33 results about "Lipase b" patented technology

The invention relates to a low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and a construction method thereof. The method comprises acquisition of a target gene, construction of a chromosome integrated vector and fermented verification of the breeding bacterial strain. The selected bacterial strain performs heterogenous secrete expression on pseudonym antarctica lipase B, compared with parent strains, the fermentation performance of the constructed saccharomyces cerevisiae is not influenced, after fermentation by sorghum raw material semi-solid liquor for 6 days, ethyl lactate content of the breeding bacterial strain is increased by 57.4%-84.5%; the high-grade alcohol production amount is obviously reduced, isoamyl alcohol is reduced by 9.5%, isobutyl alcohol is reduced by about 50.0%, and active-amyl alcohol is reduced by about 44.0%. According to the breeding bacterial strain, the production amount of ethyl lactate is obviously increased, the high-grade alcohol production amount is obviously reduced, the problem of incompatibility of flavor of the wind can be solved at certain degree, and the method has wide application prospect in the wine-related filed.

The invention relates to a technique method of immobilized candida antarctica lipase B, which adopts an organic solvent as an immobilizing medium and realizes the immobilization of lipase in microaqueous phase. The technique method comprises the following steps of: (1) pretreatment of resin; (2) preparation of immobilized enzyme: the pretreated resin is added into a reactor and an organic medium is added; and after standing, then zymogen solution is added according to the mass ratio of the candida antarctica lipase B and the resin, which is equal to 1:60-1:100; the reactor is sealed and then arranged in a shaking bath with constant temperature of 25-35 DEG C for 1h-3h; after vacuum freezing and drying, the immobilized candida antarctica lipase B is prepared. The method for preparing the immobilized enzyme has low consumption of resolvase and high immobilization efficiency and avoids the considerable waste of resolvase caused by an aqueous-phase absorption method, thereby reducing immobilization cost; a carrier has low price and is easily obtained; furthermore, the method has simple technique, mild condition, low loss in enzyme activity and enzyme activity recovery rate of 83.3 percent.

The invention discloses a method for producing biomass diesel under the catalysis of lipase, pertaining to the technical field of production of biomass diesel from fatty acids. Jatropha oil and methyl acetate are taken as raw materials, and the jatropha oil is pre-processed first; then, biomass diesel is produced by reaction between the jatropha oil and methyl acetate under the catalysis of fatty acids, methyl acetate: jatropha oil (mass ratio) = 1: 1-2, the dosage of fatty acids is 10 to 20 wt percent of the dosage of jatropha oil; the lipase is of columnar candida lipase, pubescent putrid mycete lipase, Antarctic candida yeast lipase B or thermophil fungal lipase, or of an synthase achieved by mixing two of the lipases. According to the method, methyl acetate is involved in the transesterification to provide ester-base; the reaction is free of enzyme inactivation, and the subsequent operation is convenient; the jatropha oil is easy to get and the price is low; the preparation process is simple, the reaction condition is mild and the environment is not contaminated; the yield of effective constituent (i.e. fatty-acid methyl ester) is as high as 66.27 to 87.57 percent.

The invention relates to a technology method for producing ethyl lactate from ammonium lactate. The technology method takes the ammonium lactate as a material and adopts enzyme catalysis for preparing the ethyl lactate and comprises the following steps of: adding the ammonium lactate and ethanol in a reactor, sequentially adding a solvent and an enzyme catalyst, wherein the mass proportioning ratio of the ammonium lactate to the ethanol is 1:3-11, the usage of the enzyme catalyst accounts for 14-29 percent of the mass of the ammonium lactate, and the adding quantity of the solvent in the ammonium lactate is 18-28.5ml / g; and sealing and putting the reactor in a constant temperature water bath shaker for reaction for 15-24 hours at temperature of 50-75 DEG C to obtain the ethyl lactate, wherein the enzyme catalyst is candida cylinder lipase, fuzzy humic mould lipase, candida antarctica lipase B or thermophilic fungi lipase. The method takes biological enzyme as a cytalyst and has the advantages of having moderate reaction conditions, being environment-friendly and the like, and the yield reaches over 30 percent; and the method omits lactate acidulating process of lactate salts, shortens the preparation process of the ethyl lactate and lowers the production cost of the ethyl lactate.

