157 results about "Mutant enzyme" patented technology

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

The invention relates to the technical field of biological catalysis, and discloses an L379A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: leucine of the 379th amino acid sequence is mutated into alanine; the enzyme can be applied to RA in-vitro conversion preparation of RI with higher utilization value, the conversion rate is higher than 50%, and the enzyme activity is remarkably improved by 7 times compared with the original enzyme catalytic conversion enzyme activity. The UGT76G1 mutant disclosed by the invention has the advantages that (1) the blank is filled, and a special enzyme catalyst capable of realizing efficient in-vitro synthesis of rebaudioside I (RI) is provided for the first time; 2) efficiency jump: the catalytic conversion rate is greatly increased from original about 7% to more than 50% (increase gt; and 3) stability and reliability: in the cross-scale reaction of 10mL to 5L, the catalyst has stable catalytic performance, shows excellent industrial application potential, and is suitable for popularization and application.

The invention relates to the technical field of biological catalysis, and discloses an M88V mutant enzyme for preparing rebaudioside I. The M88V mutant enzyme is obtained by the following mutations generated by UGT76G1: M88V: methionine of the 88th amino acid sequence of UGT76G1 is mutated into valine. Aiming at the technical bottlenecks of low RI yield and poor selectivity in the existing RA biotransformation process, an efficient solution is provided for the UGT76G1 mutant developed by the invention. The mutant can specifically catalyze conversion from RA to RI, and the conversion rate of a target product is remarkably increased to 65% or above from general less than 7%. The excellent catalytic performance of the catalyst is verified in milliliter-level to upgraded reaction systems, the key problems of conversion efficiency and process amplification are solved, and a foundation is laid for large-scale green manufacturing of a high-added-value product RI.

The invention relates to the technical field of biological catalysis, and discloses an M88A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: M88A: methionine of the 88th amino acid sequence of UGT76G1 is mutated into alanine; the key conversion rate of RI is greatly increased from 7% to 85% (increased by more than 12 times), and the utilization rate and economical efficiency of raw materials are remarkably improved; the method can be perfectly reproduced under any test scale (10mL, 100mL and 5L), the conversion rate is always anchored to be more than 85%, and incomparable stability and amplification potential are shown.

The invention relates to the technical field of biological catalysis, and discloses an L204A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: L204A: leucine of the 204 amino acid sequence of UGT76G1 mutates into alanine; the high-efficiency in-vitro enzymatic preparation of the rebaudioside I (RI) is realized for the first time, the catalytic efficiency of the rebaudioside I (RI) is improved by more than 7 times (from about 7% to more than or equal to 50%) compared with that of an original enzyme, the high-efficiency performance can be stably maintained under different scales of 10mL to 5L, a solid foundation is laid for industrial application of the RI, and the preparation method is simple, controllable in process condition and suitable for popularization and application.

The invention provides application of acidophilous glycosyltransferase in salidroside production, and belongs to the technical field of biological engineering. The problem of producing salidroside under the acidic condition is solved. Comprising an application of acidophilous glycosyl transferase with an amino acid sequence as shown in SEQ ID NO.1 in salidroside production under an acidic condition and an acidophilous escherichia coli engineering strain for producing salidroside. The escherichia coli engineering strain overexpresses a mutant 3-deoxy-D-arabinoheptulose-7-phosphate (DAHP) synthetase gene aroGfbr, a cyclohexadiene dehydrogenase gene tyrC, a glucose phosphate mutant enzyme gene pgm and a UDP-glucose pyrophosphorylase galU, overexpresses a phenylpyruvate decarboxylase gene ARO10 derived from saccharomyces cerevisiae, and can be used for producing a mutant 3-deoxy-D-arabinoheptulose-7-phosphate mutant enzyme. The kit comprises an ethanol dehydrogenase gene ADH6 and a glycosyl transferase gene LrUGT85AF8. The method is mainly used for producing salidroside under an acidic condition.

The application discloses a kind of fungal laccase mutant Lcc5-G and its expression strain and application.The application is based on the fungal laccase from Coprinopsis cinerea heterologous expression since, by site-directed mutagenesis, obtain mutant gene.After fungal induction expression containing mutant plasmid, obtain laccase mutant enzyme Lcc5-G with improved specific activity, stability and AFB1 removal efficiency.When ABTS is used as substrate, the specific activity of the mutant is improved by 3 times, and the stability of the mutant enzyme is improved to 5 times of the original enzyme at 45 DEG C and pH 7.The mutant has potential application value in oxidizing aflatoxin.

