83 results about "Substrate specificity" 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 D-psicose 6-phosphate phosphatase mutant and application of the D-psicose 6-phosphate phosphatase mutant in preparation of psicose, and belongs to the technical field of enzyme engineering. According to the invention, site-directed mutagenesis is carried out on an amino acid sequence of D-psicose 6-phosphate phosphatase A6PP, and a combined mutant I21V / Y73F / R115M / T180V / L203I / Q193E is constructed, so that the substrate specificity, the thermal stability and the catalytic activity of the combined mutant are all remarkably improved. The invention further provides application of the combined mutant in preparation of psicose through multi-enzyme cascade catalysis, 150 g / L of maltodextrin can be converted into 97.7 g / L of D-psicose, the conversion rate reaches 65.1%, and the application prospect of the combined mutant in the field of food is widened.

The invention relates to a UGT enzyme subtype substrate specificity prediction method and model. On the basis of a directional message passing neural network, graph structure characterization of a small molecule compound and features of specific protein binding sites of UGT enzyme are deeply fused, a bimodal prediction normal form of'molecule + protein binding sites' is designed, a deep learning model is constructed, conversion from compound center prediction to molecule-enzyme binding site comprehensive prediction is achieved, and the prediction accuracy is improved. And accurate classification prediction can be carried out on UGT enzyme substrates and non-substrates.

The invention relates to the technical field of biochemistry, in particular to an enzyme mutant and application thereof in preparation of conopeptide. According to the preparation method, a strategy of combining green chemistry and an enzyme method is adopted, seven tripeptide fragments are taken as raw materials, a conopeptide main chain is prepared through a liquid-phase synthesis method, and then enzymatic precise modification and synthesis are realized by utilizing a multiple directed evolution enzyme concerted catalysis system. Based on the efficient connection characteristic of a liquid-phase synthesis system and the high substrate specificity of an enzymatic system, the complex protection group operation in traditional solid-phase synthesis is effectively avoided, generation of by-products is remarkably reduced, meanwhile, fixed-point modification and precise assembly of a conopeptide main chain are achieved, and the application prospect is wide. And an efficient and green new path is provided for the continuous production of the cosmetic-grade high-purity conopeptide.

The invention provides chondroitin sulfate excision enzyme and application of the chondroitin sulfate excision enzyme in dermatan sulfate preparation. The amino acid sequence of the chondroitin sulfate excision enzyme is shown as SEQ ID NO: 3, and the chondroitin sulfate excision enzyme has broad-spectrum substrate specificity and can efficiently degrade hyaluronic acid and glycosaminoglycans such as chondroitin sulfate (A, C, E and D). The chondroitin sulfate excision enzyme can efficiently act on a substrate by virtue of a unique catalytic mechanism, so that efficient preparation of oligosaccharide and low-molecular-weight polysaccharide is realized, and the chondroitin sulfate excision enzyme can be particularly used for enzymatic preparation of dermatan sulfate. The chondroitin sulfate excision enzyme has efficient catalytic performance and unique treatment advantages, not only fills the technical blank in related fields, but also opens up a wide space for the development of carbohydrate chemicals in the fields of biological medicines, cosmetics, foods and the like.

The present invention discloses a transaminase and its use in preparing optically pure chiral amines. The amino acid sequence of the transaminase of the present invention has one or more mutations at positions 65, 318, 399, 419, 426, 465, and 466 compared to SEQ ID NO: 1, wherein the mutation is an addition, deletion, or substitution of an amino acid residue. The present invention also discloses a nucleic acid molecule encoding the transaminase, a nucleic acid construct comprising the nucleic acid molecule, a recombinant vector, and a host cell. The present invention also discloses an enzyme preparation comprising the transaminase of the present invention. The present invention also discloses a method for preparing chiral amines using the transaminase. In the industrial production process of optically pure chiral amines, the enzyme provided by the present invention has advantages such as substrate specificity, enantiomeric selectivity, and high conversion rate. The method for preparing optically pure chiral amines provided by the present invention has high reaction efficiency, stereoselectivity, and yield.

