17 results about "Lipase b" patented technology

The application discloses a mutant of PET hydrolytic enzyme DE7 and a preparation method and application thereof. A classification model of PET hydrolytic enzyme and a regression model of lipase B are constructed respectively, and a PET hydrolytic enzyme DE7 with high activity to BHET or PET is obtained according to the classification model and the regression model. An omnidirectional multi-point mutagenesis strategy is used to transform the PET hydrolytic enzyme DE7, and eight mutants with high catalytic activity to PET are selected, and the amino acid sequences of the eight mutants are shown in SEQ ID No. 2-9. According to the determination of hydrolytic activity, the eight mutants are all improved in the PET hydrolysis efficiency compared with DE7. According to the determination results of thermal stability, the values of four mutants in the mutants with improved catalytic activity are improved compared with the wild type DE7, and the thermal stability of the four mutants is effectively improved. The application has important application prospects in the efficient degradation and recycling of PET or BHET. T m ​

The invention belongs to the technical field of directed evolution modification and biological catalysis application of enzymes, and relates to a Candida antarctica lipase B mutant protein and application thereof, the sequence of the mutant protein comprises one or more of the mutation of the 195 S and F, the mutation of the 195 S and Y and the mutation of the 283 A and Y from the N terminal to the C terminal of the amino acid sequence shown in SEQ ID NO.1 of the original CALB; on the basis of a wild type lipase gene sequence, through rational design and site-specific mutagenesis, mutants S195F, S195Y, A283Y, S195F-A283Y and S195Y-A283Y, of which the Tm value and the optimal reaction temperature are obviously improved and the catalytic activity is improved by 1-2 times, are finally obtained; when the mutant is used for catalyzing synthesis of docosahexaenoic acid (DHA) glyceride, higher catalytic efficiency is shown, and the yield of the DHA glyceride is effectively improved.

The application discloses an enzymatic production process of diglyceride, and comprises the following steps: using low-cost lipase as a catalyst, preparing lipase A through immobilization of the enzyme, preparing lipase B through an ester exchange reaction, and then using the lipase B as a catalyst to catalyze esterification of free fatty acid and glycerol to produce diglyceride; the method can realize efficient enzymatic production of diglyceride through a series of methods by using low-cost lipase, and the production cost is reduced. The application provides an economical and efficient method for producing diglyceride by using the enzyme method, greatly reduces the use cost of lipase in the enzymatic esterification reaction, and has certain industrialization application prospect.

The present application relates to the field of genetic engineering and bioengineering technology, in particular to a Candida antarctica lipase B mutant and application thereof. The Candida antarctica lipase B mutant provided by the present application is obtained by joint mutation of the 143th and 284th positions in the amino acid sequence shown in the wild type lipase B. The Candida antarctica lipase B mutant constructed by the present application has 12.5 times higher enzyme activity than the wild type enzyme, and the protein expression amount is as high as 1.8-1.9 g / L under the scale of 5 L fermenter, which can significantly improve the yield and reduce the production cost. In addition, the Candida antarctica lipase B mutant can be used for synthesis of high value-added, non-natural substrates such as vitamin C fatty acid ester and vitamin A oleate, which lays a foundation for the application of lipase in the field of fine chemical industry.

The application discloses a Candida antarctica lipase B mutant and a preparation method thereof, and belongs to the technical field of biology. Eleven mutants with improved enzyme activity are obtained, including six unit point mutants and five combined mutants; compared with a wild type, the enzyme activity of the mutants Q11L, F71N, F118Y, V149T, L219N, T244D, S31T / F71N, F71N / F118Y, F71N / L219N, S31T / F71N / F118Y and F71N / F118Y / L219N is improved, and is respectively increased by 18.45% to 240.88%; the CALB mutant with high enzyme activity is obtained, and has great industrial application potential and economic value.

The invention relates to a mutated Candida antarctica lipase B. The invention also relates to a gene encoding the enzyme, and a vector and a host cell comprising the gene. In addition, the invention also relates to a preparation method and application of the enzyme.

The application provides a rational design method for improving thermal stability of lipase, and belongs to the field of enzyme engineering. A flexible region of the lipase to be reformed is determined through molecular dynamics simulation, a reforming region is determined through structure analysis, a reforming site is determined through comprehensive measurement of multiple calculation tools, a mutant library is established through site-directed saturation mutation using NNK degenerate codon, and an excellent mutant with improved thermal stability is screened through a microplate screening method based on spectrophotometry. The half-life of the Candida antarctica lipase B mutant of the application at 50 DEG C is 9 times that of the wild type, and the specific activity is also 6.32% higher than that of the wild type. The application breaks the trade-off between stability and activity, and realizes the improvement of the thermal stability of the lipase, which is conducive to the important role of the lipase mutant in the industrial application in the fields of food, medicine or biodiesel.