The invention belongs to the field of biotechnology and environmental governance, and particularly relates to a manganese peroxidase mutant based on high-throughput screening modification and application of the manganese peroxidase mutant to straw degradation. Aiming at the problems of low natural secretion amount, insufficient specific activity, complex preparation process and the like of the existing manganese peroxidase, the method comprises the following steps: firstly, constructing engineering bacteria based on an escherichia coli exocrine system, constructing a mutant library through error-prone PCR in combination with a directed evolution technology, and performing high-throughput screening by utilizing an ABTS colorimetric method; the recombinant manganese peroxidase mutant with high catalytic activity is obtained, and the enzyme activity is improved by 1.29 times compared with that of a wild type. Furthermore, recombinant engineering bacteria are constructed on the basis of a bacillus subtilis exocrine system, and efficient secretion of the mutant enzyme is achieved. The mutant enzyme preparation is used for wheat straw degradation, and the lignin degradation rate reaches 41.37% (improved by 30.09% compared with that of a wild type). The method is simple and convenient in process, low in cost and remarkable in effect, and has a wide industrial application prospect.

The invention relates to the technical field of biological catalysis, and discloses an M88F mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: M88F: methionine of the 88th amino acid sequence of UGT76G1 is mutated into phenylalanine; and the substrate rebaudioside A (RA) can be efficiently and directionally converted into rebaudioside I (RI) with higher value. In an optimized reaction system, the conversion rate stably reaches 40%, and the catalytic efficiency is improved by more than 5-8 times compared with the common reference enzyme. The enzyme preparation has the characteristics of high catalytic activity, mild reaction conditions, simplicity and convenience in operation and the like, shows good stability and reproducibility in laboratory research and large-scale production, and has a wide industrial application prospect.

The application discloses efficient expression of Aspergillus oryzae beta-galactoside enzyme mutant and application thereof in dairy products, and belongs to the technical fields of enzyme engineering and dairy product processing, and particularly relates to efficient expression of Aspergillus oryzae beta-galactoside enzyme mutant and application thereof in dairy products.The mutant protein can be as follows: A1) a protein with an amino acid sequence of SEQ ID No.2; A2) a fusion protein with the same function obtained by connecting a label to the N terminal and / or C terminal of A1).The mutant protein is subjected to high-density fermentation in a 5L fermentation tank, and the enzyme activity of the fermentation liquor can reach 4628U / mL, and the recovery rate of the mutant enzyme is increased by 1.7 times.Compared with the wild type, the optimal pH of the mutant is increased from 4.5 to 5.5, the optimal temperature is decreased from 60 DEG C to 50 DEG C, the lactose hydrolysis efficiency is increased by 11%, and the mutant is more suitable for the production process of low / zero lactose dairy products.

The invention relates to a carbonyl reductase mutant based on cosubstrate catalytic ability optimization and application thereof, the mutant is constructed on the basis of a carbonyl reductase BaSDRX amino acid sequence as shown in SEQ ID No.2, the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.1 in a sequence table, and the nucleotide sequence of the carbonyl reductase is as shown in SEQ ID No.2 in the sequence table. The mutant is single-point mutation or multi-point combined mutation on the basis of an amino acid sequence of carbonyl reductase BaSDRX as shown in SEQ ID No.2: threonine Thr133 at the 133 site, arginine Arg137 at the 137 site and glutamic acid Glu142 at the 142 site; when the mutant enzyme or a recombinant cell containing the mutant enzyme is applied to synthesis of (S)-1-(2, 6-difluorophenyl) ethanol through asymmetric reduction of 2, 6-difluoroacetophenone, high catalytic activity and high stereoselectivity are achieved, and high-optical-purity (S)-1-(2, 6-difluorophenyl) ethanol (eet; therefore, the method has a good industrial application and development prospect.

