66results about How to "Higher than vitality" patented technology

The invention discloses a pullulan enzymatic mutant with high specific activity and high thermal stability and a preparation method of the pullulan enzymatic mutant, and belongs to the field of genetic engineering and enzyme engineering. The invention improves the specific activity and the thermal stability of pullulan enzyme by site-specific mutagenesis, and provides a mutation scheme through which the catalytic specific activity and the thermal stability of the pullulan enzyme derived from debranch bacillus are improved. At least one property of the pullulan enzymatic mutant is changed: (1) the optimal reaction temperature is increased, (2), the thermal stability on the condition that the pH is 4.0 to 5.0 is improved; and (3) the specific activity on the condition that the pH is 4.0 to 5.0 is improved. The pullulan enzymatic mutants are more suitable for the production process of starch saccharification as compared with wild type pullulan enzyme.

The invention belongs to the fields of biochemical engineering and enzyme engineering, and relates to immobilized lipase and a preparation method and application thereof. The method comprises the steps of carrying out hydrophobic modification on surface of preprocessed mesoporous TiO2 by a silane coupling agent, so as to obtain a surface-modified mesoporous TiO2 carrier; taking modified mesoporous TiO2 as a carrier to carry out immobilized preparation of immobilized lipase on lipase; carrying out aromatic ester enzymatic synthesis and enzymatic enantioseparation of a drug intermediate by the immobilized lipase in an organic phase, and carrying out purification research on the lipase by the carrier. The carrier obtained by the preparation method can selectively absorb a lipase protein molecule from crude enzyme for lipase fermentation. Therefore, the method is simple in immobilization technology, mild in condition, less in enzyme activity loss in the immobilization process, and high in active recovery rate. The prepared mmobilized lipase has good stereoselectivity and catalytic activity in the organic phase, and has wide application prospect.

The invention belongs to the technical field of agricultural biology, and specifically relates to an alkaline protease mutant with increased specific activity and thermal stability, and a coding geneand applications thereof. The amino acid sequences of the alkaline protease mutant are shown as SEQ ID No.3. Compared with the specific activity of a wild type, the specific activity of the mutant canbe enhanced 15%; the optimum temperature of enzymatic reaction can be maintained unchanged; the optimum pH value can be reduced 0.5; and compared with the wild type, the thermostability of the mutantcan be enhanced under conditions of 55 DEG C and 60 DEG C. Thus, compared with the activity of the wild type, the alkaline protease mutant PROK-M can be higher in activity and more high temperature resistant.

The invention discloses a cytochrome P450 BM-3 (L148S/Q 229R) variant enzyme and a coding gene and use of the cytochrome P450 BM-3 (L148S/Q 229R) variant enzyme. The amino acid sequence of the variant enzyme is shown as the SEQ ID NO. 1. Two mutational sites are added on the basis of the cytochrome P450 BM-3 (F87V/A74G/L188Q) variant enzyme, i.e., the No. 148 leucine (L) is mutated into serine (S), and the No. 229 glutamine (Q) is mutated into arginine (R). Compared with a male parent, the variant enzyme disclosed by the invention is higher in affinity to substances and thermal stability.

The invention discloses an endoglucanase mutant, a genetically engineered bacterium and application of the endoglucanase mutant in degradation of cellulose. The endoglucanase mutant is obtained by mutating lysine at the 202 <nd> position of an amino acid sequence shown in SEQ ID No. 3 into arginine. The specific activity of the endoglucanase mutant disclosed is obviously improved by about 1.4 times compared with that of a wild enzyme. When microcrystalline cellulose is used as a substrate; through the common reaction of cellulase and mutant enzyme, the reaction catalytic efficiency is remarkably improved by 23.5% compared with that of single degradation of a substrate with cellulase, it can be seen that the mutant enzyme remarkably improves the enzyme activity, the economic cost of industrial application can be reduced, and an application basis is provided for the mutant enzyme in the field of industrial production of food bioethanol and the like.

The invention relates to a low-temperature beta-xylosidase mutant with improved thermal stability and specific activity. The mutant is a low-temperature beta-xylosidase mutant G110S, and an amino acidsequence of the mutant is SEQ ID NO.1; alternatively, the mutant is a low-temperature beta-xylosidase mutant Q201R, and an amino acid sequence of the mutant is SEQ ID NO.2; and alternatively, the mutant is a low-temperature beta-xylosidase mutant loop2, and an amino acid sequence of the mutant is SEQ ID NO.3. The low-temperature beta-xylosidase mutant with improved thermal stability and specificactivity is more suitable for industrial applications, and has wider application prospects in the fields of food, feed, papermaking and the like, so that the application potential of low-temperature beta-xylosidase is expanded in food, pharmaceutical, papermaking, feed and other fields.