The invention discloses polyethylene glycol terephthalate hydrolase PmPETase and application thereof.The hydrolase PmPETase is protein with the amino acid sequence shown in SEQ ID NO.1. An expression system suitable for the PmPETase is constructed, recombinase protein with catalytic activity is obtained, and the polyethylene glycol terephthalate hydrolase PmPETase can be used for preparing the polyethylene glycol terephthalate hydrolase PmPETase. The substrate specificity, the catalytic property and the potential plastic degradation capacity of the PET degrading enzyme are comprehensively analyzed, and a new theoretical basis and application reference are provided for development and utilization of the PET degrading enzyme and a high polymer material green treatment technology.

This invention provides a ketolic acid reductase mutant and its application in constructing genetically engineered bacteria that produce high levels of D-pantothenic acid. To address the technical problem of low D-pantothenic acid yield in existing microbial fermentation, this invention utilizes semi-rational enzyme design to obtain a substrate-specific ketolic acid reductase mutant, which is then applied to construct genetically engineered bacteria that produce high levels of D-pantothenic acid. Furthermore, this invention employs CRISPR-Cas9 gene editing technology to construct genetically engineered bacteria that produce high levels of D-pantothenic acid using the aforementioned ketolic acid reductase mutant, increasing D-pantothenic acid production by approximately 12.04%. Compared to the starting strain, the accumulation of valine in the fermentation broth remained almost unchanged compared to the control, but the accumulation of the branched-chain amino acid isoleucine was lower, achieving substrate-specific modification of the ketolic acid reductase.

The invention discloses a method for detecting F26G enzyme activity based on an HPLC (High Performance Liquid Chromatography) technology, and belongs to the technical field of biological enzyme activity detection. According to the method, on the basis of the characteristic that furostanol saponin 26-O-beta-D-glucosidase (F26G) hydrolyzes furostanol saponin C-26 glucosyl, a detection system which takes furostanol saponin as a substrate and quantifies substrate consumption through an HPLC technology to calculate enzyme activity is established, the method has high substrate specificity, and compared with a pNPG method, the method has the advantages that the detection efficiency is high, and the detection cost is low. The method has the advantages of high sensitivity and high accuracy, can accurately eliminate the interference of other glucosidase in the system, overcomes the defects of poor specificity, low sensitivity and difficulty in accurate determination of the enzyme activity of the traditional pNPG method, provides a new method for accurate detection of the activity of the F26G enzyme in the medicinal materials, and provides technical support for quality monitoring of the medicinal materials and decoction pieces.

This invention discloses the application of arginine-depleted engineered bacteria for the treatment of soft tissue sarcomas, including preliminary validation, obtaining qualified engineered bacteria SGR1, obtaining drug compositions and regimens, experimental conclusions, and clinical application promotion. During use, clinical soft tissue sarcoma samples are collected, including undifferentiated pleomorphic sarcoma, synovial sarcoma, and liposarcoma. Immunohistochemistry (IHC) is used to detect ASS1 protein expression, qPCR is used to detect ASS1 gene transcription levels, and the proportion of ASS1 deletion / low expression is statistically analyzed. Arginine auxotrophic sarcoma subpopulations are confirmed based on literature. Cells are cultured in arginine-containing or arginine-free media, and cell viability, colony formation, cell cycle, and apoptosis are detected. The sensitivity of ASS1-deficient cells to arginine deprivation is verified, and therapeutic targets are identified. The enzyme activity, substrate specificity, and immunogenicity of human ARG I / II, mycoplasma ADI, and Pseudomonas ADI are compared. High-activity, low-immunogenic enzymes or enzyme combinations (such as human ARG I + microbial ADI) are selected, and site-directed mutagenesis or codon optimization is performed on microbial ADI to increase expression levels.

The purpose of the present invention is to provide: a plant having an increased yield of storage tissue; and a method for producing the plant. It was discovered that the yield of storage tissue in a plant can be increased by expressing, in the storage tissue of the plant, a sucrose synthase having a substrate specificity to adenosine diphosphate.

