285 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 discloses a P450 mutant and application thereof in synthesis of calcifediol, and belongs to the technical field of bioengineering. According to the present invention, the P450 enzyme mutant enzyme is constructed, and the single plasmid three-enzyme co-expression system of the redox partner and the cofactor cycle enzyme is constructed, such that the sufficient electron transport chain is provided for the P450 enzyme, and the calcifediol can be efficiently synthesized under the mild condition by using the vitamin D3 as the substrate; the conversion rate of calcifediol can reach 63.1%, and the conversion rate of the reaction is greatly improved. By adopting the method disclosed by the invention, the catalytic efficiency of the catalyst is improved, and the reaction cost is reduced.

The present invention relates to mutant enzymes with enhanced properties and processes for oxidation of organic compound substrates using such enzymes. More specifically, provided herein are mutations in cytochrome P450 enzymes that enhance their peroxygenase activity and / or alter product selectivity compared to wild-type cytochrome P450 enzymes. Uses for such mutant cytochrome P450 enzymes are also provided, such as for biocatalytic oxidation of carbon-hydrogen bonds via a peroxygenase pathway, or for oxidizing a substrate which is an organic compound.

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 screening and application of a Candida antarctica lipase B mutant. Specifically, the Candida antarctica lipase B mutant provided by the invention is obtained by carrying out rational design and directed evolution combined strategy mutation on the basis of a wild Candida antarctica lipase B gene sequence, and the amino acid sequence of the mutant is shown as SEQ ID NO.3. The invention also relates to a preparation method of the Candida antarctica lipase B mutant. The mutant has higher enzyme activity and thermal stability, the hydrolytic catalytic activity of the mutant is improved to 2.1 times of the original hydrolytic catalytic activity, and the half-life period of the mutant enzyme at 50 DEG C is prolonged by 4 times. On the basis, pichia pastoris is used as a host to express the mutant, and a strain with hydrolytic activity improved by 52% is obtained through high resistance screening. The lipase is prepared into an immobilized enzyme to be used in a synthetic reaction of chlorogenic acid derivatives, the highest conversion rate of chlorogenic acid reaches 74% in 24 hours, and the immobilized enzyme has a good industrial application prospect.

The invention discloses a 3 '-phosphoadenosine-5'-phosphoryl sulfate synthetase mutant and an application of the 3 '-phosphoadenosine-5'-phosphoryl sulfate synthetase mutant. Compared with a wild enzyme amino acid sequence as shown in SEQ ID NO.1, the amino acid sequence of the 3 '-adenosine-5'-phosphoryl sulfate synthetase mutant has one or two mutations in the 101 site, the 207 site and the 560 site; preferably, phenylalanine at the 101 site is mutated into tryptophan; cysteine at the 207th site is mutated into glycine; phenylalanine at the 560th site is mutated into tryptophan. The invention also provides a synthesis method and application of the 3 '-adenosine-5'-phosphoryl sulfuric acid. The APS intermediate product inhibition effect of hPAPSS1 is relieved through a point mutation technology, the substrate affinity of hPAPSS1 to ATP is improved, and a mutant enzyme with a high PAPS conversion rate is obtained; and the bifunctional enzyme hPAPSS1 is synthesized by heterologous expression of PAPS in escherichia coli, so that the acquisition of the enzyme is simplified, and the synthesis efficiency of PAPS is improved.

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 belongs to the field of enzyme engineering, and relates to a glycosyl transferase mutant and application thereof in synthesis of salidroside. On the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, iterative saturated mutation is carried out through a molecular modification technology, and the enzymatic activity and catalytic efficiency of the modified glycosyl transferase mutant are greatly improved. The mutation at a specific site can significantly promote the glycosylation activity of a tyrosol substrate. The mutant enzyme is obtained by carrying out induced expression and protein purification on the obtained mutant strain. The substrate conversion rate of the transformed glycosyl transferase mutant for catalyzing the tyrosol glycosylation reaction can reach 95% or above, the yield reaches 55.0 g / L, and an efficient and green new way is provided for salidroside production.

The invention provides an L-threonine aldolase mutant and application of the L-threonine aldolase mutant in catalytic synthesis of p-methylsulfonylphenylserine, the L-threonine aldolase mutant is derived from pseudomonas, threonine and p-methylsulfonylbenzaldehyde can be used as substrates, and the L-threonine aldolase mutant can be synthesized by using a one-step method. According to the present invention, L-threonine aldolase is adopted as a raw material, pyridoxal phosphate and reduced nicotinamide adenine dinucleotide are adopted as coenzymes, the synthesis of florfenicol intermediate product p-methylsulfonyl phenyl serine is obtained through catalysis, and compared with the wild type L-threonine aldolase, the mutant enzyme has high activity, can effectively improve the conversion efficiency, and can shorten the conversion time; the enzyme and the technical method provided by the invention have the advantages of mild reaction conditions and less pollution, and have good industrial prospects in the production of florfenicol synthesis intermediates.