The invention relates to the technical field of agricultural biology, in particular to a method for improving the mannase activity of bifunctional cellulase, a cellulase mutant RMX-M and an application. An E218H mutant is obtained by implementing site-specific mutagenesis on an E218 site of wild cellulase with an amino acid sequence as shown in SEQ ID NO:1. Results show that the optimum pH value of an enzymatic reaction is not changed and the optimum temperature is reduced by 5 DEG C when two substrates, namely sodium carboxymethylcellulose and carob bean gum, are used for determination; whenthe carob bean gum is used as the substrate, the mannan specific activity of the mutant is improved by about 80% compared with that of the wild cellulase; and when the sodium carboxymethylcellulose isused as the substrate, compared with the cellulose specific activity of wild RMX, the specific activity of the mutant RMX-M is slightly reduced, and the capability of degrading the hemicellulose substrate mannan is improved on the basis of relatively small cellulase activity loss.

The invention relates to the field of gene engineering, and in particular relates to an alkaline protease BmP mutant with the improved specific activity and a coding gene of the alkaline protease BmPmutant. The mutation site of the alkaline protease BmP mutant refers to that the 178th site of the alkaline protease BmP as shown in amino acid SEQ ID NO.7 mutates into D,M,G,Q or T from S. The obtained mutant has higher specific activity than the original enzyme.

The invention belongs to the field of biomedicine, and relates to a preparation method of a lactococcus lactis product for expressing phenylalanine ammonialyase. The method sequentially comprises the following steps of: 1) acquiring genes for expressing the phenylalanine ammonialyase; 2) connecting the genes for expressing the phenylalanine ammonialyase and expression vectors suitable for transforming lactococcus lactis to form recombinant expression vectors; 3) transforming the recombinant expression vectors into competent lactococcus lactis; 4) screening lactococcus lactis strains for obtaining efficiently expressed phenylalanine ammonialyase; 5) fermenting the lactococcus lactis strains, and inducing the expressed phenylalanine ammonialyase; and 6) drying the strains obtained by fermentation, wherein the culture medium used in the fermentation of the step 5) is obtained by mixing an intermediate culture medium and an M17 broth culture medium in a volume ratio of 4:6-6:4; and regulating the pH value to be between 6.5 and 7.5. By adopting the technical scheme, the viable count of the obtained lactococcus lactis strains and the specific activity of the phenylalanine ammonialyase are greatly improved, wherein the viable count can reach over 10<12>CFU/ml, and the specific activity can reach over 80U/g.

The invention relates to construction of a free non-methanol induced pichia pastoris expression vector and an application of the expression vector in enzyme recombination expression, and belongs to the technical field of genetic engineering. The non-methanol induced pichia pastoris expression vector is obtained by inserting a kluyveromyces lactis autonomous replication sequence PARS2 and a pichiapastoris promoter PGCW14 sequence into a vector framework, and constructing a free vector with a self-replication sequence. Compared with the general integrated recombinant pichia pastoris engineeringbacteria, the free recombinant pichia pastoris engineering bacteria constructed by the vector have obviously-improved capability of secreting and producing recombinant enzymes. In addition, the fermentation raw materials have wide sources, and the production cost is low.

The invention relates to the technical field of protein engineering modification, and particularly provides a high-specific-activity mannase mutant. Compared with a wild type, the mannase mutant provided by the invention has the advantages that the specific activity of the mannase mutant is generally improved by 4.4%-38.97%. The specific activity of the mannase mutant is improved, the production cost of mannase is reduced favorably, wide application of the mannase in the field of feed is accelerated, and the market prospect is wide.

The invention discloses a polyethylene glycol-modified aprotinin and a preparation method thereof. The polyethylene glycol-modified aprotinin is characterized in that an amino group of aprotinin is connected with a chain of polyethylene glycol or a derivative thereof. The polyethylene glycol-modified aprotinin can improve the specific activity of the aprotinin, dramatically lowers the immunogenicity of the aprotinin and improves the stability of the aprotinin, thereby improving pharmacokinetic properties of the aprotinin and the tolerance, and reducing drug delivery times.

The invention provides an enzymology modification method for improving the vitality of a lipase. The method comprises the following steps: an enzyme solution with a certain concentration is prepared from trypsin and an HCl solution; a free lipase or an immobilized lipase and a protease which are added to a buffer solution with a certain pH value according to a certain proportion and well mixed are incubated for a period of time in a thermostatic water bath; the vitality of the lipase is determined by an olive oil emulsification process at a temperature of 37 DEG C; and the vitality improvement rate of a modified lipase is obtained through comparing with the activity of the lipase which is not processed by the protease. The method of the present invention has the advantages of simple step, easy operation, substantial improvement of the enzyme activity of the free lipase and the immobilized lipase (the vitality improvement rate of the free lipase reaches 32%, and the vitality improvement rate of the immobilized lipase reaches 38%), and mild reaction condition, and does not cause the denaturalization and the deactivation of the lipase because of extreme conditions.

