105 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 genetically modified cell, and belongs to the technical field of biological genetic engineering. The invention provides a genetically modified cell, which contains a heterologous nucleic acid sequence of an alpha-1, 3-fucosyltransferase polypeptide. The alpha-1, 3-fucosyltransferase polypeptide further comprises but is not limited to derived polypeptides M1-M21 of the polypeptide with the amino acid sequence as shown in SEQ ID NO: 1, and compared with the polypeptide as shown in SEQ ID NO: 1, the 3-FL catalytic synthesis capability of the derived polypeptides and the specificity of a catalytic substrate are both improved.

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 discloses a carboxylic acid reductase DeCAR recombinant plasmid and application of the carboxylic acid reductase DeCAR recombinant plasmid in preparation of aldehyde spices, belongs to the technical field of bioengineering, and further provides a recombinant bacterium containing the recombinant plasmid, and the carboxylic acid reductase DeCAR is further produced by using the recombinant bacterium, or the recombinant bacterium is directly applied to production of the aldehyde spices. Compared with other biocatalysts for preparing aldehydes, the carboxylic acid reductase and the recombinant bacteria thereof provided by the invention have the advantages of wide substrate spectrum, high catalytic activity, strong substrate specificity and the like, and have wide application prospects in industrial application.

The invention relates to an inositol dehydrogenase mutant and application of the inositol dehydrogenase mutant in synthesis of D-chiral inositol. The invention discloses an inositol dehydrogenase mutant, nucleic acid for coding the inositol dehydrogenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, and a recombinant inositol dehydrogenase mutant catalyst. The invention also discloses an application of the inositol dehydrogenase mutant or the recombinant inositol dehydrogenase mutant catalyst in catalysis of biotransformation of inositol to synthesize D-chiral inositol. Compared with wild-type inositol dehydrogenase and literature reports, the inositol dehydrogenase disclosed by the invention has the characteristics of high enzyme activity and strong substrate specificity, and has the advantages of mild reaction conditions, small coenzyme addition amount, economy, environmental protection and the like when being used for catalytically preparing D-chiral inositol. Therefore, the inositol dehydrogenase mutant disclosed by the invention has a very good application prospect in actual production of the health care medicine D-chiral inositol.

The invention relates to peptides comprising or consisting of an amino acid sequence of 5 to 50 amino acids which has, in N- to C-terminus orientation, the amino acid sequence X5X6X7X8X9, where each of the five residues X represents an amino acid of a particular group, and / or the amino acid sequence has at least 85% sequence identity to one of the amino acid sequences mentioned in SEQ ID NOs: 1 - 17.

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.

Describes herein is a lipase comprising a peptide sequence with at least 80% sequence similarity to SEQ ID NO: 3 and at least one mutation of SEQ ID NO: 3 selected from Q9, N11, A14, A23, K24, N26, A30, G31, A32, T35, R37, I40, S39, M58, G59, E87, G91, N92, I93, D96, D102, S105, G106, K108, D111, S115, N122, T123, T125, R133, A152, D158, G161, N162, F169, A180, A187, L193, R209, E210, S216, T223, T226, L227, G245, N248, P250, N251, T252, A256, L259, and any combinations thereof, or the peptide sequence comprises SEQ ID NO: 3. The lipase may be mixed with a fatty acid or a triacylglycerol and an alcohol under suitable conditions to produce a fatty acid ester. The substrate specificity of a Thermomyces dupontii lipase (TDL) may be enhanced by introducing a L259 mutation into a mature peptide sequence of TDL and expressing the L259 mutant.

The present invention discloses a genetically engineered bacterium expressing oligosaccharide debranching enzyme and its application, belonging to the technical field of microbial engineering. The present invention provides an oligosaccharide debranching enzyme with a specific base sequence, and successfully heterologously expresses the oligosaccharide debranching enzyme in Escherichia coli. By using the method of the present invention, the catalytic activity of the crude enzyme solution of the expressed oligosaccharide debranching enzyme can reach 258.83 U / mL. The oligosaccharide debranching enzyme provided by the present invention is a novel oligosaccharide debranching enzyme from a new source, which has not been reported before, has a high specific enzyme activity, and strong substrate specificity. The oligosaccharide debranching enzyme of the present invention can debranch short chain segments with DP<6 in maltodextrin, filling the blank in the substrate specificity of commonly used debranching enzymes at present, and has high potential application prospects in industrial fields such as starch sugar, resistant starch, beer production and ethanol fuel.

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 present invention discloses a method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid, which relates to the technical field of bioengineering. The method for regulating the transport activity of a strain to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues and the tolerance of the strain to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues is to transfer the LeuE gene into Escherichia coli to obtain a recombinant Escherichia coli strain with corresponding functions. The present invention discloses for the first time the application of LeuE protein and LeuE gene in regulating the transport activity of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues and the tolerance of Escherichia coli to 1-aminocyclopropane-1-carboxylic acid and / or methionine analogues. The present invention discloses for the first time that LeuE is the best candidate gene for constructing ACC-producing bacteria, and its substrate-specific transport meets the purpose of enhancing cell ACC tolerance without affecting precursor supply.

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 belongs to the technical field of biomaterial chemistry, and particularly discloses a preparation method and application of functional escherichia coli for expressing laccase. The method comprises the following steps: firstly, according to a full-length cDNA (complementary deoxyribonucleic acid) sequence of a laccase gene, integrating a cotA target gene into a vector plasmid and constructing a recombinant expression plasmid; and introducing the recombinant expression plasmid into escherichia coli to prepare functionalized recombinant escherichia coli. Wherein the active center of the laccase contains copper ions [Cu (II)]. The binding domain of the laccase is mainly composed of histidine, cysteine, methionine and the like, and the laccase can utilize molecular oxygen as an electron acceptor to oxidize various phenolic and non-phenolic compounds and increase active sites except cytochrome c in escherichia coli. Meanwhile, when the substrate is oxidized, the method has the advantages of substrate specificity, mild reaction conditions, harmlessness to the environment and the like, molecular oxygen in air is used as an electron acceptor, only water is generated as a byproduct, the principle of environmental protection is met, and the oxygen reduction capacity of the microbial fuel cell is accelerated.

The invention belongs to the technical field of glutamic acid content detection, and particularly relates to a method for detecting the glutamic acid content based on glutamine synthetase isoenzyme and application of the method. The invention relates to a glutamine synthetase isoenzyme TaGS1; the invention discloses application of glutamine synthetase TaGS1, TaGS2, TaGS2, TaGS2, TaGS2, TaGS2, TaGS2, TaGS2; the amino acid sequence of 3 is as shown in SEQ ID No.1 in the table. The method has the advantages that (1) the TaGS1 is disclosed through a multi-dimensional experiment system; the invention relates to catalytic characteristics and application potential of TaGS1, TaGS2, TaGS2, TaGS2; the temperature adaptability and substrate specificity of the enzyme 1, 2, 3 show high consistency with that of an HPLC (High Performance Liquid Chromatography) detection method in a cross validation experiment; (2) the method for detecting the content of glutamic acid is obviously superior to other glutamate dehydrogenase detection methods, for example, GDH is often interfered by alpha-ketoglutaric acid; and (3) the method for detecting the content of glutamic acid does not need complex sample pretreatment or expensive instruments, the single detection cost is about 10% of that of an HPLC method, and the flux is high.