39 results about "Nitrile hydratase" patented technology

A nitrile hydratase mutant and the use thereof. The nitrile hydratase mutant is a wild-type nitrile hydratase having a mutation of an amino acid residue in the flexible loop domain, and comprises any one of or a combination of mutations 1-3, wherein mutation 1 is a polar amino acid mutation located in the flexible loop between secondary domains S1 and S2 of the β subunit of the wild-type nitrile hydratase; mutation 2 is a polar amino acid mutation located in the flexible loop between secondary domains H6 and H7 at the starting end of the β subunit of the wild-type nitrile hydratase; and mutation 3 is a hydrophobic amino acid mutation in starting flexible loop L1 of the β subunit of the wild-type nitrile hydratase. On the basis of the wild-type nitrile hydratase, the nitrile hydratase mutant can comprise any one of or a combination of the mutations described above, which can exert regulatory effects alone or synergistically, has a certain enzyme activity, and has good thermal stability and tolerance.

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 discloses a nitrilase mutant and application thereof in synthesis of a gamma-aminobutyric acid derivative, the nitrilase mutant with improved catalytic activity and stereoselectivity is constructed to efficiently catalyze hydrolysis of 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid, and the gamma-aminobutyric acid derivative is further synthesized through hydrogenation. And a foundation is laid for industrial production of gamma-aminobutyric acid drugs synthesized by a nitrilase method.

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 invention provides an engineered nitrile hydratase and application thereof. Based on wild type nitrile hydratase, the engineered nitrile hydratase is characterized in that an amino acid residue located at the 85 site of an alpha subunit is mutated into any one of methionine, arginine and lysine, the wild type nitrile hydratase comprises the alpha subunit, the amino acid sequence of the alpha subunit is as shown in SEQ ID NO: 1, and the amino acid sequence of the alpha subunit is as shown in SEQ ID NO: 2. The engineered nitrile hydratase provided by the embodiment of the invention has good catalytic activity and product tolerance.

The present invention belongs to the technical field of enzyme engineering, and in particular relates to a nitrile hydratase mutant and its application, wherein the nitrile hydratase mutant is obtained by performing multi-point mutations at multiple sites in the 98th position of the amino acid sequence shown in SEQ ID NO.1 and the 41st, 51st and 55th positions of the amino acid sequence shown in SEQ ID NO.2. The present invention performs iterative saturation mutation on nitrile hydratase, and provides multiple nitrile hydratase mutants with significantly improved product tolerance, and these mutants can be used to produce a variety of amide compounds, and compared with the starting type, the specific enzyme activity and catalytic performance are significantly improved.

A mutant nitrile hydratase, being a nitrile hydratase having an α subunit with a sequence identity of 90% or more with respect to an amino acid sequence represented by SEQ ID NO: 1 and a β subunit with a sequence identity of 90% or more with respect to an amino acid sequence represented by SEQ ID NO: 2, the nitrile hydratase including at least one substitution to a specific amino acid residue from an amino acid residue at a position selected from the group consisting of 23th, 105th, 142nd, 145th, 152th, 153rd, 180th and 188th from the N-terminus in the α subunit, and 89th, 122nd, 149th, 153rd, 189th, 207th, 232nd, 233rd and 50th from the N-terminus in the β subunit.

The present invention belongs to the field of detection technology and relates to a method for detecting organic cyanides by using an inverse opal thin film, which comprises the following steps: Step 1, put the inverse opal thin film into an alkaline solution, observe the photonic crystal reflection peak and its color after equilibrium and stability are achieved, and obtain an inverse opal photonic crystal system; Step 2, oscillate and mix the organic cyanide solution and the nitrile hydratase solution to obtain a mixed solution; put the mixed solution into the inverse opal photonic crystal system, and realize the detection of organic cyanides by observing the color change of the inverse opal photonic crystal system and the movement of the photonic crystal reflection peak. The method for realizing the visual detection of organic cyanides by combining the inverse opal thin film with the enzymatic catalytic reaction breaks through the barriers of poor portability, complex operation and inability to perform on-site analysis of traditional detection technologies. This method is easy to operate, highly portable, highly sensitive, capable of naked-eye observation, and the lowest detection limit can reach 2×10<supgt;‑8< / supgt; g / L.

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 invention relates to a nitrile hydratase catalysis application process for efficiently preparing acrylamide, and relates to the technical field of acrylamide preparation, and the nitrile hydratase catalysis application process comprises the following steps: adding a nitrile hydratase strain into a seed culture medium for culturing to obtain a primary culture solution; adding the primary culture solution into a fermentation culture medium for fermentation to obtain a fermentation solution; preparing a nitrile hydratase catalyst; the method comprises the following steps: adding acrylonitrile into water, adding a nitrile hydratase catalyst to obtain a reaction solution, and carrying out hydration reaction to obtain a product mixed solution; removing the nitrile hydratase catalyst from the product mixed solution through magnetic separation to obtain an acrylamide hydrated solution; and filtering the acrylamide hydration liquid through an ultrafiltration membrane to obtain an acrylamide crude product liquid, refining the crude product liquid, and concentrating to obtain an acrylamide finished product. The method can be used for efficiently preparing acrylamide, and has the effect of improving the yield of acrylamide.

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.