The invention discloses a phospholipase D mutant with improved thermal stability and a preparation method thereof. The amino acid sequence of the phospholipase D mutant is as shown in SEQ ID NO.4; according to the phospholipase D mutant A426P with improved thermal stability, the mutant is incubated for 30 min at 55 DEG C and 60 DEG C respectively, the residual enzyme activity is 85% and 58% respectively, the activity of wild-type enzyme is only 56% and 7% under the same treatment condition, the thermal stability of the mutant enzyme is remarkably improved, and the phospholipase D mutant A426P can be used as a phospholipase D mutant. The problem that phospholipase D is not ideal in thermal stability in biological catalytic reaction is solved.

The invention belongs to the technical field of enzyme mutants, and discloses a mutant of lycopene cyclase and application of the mutant, glutamic acid at the 321 site of wild type lycopene cyclase which is derived from arabidopsis and has an amino acid sequence as shown in SEQ ID NO.1 is mutated into lysine, phenylalanine at the 319 site of the wild type lycopene cyclase is mutated into leucine, cysteine at the 323 site of the wild type lycopene cyclase is mutated into alanine, and the mutant of the lycopene cyclase is obtained. 1, and the lycopene cyclase mutant with the amino acid sequence as shown in SEQ ID NO. 2 is obtained. BTS1, CrtB and CrtI are integrated into a saccharomyces cerevisiae BY4741 strain, and a chassis strain ZA1 for stably producing alpha-carotene precursor lycopene is constructed; mutant expression plasmids are constructed, positive clone strains are screened out, and the epsilon-cyclization ability of mutant enzymes is remarkably higher than the beta-cyclization ability; the method comprises the following steps: by taking lycopene as a substrate, constructing an expression vector lipid droplet surrounding protein PET10; gene PAH1, DGA1 and Cat2 related to TAG synthesis and perilipid droplet protein PET10 are constructed on an expression vector and converted into ZA1, and when lycopene is used as a substrate, the efficiency of catalytic production of alpha-carotene is improved.

This invention discloses a laccase mutant, Lcc5-I479T, its expression strain, and its applications. Using laccase from *Coprinopsis cinerea* as the starting enzyme, this invention obtained the mutant gene through molecular docking and positional analysis. After induction expression in engineered bacteria containing the mutant gene, the stable laccase mutant enzyme Lcc5-I479T was obtained. Using ABTS as a substrate, the mutant enzyme Lcc5-I479T showed a stable increase in activity to 8-20 times that of the starting enzyme at 35-50℃. This mutant enzyme has potential application value in the oxidative transformation of aflatoxin.

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to proline hydroxylase mutants and their application and methods in hydroxyproline production. This invention provides p4h... G141A p4h S85A p4h V126A p4h H125A p4h S205A p4h D122A p4h S211A Mutations at specific sites significantly increased the production of hydroxyproline and decreased the proportion of proline in the mutant enzyme. Further saturation mutations at these sites, combined with the dominant mutations, yielded strains with even better performance, such as p4h. S85Q‑V126A p4h S85A‑V126T p4h G141A‑H125S p4h G141A‑H125T Compared with traditional chemical methods, this process has lower production costs, is more environmentally friendly, and allows for large-scale continuous production.

The present application relates to a deoxyribonuclease mutant and its application in preparing PDRN. The amino acid sequence of the deoxyribonuclease mutant is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. The present application also provides the application of the deoxyribonuclease mutant and / or the encoding gene of the deoxyribonuclease mutant in preparing PDRN, and the specific application method. The present application is based on the deoxyribonuclease expressed by the DNASE1 gene of bovine pancreas tissue, and the amino acids at positions 69, 132 and 215 are selected for iterative saturation mutation. Compared with the wild-type enzyme, the enzyme activity of the deoxyribonuclease mutant A69H-S132R-Q215C is increased by 2.15 times, reaching 4.1x10 6 U / mL, and the DNA extraction rate of salmon PDRN prepared by using the mutant enzyme prepared by the present application is 15.2%, the DNA content is 99.1%, and the molecular weight is 1.05 million Daltons, which is significantly improved compared with the wild-type enzyme, and the PDRN in this molecular weight range has more significant biological activity.

This invention discloses a mutant of raw starch hydrolase with improved stability and its applications. Using α-amylase AmyZ2 as the starting enzyme, and through computer-aided design to clarify the mutation site, a mutant was heterologously expressed in Bacillus subtilis, resulting in an α-amylase with improved stability. When using raw corn starch as a substrate, the mutant exhibits 1.57 times the specific enzyme activity and 2 times the stability of the starting enzyme. While maintaining the specific enzyme activity, the mutant enzyme shows a significant improvement in thermostability. This mutant has potential application value in the production of starch sugars using raw corn starch as a substrate.

