18 results about "Substrate channeling" patented technology

The invention relates to the fields of genetic engineering and microbial fermentation, and discloses an ergothioneine synthetic fusion enzyme, and a coding gene, a recombinant vector and a recombinant strain thereof, and applications of the ergothioneine synthetic fusion enzyme and the coding gene, the recombinant vector and the recombinant strain. The ergothioneine synthetic fusion enzyme is an enzyme with an amino acid sequence as shown in SEQ ID NO: 1. The original strain of the recombinant strain is Yarrowia lipolytica, and the original strain of the recombinant strain is Yarrowia lipolytica; the method for producing the ergothioneine through fermentation comprises the following steps: inoculating a recombinant strain containing the ergothioneine synthesis fusion enzyme into a fermentation culture medium for fermentation. The ergothioneine synthesis fusion enzyme can promote a substrate channel while exerting the efficient expression and catalysis effects of ergothioneine synthetase, reduces the accumulation of intermediate metabolites, relieves the cell burden, and further improves the fermentation yield of ergothioneine.

The invention discloses a method for improving L-histidine production efficiency of recombinant escherichia coli based on a substrate channel strategy and application, and belongs to the technical field of biology. The invention provides a recombinant escherichia coli, the recombinant escherichia coli is fermented in a 5L fermentation tank at the temperature of 37 DEG C, the pH value of 7.0-7.2 and the dissolved oxygen of 25-35% for 36 hours, the yield of L-histidine reaches 57.8 g / L, the average production intensity is 1.6 g / L / h, and the conversion rate is 0.23 g / g. Therefore, the L-histidine produced by fermenting the recombinant escherichia coli has the advantages of short fermentation period, high production efficiency and easiness in operation.

The invention relates to the field of enzyme engineering, in particular to a mutant of nylon degrading enzyme and application of the mutant. The mutant of the nylon degrading enzyme is obtained by mutation at one or more key sites through stability modification guided by machine learning, substrate channel inlet loop region modification and tetramer interface modification on the basis of a wild type nylon degrading enzyme NylCp2-TS as shown in SEQ ID NO: 1; the mutant enzyme can efficiently degrade nylon materials in various forms, including films, particles and industrial fibers of nylon 6 and nylon 66, and has a wide application prospect in the field of nylon waste biodegradation.

The application discloses a kind of biological immobilized enzyme based on yeast surface display technology and the application of synthesis 2-phenylethanol, using yeast surface display technology, for the first time with Saccharomyces cerevisiae surface protein a-lectin as anchor protein, the key enzyme (transaminase, decarboxylase, ethanol dehydrogenase) involved in 2-PE producing Ehrlich pathway in Saccharomyces cerevisiae is separately or jointly carried out biological fixation, enhance substrate channel effect and proximity effect, in vitro one-step catalysis L-phenylalanine synthesis 2-PE, provide yield.The method for fixing multiple enzymes of Ehrlich pathway in the application can be used to reconfigure the biosynthesis pathway of other natural products in vitro, to provide feasible scheme for the rational design of cell factory.

The application provides a method for analyzing the structure and dynamic behavior of kappa-carrageenase in a solution, which utilizes the HolE to analyze the changes of the substrate channel of wild-type kappa-carrageenase and mutant N205C-G239C; utilizes the JEM-1400 Flash transmission electron microscope to observe the reaction of wild-type kappa-carrageenase and mutant N205C-G239C in an environment containing a substrate kappa-neo-carrageenan tetrasaccharide under an electron energy of 100 keV and a dose rate; wherein the sample is photographed with an exposure time of 0.1 seconds, and the image is processed and analyzed by using ImageJ. The method utilizes the mutant with higher stability and designs the observation system and conditions, can reveal the structure and function relationship of the protein, research the dynamic behavior of the protein, and provides a theoretical basis for the application of liquid phase electron microscopy in the field of protein observation.

