108 results about "Wild type enzyme" patented technology

The invention discloses a method for preparing butanediamine and spermidine by an enzyme method, and belongs to the field of bioengineering. The novel arginine decarboxylase mutant is successfully provided, the mutant shows excellent catalytic performance under the alkaline condition, and the specific enzyme activity of the mutant reaches 24.9 U / g and is improved by 8.3 times compared with that of wild type enzyme. The invention develops a two-step synthesis process based on the constructed arginine decarboxylase mutant, and the two-step synthesis process is used for efficiently preparing spermidine. Experimental results show that the process successfully realizes the remarkable increase of the yield of spermidine, and the final yield reaches 200.4 mg / L.

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.

The invention discloses a lactic dehydrogenase LdLDH mutant and application thereof, a wild type LdLDH enzyme derived from Lactobacillus delbrueckii subsp. Bulgaricus is taken as a research object, the amino acid sequence of the wild type LdLDH enzyme is designed and modified to obtain a mutant protein, the mutant protein is named as LdLDH-M, the amino acid sequence of the mutant protein is shown as SEQ ID NO.2, the Tm value of the LdLDH-M is 77.1 DEG C, the Tm value of the LdLDH-M is 77.1 DEG C, and the Tm value of the LdLDH-M is 77.1 DEG C; compared with the wild type LdLDH enzyme, the Tm value of the LdLDH enzyme is increased by 27.4 DEG C, and the enzyme activity of the LdLDH enzyme is increased by 1.4 times. Therefore, compared with the wild type LdLDH enzyme, the LdLDH-M has remarkably improved thermal stability and enzymatic activity, and is more beneficial to widening the application range of the LdLDH-M in the fields of high-temperature biological research, industrial biological catalysis and the like.

The application discloses an acyltransferase for synthesizing polyester intermediates and application thereof. The acyltransferase is specifically a wild-type enzyme or a mutant acyltransferase variant with catalytic acyltransferase activity, which is obtained by substituting at least one of amino acid residues at positions 209, 166 and 119 in the amino acid sequence of the wild-type enzyme. The acyltransferase variant is specifically a protein with improved acyltransferase activity. Both the wild-type enzyme and the acyltransferase variant provided by the application have acyltransferase activity. The application has important application value.

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 enzyme mutant and application of an engineering bacterium thereof in synthesis of calcifediol, the mutant is one or more point positions in an amino acid sequence based on a wild type P450 enzyme Vdh being mutated; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1. Through systematic molecular dynamics simulation, conservation analysis and substrate binding pocket engineering on the Vdh enzyme, multiple key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I114R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, and the pure enzyme activity can reach 9.8 times of the wild type enzyme, greatly improving the conversion rate from vitamin D3 to calcifediol.

This invention discloses a pullulanase mutant with enhanced catalytic activity against highly branched dextrin substrates, its construction method, and its applications, belonging to the fields of enzyme engineering and biotechnology. This invention obtains two mutants, D139A and D139E, by site-directed mutagenesis of the pullulanase derived from Klebsiella pneumoniae at position 139 aspartic acid residue. Compared with the wild-type enzyme, both mutants maintain high hydrolytic activity against pullulan polysaccharides while significantly improving hydrolytic activity against dextrin substrates with high α-1,6 glycosidic bond content. Specifically, the hydrolytic activity against dextrin substrates with 6% and 10% α-1,6 bond content is increased by up to 242% and 110%, respectively. The pullulanase mutants obtained in this invention exhibit excellent catalytic performance in the enzymatic debranching reaction of highly branched dextrin substrates, showing promising application prospects in starch deep processing and related biomanufacturing fields.

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.

The application discloses a method for preparing putrescine and spermidine by using an enzyme, and belongs to the field of bioengineering.The application successfully provides a novel arginine decarboxylase mutant, the mutant exhibits excellent catalytic performance under alkaline conditions, and the specific enzyme activity reaches 24.9 U / g, which is 8.3 times higher than that of a wild-type enzyme.Based on the constructed arginine decarboxylase mutant, the application develops a two-step synthesis process for efficiently preparing spermidine.The experimental results show that the process successfully realizes significant improvement of the yield of spermidine, and the final yield reaches 200.4 mg / L.

