12results about How to "Increased specific enzyme activity" patented technology

The invention discloses a T7 RNA polymerase mutant capable of being used for preparing mRNA vaccines and improving thermal stability and transcription performance and a preparation method of the T7 RNA polymerase mutant, relates to the technical field of biology, and particularly discloses a T7 RNA polymerase mutant containing mutation sites K8G, H27K, A70N, K180D and S192P / S192G relative to wild type T7 RNA polymerase. The amino acid sequence of the wild type T7 RNA polymerase is as shown in SEQ ID NO: 1. Compared with wild type T7 RNA polymerase, the T7 RNA polymerase has higher specific enzyme activity and heat stability, the heat resistance of one mutant is improved, a large number of transcription products can still be obtained at 45 DEG C, and transcription by-products of the other mutant are reduced. The mutant T7 RNA polymerase disclosed by the invention has a good application prospect in the fields of RNA vaccines and medicines, in-vitro transcription, gene editing and the like.

The invention discloses a mutant with improved enzyme activity of sucrose isomerase and application of the mutant, and belongs to the technical field of genetic engineering and enzyme engineering in molecular biology. A known sucrose isomerase parent is subjected to molecular modification by utilizing a site-directed mutagenesis technology to obtain mutant enzymes T522S / K340D, D222H / K340D, G467N / K340D, T522S, K340D, D222H and G467N, and the catalytic activity of the series of mutants provided by the invention is increased by 10%-25% compared with that of original bacteria; the isomaltulose conversion rates of the combined mutants T522S / K340D, D222H / K340D and G467N / K340D are respectively increased by 2.3%, 1.5% and 1.3% compared with those of original bacteria, and the strain has good stability, can be used for biological preparation of isomaltulose and has potential application prospects.

The application belongs to the technical field of bioengineering, and provides a mutant of pyrethroid hydrolase PytH, a coding gene thereof and purposes, and the amino acid sequence of the mutant is shown as SEQ ID NO. 4. The mutant PytH-M5 provided in the application has a significantly improved degradation efficiency of various pyrethroid pesticides compared with the wild type PytH, can be used for removing pyrethroid pesticide residues in soil and water, can also be used for constructing pyrethroid pesticide-resistant transgenic crops, and has very important theoretical and application values in the fields of pesticide residue bioremediation and environmental protection.

The invention provides a novel alpha-amylase for specifically hydrolyzing gelatinized starch. The amino acid sequence of the novel alpha-amylase is shown as SEQ ID NO.2 or SEQ ID NO.3. The invention also provides a preparation method of the novel alpha-amylase. The invention also provides a coding gene of the alpha-amylase as well as a recombinant expression vector and recombinant engineering bacteria containing the gene. Meanwhile, the invention also provides application of the novel alpha-amylase in preparation of dextrin with a low DE value. According to the invention, RoseTTAFold software is used for modeling wild type alpha-amylase, the structure of the wild type alpha-amylase is analyzed, and finally mutants S263A and I236R / S263A with better starch hydrolysis and gelatinization effects are screened by combining a mutation technology; the two optimized alpha-amylase mutants are used as novel alpha-amylase, not only can specifically hydrolyze gelatinized starch, but also have a better hydrolysis effect compared with a wild type.

The present invention relates to a starch branching enzyme mutant with improved extracellular secretion ability and enzyme activity. The present invention performs site-directed mutation on starch branching enzyme derived from Geobacillus thermoglucosidans, wherein the mutation sites are lysine at positions 159, 374, 393, 426 and 432, and after detecting the crude enzyme activities of the mutants, it is found that the extracellular expression of mutants K159A and K159H in Bacillus subtilis is significantly improved compared with the wild type. The crude enzyme solution is further purified using a hydrophobic column and an anion column, and the pure enzyme specific enzyme activities of mutants K159A and K159H reach 1745.18U / mg and 1873.78U / mg, respectively. The starch branching enzyme mutant provided by the present invention has improved extracellular expression ability and specific enzyme activity on the basis of the wild type, and can be applied to the modification of potato amylopectin, which is beneficial to the industrial application of starch branching enzyme.

The invention discloses an acyl coenzyme A synthetase mutant ACS01-Des4 as well as a coding gene, an expression strain and application thereof. According to the invention, acyl-coenzyme A synthetase from marine uncultured microorganisms is taken as a basis, mutation sites are designed through PROSS, and then whole-gene synthesis is carried out, so that mutant genes are obtained. After the engineering bacteria containing the mutant plasmids are subjected to induced expression, the acyl-coenzyme A synthetase with improved stability and specific enzyme activity is obtained. Under the condition of 37 DEG C, the stability of the mutant is improved by 3 times. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is improved by 1.14 times. The mutant has potential application value in an in-vitro diagnostic kit for detecting free fatty acid by an enzyme method.

This invention discloses a method with NMN + A preferred glucose-6-phosphate dehydrogenase mutant and its preparation method were investigated. Using the TmG6PDH-R7 mutant as the starting protein, random mutations were performed using error-prone PCR to construct a random mutant library. A high-throughput screening method for positive clones on plates was used to obtain the clones with the most obvious color development. Recombinant bacteria were cultured and purified to obtain the mutants. After three rounds of screening, NMN with amino acid sequences as shown in SEQ ID NO.6, SEQ ID NO.8, or SEQ ID NO.10 was obtained. + A glucose-6-phosphate dehydrogenase mutant with enhanced preference, achieving NMN + The specific enzyme activity of TmG6PDH coenzyme is increased. The mutant with the amino acid sequence shown in SEQ ID NO.10 has more than 5 times higher specific enzyme activity at 37 degrees compared with the R7 mutant with the highest specific enzyme activity, and has good application prospects in industrial production.

