19 results about "Amidohydrolase" patented technology

The invention belongs to the field of enzyme catalysis, and particularly relates to an amide hydrolase mutant, a immobilized enzyme, a coding gene, an expression plasmid, a preparation method of the amide hydrolase mutant, the immobilized enzyme, the coding gene and the expression plasmid, and application of the amide hydrolase mutant, the immobilized enzyme, the coding gene and the expression plasmid in catalysis of amide hydrolysis. The gene is characterized in that the gene is an amino acid sequence with at least one mutation of L85Y and E209R in a wild amino acid sequence as shown in SEQ ID NO.1. Researches show that the mutant can significantly enhance the catalytic activity of enzyme, and the reaction conversion rate can be effectively increased when the mutant is used for enzymatic amide hydrolysis reaction; for example, the compound is innovatively used for amide hydrolysis of ASG, and has excellent degradation capacity.

The invention provides an application of an HDAH inhibitor in preparation of antibiotics. According to the invention, research finds that escherichia coli has Zn < 2 + >-dependent histone deacetylase-like amide hydrolase-HDAH, and the HDAH can directly influence the enzyme activity of metabolic enzyme through acetylation modification so as to regulate and control the metabolic level of bacteria; and the transcription level of metabolism-related genes can be indirectly regulated by acting on a transcription regulation factor (such as histone-like protein HU), and the metabolism of escherichia coli is synergistically regulated from two levels of post-translational modification and transcription. The invention also finds that the vorinostat (SAHA), the trichosaliocin (TSA) and the like can be combined with the HDAH and inhibit the deacetylation activity of the HDAH, are HDAH inhibitors, and inhibit the growth and migration ability of escherichia coli after being administered to the escherichia coli.

The application discloses a novel and powerful inhibitor of fatty acid amide hydrolase (FAAH), pyridinone compound (formula I) and therapeutic effect thereof in related diseases, and belongs to the technical field of biological medicine. The inhibitor can inhibit the biological activity of fatty acid amide hydrolase (FAAH), increase the physiological concentration of endogenous arachidonic acid ethanolamine, and play the roles of regulating immunity and resisting inflammation; for example, in inflammatory bowel disease, the inhibitor can significantly improve various pathological and physiological indexes of animals in an inflammatory bowel disease (IBD) model, slow down the progress of the disease, and be effectively used for treating multiple types of inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn's disease (CD).

The invention discloses an amide hydrolase mutant and an application in production of nicotinic acid, and provides two novel amide hydrolase mutants, which can significantly improve the production efficiency and yield of nicotinic acid, reduce the operation steps of nicotinic acid synthesis, realize one-step reaction synthesis from nicotinamide to nicotinic acid after cell disruption, break the bottleneck of the prior art, and improve the production efficiency and yield of nicotinic acid. The synthesis time is greatly shortened while the production cost of the nicotinic acid is reduced, and the production of the nicotinic acid is promoted to develop towards a more efficient, more economic and more environment-friendly direction.

The invention discloses application of amide hydrolase OtaH in degradation of ochratoxin A, and relates to the technical field of enzyme engineering and food safety biology. The amino acid sequence of the amide hydrolase OtaH is as shown in SEQ ID NO.2, and the coding gene sequence of the amide hydrolase OtaH is as shown in SEQ ID NO.1. The invention provides the application of the amide hydrolase OtaH in degradation of ochratoxin A. The invention further provides the application of the amide hydrolase OtaH in degradation of ochratoxin A. The amide hydrolase OtaH can specifically hydrolyze an amido bond of ochratoxin A (OTA) to generate almost non-toxic ochratoxin alpha, has high catalytic efficiency, can completely degrade 12.4 mu M OTA within 5 minutes in the enzyme amount of 5.0 mu g / mL, still keeps more than 50% of activity after being incubated for 1 hour at 65 DEG C, shows excellent thermal stability, can be widely applied to detoxification treatment of OTA in feed and food, and has a wide application prospect in the treatment of ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha, ochratoxin alpha and ochratoxin alpha. The harm of OTA to human beings and animals is reduced.