The present application belongs to the technical field of genetic engineering, and relates to a CrtI enzyme mutant, a recombinant plasmid, a recombinant strain and application thereof. The CrtI enzyme mutant provided by the present application is obtained by random mutation of a wild-type CrtI gene through error-prone PCR, and the amino acid sequence thereof is shown as SEQ ID NO. 1. The obtained mutant has multiple site variations in the amino acid sequence, thereby significantly improving the catalytic efficiency and substrate affinity of the enzyme. The present application further constructs a recombinant expression vector containing the CrtI enzyme mutant gene, and transforms the recombinant expression vector into a red Phaffia yeast competent cell, and obtains a high-yield astaxanthin recombinant strain through resistance screening and pigment phenotype screening. Compared with the wild-type CrtI enzyme, the CrtI enzyme mutant of the present application significantly improves the conversion rate of beta-carotene to astaxanthin in the red Phaffia yeast.

The present invention relates to variants of TEV protease that have—compared to the wildtype enzyme—increased stability and catalytic activity as well as altered substrate specificity. The invention further relates to compositions comprising these variants as well as uses thereof and methods in which these variants are employed.

The invention relates to the technical field of enzyme engineering and biology, and discloses an olefin reductase mutant as well as a coding gene, a recombinant vector, a recombinant strain, an enzyme preparation and application thereof. The olefin reductase mutant provided by the invention is obtained by mutating at least one of F297V, F297V / F124N and F297V / F124N / M40F on an enzyme with an amino acid sequence as shown in SEQ ID NO.1. Compared with a wild enzyme, the olefin reductase mutant has the advantages that the efficiency of catalyzing p-hydroxybenzylideneacetone to generate raspberry ketone is obviously improved, complicated reaction environment control equipment is not needed, the cost is low, and the production cost is low. And an efficient and feasible enzyme catalyst scheme is provided for industrial green production of raspberry ketone.

The present invention relates to an improved cysteine-producing strain, in particular to a microbial strain suitable for the fermentative production of L-cysteine, characterized in that the relative enzymatic activity of the enzymes identified by the number EC 2.7. 9.2 in the KEGG database is inactivated or the specific activity is reduced based on the wild-type enzymes, and the enzyme represented by the number EC 2.7. 9.2 in the KEGG database forms an increased amount of L-cysteine as compared to a microbial strain having a wild-type enzymatic activity in which the gene encoding the enzymatic activity is represented by ppsA. The invention also provides methods of producing L-cysteine using these microbial cells.

The invention provides an improved engineered phenylalanine ammonia lyase which can be used for preparing a chiral amine compound, in particular for asymmetrically synthesizing L-2-chloro-phenylalanine. Compared with a wild-type enzyme corresponding to SEQ ID NO: 2, the engineered phenylalanine ammonia lyase polypeptide has better activity and / or stability, can be used for asymmetrically preparing chiral amine compounds with extremely high stereoselectivity, particularly can be used for more efficiently catalyzing o-chloro-cinnamic acid to prepare L-2-chloro-phenylalanine, can accept various ammonia donors, and can be used for preparing the chiral amine compounds. Such as ammonium carbonate, ammonia water and ammonium chloride.

The invention belongs to the technical field of biochemical engineering, and particularly relates to application of a P450 enzyme mutant and engineering bacteria thereof in synthesis of calcifediol, and the mutant is obtained by mutating one or more sites in an amino acid sequence based on a wild type P450 enzyme Vdh; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to the accession number of GenBank, namely CP069288.1. Through systematic molecular dynamics simulation, conservative analysis and substrate binding pocket engineering on the Vdh enzyme, a plurality of key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I144R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, the pure enzyme activity of the combined mutant can reach 9.8 times that of a wild type enzyme, and the conversion rate from vitamin D3 to calcifediol is greatly increased.