The invention discloses a polypeptide asparagine ligase VdiPAL1 and an application of the polypeptide asparagine ligase VdiPAL1. The polypeptide asparagine ligase VdiPAL1 is newly found from viola plants, 12 mg / L zymogen protein is obtained through expression of an escherichia coli prokaryotic system, and compared with homologous enzymes VyPAL2 and VdiPAL1 which are widely applied at present, the polypeptide asparagine ligase VdiPAL1 has the advantages of high expression, high catalytic efficiency and substrate specificity and has good development and application potential. The invention also shows the value of the VdiPAL1 for guiding homologous enzyme modification, and particularly discloses the mutant protein VyOPT of the polypeptide asparagine ligase VyPAL2 and a coding cDNA (complementary deoxyribonucleic acid) sequence of the mutant protein VyOPT. The invention also discloses a mutant VyOPT, which comprises five mutations obtained by modification guided by the VdiPAL1. Compared with the expression quantity of the natural VyPAL2, which is about 0.1 mg / L, the expression quantity of the VyOPT reaches 12 mg / L.

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 discloses an ingenol ABC transport protein ElABCG39 as well as a coding gene and application thereof. The protein is positioned in a cytoplasmic membrane, and expression of the protein in yeast proves that the protein has ingenol transmembrane efflux transport function, improves the ingenol tolerance of the yeast, and proves that the ElABCG39 has certain substrate specificity in the yeast. The ingenol ABC transporter ElABCG39 can be used for producing ingenol by fermentation of microorganisms or plant tissues.

The application provides a hot liquid source quorum quenching strain Shiquan Haeyuan bacteria and application thereof, the strain screened can inhibit pathogenic bacteria and is used for preparing bacteriostatic drugs; the preservation number of the Shiquan Haeyuan bacteria screened is CCTCC NO: M 20251224.The application also provides an AHL degradation enzyme Aii937 protein separated from the YESM3 strain, characterized in that the amino acid sequence of the protein is SEQ ID NO: 2.The YESM3 strain of the application can block the QS path of pathogenic bacteria through AHL lactonase Aii937, reduce the secretion of virulence factors of pathogenic bacteria; the strain YESM3 has no pathogenicity.AHL lactonase does not have broad spectrum, has strong substrate specificity, and only degrades C6-HSL and 3OC-C 14 -HSL.

The current invention relates to a computer-implemented method for ranking and / or identifying substrate-enzyme combinations according to the likelihood that a substrate undergoes catalytic conversion by an enzyme or inhibits the enzyme; to a computer system; and a computer program product. The invention also relates to uses of the method for predicting the substrate range of an enzyme X, for screening enzyme variants with altered substrate specificity and / or catalytic activity in relation to a substrate of interest, and for predicting biological phenotypes associated with an enzyme X.

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

The present invention provides an in vitro method for detecting cell-cell interactions and related kits and uses, wherein the method comprises: (a) at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically different; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; The first and second secondary binders are conjugated or fused to DNA sequences, wherein the DNA sequences are different and connected to form a circle and are amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binder is labeled with a FRET donor and the second secondary binder is fused to an enzyme, which reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich portion on the surface of a molecule adjacent to the enzyme; wherein the method comprises: i. contacting an isolated sample containing cells with the at least two primary binders; ii. contacting the sample with the at least two secondary binders; iii. performing a washing step; iv. detecting the interaction between the secondary binders.

The application provides a ketol-acid reductoisomerase mutant and application thereof in construction of a genetically engineered bacterium with high D-pantothenic acid yield. In order to solve the technical problem of low yield of D-pantothenic acid synthesized by microbial fermentation in the prior art, the application uses semi-rational design of enzymes to obtain a ketol-acid reductoisomerase mutant with substrate specificity, and applies the mutant to construction of a genetically engineered bacterium with high D-pantothenic acid yield. The application also uses the ketol-acid reductoisomerase mutant to construct a genetically engineered bacterium with high D-pantothenic acid yield by CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04 %, and compared with a starting strain, the accumulation amount of valine in the obtained fermentation liquor is almost unchanged compared with a control, but the accumulation of branched-chain amino acid isoleucine is less, achieving modification of the substrate specificity of the ketol-acid reductoisomerase.