The present application relates to the technical field of oil and fat chemistry, and particularly relates to a preparation method and application of a diglyceride oil with the function of improving cardiovascular and cerebrovascular health. The preparation method of the diglyceride oil comprises the following steps: (1) mixing oil and water, adding a first hydrolytic enzyme to perform first hydrolysis, and then adding a second hydrolytic enzyme to perform second hydrolysis, to obtain a hydrolysis product; (2) adding the hydrolysis product to glycerol and a lipase to perform reaction, and then distilling, to obtain the diglyceride oil; in step (1), the first hydrolytic enzyme is Lipozyme RM IM, and the second hydrolytic enzyme is Candida antarctica lipase B; in step (2), the lipase is a mixture of lipase Lipase G50 and Rhizomucor niveus lipase, and the mass ratio of the two is 1:1-3. The diglyceride oil prepared by the method has the function of improving cardiovascular and cerebrovascular health.

The invention discloses a lipase hydrolysis performance modification method based on stability constraint and channel dynamic configuration analysis and a mutant. Aiming at the limitation that only a static structure or a rigid framework of a channel is concerned in the prior art, through combination of molecular dynamics simulation and dynamic analysis of the channel, a flexible key site of the channel with the occurrence probability larger than 60% is captured, multiple thermodynamic stability prediction (PoPMuSiC / HoTMuSiC and the like) is innovatively introduced to serve as a rigid filtering index, and a modification site is accurately locked. The hydrolysis activity of the Candida antarctica lipase B mutant (such as D145Q-A281W-A282P) obtained by the method on a long-chain substrate (pNPP) at 40 DEG C is improved by 30-40 times compared with that of a wild type, and the hydrolysis activity of the Candida antarctica lipase B mutant is obviously superior to that of the existing Candida antarctica lipase B mutant on esterification activity or local area modification. The invention effectively solves the problem of low hydrolysis efficiency of lipase on large steric hindrance long-chain substrate, and has wide application prospects in the fields of food, medicine and biochemical engineering.

The invention relates to the technical field of genetic engineering and biological catalysis application, in particular to a Candida antarctica lipase B mutant as well as a coding gene and application thereof. On the basis of a wild type Candida antarctica lipase B gene sequence, through rational design and site-specific mutagenesis, Candida antarctica lipase B mutants with higher thermal stability and higher enzyme activity, including lipase B mutants such as A282Y, A282M, Q231F, A89K, A275M, A275L, I255P, V236I and the like, are obtained, the Tm values of the mutants are respectively increased by 1.9 DEG C, 2.8 DEG C, 9.8 DEG C, 1.3 DEG C, 0.7 DEG C, 0.7 DEG C, 0.5 DEG C and 1.8 DEG C compared with those of the wild type, and the Tm values of the mutants are respectively increased by 1.9 DEG C, 2.8 DEG C, 9.8 DEG C, 1.3 DEG C, 0.7 DEG C, 0.7 DEG The highest catalytic reaction efficiency of the mutant is 4.5 times that of a wild type, when the mutant is used for synthesis of DHA glyceride, higher catalytic efficiency and thermal stability are shown, and the yield of DHA glyceride can be effectively increased.

The invention discloses a Candida antarctica lipase B mutant and application thereof, and belongs to the technical field of biology. According to the invention, 11 mutants with improved enzyme activity are obtained, including 6 single site mutants and 5 combined mutants; wherein compared with a wild type, the enzyme activities of the mutants Q11L, F118Y, V149T, L219N and T244D are all improved by 76.39%, 20.97%, 20.29%, 18.45% and 34.06% respectively, and the enzyme activities of the mutants Q11L, F118Y, V149T, L219N and T244D are respectively improved by 76.39%, 20.97%, 20.29%, 18.45% and 34.06%; according to the invention, the CALB mutant with high enzymatic activity is obtained, and the CALB mutant has great industrial application potential and economic value.