The invention discloses short-chain dehydrogenase for synthesis of (S)-1-(2-methoxy-3-bromophenyl) ethanol as well as a mutant, a coding gene, a plasmid, a genetically engineered bacterium and application of the short-chain dehydrogenase, and belongs to the technical field of biocatalysts. The mutant is obtained by mutating methionine at the 97th site into isoleucine on the basis of a wild type enzyme shown in SEQ ID NO.1 (SEQ ID NO.3). The invention further discloses a preparation method of the short-chain dehydrogenase. The invention also provides a coding gene, an expression vector, a genetically engineered bacterium and a double-enzyme catalytic system containing the mutant and formate dehydrogenase. The system can efficiently and highly selectively catalyze and synthesize the target chiral alcohol under mild conditions, and the conversion rate is gt; 95%, eet; 99% of the method is suitable for green preparation of the key intermediate of the medicine lutrombopag.

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

The application belongs to the technical field of biotechnology and cosmetics, and discloses a polypeptide with skin barrier repair activity and application thereof. The polypeptide is an enzymatic fragment derived from Bacillus subtilis neutral metalloproteinase A7UAM1, and the amino acid sequence is shown as SEQ ID NO:1. The mutant is a site-directed mutant of the wild-type A7UAM1, and is preferably a H142A / E143A double mutant. Compared with the wild-type enzyme, the proteolytic activity of the polypeptide and the mutant is reduced by greater than or equal to 90%, but the activity of promoting keratinocytes to express filaggrin (FLG) and loricrin (LOR) is not reduced or is significantly improved, and the application realizes the decoupling of the functions of 'gentle exfoliation' and 'active barrier repair'. The polypeptide or the mutant can be applied to the preparation of cosmetics or drugs with the effects of skin barrier repair, anti-sensitivity and anti-photoaging, and is especially suitable for sensitive skin and post-medical aesthetic repair.

This invention discloses a desmethylmenoprenone methyltransferase mutant, its construction method, and its applications, belonging to the field of biotechnology. Desmethylmenoprenone methyltransferase is a key enzyme in the MK-7 biosynthetic pathway, responsible for catalyzing the methylation reaction of desmethylmenoprenone to generate a series of methylnaphthoquinone (MK) products, ultimately forming MK-7. This invention, through metabolic network regulation and rational design, successfully obtained 19 single-point mutants, among which 5 mutants showed significantly higher catalytic efficiency than the wild-type enzyme. Experimental results show that the recombinant engineered bacteria constructed based on these mutants exhibit significant advantages in the whole-cell catalytic production of vitamin K2 (MK-7), with MK-7 yield increased by more than 20% compared to the wild-type strain. The excellent catalytic performance of the menG mutant provided by this invention provides strong technical support for the industrial production of vitamin K2, and has broad application prospects and market value.

The invention provides a transferase and a green enzymatic carnosine production method. The high-activity mutants of DmpA-2 and DmpA-6 are obtained by performing single-point or double-point mutation on the 86th site (E86V) and the 259th site (D259A) of wild type DmpA enzyme, so that the catalytic efficiency of the beta-amino acid ester acyltransferase is remarkably improved. According to the method, beta-alanine and L-histidine serve as substrates, recombinant bacteria are used for expressing mutant enzyme, carnosine is catalytically synthesized under the mild condition, the product concentration reaches 5.36 g / L and is 3.53 times that of a wild type, and the method has the advantages of being green in reaction condition, high in product purity and the like and is suitable for industrial production.