The invention discloses a self-assembly double-enzyme compound, a preparation method and application of the self-assembly double-enzyme compound in synthesis of a prednisone intermediate, and the self-assembly double-enzyme compound is obtained by fusing 11 alpha-hydroxylase and 1, 2-dehydrogenase on the basis of a SpyCatcher / SpyTag system. According to the method, a self-assembled double-enzyme compound of 11alpha-hydroxylase and 1, 2-dehydrogenase is constructed through a SpyCatcher / SpyTag system, 17alpha-hydroxyprogesterone is taken as a substrate, double-enzyme cascade one-step catalytic reaction is realized, the reaction process is simplified, and separation and purification operation of an intermediate product is omitted. The self-assembled double-enzyme compound constructed by the invention forms a high-efficiency substrate channel, accelerates the rapid transfer of an intermediate from a catalytic activity center of a first enzyme to a catalytic activity center of a second enzyme, improves the catalytic efficiency, is suitable for industrial application, and has a conversion rate of 99.6%.

The invention relates to a protein scaffold mediated multienzyme cascade system and application thereof in preparation of D-tagatose. The protein scaffold mediated multi-enzyme cascade system provided by the invention comprises fructokinase, D-tagatose-6-phosphate 4-epimerase and D-tagatose-6-phosphate phosphatase, and the protein scaffold mediated multi-enzyme cascade system comprises fructokinase, D-tagatose-6-phosphate 4-epimerase and D-tagatose-6-phosphate phosphatase, the D-tagatose-6-phosphoric acid 4-epimerase and the D-tagatose-6-phosphoric acid phosphatase are immobilized on the protein support SH3, and the D-tagatose-6-phosphoric acid 4-epimerase is immobilized on the protein support SH3. According to the system, a plurality of key enzyme spaces in a reaction path are positioned on a protein scaffold through a protein-protein interaction structural domain (SH3), so that a substrate channel and a spatial proximity effect are enhanced, and the yield of D-tagatose is increased. According to the method disclosed by the invention, the yield of the D-tagatose is 3.2-7.7 times that of the original D-tagatose by optimizing reaction conditions.

The present invention belongs to the technical field of enzyme engineering, and specifically relates to a chitosanase mutant with improved catalytic activity by changing the acidity and alkalinity of the substrate channel. Based on the substrate channel-regulated Bacillus subtilis chitosanase BsCsn46A, the 49th alanine of the chitosanase with the amino acid sequence shown in SEQ ID NO: 1 is mutated to the basic amino acid lysine, and the mutant is A49K. Compared with the wild-type chitosanase, the chitosanase mutant of the present invention uses chitosan as a substrate, and its catalytic activity is 1.13 times higher than that of the wild-type.

The invention discloses a genetically engineered bacterium for producing D-pantothenic acid as well as a construction method and application of the genetically engineered bacterium, wild type C. glutamicum ATCC 13032 is taken as a chassis bacterium, an ilvE gene, an avtA gene, an ilvA gene and a pqo gene in an original strain genome are knocked out, ilvBNC from different sources and mutants thereof are screened, an optimized mutant ilvBNC-VWBGTG / ATG is determined, the gene cluster and promoters Ptuf and Pefu are connected and integrated to a strain DPAe-2, and the D-pantothenic acid is produced. An engineering strain DPAg-11 capable of efficiently accumulating KIV is obtained, then a promoter of an aceE gene is replaced by PdapA A16, KIV accumulated by the obtained engineering strain DPAg-15 reaches 3.99 g / L, panB, panC, panE or ilvC from different sources are screened, it is determined that BspanBC + EcpanE has the best effect, BspanBC + EcpanE is constructed on plasmids and then converted into various engineering strains, a promoter of an EcpanE gene is replaced by PdapB, and KIV accumulated by the engineering strain DPAg-11 and PdapA A16 reaches 3.99 g / L. The method has the advantages that the method is simple and easy to implement, a substrate channel can be built between dihydroxy acid dehydratase (DHAD) and KPR by polypeptide pair RIAD-RIDD, D-PA produced by the finally obtained strain reaches 3.21 g / L and is increased to 97.0 times that of DPA produced by a wild strain, and production of D-PA can be effectively promoted by the strategy of the built substrate channel.

