20 results about "Nitrile hydratase" patented technology

The invention discloses a construction method of nicotinamide engineering bacteria for cosmetics, which comprises the following steps: amplifying target DNA, recovering the target DNA, carrying out seamless cloning or CPEC connection on the recovered DNA fragment and a vector, carrying out seamless cloning by using a seamless cloning kit of an all-type gold company, carrying out connection by using a sterile ultrapure water complementing system, and carrying out plasmid construction. The method comprises the following steps: inoculating a liquid culture medium with a strain which is verified to be correct, carrying out plasmid extraction on the obtained bacterial liquid by using an AxyPrep plasmid small extraction kit, carrying out shake flask small test on enzyme activity, thallus propagation and protein expression after chassis strain gene editing, adding a substrate 3-cyanopyridine solution into the crushed thallus for reaction, and carrying out HPLC (High Performance Liquid Chromatography) detection on nicotinamide. According to the method, the nitrile hydratase is expressed through metabolism of microorganisms, conversion from 3-cyanopyridine to nicotinamide is achieved through one-step reaction after cell disruption, the enzyme activity of the nitrile hydratase is improved, and meanwhile the generation amount of the by-product nicotinic acid is reduced.

The invention belongs to the technical field of green chemistry, and particularly relates to a method for modifying a flexible ring region of nitrile hydratase and application of the method. The specific method comprises the following steps: carrying out double mutation on nitrile hydratase sites alpha D38 and alpha I152, or carrying out double mutation on nitrile hydratase sites alpha E157 and beta G197, or carrying out quadruple mutation on the nitrile hydratase sites alpha D38, alpha I152, alpha E157 and beta G197, so as to construct the nitrile hydratase mutant with improved thermal stability. The thermal stability of the nitrile hydratase modified by mutation is greatly improved. The mutant nitrile hydratase can keep the conformation and activity for a longer time, the resistance to external thermal interference is enhanced, and the half-life period for catalyzing an adiponitrile reaction is remarkably prolonged, which is the inherent excellent attribute of the mutant nitrile hydratase as a catalyst, and the mutant nitrile hydratase has important reference significance for research on improvement of the thermal stability of the nitrile hydratase.

The application discloses a nitrile hydratase mutant and application thereof in synthesis of nicotinamide, and belongs to the field of biological catalysis. Am NHase-beta-I106A / K19D / gamma-R8S as a catalyst, 300 g / L of 3-cyanopyridine is converted to obtain 348.5 g / L of nicotinamide under the conditions of 30 DEG C and pH 7.0 for 5 h, while the unmodified nitrile hydratase Am The nitrile hydratase catalyzes generation of nicotinamide to be only 206.3 g / L, and the conversion rate is increased from the original 58.6 % to 99 %. The application further provides a process for preparing immobilized cells from whole cells, which can significantly reduce generation of nicotinic acid byproducts while maintaining high conversion rate, is suitable for industrial amplification production, and provides an excellent enzyme catalyst and process for efficient and green synthesis of nicotinamide.

The present invention relates to a variant nitrile hydratase which is engineered to comprise greater activity and / or stability, nucleic acids encoding said nitrile hydratase, and microbia engineered to express said novel nitrile hydratase. Additionally the invention relates to the use of this nitrile hydratase and microbia which express said nitrile hydratase as a biocatalyst, particularly in methods for producing an amide compound from a nitrile compound, preferably for use in converting acrylonitrile to acrylamide.

The application discloses an engineered nitrile hydratase and application thereof. The engineered nitrile hydratase is based on a wild-type nitrile hydratase and comprises a random coil domain located near an active center and having a sequence of SEQ ID NO. 1, wherein methionine in the random coil domain is replaced by any one of glycine, leucine, isoleucine, glutamine, threonine, glutamic acid, arginine and lysine. The engineered nitrile hydratase with any one of the above mutations has higher enzyme activity and improves catalytic efficiency.

The present invention relates to a variant nitrile hydratase which is engineered to comprise greater activity and / or stability, nucleic acids encoding said nitrile hydratase, and microbia engineered to express said novel nitrile hydratase. Additionally the invention relates to the use of this nitrile hydratase and microbia which express said nitrile hydratase as a biocatalyst, particularly in methods for producing an amide compound from a nitrile compound, preferably for use in converting acrylonitrile to acrylamide.

The invention belongs to the field of bioengineering, and particularly relates to a nitrile hydratase mutant, a mutant encoding gene, a recombinant vector containing the mutant encoding gene, a recombinant genetic engineering bacterium containing the mutant encoding gene and application of the nitrile hydratase mutant in catalytic degradation of nitrile toxic compounds. The nitrile hydratase mutant is obtained by performing single-point or combined mutation on the 48th and 72nd sites of a beta subunit and the 127th site of an alpha subunit of wild type nitrile hydratase. Compared with wild type nitrile hydratase, the nitrile hydratase mutant shows remarkably enhanced catalytic activity and stability when facing nitrile compounds such as ricinine in castor cake meal and 2-cyanobiphenyl in industrial sewage, the specific enzyme activity of the nitrile hydratase mutant is 320 times and 82 times that of the wild type nitrile hydratase, and the nitrile compounds can be efficiently degraded. The method effectively solves the technical problem of detoxification treatment of toxic nitrile compounds in the fields of industry and environmental protection, and has a wide market application prospect.