The invention discloses a PET hydrolase DE7 mutant as well as a preparation method and application thereof. According to the invention, a classification model of the PET hydrolase and a regression model of lipase B are respectively constructed, so that the PET hydrolase DE7 with relatively high activity to BHET or PET is obtained through mining and identification according to the classification model and the regression model; an omni-directional multipoint mutagenesis strategy is adopted to modify and optimize eight mutants with relatively high catalytic activity on PET, and the amino acid sequences of the eight mutants are respectively shown as SEQ ID No.2-9; according to hydrolytic activity determination, compared with DE7, the eight mutants are improved in the aspect of PET (Polyethylene Terephthalate) hydrolysis efficiency. A thermal stability determination result shows that in the mutants with improved catalytic activity, Tm values of four mutants are improved compared with that of wild type DE7, so that the thermal stability of the mutants is effectively improved. The method disclosed by the invention has an important application prospect in the aspects of efficient degradation and cyclic utilization of PET or BHET and the like.

The invention relates to the technical field of genetic engineering and bioengineering, in particular to a Candida antarctica lipase B mutant and application thereof. The Candida antarctica lipase B mutant provided by the invention is obtained by combined mutation of the 143rd site and the 284th site on an amino acid sequence shown by wild type lipase B. The Candida antarctica lipase B mutant provided by the invention is a Candida antarctica lipase B mutant. Compared with a wild type enzyme, the enzyme activity of the constructed Candida antarctica lipase B mutant is improved by 12.5 times, the protein expression quantity is as high as 1.8-1.9 g / L under the condition of a 5L fermentation tank scale, the yield can be remarkably improved, and the production cost can be reduced. In addition, the Candida antarctica lipase B mutant can be used for synthesis of non-natural substrates with high added values, such as vitamin C fatty acid ester and vitamin A oleate, and lays a foundation for application of lipase in the field of fine chemical engineering.

The application discloses a method for preparing chiral isoquinoline carboxylic acid (I), which comprises the following steps: reacting a racemate of compound I in an aqueous buffer solution under catalysis of Candida antarctica lipase B to generate a compound shown in formula (I), and performing the reaction at a preset pH value of 7.8-8.2. The method has the characteristics of mild reaction condition, high stereoselectivity, high reaction efficiency, relatively simple process and the like, and has an industrial application prospect.

The invention relates to the technical field of grease chemistry, in particular to a preparation method and application of diglyceride oil with the effect of improving cardiovascular and cerebrovascular health. The preparation method of the diglyceride oil comprises the following steps: (1) mixing grease and water, adding a first hydrolase for primary hydrolysis, and then adding a second hydrolase for secondary hydrolysis to obtain a hydrolysate; and (2) adding glycerol and lipase into the hydrolysate to react, and distilling to obtain the product, the first hydrolase in the step (1) is Lipozyme RM IM, and the second hydrolase in the step (1) is candida antarctica lipase B; the lipase in the step (2) is a mixture of lipase Lipase G50 and rhizopus nidus lipase, and the mass ratio of the lipase Lipase G50 to the rhizopus nidus lipase is 1: (1-3). The diglyceride oil prepared by the invention has a better effect of improving the health of heart and cerebral vessels.

The invention provides a rational design method for improving the hydrolytic activity of lipase, and belongs to the field of enzyme engineering. It is found through molecular simulation that whether the initial conformation of a substrate entering Tunnel is reasonable or not is likely to be an important factor affecting the activity of the substrate. On the basis, key residues at a Tunnel inlet of the lipase are recognized through CaverWeb, NNK degenerate codons are adopted for site-directed saturation mutation to establish a mutant library, and excellent mutants with improved hydrolytic activity are screened by adopting a microwell plate screening method based on a spectrophotometric method. The hydrolytic activity of a plurality of single-region mutants of the candida antarctica lipase B obtained by the invention on 4-p-nitrophenol palmitate at 40 DEG C is improved by more than 10 times compared with that of a wild type; the hydrolysis activity of a plurality of multi-region mutants to pNPP at 40 DEG C is improved by nearly 20 times compared with that of a wild type. A lipase Tunnel inlet is modified, so that the hydrolytic activity of the lipase is greatly improved.

The invention provides a Candida antarctica lipase B mutant and application thereof, and compared with an amino acid sequence of a wild type lipase B as shown in SEQ ID NO.1, the mutant has mutation of a 143rd site, a 278th site, a 285th site or a 286th site. According to the mutant, the DHA doping amount of phospholipid type DHA produced through enzyme catalysis is increased, meanwhile, reduction of hydrolysis side reactions during synthesis reaction is facilitated, the yield is increased, and the mutant has higher industrial application value.