The invention discloses a PET hydrolase mutant as well as a preparation method and application thereof, and belongs to the field of enzyme engineering. On the basis of FAST-PETase, in combination with deep learning prediction and enzyme engineering optimization strategies, a plurality of mutants with specific performance advantages are systematically constructed and screened, the method is suitable for application scenes with different temperatures, substrate loads and degradation efficiency requirements, and experimental basis and technical support are provided for constructing a diversified and combinable PET degrading enzyme system. The mutants show diversified advantages in different performance dimensions, for example, the enzyme activity of the K233R mutant is 1.62 times that of FAST-PETase, and the K233R mutant still keeps good thermal stability (1.03 times) after being subjected to heat treatment for 2 hours at the temperature of 50 DEG C; q224K is more remarkably improved in the aspect of thermal stability, and the residual activity after heat treatment is 1.64 times that of FAST-PETase.

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 present invention discloses a pectin methylesterase mutant ΔDdPemA, its encoding gene, preparation method, and application. The pectin methylesterase mutant ΔDdPemA is obtained by mutating the amino acid at position 200 in the center of the substrate binding pocket of the wild-type pectin methylesterase DdPemA from a high-molecular-weight, branched valine to a low-molecular-weight alanine, and by mutating the amino acid at position 339 at the substrate inlet and outlet of the substrate binding pocket from a high-molecular-weight, branched valine to a basic lysine. The amino acid sequence and nucleotide sequence of the wild-type pectin methylesterase DdPemA are SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The mutant enzyme provided by the present invention has significantly improved enzyme activity under weakly acidic conditions, solving the problem of insufficient catalytic activity of the wild-type pectin methylesterase under weakly acidic conditions and creating favorable conditions for the application of the enzyme in the fields of food, textiles, and feed processing.

The invention discloses a glutamate decarboxylase multi-site combined mutant and application thereof, and relates to the technical field of enzyme engineering and gene engineering. Amino acid residue sites needing to be mutated are determined on the basis of amino acid sites near key amino acids of a coenzyme 5 '-phosphopyridoxal binding pocket, and experimental verification is performed through a site-directed mutagenesis technology in combination with a high performance liquid chromatography screening method. Compared with a chemical synthesis method, the compound is synthesized by utilizing glutamic acid decarboxylase through biological catalysis, the synthesis condition is mild, the operation is simple, and separation and extraction are easy. According to the method, the screening probability of mutant amino acid sites can be effectively improved, the experiment efficiency and feasibility are improved, and the mutant enzyme of which the enzyme activity is obviously superior to that of the parent enzyme is obtained through screening.

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 invention relates to a nicotinamide ribose phosphate transferase mutant, a genetically engineered bacterium and application of the nicotinamide ribose phosphate transferase mutant and the genetically engineered bacterium. Compared with a wild enzyme strain NMN, the production capacity of a mutant enzyme strain is greatly improved. The saccharomyces cerevisiae genetically engineered bacterium capable of stably inheriting and efficiently synthesizing the NMN is obtained, and safe, efficient and green synthesis of the NMN is achieved.

The invention relates to the field of bioengineering, in particular to an enzyme composition, a product and application of the enzyme composition in preparation of Ectoine. The invention provides an enzyme composition. The enzyme composition comprises a mutant of 2-hydroxy-4-aminobutyric acid oxidase, a mutant of keto transaminase, a mutant of hydroperoxide hydrolase, a mutant of Ectoine synthetase, a mutant of pyruvic acid-CoA oxidase and lactobacillus helveticus lactic dehydrogenase. According to the method disclosed by the invention, a plurality of mutant enzymes are utilized for combined transformation for the first time, and cheap 2-hydroxy-4-aminobutyric acid is taken as a starting raw material to quickly and efficiently produce Ectoine; the enzyme method has the characteristics of easiness in control, short conversion time, few generated impurities and the like, so that the method has unique advantages in the aspects of low-cost and large-scale mass production of Ectoin.

The invention belongs to the technical field of bioengineering, and particularly relates to an activity-improved tyrosinase mutant obtained by site-directed mutagenesis through an overlapping PCR technology, and preparation and application thereof. The tyrosinase mutant is obtained by mutation containing F337I / F352I on the basis of wild type tyrosinase as shown in SEQ ID NO.1, the enzyme activity of the tyrosinase mutant is obviously improved compared with that of the wild type tyrosinase, the F337I / F352I mutant gene is used for constructing a recombinant vector, and the recombinant vector is successfully expressed in bacillus subtilis WB600, bacillus amyloliquefaciens CGMCC No.11218 and bacillus licheniformis 2709, so that the tyrosinase mutant is obtained. The recombinant strain with improved enzyme production activity is further optimized through a fermentation process to obtain the novel tyrosinase.