The application relates to the technical field of biological catalysis, and particularly discloses a catalyst for acrylamide production and a preparation method thereof. The preparation method comprises the following steps: selecting a nitrile hydratase producing bacterium to perform fed-batch fermentation culture, and centrifuging to obtain wet sludge; sodium alginate and nano-titanium dioxide modified by a silane coupling agent are prepared into a composite carrier colloid; a bacterium suspension and the carrier colloid are mixed at a ratio of 1:3, and then are dropped into a calcium chloride solution containing glutaraldehyde to form; cross-linking is performed at low temperature by using glutaraldehyde; after washing, surface activation is performed in two steps; and finally, freeze-drying is performed to obtain solid catalyst particles. The method optimizes the whole process, the prepared catalyst has high reusability and high stability, is easy to separate from a product, and is suitable for continuous operation, solves the core defects of a traditional free bacterium catalyst, and takes into account the catalytic performance and industrial production efficiency.

The invention discloses a high-expression nitrile hydratase fermentation process which comprises the following steps: marking a strain on an LB plate culture medium, and culturing for 16 hours in a constant-temperature incubator at 37 DEG C; selecting monoclonal cells, transferring the monoclonal cells into a 40ml LB culture medium shake flask, and carrying out shake flask culture at 37 DEG C and 220rpm for 8 hours; transferring 40mL of the primary seed solution into a 150ml secondary seed culture medium shake flask, and carrying out shake cultivation for 8 hours at 37 DEG C and 220rpm (revolutions per minute); transferring 150mL of the secondary seed solution into a 10L fermentation tank filled with a fermentation culture medium, carrying out fermentation culture at 37 DEG C until OD60060 or so, cooling to 24 DEG C, and adding an inducer for induction; fermenting and culturing in a tank: detecting the OD value of the fermentation liquor, sampling every 6-8 hours, detecting the OD value and the enzyme activity of the fermentation liquor, and putting in the tank until the enzyme activity does not rise any more. According to the method, fermentation conditions of screened strains are optimized, so that the unit enzyme activity of nitrile hydratase is improved, the use amount of bacterial sludge is reduced, the fermentation period is shortened, and the production cost is reduced.

The invention belongs to the technical field of green chemistry, and particularly relates to a method for modifying a flexible ring region of nitrile hydratase and application of the method. The specific method comprises the following steps: carrying out double mutation on nitrile hydratase sites alpha D38 and alpha I152, or carrying out double mutation on nitrile hydratase sites alpha E157 and beta G197, or carrying out quadruple mutation on the nitrile hydratase sites alpha D38, alpha I152, alpha E157 and beta G197, so as to construct the nitrile hydratase mutant with improved thermal stability. The thermal stability of the nitrile hydratase modified by mutation is greatly improved. The mutant nitrile hydratase can keep the conformation and activity for a longer time, the resistance to external thermal interference is enhanced, and the half-life period for catalyzing an adiponitrile reaction is remarkably prolonged, which is the inherent excellent attribute of the mutant nitrile hydratase as a catalyst, and the mutant nitrile hydratase has important reference significance for research on improvement of the thermal stability of the nitrile hydratase.

The application discloses a nitrile hydratase mutant, a genetically engineered bacterium and application thereof in preparation of acrylamide. Through a semi-rational design method, the molecular of a beta subunit and an alpha subunit are reformed, and the stability of the modified nitrile hydratase is obviously improved. With acrylonitrile as a substrate, the product tolerance of the nitrile hydratase mutant of the application is all enhanced, and the enzyme activity of some mutants is also improved. The initial type NHAM and the mutants B-G41D, B-S51E, B-R55C and B-R98I with the most obvious improvement in enzyme activity and stability are simultaneously subjected to catalytic reaction, and it is found that after the product tolerance of the mutants is improved, the final concentration of the product of B-R98I can reach 523 g / L, which is obviously improved compared with 322 g / L of the initial type, and the application provides convenience for industrial application.

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 relates to an azotobacter and application thereof in repairing co-pollution of insecticides acetamiprid and sulfoxaflor, belongs to the technical field of microorganisms, and provides an Ensifer sp. DA6. Resting cells of the Ensifer sp. DA6 can degrade the insecticides acetamiprid and sulfoxaflor at the same time in phosphate buffer. Immobilized cells of the strain can effectively repair co-pollution residues of insecticides acetamiprid and sulfoxaflor in surface water; the strain can effectively repair the co-pollution residues of two insecticides, namely acetamiprid and sulfoxaflor, and a microbial remediation method is provided for repairing the co-pollution residues of the insecticides, namely acetamiprid and sulfoxaflor; meanwhile, the strain has nitrogen fixation activity, and can be used as a plant growth-promoting bacterium while repairing the co-pollution of acetamiprid and sulfoxaflor; the invention further provides a new nitrile hydratase derived from Ensifer sp. DA6, and the nitrile hydratase can degrade acetamiprid and sulfoxaflor when the acetamiprid and the sulfoxaflor coexist.