The invention discloses a Candida antarctica lipase B mutant as well as a transformation method and application thereof, belonging to the technical field of bioengineering. According to the transformation method, a key locus capable of decreasing dynamics of the conformation of a pocket is found by virtue of conformational dynamics engineering of a combined pocket of CALB through molecular dynamics simulation, and an optimal mutant is designed, so that the limitation that previous enzyme EF5 only presents high enantio-selectivity at a relatively low temperature is overcome, and the optimal mutant is experimentally verified. Finally, the obtained optimal mutant D223V / A281S is used for catalyzing and preparing (R)-3-substituted glutaric acid alkyl monoester compounds, an eeR value of the optimal mutant can reach above 99% under an industrially acceptable temperature (20-55 DEG C), the yield is more than 80.0%, the space time yield can reach 107.54g.L<-1>d<-1>, the cost caused by a previous low-temperature condition is greatly lowered, the production period is shortened, and foundation is laid for the industrialization of the (R)-3-substituted glutaric acid alkyl monoester compounds.

The invention discloses a process method for immobilizing lipase B from candida antarctica fermented supernatant fluid. According to the method, the immobilized candida antarctica lipase B is prepared by using a cheaper vector, adopting a water-phase immobilization mode and cooperating with a post-treatment process, therefore, the enzyme activity recovery rate under water-phase adsorption is increased, the cost and the using effect are balanced, and the important significance on industrial application of the CALB is achieved.

The invention discloses a method for preparing methylene carbonic ester by using an enzymic method, which belongs to the field of biochemical engineering, and. The method for preparing the methylene carbonic ester comprises the following steps: adding diol and dimethyl carbonate in a mole ratio of 1:(1-20) into a reaction container taking an ion liquid as a reaction medium or without a solvent, further adding candida antarctica lipase B which accounts for 1-25% of the total mass of the diol and dimethyl carbonate, stirring for reaction in air for 1-160 hours at 25-75 DEG C, performing transesterification reaction, and after the reaction, treating to obtain a product, namely, the methylene carbonic ester. The method has the advantages of high conversion rate, high selectivity, high universality, simple process, environment-friendliness and the like.

The invention relates to a technique method of immobilized candida antarctica lipase B, which adopts an organic solvent as an immobilizing medium and realizes the immobilization of lipase in microaqueous phase. The technique method comprises the following steps of: (1) pretreatment of resin; (2) preparation of immobilized enzyme: the pretreated resin is added into a reactor and an organic medium is added; and after standing, then zymogen solution is added according to the mass ratio of the candida antarctica lipase B and the resin, which is equal to 1:60-1:100; the reactor is sealed and then arranged in a shaking bath with constant temperature of 25-35 DEG C for 1h-3h; after vacuum freezing and drying, the immobilized candida antarctica lipase B is prepared. The method for preparing the immobilized enzyme has low consumption of resolvase and high immobilization efficiency and avoids the considerable waste of resolvase caused by an aqueous-phase absorption method, thereby reducing immobilization cost; a carrier has low price and is easily obtained; furthermore, the method has simple technique, mild condition, low loss in enzyme activity and enzyme activity recovery rate of 83.3 percent.

The invention discloses a method for splitting and preparing optically pure 1-(1-naphthyl)ethylamine by an immobilized enzyme method. Selective esterification of ‑1‑(1‑naphthyl)ethylamine followed by separation of (R)‑1‑(1‑naphthyl)ethylamine from (S)‑1‑(1‑naphthyl)ethylamine , to obtain (S)‑1‑(1‑naphthyl) ethylamine, and finally (R)‑1‑(1‑naphthyl) ethylamine ester hydrolysis to obtain (R)‑1‑(1‑naphthyl) Ethylamine. The catalyst used for selective esterification is the cross-linked Candida antarctica lipase B aggregate, which first dissolves Candida antarctica lipase B in water to obtain an enzyme solution, and then uses a precipitant to precipitate the enzyme protein from the enzyme solution Come out, then add a bifunctional cross-linking agent for cross-linking, and finally dry it. The cross-linked enzyme aggregate of the invention has high reutilization rate and strong operation stability in pure organic solvent.