The application discloses a cytochrome P450 enzyme mutant and a method for catalytically synthesizing betulinic acid, and relates to the technical fields of genetic engineering and biological enzyme catalysis.The reported RoCYP01 from Litsea cubeba is used as a mutation template, site-directed mutation is carried out, the substrate channel for the entry and exit of the enzyme catalytic center of a substrate and a product is changed, and a mutant with improved catalytic efficiency for the synthesis of betulinic acid from betulonic acid is obtained.Under the same catalytic conditions, the catalytic activity of the mutant is increased by 198% compared with the wild type, which has important guiding significance for the construction of a betulinic acid yeast engineering strain.

The invention provides a novel reverse aldolase and reductase cascaded Bi-BDO independent synthesis pathway, and relates to the technical field of bioengineering, the pathway constructs a non-natural metabolism module composed of a modified reverse aldolase RA variant, 4-hydroxybutyraldehyde reductase AHR and a coenzyme cyclic regeneration enzyme, the RA variant performs hydrophobic modification on a Lys146 site, and the reverse aldolase and reductase cascaded Bi-BDO is obtained. Aldol condensation of a non-phosphorylated substrate hydroxyacetaldehyde is realized to generate a C4 intermediate, RA and AHR are physically anchored by utilizing an artificially synthesized scaffold protein, toxicity of the intermediate is avoided through a substrate channel effect, the intermediate is instantly reduced into 1, 4-butanediol, engineering bacteria of aldA and gapA genes are knocked out, a precursor is generated by utilizing a xylose way, and the 1, 4-butanediol is obtained. The method has the advantages that the method is simple in operation, the reduction reaction is driven by in-situ regeneration of NADPH by adding formate, finally, the high-purity product is obtained through macroporous adsorption resin enrichment and vacuum rectification, the TCA circulation decarboxylation step is omitted, the carbon conversion rate is close to the theoretical limit, effective decoupling of production and growth is achieved, and the yield and purity of Bi-BDO are remarkably improved.

The invention discloses a 4, 3-alpha-glycosyltransferase mutant for regulating and controlling product specificity, and belongs to the technical field of enzyme engineering and starch modification. According to the method, key amino acid residues are determined through a substrate channel and conservative analysis, glutamine at the 782nd position of Lf2970GtfB 4, 3-alpha-glycosyl transferase is mutated into proline, serine, aspartic acid, asparagine, leucine, phenylalanine or tryptophan, and then transglutaminase at the 782nd position of the Lf2970GtfB 4, 3-alpha-glycosyl transferase is mutated into glutamine at the 782nd position. By changing the interaction between the enzyme and the substrate, the regulation and control of the specificity such as the bond type proportion and the molecular weight of the product are realized. The mutant obtained by the invention can realize the preparation of alpha-glucan with multi-bond proportion and molecular weight distribution, and provides an effective enzyme tool for the preparation and structural regulation of functional alpha-glucan.

The present invention discloses a nitrile hydratase mutant with enhanced activity, and specifically relates to the modification of the amino acid motif of the nitrile hydratase substrate channel for the preparation of acrylamide, which belongs to the field of bioengineering technology. The present invention finds that the amino acid residue at position 177 on the β subunit of Pt-NHase may be in the key structural domain of nitrile hydratase and play an important role in the catalytic activity of nitrile hydratase. The present invention provides a Pt-NHase mutant, wherein the mutant is mutated to isoleucine at position 177 on the β subunit relative to the wild type, to obtain mutant I177K. For acrylonitrile catalysis, the enzyme activity of the mutant is significantly improved compared with the wild type, which is increased by more than 1.4 times.

The invention discloses a 3D nano double-enzyme cascade reactor based on a Dps scaffold and application of the 3D nano double-enzyme cascade reactor based on the Dps scaffold. ATA117 is connected with SpyCatcher and Dps so that the ATA117 and the SpyCatcher can be fixed to the Dps protein scaffold, a fusion protein SpyCatcher-Dps-ATA117 is obtained, then ADH is connected with SpyTag to obtain a fusion protein SpyTag-ADH, a SpyTag / SpyCatcher system further promotes double enzymes to complete self-assembly, and the stable and efficient 3D nano double-enzyme cascade reactor is formed. The 3D nano multi-enzyme cascade reactor is used for preparing R-BPA by asymmetrically converting BPO in an oil-water two-phase system, and the reaction yield is further improved by providing a space structure and constructing a more efficient substrate channel and improving the stability and reuse frequency of a catalyst.