The invention discloses a ruminococcus torsionosus-sourced 7beta-HSDH enzyme mutant and application thereof, the ruminococcus torsionosus-sourced 7beta-HSDH enzyme is taken as a research object, a series of single-point mutation and combined mutation of the 7beta-HSDH enzyme are provided, and compared with wild type 7beta-HSDH enzyme, the thermal stability of the 7beta-HSDH enzyme mutant is improved by 1-20 DEG C, and the 7beta-HSDH enzyme mutant has the advantages that the 7beta-HSDH enzyme mutant has the advantages that the 7beta-HSDH enzyme mutant can be used for preparing the 7beta-HSDH enzyme mutant; compared with a wild type 7beta-HSDH enzyme, the 7 beta-HSDH enzyme has the advantages that the Tm value of the (C217I and E252K) mutant is improved by 1.2-4.2 times, the enzyme activity of the (C217I and E252K) mutant is 4.2 times that of the wild type 7beta-HSDH enzyme, and the (T103I, D251K, L229K and S161A) mutant has ultrahigh heat stability, the Tm value is close to 68 DEG C, and 50% of enzyme activity is maintained, so that the 7 beta-HSDH enzyme can be used for preparing the 7 beta-HSDH enzyme. Compared with a wild type 7 beta-HSDH enzyme, the 7 beta-HSDH enzyme mutant provided by the invention has wider application conditions, is more suitable for efficiently producing ursodesoxycholic acid from 3 alpha-hydroxy-7-oxo-5beta-cholanic acid (7-ketolithocholic acid, 7-KLCA) by a biological conversion method, and is beneficial to large-scale production and industrial application.

The invention provides novel hyaluronidase and a preparation method of hyaluronic acid with specific molecular weight. According to the novel hyaluronidase, at least one mutation site exists in N266R, K349R, S180P, V417I and T488A in an amino acid sequence on the basis of a wild type enzyme of SEQ ID No. 1. Compared with wild hyaluronidase, the hyaluronidase provided by the invention has significantly improved catalytic activity and / or thermal stability. The method is used for enzymolysis of high-molecular-weight hyaluronic acid, and the molecular weight of a degradation product can be accurately controlled.

The application relates to the technical field of enzyme engineering, and discloses a 7beta-HSDH mutant with alkali resistance and application thereof. In order to improve the alkali resistance of 7beta-HSDH, the 7beta-HSDH from Collinsella aerofaciens is subjected to site-directed mutation modification, and a plurality of 7beta-HSDH mutants with alkali resistance are obtained. The 7beta-HSDH from Collinsella aerofaciens is used as a wild type, and the 7beta-HSDH mutant provided by the application is a single-point mutant or a multi-point combination mutant of E26K, P104S, N113Y and M144T. The optimum pH of the wild type enzyme is 5.5, the optimum pH of the 7beta-HSDH mutant provided above is improved from 5.5 to 7.0 after mutation, and the enzyme activity of the 7beta-HSDH mutant is much higher than that of the wild type enzyme under the condition of pH 7.0; under the alkaline condition of pH 8.0-8.5, the 7beta-HSDH mutant still has relatively high enzyme activity.

The invention discloses a colletotrichum linum P450 enzyme mutant and application thereof, the mutant is obtained by mutating 121st aspartic acid of an amino acid sequence of P450 enzyme CYP68J derived from a colletotrichum linum ST-1 strain into histidine, and the amino acid sequence is shown as SEQ ID NO: 1. Compared with a wild type P450 enzyme, the P450 enzyme mutant disclosed by the invention has the advantages that dehydroepiandrosterone can be efficiently converted into 7 alpha-hydroxy-dehydroepiandrosterone, and the ratio of 7 alpha-hydroxy-dehydroepiandrosterone to 7 alpha, 15 alpha-dihydroxy-dehydroepiandrosterone in a hydroxylation product can be increased from about 4: 5 to about 14: 1; an important way is provided for the biological conversion of the 7alpha-hydroxyl-dehydroepiandrosterone, and the method has important significance on the industrial production of the 7alpha-hydroxyl-dehydroepiandrosterone.

The invention provides a beta-1, 3-glucan hydrolase mutant and application thereof, and relates to the technical field of biology. The mutant provided by the invention is obtained by carrying out site-directed mutagenesis by taking beta-1, 3-glucan hydrolase GH3 as a wild type enzyme. Compared with a wild enzyme, the enzyme activity of the D446W mutant is improved by 54%, so that the application potential and commercial value of the D446W mutant in the fields of medicines, health care products, functional foods and the like are favorably improved.