This invention discloses a polyethylene terephthalate (PET) hydrolase, PmPETase, and its applications. The hydrolase PmPETase is a protein with the amino acid sequence shown in SEQ ID NO:1. This invention constructs an expression system suitable for PmPETase, obtains a recombinant enzyme protein with catalytic activity, and comprehensively analyzes its substrate specificity, catalytic properties, and potential plastic degradation ability. This provides a new theoretical basis and application reference for the development and utilization of PET degrading enzymes and green treatment technology for polymer materials.

The invention discloses a transaminase mutant and its application in the preparation of (S)-chroman-4-amine, the transaminase mutant is obtained by directed evolution of wild-type transaminase, including any one of C60W, V242A, and L272M mutations or a combination thereof. The transaminase mutant disclosed in the present invention can substrate-specifically identify chroman-4-one, efficiently synthesize (S)-chroman-4-amine, and achieve a significant increase in stereoselectivity and yield; the invention discloses a product for preparing (S)-chroman-4-amine, the product including a transaminase mutant or its encoding gene or corresponding expression vector or corresponding recombinant cell with high yield and high stereoselectivity, the temperature conditions and pH conditions of the product application are easy to achieve, with an industrial application basis; the invention also discloses a preparation method of the mutant and its application, which contributes to the industrial production and application of (S)-chroman-4-amine.

The invention provides a cellobiose phosphorylase mutant capable of synthesizing laminobiose, which is characterized in that substrate specificity of cellobiose phosphorylase from thermophilic archaea is changed, wild cellobiose phosphorylase with an amino acid sequence as shown in SEQ ID NO: 2 is used as starting protein, multiple rounds of site-directed mutagenesis are carried out on the starting protein through rational design, and the cellobiose phosphorylase mutant capable of synthesizing laminobiose is obtained. The mutant and a wild type CBP amino acid sequence SEQ ID NO: 2 have at least 90% identity, the mutant has the activity of catalyzing glucose and glucose 1-phosphoric acid to synthesize laminobiose, the mutant is super-heat-resistant laminobiose phosphorylase, the dominant mutant is applied to an in-vitro multi-enzyme system for catalytically producing laminobiose by taking starch as a substrate, and the yield of the laminobiose is improved. And the laminobiose can be synthesized at high temperature, high conversion rate and high efficiency.

The application provides a glucose dehydrogenase mutant pTrc99A-6, a preparation method and application thereof, and blood glucose detection test paper, and relates to the technical field of biology.The glucose dehydrogenase mutant pTrc99A-6 provided by the application has an amino acid sequence as shown in SEQ ID NO.1.The glucose dehydrogenase mutant has better stability than a wild type, is more specific in substrate specificity, and can be used for preparing a blood glucose detection product.

The invention relates to the technical field of catecholamine detection, in particular to a copper-doped carbon dot with similar catechol oxidase activity as well as a preparation method and application of the copper-doped carbon dot. The copper-doped carbon dot provided by the invention can be used as a nano-enzyme for specific catalytic oxidation of catecholamine, so that catecholamine is detected by a colorimetric method. Copper atoms in the copper-doped carbon dots are dispersed in a monatomic form, and a local coordination structure is Cu-N3O which is similar to metal active sites in natural enzyme; according to the structure, the electron transfer capability and the redox activity of a Cu center are remarkably enhanced, so that the structure shows efficient catalytic activity and substrate specificity in a catecholamine oxidation reaction.

This invention provides libraries of Botulinum neurotoxin A (BoNT / A) protease variants for stepwise evolution of the enzyme to generate BoNT / A variants that can specifically cleave a desired cleavage site in a target protein. Related methods for performing stepwise evolution of BoNT / A with the BoNT / A variant libraries and simultaneous stepwise evolution of a desired substrate sequence in a target protein are also provided by the disclosure. Additionally provided in the disclosure are specifically evolved BoNT / A variant enzymes and conjugate proteins that specifically degrade intrinsically disordered proteins (IDPs) that are involved in human deceases, e.g., a-Synuclein. Polynucleotide sequences encoding the engineered BoNT / A proteases, expression vectors and related pharmaceutical compositions are also provided in this disclosure. Further encompassed by the invention are therapeutic methods that utilize the engineered BoNT / A enzymes in the treatment of various synucleinopathies.