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 invention discloses efficient synthesis of ursodesoxycholic acid by improving the positive enzyme activity of 7 beta-hydroxysteroid dehydrogenase through directed evolution, and belongs to the field of biological catalysis engineering. According to the invention, the 7beta-hydroxysteroid dehydrogenase mutant TEAE / F152L / W101N with improved positive reaction activity and improved affinity with a substrate is obtained. Compared with TEAE, the forward enzyme activity of the mutant enzyme is improved by 2.9 times, the reverse activity of the mutant enzyme is reduced by 3.6 times, the catalytic efficiency kcat / Km of the mutant enzyme to 7K-LCA is improved by 4.3 times, and compared with a wild type mutant TEAE / F152L / W101N, the enzyme activity of the mutant enzyme is improved by 206.23 times, and the catalytic efficiency of the mutant enzyme to 7K-LCA is improved by 58.56 times. The invention provides a universal technical platform for directed evolution of hydroxysteroid dehydrogenase, provides an excellent mutant enzyme for biological efficient synthesis of ursodesoxycholic acid, and lays a solid research foundation for efficient and low-cost preparation of ursodesoxycholic acid.

The present disclosure provides a new PH20 mutant enzyme which shows significantly enhanced aggregation stability, can be used for the development of drug subcutaneous injection preparations, facilitates the diffusion of drugs in subcutaneous tissues, reduces subcutaneous administration volume limitations, and alleviates swelling or pain caused by large-volume subcutaneous injection.

The invention provides a strain of high-yield gamma-cyclodextrin glucosyltransferase and application of the strain. Specifically, the invention relates to a gamma-cyclodextrin production method, which comprises the following steps: (a) in the presence of a gamma-cyclodextrin glucosyltransferase mutant, carrying out enzymatic reaction on a substrate starch so as to form gamma-cyclodextrin. The invention also provides a strain for high yield of gamma-cyclodextrin glucosyltransferase, and a gamma-cyclodextrin glucosyltransferase mutant gene is integrated in a strain genome. The mutant enzyme and the strain provided by the invention can be used for generating high-yield gamma-cyclodextrin.

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 discloses efficient expression of an aspergillus oryzae beta-galactosidase mutant and application of the aspergillus oryzae beta-galactosidase mutant in dairy products. The invention belongs to the technical field of enzyme engineering and the technical field of dairy product processing, and particularly relates to efficient expression of an aspergillus oryzae beta-galactosidase mutant and application of the aspergillus oryzae beta-galactosidase mutant in dairy products. The mutant protein disclosed by the invention can be as follows: A1) a protein with an amino acid sequence of SEQ ID No.2; a2) a fusion protein which is obtained by connecting a label to the N end and / or the C end of A1) and has the same function. The mutant protein is subjected to high-density fermentation in a 5L fermentation tank, the enzyme activity of fermentation liquor can reach 4628U / mL, and the recovery rate of mutant enzyme is increased by 1.7 times. Compared with a wild type, the mutant has the advantages that the optimum pH is increased from 4.5 to 5.5, the optimum temperature is reduced from 60 DEG C to 50 DEG C, the lactose hydrolysis efficiency is improved by 11%, and the mutant is more suitable for a production process of low / zero lactose dairy products.

The invention discloses an aldose reductase mutant and application thereof in production of D-tagatose, and belongs to the technical field of genetic engineering. The aldose reductase mutant disclosed by the invention has relatively high catalytic activity, and can be coupled with galactitol 2-dehydrogenase to realize cyclic regeneration of NADH (nicotinamide adenine dinucleotide), so that the preparation cost of D-tagatose is greatly reduced. When the aldose reductase mutant is used for preparing D-tagatose, 98.2 g / L of D-tagatose can be obtained through conversion within 5 h, the yield reaches up to 98.2%, and the conversion effect is better than that of wild type and other mutant enzymes, so that the aldose reductase mutant has good industrial application prospects.

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 discloses a modified 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), which has higher activity and stability in the synthesis process of ursodesoxycholic acid (UDCA), and takes NAD (P) H as a coenzyme. According to the 7beta-hydroxysteroid dehydrogenase mutant, the V104 site in the 7beta-hydroxysteroid dehydrogenase is subjected to saturated mutation modification, and the mutant with enhanced activity and changed coenzyme specificity is screened out. The mutant enzyme can keep high catalytic efficiency under the condition of taking NADH as a coenzyme, so that the synthesis yield of UDCA is remarkably increased, and the production cost is reduced. The enzyme mutant disclosed by the invention has a wide application prospect in industrial production, is particularly suitable for the production process of large-scale biosynthesis of UDCA, can effectively reduce the production cost, improve the product yield and reduce the environmental pollution and the production cost, and provides a technical support for wide application of UDCA.

The invention relates to the technical field of bioengineering, and discloses Burkholderia lipase with T251S mutation and a recombinant expression vector and application thereof, threonine of the 251 amino acid sequence of LipA of wild Burkholderia lipase is mutated into serine. The enzyme activity of the mutant enzyme is remarkably improved by 23%, meanwhile, the hydrolysis capacity on ethyl ester type fish oil is remarkably improved, the selectivity on EPA is improved, and excellent EPA purification and enrichment effects are achieved. According to the mutant, the preparation cost is reduced, the hydrolysis capability and selectivity are improved, and the mutant has great popularization and application values.