The invention relates to the technical field of biological enzyme engineering, in particular to a 3beta-hydroxysteroid dehydrogenase mutant and application thereof. The 3beta-hydroxysteroid dehydrogenase mutant provided by the invention is obtained by carrying out point mutation on an amino acid sequence shown by wild type 3beta-hydroxysteroid dehydrogenase. Compared with a wild enzyme, the 3beta-hydroxysteroid dehydrogenase mutant constructed by the invention has the advantages that the enzyme activity of the mutant is greatly improved and is 1.68-3.30 times that of a parent, and the use amount of an industrial enzyme can be remarkably reduced. Meanwhile, the mutant can specifically oxidize 3beta-ursodesoxycholic acid to generate 3ketoursodesoxycholic acid, and then the 3ketoursodesoxycholic acid is converted into ursodesoxycholic acid under the action of 3alpha-hydroxysteroid dehydrogenase, so that the defect that 3beta-ursodesoxycholic acid impurities generated in the ursodesoxycholic acid chemical synthesis process are difficult to remove is overcome; and a wide application prospect is provided for industrial production of ursodesoxycholic acid.

The application discloses a monomerized pyrimidine nucleoside phosphorylase mutant and a construction method and application thereof, and belongs to the field of enzyme engineering and biological catalysis technology.The mutant is obtained by amino acid substitution mutation of wild-type pyrimidine nucleoside phosphorylase shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.The mutant is transformed into a stable monomer form by dimer interface remodeling and hinge region stabilization of the closed active conformation, the natural dimer PyNP is transformed into a stable monomer form, and the catalytic activity of the mutant to uridine, 2'-deoxyuridine and similar substrates is maintained in the monomer state.Compared with the wild-type enzyme, the mutant has lower oligomer dependence, better temperature stability, pH stability and storage stability, and can be used for biological catalytic synthesis of nucleosides and nucleoside analogs.

The present application provides a mutant glucanase, which has a mutation at the amino acid position corresponding to position 121 of SEQ ID No. 2 compared to the parent glucanase. The enzyme activity of the mutant glucanase of the present application is increased by 68% compared to the wild-type enzyme, and has a wide application prospect in degrading materials containing glucan (for example, Aureobasidium pullulans residues produced in the production of pullulan).

This invention discloses a highly efficient method for the biosynthesis of butanediamine, belonging to the field of bioengineering. The arginine decarboxylase mutant provided by this invention exhibits a specific enzyme activity of 25.1 U / g under alkaline conditions, approximately 8 times higher than the wild-type enzyme, significantly enhancing the enzyme's catalytic efficiency and providing a highly efficient enzyme catalyst for the biosynthesis of butanediamine. Recombinant strain E. coli BL21(DE3) speA ‑ E482R ‑ E467K ‑ H736E ‑ speB In the catalytic synthesis of butanediamine, there is no need to collect the bacterial cells by centrifugation. The bacterial cells will self-sediment in the culture medium, and complete sedimentation can be achieved within 20-30 minutes. Moreover, the bacterial cell content in the supernatant is only 1% (OD600). By reusing the bacterial cells in the fermentation broth, continuous catalytic reaction can be achieved, significantly increasing the yield of butanediamine.

The application discloses a trehalose-6-phosphate phosphatase and a mutant and application thereof, and belongs to the technical field of biology. The amino acid sequence of the wild-type LcTre6PPase is shown as SEQ ID NO:1, and the encoding nucleotide sequence is shown as SEQ ID NO:2. The mutant is obtained by mutating the isoleucine at the 571th position of the wild-type enzyme into leucine (I571L), the amino acid sequence is shown as SEQ ID NO:3, and the corresponding encoding nucleotide sequence is shown as SEQ ID NO:4. Bidirectional enzyme activity analysis shows that, compared with the wild type, the I571L mutant is improved by about 1.2 times in the direction of catalyzing glucose-6-phosphate and beta-D-glucose-1-phosphate to synthesize trehalose-6-phosphate, and the activity in the direction of catalyzing trehalose-6-phosphate to phosphorolyze is reduced to 0.8 times of the wild type. The mutant shows good application potential in the efficient synthesis of trehalose-6-phosphate, and provides a new enzyme tool for preparing trehalose-6-phosphate.