233 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 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 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 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 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 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 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 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.

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.

Provided herein is a mutant enzyme comprising a variant of the amino acid sequence shown in SEQ ID NO: 1, as well as compositions comprising said enzyme; and applications of said enzyme and compositions in both therapeutic and non-therapeutic applications.

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.

The invention discloses an N-acetyl glucosamine 2-epimerase mutant as well as preparation and application of the N-acetyl glucosamine 2-epimerase mutant. The mutant enzyme comprises an amino acid sequence as shown in SEQ ID No.3. The preparation method comprises the following steps: by taking a recombinant vector containing a Sumo enhancing element and a wild type N-acetyl glucosamine 2-epimerase gene as a template, carrying out PCR (Polymerase Chain Reaction) by adopting a mutation primer to obtain a mutant gene segment; transforming the mutant gene segment into an engineering bacterium to obtain a recombinant bacterium for expressing the N-acetyl glucosamine 2-epimerase mutant protein; culturing recombinant bacteria, collecting and crushing thalli, centrifuging thallus crushing liquid, and taking supernatant, so as to obtain crude enzyme liquid containing the enzyme mutant; the thermal stability of the wild-type enzyme is improved through point mutation of specific amino acid sites, and the substrate affinity and the catalytic efficiency of the mutant are superior to those of the wild-type enzyme. The mutant can be used for efficient catalytic preparation of lactulose, and has the advantages of few byproducts, high product yield and the like.

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.

The present invention discloses a polyphosphate kinase mutant and its application in glutamine synthesis, belonging to the field of biotechnology. The present invention has screened a polyphosphate kinase mutant with significantly improved enzyme activity. The catalytic activity of this mutant enzyme has increased by 182.2% compared to the original enzyme, enabling efficient regeneration of ATP and reducing the cost of biocatalysis. Using the polyphosphate kinase mutant constructed in the present invention for the production of glutamine significantly improves the sustainability of ATP regeneration. The glutamine yield can reach 94.4 mM after 8 h of reaction, and the conversion rate is 94.4%. The yield of glutamine produced by coupling with the original enzyme is increased by 41.5%.

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.