Disclosed herein, inter alia, are mutant enzymes, kits, and methods of use thereof.

The invention discloses a phytase mutant AppA as well as a coding gene, a preparation method and application thereof. The phytase mutant AppA is obtained by mutating amino acids at the 137th site, the 185th site and the 255th site of wild type phytase AppA from asparagine, aspartic acid and tyrosine into valine, asparagine and aspartic acid respectively; the amino acid sequence and the nucleotide sequence of the gene are SEQ ID NO.1 and SEQ ID NO.2 respectively. The catalytic efficiency of the mutant enzyme AppA provided by the invention on sodium phytate is improved by 2.5 times compared with that of a wild enzyme AppA; under the condition of 70 DEG C, the half-life period of the mutant enzyme AppA is 3.4 times that of the wild enzyme AppA. The mutant enzyme AppA has the characteristic of high enzyme activity under a high-temperature condition, which indicates that the mutant enzyme AppA has an important application prospect in industrial production of a feed in which phytic acid needs to be degraded under the high-temperature condition.

Disclosed herein, inter alia, are mutant enzymes, kits, and methods of use thereof.

The application discloses a glycerol dehydrogenase mutant and application thereof, and the glycerol dehydrogenase is derived from Klebsiella pneumoniae and is encoded by gene dhaD. The application improves the molecular structure of the glycerol dehydrogenase by rational design and a site-directed mutagenesis technology to obtain a mutant enzyme F245Q / GDH, and the mutant enzyme is back-supplemented into Klebsiella pneumoniae which is deficient in glycerol dehydrogenase. Compared with a wild-type strain, the yield of a main product 1,3-propanediol of the back-supplemented mutant enzyme F245Q / GDH strain is improved, and the yield of a by-product 2,3-butanediol is significantly reduced.

The invention discloses a beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H and an application thereof, and belongs to the technical field of functional enzymes. And the amino acid sequence of the beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H is as shown in SEQ ID NO. 3. The invention further discloses the application of the beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H in the preparation of lactose-N-tetrasaccharide. According to the invention, 1, 3-galactosyl transferase Pm beta 3GalT is subjected to mutation modification to obtain a mutant enzyme, and compared with a wild enzyme, the enzyme activity of the mutant enzyme is improved by 157.21% and is obviously improved, and the thermal stability and alkali resistance are also obviously improved. The beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H disclosed by the invention has important theoretical value and application value on the industrial production of LNT (Lipoxystrobin).

The present invention relates to mutants of the bumblebee Yarrowia SBLE enzyme having modulated transesterification and / or esterification activity compared to the bumblebee Yarrowia SBLE enzyme, and to homologues of the bumblebee Yarrowia SBLE enzyme, such as the C. bogoriensis LE enzyme, the B. batistae LE enzyme and the R. riograndensis LE enzyme, having transesterification and esterification activity on bolaform glycolipids. The present invention also provides genetically modified yeast strains comprising a mutant LE enzyme or a bumblebee Yarrowia SBLE enzyme homologue of the present invention. The present invention further provides the use and methods of the mutant LE enzymes, the bumblebee Yarrowia SBLE enzyme homologues and the modified yeast strains of the present invention for the production of acidic glycolipids, lactone-form glycolipids and / or glycolipid oligomers / polymers / esters. The present invention also provides the three-dimensional structure of the bumblebee Yarrowia SBLE enzyme and its use for the design of mutants having modulated hydrolysis and / or transesterification activity.

The present invention relates to mutant enzymes with enhanced properties and methods of using such enzymes to oxidize organic compound substrates. More specifically, provided herein are mutations in cytochrome P450 enzymes that enhance their peroxygenase activity and / or alter product selectivity as compared to wild-type cytochrome P450 enzymes. Also provided are uses of such mutant cytochrome P450 enzymes, e.g., for biocatalytic oxidation of carbon-hydrogen bonds by a peroxygenase pathway, or for oxidation of organic compound substrates.