The application discloses a sulfonylurea herbicide hydrolytic enzyme TsmE mutant, a coding gene thereof and application. The sulfonylurea herbicide hydrolytic enzyme TsmE mutant has an amino acid sequence shown in SEQ ID NO. 4. The application also discloses a gene coding the mutant. The application further discloses application of the mutant in degrading sulfonylurea herbicides. The application takes wild-type TsmE as a starting template, and obtains a mutant M7 (S179A) with significantly improved enzyme activity through a directional evolution technology. Compared with the wild type, the specific enzyme activity of the mutant M7 to metsulfuron methyl, mesosulfuron methyl, tribenuron, bensulfuron methyl, sulfometuron methyl and chlorimuron ethyl is respectively increased by 1.7, 1.6, 2.5, 1.3, 1.4 and 1.2 times. The mutant and the coding gene thereof can be used for constructing a sulfonylurea herbicide-resistant transgenic crop, and can also be used for removing sulfonylurea herbicides in soil and water, and has very important theoretical and application values.

The invention relates to a serine hydroxymethyltransferase mutant and a method for synthesizing L-glycine by using the serine hydroxymethyltransferase mutant through a biological method. According to the method, serine hydroxymethyltransferase glyA is over-expressed in host bacteria, and L-glycine is produced through whole-cell catalysis; through site-directed mutagenesis, the catalysis direction of the enzyme is changed, so that catalysis is facilitated to be carried out towards the direction of producing L-glycine; the specific enzyme activity of the L-glycine generated through catalysis of the mutant enzyme glyAA292V-D392N is improved to 1.52 U / mg and is improved by 204% compared with that of an original enzyme; the conversion rate of L-glycine produced by whole-cell conversion of the mutant enzyme glyAA292V-D392N can reach 61.8%, which is 2.8 times higher than that of the original enzyme glyA; after serine hydroxymethyltransferase and tetrahydrofolic acid are circularly coupled in a whole-cell catalysis system, only 5-10mM of tetrahydrofolic acid is added, the yield of L-glycine is 14.03 g / L in 12 hours, the substrate conversion rate is increased to 93.2%, the addition amount of the substrate tetrahydrofolic acid is effectively reduced, the catalysis cost is reduced, and the production efficiency of L-glycine is greatly improved. The method disclosed by the invention is of great significance for synthesizing the L-glycine by a biological method.

The invention provides a 5 '-methylthio adenosine phosphorylase mutant, the amino acid sequence of the 5'-methylthio adenosine phosphorylase mutant is obtained by mutating a sequence as shown in SEQ ID No.3 in a sequence table, and the mutation is selected from at least one of the following components: H64A, P131D, W114N, L206S, E168Q, F217V, F217S, F217W, R219A, R219Q, R219F, V184A and V184T. The specific enzyme activity of the mutant H64A / W114N / L206S reaches 1.31 U / g, the specific enzyme activity of an ApMTAP non-mutated enzyme strain is 0.11 U / g, and the specific enzyme activity is improved by about 12 times, that is, the yield of 2 '-fluoro-2'-deoxyadenosine is greatly improved, and the mutant H64A / W114N / L206S is suitable for the field of enzyme catalysis.

The application discloses an acyl-CoA synthetase mutant, a coding gene thereof, an expression strain and application thereof. The application is based on an acyl-CoA synthetase from a marine uncultured microbial source, a mutation site is designed by using Funclib, and then a whole gene is synthesized to obtain a mutant gene. After expression of an engineering bacterium containing the mutant plasmid, an acyl-CoA synthetase with improved specific enzyme activity and stability is obtained. When palmitic acid is used as a substrate, the specific enzyme activity of the mutant is improved by 7.0 times, and the mutant has potential application value in an enzyme method for detecting free fatty acids in vitro.

The invention relates to a dicamba monooxygenase mutant DicX4M2 and application of the dicamba monooxygenase mutant DicX4M2. In particular to a dicamba monooxygenase mutant DicX4M2, a gene for coding the dicamba monooxygenase mutant DicX4M2, a recombinant vector inserted with the gene, a transformant transformed with the gene, a method for improving the activity of the dicamba monooxygenase, a method for preparing the dicamba monooxygenase mutant DicX4M2 and application of the dicamba monooxygenase mutant DicX4M2 and the dicamba monooxygenase mutant DicX4M2. Compared with the wild type dicamba monooxygenase, the dicamba monooxygenase mutant DicX4M2 obtained after the 82nd histidine of the wild type dicamba monooxygenase is replaced with isoleucine and the 190th phenylalanine of the wild type dicamba monooxygenase is replaced with leucine has the advantages that the enzyme activity is obviously improved, and the dicamba monooxygenase mutant DicX4M2 has a better dicamba degradation effect; the dicamba-resistant crops can be prepared, and the dicamba resistance of the crops is improved.