The present invention relates to a biocatalytic method of preparing a mono-acylated polyol catalyzed by triacylglycerol lipase mutants, as for example derived from Candida antarctica lipase B (CALB); a biocatalytic method of enantioselectively preparing an asymmetric mono-acylated polyol, catalyzed by the same enzyme mutants; as well as the use of a mutated triacylglycerol lipase in a method of preparing mono-acylated polyols. The invention also provides novel mutants, coding sequences thereof, and recombinant microorganisms carrying said coding sequences.

The invention discloses a method for immobilizing Candida antarctica lipase B. In the method, dimethylimidazole aqueous solution, zinc nitrate aqueous solution and Candida antarctica lipase B are mixed under the conditions of 25° C. and 200 rpm magnetic stirring React for 30 minutes, then add non-polar macroporous adsorption resin and cross-linking agent, conduct cross-linking reaction at 15-45°C and 200 rpm water bath for 5 hours, filter with vacuum filter, and dry the filter cake to obtain immobilized Candida antarctica Lipase B: Compared with the existing commodity immobilized lipase on the market, the immobilized lipase prepared by the present invention has higher enzyme activity, so the enzyme immobilization method has a good industrial application prospect.

The invention discloses an aspergillus niger recombinant expression strain of high-yield lipase B as well as a construction method and application of the aspergillus niger recombinant expression strain. According to the method for constructing the aspergillus niger recombinantexpression strain, a recombinant expression cassette containing the lipase B is constructed, and the recombinant expression cassette is a gene segment containing a lipase B gene sequence, a promoter, a terminator, a selection marker, upstream and downstream homologous sequences and other elements. The lipase B-producing aspergillus niger recombinant expression strain is constructed according to the method disclosed by the invention. The invention also discloses the application of the lipase B-producing aspergillus niger recombinant expression strain in expression of the lipase B. According to the invention, the expression cassette of the lipase B is constructed by means of genetic engineering, and the expression cassette is introduced into aspergillus niger expression host bacteria, so that efficient secretory expression of the lipase B is realized, and the high-yield lipase B expression strain is obtained. Through optimization of fermentation conditions, the expression level of supernatant crude enzyme liquid obtained through liquid fermentation of the strain can reach 4 g / L, and the synthetic enzyme activity of the lipase B can reach 10600unit / L.

The invention relates to candida Antarctica lipase B gene and applications thereof in yeast display. Coded protein of improved candida Antarctica B and wild type candida Antarctica lipase protein havethe same function on the amino acid level; the heat resistance capacity of the enzyme is 50-80 DEG C, and the half-lift is 3-24 hours; a nucleotide sequence is hybridized with SEQ.ID.NO2 from 1st to978th of nucleotide under the moderate precise condition; and a preservation number of colon bacillus DH5Alpha / Puc57-CALB (Escherichia coliDH5Alpha / pUC57-CALB) which carries the plasmids is CCTCC M 209081. The candida Antarctica lipase B gene is transferred into pichia pastoris host bacteria so as to realize the high-efficient display expression of the candida Antarctica lipase B in pichia pastoris; and the provided pichia pastoris bacteria can effectively display candida Antarctica lipase B, can be widely applied to the synthesis of ethyl caproate, has different melting points and does not contain triglyceride of various fatty acids, a plurality of structured lipids, and the like.

A method for preparing methyl-p-benzoquinone catalyzed by lipase is characterized in that butylamine is reacted with methyl-p-benzoquinone catalyzed by immobilized Candida antarctica lipase B to prepare 2-methyl-5-butane Amine p-benzoquinone. The enzyme-catalyzed method used in the present invention prepares 2-methyl-5-butylamino-p-benzoquinone. Due to the high degree of steric selectivity of the enzyme, it can avoid the poor selectivity of the chemical synthesis process and the difficulty of product separation, and is an environmentally friendly and efficient synthetic method. .