This invention discloses an engineered astaxanthin-producing probiotic yeast strain, its construction method, and its applications, belonging to the field of synthetic biology. The engineered strain uses β-carotene-producing *Saccharomyces cerevisiae* as the substrate cell. Its genome integrates a fusion gene selected through adaptability screening. This gene is composed of *BDC263crtW* from *Saccharomyces cerevisiae*, a flexible linker peptide, and *AspcrtZ* from *Alcaligenes aeruginosa*, linked sequentially. Simultaneously, based on systematic expression screening of multiple key genes, the endogenous acetyl-CoA synthesis key gene ACS1 was optimized and overexpressed, while the acetic acid metabolism negative regulatory gene YPL062W was knocked out. The construction method includes plasmid construction of the aforementioned fusion expression module and ACS1 expression module, and targeted integration into the genome. This invention significantly improves astaxanthin synthesis levels and enhances the engineered strain's tolerance to the simulated gastrointestinal environment and its antioxidant capacity through enzyme source optimization and substrate channel effects. This engineered strain can be directly used as a functional live bacteria preparation for food and feed additives, with broad prospects for industrial application.

The invention discloses a molecular docking-based glycosyl transferase substrate catalytic specificity multi-dimensional evaluation method, system, equipment and medium, and belongs to the technical field of protein structure simulation evaluation, and the method comprises the following steps: determining glycosyl transferase, and predicting a corresponding protein three-dimensional structure; performing molecular docking simulation based on the predicted protein three-dimensional structure; the catalytic specificity of different substrates is evaluated according to the difference between receptor substrate channels and ligands; and displaying a docking result through visual software. According to the method, a set of complete standardized operation program is formed, a clear methodology guide is provided for similar research, on the basis of traditional docking scoring, three-dimensional space size measurement analysis of the acceptor and the ligand is creatively used as a key screening index, multi-dimensional evaluation from the bonding strength to the space suitability is achieved, and the evaluation accuracy is improved. A new visual angle is provided for understanding an enzyme substrate recognition mechanism.

The invention discloses a recombinant Yarrowia lipolytica engineering strain for producing ergosterol and application of the recombinant Yarrowia lipolytica engineering strain, and belongs to the technical field of genetic engineering and bioengineering. According to the method, sterol synthesis route genes are screened, the speed limiting step is determined, high-yield ergosterol is preliminarily achieved through combined expression, the flow direction of carbon metabolic flow is further pulled through lipid droplet engineering and cell area chamber engineering, and the yield of ergosterol is increased. On the basis of enzyme modification engineering, the catalytic efficiency of the ERG11 on the lanosterol is improved through substrate channel engineering and proton-dependent catalytic path construction. Through multi-copy integration of the genes in the speed limiting step, the yield of ergosterol in a shake flask of the constructed engineering strain reaches 433.1 mg / L, and the yield of ergosterol in a 5L fermentation tank system reaches 4.58 g / L.

The invention belongs to the technical field of green chemistry, and particularly relates to a method for bi-directionally synthesizing chiral lactam through bi-enzyme cascade-chemical catalysis. According to a methylpiperidone synthesis route taking 2-methyl glutaronitrile as a raw material, nitrile hydratase is subjected to iterative saturated mutation, substrate channel residues and active site residues are subjected to semi-rational engineering transformation, and 4-cyano valeramide and 4-cyano-2-methyl butyramide are generated with high selectivity. And carrying out cascade reaction on the modified nitrile hydratase and amidase, so as to directionally generate the corresponding mono-cyano carboxylic acid. Then, a Raney nickel catalyst hydrogenation reaction is adopted to treat the whole-cell catalytic solution, and two regioselectivity products can be obtained, namely 5-methyl-2-piperidone (the yield is 96.9%, and the regioselectivity is 98.3%) and 3-methyl-2-piperidone (the yield is 67.9%, and the regioselectivity is 70.4%).