The invention discloses a nitrile hydratase mutant, and particularly relates to a nitrile hydratase lysine mutant HBA-K2H2R, a coding gene of the nitrile hydratase lysine mutant HBA-K2H2R, a plasmid and a recombinant bacterium containing the coding gene of the nitrile hydratase lysine mutant HBA-K2H2R, and an application of the nitrile hydratase lysine mutant HBA-K2H2R in catalyzing an organic nitrile compound to prepare an amide compound. The amino acid sequence of the nitrile hydratase lysine mutant HBA-K2H2R is as shown in SEQ ID NO. 1, the most suitable pH value of the nitrile hydratase lysine mutant HBA-K2H2R is 9.0, and the most suitable temperature 35 DEG C. Compared with a wild enzyme HBA, the nitrile hydratase lysine mutant HBA-K2H2R has the improved alkaline activity, and can be applied to the biotechnical fields of production of acrylamide, nicotinamide and other amide compounds with high added values through biological catalysis, chemical fiber surface modification, alkaline sewage treatment and the like.

The invention relates to the field of gene engineering, and discloses a high-specific-enzyme-activity xylanase mutant and application thereof. According to the high-specific-enzyme-activity xylanase mutant, xylanase XYN from the GH11 family of Bacillus subtilis Lucky9 is used as a female parent and is expressed after being subjected to amino acid mutation by adopting a molecular biotechnology. Compared with original xylanase XYN,the mutant N36Y has the advantages that the specific enzyme activity is increased by 180% and reaches 150 U/mg, and an unexpected technical effect is achieved. The increase of the specific activity of the xylanase mutant is beneficial to reducing the production cost of xylanase and further expanding broad application of the enzyme in the field of functional sugar,so that the xylanase mutant has a wide market prospect.

The invention discloses a glyceride lipase mutant G28C-P206C as well as a coding gene and application thereof. According to the glyceride lipase mutant G28C-P206C, a disulfide bond is introduced intoglyceride lipase SMG1, the amino acid sequence of the mutant is shown as SEQ ID NO.3, and the nucleotide sequence of the mutant is shown as SEQ ID NO.4. Compared with a wild type, the Tm of the glyceride lipase mutant G28C-P206C obtained by the invention is increased by 9.0 DEG C, the half-life period at 50 DEG C is increased by 64.8 times, the thermal stability is remarkably improved, and compared with the wild type, the glyceride lipase mutant G28C-P206C has better enzymatic activity and is more suitable for application in the industrial field.

The invention discloses a nitrile hydratase mutant, and in particular relates to a nitrile hydratase lysine mutant HBA-K2H1 and a coding gene thereof, a plasmid and a recombinant bacterium containing the coding gene of the mutant, and application of the nitrile hydratase lysine mutant HBA-K2H1 in catalyzing an organic nitrile compound to prepare an amide compound. The amino acid sequence of the mutant HBA-K2H1 is as shown in SEQ ID NO.1, the optimum pH value of the mutant HBA-K2H1 is 8.5, and the optimum temperature of the mutant HBA-K2H1 is 35 DEG C. Compared with a wild enzyme HBA, the alkaline activity of the mutant nitrile hydratase HBA-K2H1 is improved, and the mutant nitrile hydratase HBA-K2H1 can be applied to the biotechnical fields of production of acrylamide, nicotinamide and other amide compounds with high added values through biological catalysis, chemical fiber surface modification, alkaline sewage treatment and the like.

The method for extracting and purifying glutaryl-7-aminocephaphytanic acid acylase includes the following steps: firstly, using dilute hydrochloric acid to regulate pH of fermented liquor produced by fermentation, then adding calcium chloride solution, and adding chitosan, and using dilute alkali to regulate pH to 7.6-7.8, centrifuging to obtain centrifuged bacterial liquor, controlling activity of the bacterial liquor to 5-8 a/ml, adding surfactant, naturally stirring and soaking for 3-5 hr., and its purification process includes the following steps: using calcium chloride solution to downward regulate pH of soaked bacterial liquor, plate-frame filtering and collecting enzyme clear liquor, using sodium hydroxide solution to upward regulate pH of enzyme clear liquor to 7.6-80, and plate-frame filtering and collecting clear liquor.