The invention provides a glycosidase mutant and application thereof, the amino acid sequence of the mutant is compared with the amino acid sequence SEQ ID NO.1 of a wild enzyme, in the amino acid sequence SEQ ID NO.1, five sites of M102K, P171L, V203G, T269Y and K299D are respectively subjected to single mutation, pairwise combined mutation, three combined mutation, four combined mutation or one of five combined mutation; the novel glycosidase mutant industrial enzyme is used for synthesis and preparation of hesperetin. The glycosidase mutant enzyme constructed by the invention has the characteristics of low enzyme cost, short conversion time, simple process operation and the like, and has a wide prospect of large-scale industrial application.

The invention discloses a beta-glucosidase mutant, an immobilized enzyme of the beta-glucosidase mutant and application of the beta-glucosidase mutant in efficient synthesis of kinsenoside, and belongs to the field of enzyme engineering and natural product biosynthesis. According to the mutant, one or more mutations of W177Y, W179F, Q242A, F245G, R298S and R313T are introduced at key sites of an amino acid sequence of wild type beta-glucosidase, so that the yield of catalytic synthesis of kinsenoside is remarkably improved. Furthermore, a metal organic framework material UiO-66-NH2 is adopted as an immobilization carrier, enzyme immobilization is achieved in a physical adsorption mode, and the obtained immobilized enzyme is excellent in reusability. Under the same reaction conditions, the immobilized mutant enzyme is used for catalyzing synthesis of kinsenoside, and the yield of the immobilized mutant enzyme is 3 times that of a wild enzyme. The invention provides an efficient, green and sustainable kinsenoside biocatalytic synthesis scheme, and the kinsenoside biocatalytic synthesis method has a good industrial application prospect.

According to the invention, beta-glycosidase which has a catalytic effect on vincoside, loganin and strictosamide is selected, and a foundation is laid for diversity and possibility of an aglycone production process. By researching the three-dimensional structure information of the Gelsemium elegans Beta-glycosidase, a zymoprotein structure information basis is provided for molecular modification of the Gelsemium elegans Beta-glycosidase, enzyme variants capable of improving substrate selectivity, thermal stability and catalytic efficiency are obtained more efficiently, and the requirements of industrial application are met; the invention provides a truncated mutant enzyme with improved enzymatic activity, and through induced expression and activity screening, it is found that the enzyme activity of the obtained mutant enzyme to loganin and strictosamide is about two times higher than that of a wild enzyme.

This invention discloses a creatine amidoside hydrolase mutant for creatinine detection, along with methods for selecting, purifying, and detecting the mutant site. The mutant is a single-point mutation of the wild-type full-length amino acid sequence of the creatine amidoside hydrolase protein (Af-CRE) from Alcaligenes faecalis. This invention utilizes machine learning-assisted site-directed mutagenesis to create a creatine amidoside hydrolase mutant with enhanced activity, obtaining a mutant enzyme with improved activity and certain thermostability. Furthermore, it provides methods for purifying and detecting the mutant, overcoming the shortcomings of existing creatine amidoside hydrolases with poor activity that cannot meet the requirements for reagent applications. This lays the foundation for further expanding the industrial applications of creatine amidoside hydrolases.

The application belongs to the technical field of bioengineering, and particularly relates to a high-temperature-resistant laccase mutant obtained by site-directed mutation through an overlapping PCR technique, and preparation and application thereof. The laccase mutant is obtained on the basis of a wild-type laccase shown in SEQ ID NO. 1, and at least one of D221Y, E231D, Y441H or Y441F mutations occurs, and the mutant enzyme activity and the thermal stability at 80 DEG C are both improved to different degrees relative to the wild type, which is beneficial to expand the application in the industrial fields of degrading lignocellulose, paper pulp bleaching and the like.

The invention relates to the technical field of biological catalysis, and discloses a mutant enzyme for preparing rebaudioside I. The enzyme is a UGT76G1 mutant, and the mutation points of the mutant are L379V and L126A. The mutated enzyme realizes efficient in-vitro enzymatic synthesis of RI (the conversion rate is 45-60%), the problem of low efficiency (-7%) of an existing path is solved, and the production process has economic feasibility; secondly, the catalytic efficiency is improved by 6-9 times compared with the original level, and the fundamental improvement means that the raw material utilization rate and the production flux are greatly improved; and meanwhile, the seamless connection capability from a laboratory to large-scale production is realized, and a key technical foundation is laid for constructing a stable and controllable industrial production line.