The invention belongs to the technical field of biology, and particularly relates to recombinant glucoside hydrolase and application thereof in preparation of rare ginsenoside F2. The recombinant glucoside hydrolase provided by the invention is a further modified product on the basis of specific fusion protein. The recombinant glucoside hydrolase provided by the invention can efficiently catalyze a reaction under a relatively mild condition, and has the characteristics of strong substrate specificity and high conversion rate. In the aspect of reaction efficiency, the enzyme shows excellent catalytic performance: a large amount of ginsenoside Rb1 substrate can be converted into the rare ginsenoside F2 in a short time, the performance is excellent, and the efficient and high-purity preparation of the rare ginsenoside F2 is facilitated.

The present application belongs to the technical field of biotechnology and enzyme engineering, and relates to a molecular modification method for improving substrate specificity of aldehyde ketone reductase, which is achieved by constructing a substrate library of aldehyde ketone reductase AKR13B3 and analyzing kinetic parameters, determining a mutation site by performing loop structure analysis and amino acid sequence alignment on a complex structure of aldehyde ketone reductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON, and screening a mutant with increased substrate specificity, the present application designs the geometric shape of the entrance loop region of the substrate binding pocket of AKR13B3 to change substrate preference and realize the increase of catalytic activity of the enzyme on specific substrates, in addition, the present application provides a feasible scheme for designing aldehyde ketone reductase with good substrate specificity, and provides great hope for developing high-efficiency enzyme preparations.

The invention discloses a DNA methylation level detection method based on time-of-flight mass spectrometry. According to the DNA methylation level detection method based on the time-of-flight mass spectrometry, the SAP purification step is omitted, the experiment operation time is shortened, and the experiment process is simplified; according to the method disclosed by the invention, a PCR reaction system is optimized, and an anti-pollution system of dUTP + UDG enzyme is introduced, so that the success rate of an experiment is remarkably increased, and the interference of the environment on the experiment is reduced; besides, system optimization is carried out in a mass spectrum on-machine pretreatment stage, and Mn < 2 + > is added, so that the substrate specificity of polymerase can be reduced, the binding uniformity of a modified substrate in a polymerization process is improved, and the instability of subsequent experiments caused by incomplete transcription is reduced. The method is simple and convenient to operate, has the advantages of high efficiency, high sensitivity and high accuracy, is suitable for various scientific research and has very high clinical application value.

Provided is a technique that can further improve substrate specificity to an L-form during L-menthol and / or L-menthol ester production. A polypeptide that: has A120G, Q88A, Q88G, Q88D, Q88M, Q88L variants of a polypeptide comprising the amino acid sequence represented by sequence no. 1, and in said variants, has a random different moiety other than an amino-acid residue into which a substitution has been introduced; has esterification activity for L-menthol and / or hydrolyzing activity for L-menthol ester, and improves substrate specificity to L-menthol and / or L-menthol ester compared to a polypeptide comprising the amino acid sequence represented by sequence no. 1. During L-menthol and / or L-menthol ester production, the polypeptide can further improve the optical purity of the product.

The present invention provides an enzyme useful for establishing an excellent N-acyl-amino group-containing compound production system, and the like. More specifically, the present invention provides a modified enzyme comprising: (A) a modified amino acid sequence consisting of an amino acid sequence comprising mutations of one or more certain amino acid residues in an amino acid sequence of a wild type enzyme having an N-acylation activity; (B) an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several additional amino acid residues in the modified amino acid sequence; or (C) an amino acid sequence comprising additional mutations of one or more amino acid residues in the modified amino acid sequence and having 90% or more identity to the modified amino acid sequence, having an N-acylation activity, and having an improved N-acylation activity to L-glutamic acid or L-aspartic acid or an improved substrate specificity to L-glutamic acid as compared with the wild type enzyme, and the like.