The application provides an engineered nitrile hydratase and application thereof. Based on a wild-type nitrile hydratase, the engineered nitrile hydratase comprises that an amino acid residue at a 85 site of an alpha subunit is mutated into any one of methionine, arginine and lysine, wherein the wild-type nitrile hydratase comprises the alpha subunit, the amino acid sequence of the alpha subunit is shown as SEQ ID NO: 1, and the engineered nitrile hydratase provided in the application has good catalytic activity and product tolerance.

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a nitrile hydratase mutant and application thereof, wherein the mutant is obtained by single mutation or multiple mutations of positions 22, 42, 63, 73, 105, 131, 145 and 163 of the amino acid sequence shown in SEQ ID NO. 1. The nitrile hydratase mutant of the application can be expressed in a high-density fermentation host bacterium in a heterologous manner, can improve the substrate catalytic concentration to 1000 g / L, is applied to catalytic synthesis of 4-acetyl-2-methylbenzamide, is green, environmentally friendly, non-polluting, has high catalytic efficiency and high product conversion rate.

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%).

This invention relates to the field of enzyme engineering technology, and discloses a heat-resistant chaperone protein PGI and its application in improving enzyme thermostability. Addressing the difficulties in designing linker peptides in traditional fusion protein construction and the limited applicability of existing stabilization tags, this invention provides a heat-resistant chaperone protein PGI, with the amino acid sequence shown in SEQ ID NO.1. Using the SpyTag / SpyCatcher system, the target enzyme is covalently linked to PGI in vitro, effectively improving the thermostability of the target enzyme while retaining its background catalytic efficiency. Furthermore, this invention reveals for the first time that stabilization efficiency is positively correlated with complex size, exhibiting good applicability to multimeric enzyme systems. Taking nitrile hydratase as an example, after covalent linking with PGI, it exhibits stable and sustained catalytic activity in the catalytic synthesis of acrylamide. This invention provides an effective technical strategy for modifying the thermostability of enzyme preparations.

The invention belongs to the technical field of gene engineering, and particularly relates to a nitrile hydratase mutant and application thereof. On the basis of wild type nitrile hydratase from rhodococcus ruber, a mutation site which has a remarkable influence on the activity of the nitrile hydratase is determined by predicting, screening and verifying key active sites, and the nitrile hydratase mutant with improved activity and application of the nitrile hydratase mutant in production of amide compounds are provided. The nitrile hydratase mutant provided by the invention has the advantages that the unit thallus dry weight enzyme activity reaches up to 3649.95 U / g DCW, which is 142.98% higher than that of a wild type; the specific enzyme activity of the purified nitrile hydratase pure enzyme can reach 3589.44 U / mg, and the efficiency of converting nitrile substances into amide products is improved. When the mutant provided by the invention is used for conversion, 2, 6-difluorobenzamide, nicotinamide, cinnamamide, acrylamide and other amide substances can be rapidly produced, and the mutant has higher space-time conversion rate and shorter conversion time.

This invention belongs to the fields of enzyme engineering and genetic engineering, specifically relating to nicotinamide enzyme mutants and their application in a method for producing nicotinic acid based on a nitrile hydratase-nicotinamide enzyme cascade catalytic system. The invention first identifies nicotinamide enzyme as the rate-limiting enzyme in the reaction pathway. Through directed evolution, a saturated mutant library is created, yielding a series of nicotinamide enzyme mutants with high catalytic efficiency. Among them, the A79S mutant shows the most significant increase in enzyme activity, reaching 759.2 U / mg, a 4.74-fold increase compared to the wild type. Subsequently, using a cascade reactor model, resting *E. coli* cells containing nitrile hydratase and nicotinamide enzyme mutant genes are connected in series. By controlling the optimal conditions for both strains, highly efficient continuous synthesis of NA is achieved, with a nicotinic acid yield of 298.4 g / L. Both nitrile hydratase and nicotinamide enzyme exhibit maximum catalytic efficiency, and both catalytic reactions are complete, while substrate inhibition is avoided.

The invention discloses a genetic engineering strain for efficiently expressing nitrile hydratase as well as a construction method and application of the genetic engineering strain. On the basis of a nitrile hydratase (PtNHase) gene of pseudonocardia thermophila, the following optimization and transformation are carried out: (1) carrying out complete sequence optimization on a gene sequence according to codon preference of escherichia coli; (2) replacing a ribosome binding site (SD sequence) at the upstream of the alpha subunit; and (3) carrying out site-specific mutagenesis on tryptophan (Trp) at the 72nd site of the beta subunit to obtain phenylalanine (Phe). The nitrile hydratase produced by the engineering strain constructed by the invention has significantly improved enzyme activity, thermal stability and tolerance to a substrate / product, can be used for industrial biosynthesis of acrylamide and nicotinamide and degradation of nitrile-containing pollutants, is suitable for the fields of industrial large-scale production and environmental governance, and has broad application prospects.