis to obtain the required engineeringbacteria. The engineering bacteria are amplified and fermented, and the mutant enzyme with improved maximum conversion rate of 7-ADCA and the ratio of synthetic product / hydrolysate can be obtained after the engineering bacteria are purified.
The invention provides a penicillin G acylase mutant for synthesis and an application thereof in the preparation of amoxicillin. Penicillin G acylase of Achromobacter xylosoxidans origin is mutated by computer aided design in connection with semi-rational design of site-saturation mutagenesis technique and enzymeengineering modification of orthogenesis, thus acquiring the penicillin G acylase mutant lower in hydrolytic activity, better in synthetic activity, higher in synthesis-hydrolysis ratio (S / H), higher in acid resistance and better in stability, and amoxicillin can be catalytically synthesized more effectively and quickly. Immobilized enzyme hydrolytic activity of the mutant SPGA-4 obtained is decreased by 8.7 times, synthetic activity is increased by 5.6 times, the S / H ratio is increased by 8 times, the activity remains at 79% for 60 min under the condition of pH 2.0, amoxicillin is catalytically synthesized by a solid method at 10 DEG C or 20 DEG C, substrates 6-APA and D-HPM are directly charged in a solid form without dissolving, reaction pH need not be controlled, substrate conversion rate is higher than 99%, continuous use is available in more than 300 batches, and good operational stability is given.
The invention discloses a technology for preparing ampicillin by adopting an enzymic method. The technology comprises the following steps of (a) mixing 6-aminopenicillanic acid, a phenylglycine derivative and penicillin G acylase into water to obtain mixed liquid; (b) adjusting and controlling the pH value of the mixed liquid to be 5.5-6.1 through acid or alkaline, and reacting under the temperature of 0-40 DEG C; (c) after the reaction is finished, performing separation, cleaning reaction liquid containing a coarse ampicillin product through the acid, crystallizing the reaction liquid through the alkaline, and performing grain cultivation, washing and drying to obtain an ampicillin product. The preparation technology provided by the invention is simple, convenient to operate, low in energy consumption, high in safety and high in stability; furthermore, the pH value required by a reaction system is easy to control, the reaction time is short, the conversion rate is up to over 99 percent, the product purity is high, and a solvent does not need to be recycled; and therefore, the low-cost and high-efficiency production technology can be popularized and applied in the industrial production.