The invention discloses a 3, 4-dihydropyrimidine-2(1H)-ketone and derivatives thereof and synthesis method and application thereof. The method comprises the following steps: taking biocatalyst Candida antarctica lipase B as a catalyst and taking vinyl acetate and isopropanol as raw materials to generate acetaldehyde in situ, and adding beta-dicarbonyl compound, urea and water to react with the in-situ generated acetaldehyde to manufacture 3, 4-dihydropyrimidine-2(1H)-ketone and derivatives thereof. By use of the method disclosed by the invention, the biocatalyst Candida antarctica lipase B is used as the catalyst, the vinyl acetate and isopropanol are used as the raw material to generate the acetaldehyde in situ; the in-situ generated cetaldehyde is simultaneously reacted with the beta-dicarbonyl compound, urea and water; the reaction method is simple and environment-friendly; the reaction efficiency is high, and the byproduct is less; the method is an environment-friendly, sustainable, simple and effective method for synthesizing 3, 4-dihydropyrimidine-2(1H)-ketone and derivatives thereof.

A preparation technique of chitosan microspheres immobilization lipase comprises the steps that: (1) lipase from candida SP is dissolved in phosphate buffer solution by taking chitosan microspheres as an immobilization carrier and adopting glutaraldehyde cross-linking, lipase A is obtained. (2) candida SP lipase solution of 0.70-0.80mg / mL prepared by phosphate buffer solution b with a pH value of6.5-7.5 is added with immobilization lipase A (final concentration is 0.005-0.0055g / ml) and after being stirred, ethyl (3-dimethylamino propyl) carbodiimide hydrochloride solution is added, the mixture is then stirred to be fully immobilized, chitosan microspheres immobilization lipase B is obtained after the treatment of the mixture. The chitosan microspheres immobilization lipase prepared by the preparation technology of the invention improves apoenzyme combination rate and activity recovery, has powerful immobilization lipase activity and specific activity and the immobilization lipase canbe recycled for six times.

The invention discloses a method for producing Candida antarctica lipase B and the specific DNA molecule used therefor. The nucleotide sequence of the specific DNA molecule is shown in SEQ ID NO:3. Experiments have shown that the recombinant plasmid pET30a‑CALB prokaryotically expresses CALB inclusion bodies, and then undergoes renaturation to obtain high-purity (over 95%) and high-activity CALB protein; the enzyme activity of CALB protein obtained from 1L of culture medium is about 21252U, and the specific activity About 253U / mg; much higher than the specific activity of the CALB protein prepared in the existing literature. It can be seen that the present invention significantly improves the output of CALB, and the production process is simple, and the completion can be applied to actual production. The invention has important application value.

The invention discloses a method for using Candida antarctica lipase B to produce a ticagrelor chiral medicine intermediate, and belongs to the technical filed of enzyme engineering. The Candida antarctica lipase B (called CALB for short) is used for converting racemic ticagrelor with the structural formula represented by formula (a) to a single configuration intermediate of a formula (b). In the formula (a) and (b), R is CnH2n+1CO or CnH2n-1CO (n is 2-6), and R' is Cbz or Boc. The configuration of the product (b) obtained through splitting the racemic medicine intermediate by the CALB is (1S,4R). The method using the CALB to split the above substrate has a high catalysis efficiency, and the purity of the product of the formula (b) obtained in the invention is high.

This invention provides a nucleotide sequence of Candida Antarctica lipase B (CALB) gene. In this invention, CALB gene is artificially synthesized according to Pichia pastoris preferred codon. The obtained CALB nucleotide sequence can code the protein with the same function as wild-type CALB protein, and can hybridize with the 1st-957th nucleotide sequence of SEQ ID No.1 under moderate stringencyconditions. CALB gene can effectively express lipase in Pichia pastoris, while the lipase expression level can reach 21.6 mg / L. This invention can be used in lipase mass production.

The invention discloses a process method for immobilizing lipase B from candida antarctica fermented supernatant fluid. According to the method, the immobilized candida antarctica lipase B is prepared by using a cheaper vector, adopting a water-phase immobilization mode and cooperating with a post-treatment process, therefore, the enzyme activity recovery rate under water-phase adsorption is increased, the cost and the using effect are balanced, and the important significance on industrial application of the CALB is achieved.