A mutant nitrile hydratase which is derived from Pseudonocardia thermophila and has an alpha subunit having a sequence identity of 90% or more with SEQ ID NO: 1 and a beta subunit having a sequence identity of 90% or more with SEQ ID NO: 2, the mutant nitrile hydratase contains a substitution in which an amino acid residue at at least one position selected from the group consisting of the 23rd position, the 105th position, the 142 position, the 145th position, the 152nd position, the 153rd position, the 180th position and the 188th position from the N-terminal of the alpha subunit and the 89th position, the 122nd position, the 149th position, the 153rd position, the 189th position, the 207th position, the 232 position, the 233th position and the 50th position from the N-terminal of the beta subunit is substituted by a specific amino acid residue.

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 present invention relates to variant nitrile hydratases engineered to contain higher activity and / or stability, nucleic acids encoding said nitrile hydratases, and microorganisms engineered to express said novel nitrile hydratases. Additionally, the present invention relates to the use of said nitrile hydratases and microorganisms expressing said nitrile hydratases as biocatalysts, particularly in processes for producing amide compounds from nitrile compounds, preferably for use in converting acrylonitrile to acrylamide.

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 invention provides a nitrile hydratase mutant and application thereof. The nitrile hydratase mutant is obtained by mutation of amino acid residues on a flexible ring structural domain of the wild type nitrile hydratase and comprises any one or a combination of mutations 1-3: mutation 1: polar amino acid mutation on a flexible ring between secondary structural domains S1 and S2 of a beta subunit of the wild type nitrile hydratase, and mutation 2: polar amino acid mutation on a flexible ring between the secondary structural domains S1 and S2 of the beta subunit of the wild type nitrile hydratase, and mutation 3: polar amino acid mutation on a flexible ring between the secondary structural domains S1 and S2 of the beta subunit of the wild type nitrile hydratase; the 152-site amino acid site of the beta subunit is mutated; mutation 2: polar amino acid mutation on a flexible ring between secondary structural domains H6 and H7 located at the starting end of the beta subunit of the wild nitrile hydratase, and mutation of the 110-site amino acid site of the beta subunit; mutation 3: a hydrophobic amino acid located on an initial flexible ring L1 of a beta subunit of the wild nitrile hydratase is mutated, and a 17-site amino acid site of the beta subunit is mutated. The nitrile hydratase mutant provided by the invention singly or plays a synergistic regulation effect, has certain enzyme activity, and has good thermal stability and tolerance.

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 invention discloses a modification of the amino acid motif of a nitrile hydratase substrate channel for the preparation of cinnamamide, and belongs to the field of bioengineering technology. The present invention obtains mutants F37P‑L48A, F37P‑L48P, F37P‑L48T and Q89N by mutating the phenylalanine at position 37, the leucine at position 48 and / or the glutamine at position 89 on the α subunit of the nitrile hydratase, and the enzyme activity of cinnamonitrile catalyzed by the mutants F37P‑L48A, F37P‑L48P, F37P‑L48T and Q89N reaches 46.5U / mL to 53.6U / mL, which is 9.3 to 10.72 times higher than the enzyme activity of the wild-type nitrile hydratase. The mutants F37P‑L48A, F37P‑L48P, F37P‑L48T and Q89N in the present invention have good catalytic activity for cinnamonitrile and have good industrial application potential.

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 present invention relates to a variant nitrile hydratase, a nucleic acid encoding said nitrile hydratase and a microorganism engineered to express said novel nitrile hydratase, said variant nitrile hydratase being engineered to comprise higher activity and / or stability. Furthermore, the present invention relates to the use of such a nitrile hydratase and a microorganism expressing said nitrile hydratase as a biocatalyst, in particular in a method for producing amide compounds from nitrile compounds, preferably for the conversion of acrylonitrile to acrylamide.

The invention belongs to the technical field of enzyme engineering and gene engineering, and particularly relates to a nicotinamide enzyme mutant and application of the nicotinamide enzyme mutant in a method for producing nicotinic acid based on a nitrile hydratase nicotinamide enzyme cascade catalytic system. A rate-limiting enzyme of a reaction channel is firstly determined to be nicotinamide enzyme, a saturated mutant library is created through directed evolution, a series of nicotinamide enzyme mutants with high catalytic efficiency are obtained, and the A79S enzyme activity is improved most obviously and reaches 759.2 U / mg and is improved by 4.74 times compared with a wild type. Escherichia coli resting cells respectively containing nitrile hydratase and nicotinamide mutant genes are connected in series by utilizing a cascade reactor model, the optimal conditions of the two strains are controlled, finally efficient continuous synthesis of NA is realized, and the yield of nicotinic acid reaches 298.4 g / L. The nitrile hydratase and the nicotinamide enzyme can play the highest catalytic efficiency, the two catalysis reactions are complete, and the substrate inhibition effect is avoided at the same time.

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%).

The invention discloses engineered nitrile hydratase and application thereof, based on wild type nitrile hydratase, the engineered nitrile hydratase comprises a random coil structural domain which is located near an active center and has a sequence of SEQ ID NO.1, methionine in the random coil structural domain is mutated into any one of glycine, leucine, isoleucine, glutamine, threonine, glutamic acid, arginine and lysine; the engineered nitrile hydratase containing any mutation has higher enzyme activity, and the catalysis efficiency is improved.