The invention discloses an amide hydrolase mutant and application of the amide hydrolase mutant in preparation of chiral gamma-amino acid drug intermediates, and belongs to the technical field of bioengineering. Wild type GsHaeWT with an amino acid sequence as shown in SEQ ID No.1 is used as a starting enzyme, further molecular modification is performed on the starting enzyme through a protein engineering means to obtain a plurality of amide hydrolase mutants with remarkably improved catalytic activity and stability, the amide hydrolase mutants and nucleic acid for coding the amide hydrolase mutants are provided, and the amide hydrolase mutants and the nucleic acid for coding the amide hydrolase mutants are used for preparing the amide hydrolase. The invention relates to a nucleic acid, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the expression vector, a recombinant amide hydrolase mutant catalyst and application of the amide hydrolase mutant or the recombinant amide hydrolase mutant catalyst in preparation of chiral gamma amino acid drug intermediates. The amide hydrolase mutant provided by the invention has the advantages of high protein expression quantity, high catalytic activity, good thermal stability, high product optical purity and the like, and shows excellent industrial application prospects.

The application belongs to the field of synthesis of pharmaceutical intermediates and discloses a lactam hydrolase mutant, a decarboxylase mutant and application of the lactam hydrolase mutant and the decarboxylase mutant in preparation of 5-methoxytryptamine. The lactam hydrolase mutant and the decarboxylase mutant with high activity are obtained by mutating specific positions of the lactam hydrolase and the decarboxylase. When the lactam hydrolase mutant and the decarboxylase mutant are used in preparation of 5-methoxytryptamine, the conversion rate and the yield are improved, the conditions are mild, and the use of acid and alkali reagents is reduced.

The invention discloses an amide hydrolase and an application of the amide hydrolase in degradation of ochratoxin. The invention provides a protein or a mutant thereof. The protein or the mutant thereof comprises a protein as shown in an amino acid sequence of SEQ ID NO: 1; or a protein which is obtained by mutating the 76th amino acid residue S on the amino acid sequence as shown in SEQ ID NO: 1 into E and has the same function; or a protein which is obtained by mutating the 334th amino acid residue V on the amino acid sequence as shown in SEQ ID NO: 1 into E and has the same function as the amino acid residue V on the amino acid sequence as shown in SEQ ID NO: 1; or the N end or / and C end of each protein is / are connected with a tag to obtain a protein shown as a sequence. The invention finds that the amide hydrolase ThADH and the mutant can efficiently degrade OTA (Ochratoxin A) and realize OTA detoxification.

The invention discloses a method for preparing a niraparib chiral intermediate by a chemical-enzyme method. According to the method disclosed by the invention, a compound II is taken as an initial raw material, a compound V is obtained through esterification, Michael addition and cyclization reaction, then a chiral compound VI is obtained under the action of amide hydrolase (AMD), the conversion rate and ee value can reach 99%, and finally the niraparib chiral intermediate I is obtained through reduction reaction of the compound VI. The route is simple to operate and low in cost, and can be used for industrial production.

The application belongs to the technical field of biological medicine, and particularly discloses a method for preparing L-histidine from L-carnosine mother liquor. The method mainly comprises the following steps: A. vacuum concentrating the L-carnosine mother liquor into a mother liquor concentrate, adding a strong acid, a strong base or an amide hydrolase into the mother liquor concentrate to hydrolyze the peptide component into a hydrolysate mainly containing amino acids; B. removing the acid, the base or the enzyme from the hydrolysate, and then concentrating; after the concentration, sample loading column chromatography is performed; C. sequentially using purified water and gradient concentration ammonia water for elution, collecting the eluate, and using HPLC for tracking detection of the purity of L-histidine in the eluate, and collecting the eluate with the purity of L-histidine of 94% to 99%; D. combining the eluate, vacuum concentrating the eluate into a solution with the weight concentration of L-histidine of 30 to 40%, and adding simple alcohol for crystallization; E. filtering the precipitated L-histidine crystals, washing the filter cake with simple alcohol, drying by suction and drying, and then the L-histidine crystals can be obtained.

The application discloses application of an amide hydrolase OtaH in degrading ochratoxin A, and relates to the technical field of enzyme engineering and food safety biotechnology. The amino acid sequence of the amide hydrolase OtaH is shown as SEQ ID NO. 2, the coding gene sequence of the amide hydrolase OtaH is shown as SEQ ID NO. 1, and the application provides application of the amide hydrolase OtaH in degrading ochratoxin A. The amide hydrolase OtaH can specifically hydrolyze the amide bond of ochratoxin A (OTA) to generate almost non-toxic ochratoxin alpha, the enzyme has high catalytic efficiency, 5.0 mu g / mL of the enzyme amount can completely degrade 12.4 mu M OTA in 5 minutes, and more than 50% activity is still maintained after incubation at 65 DEG C for 1 hour, thereby showing excellent thermal stability. The amide hydrolase OtaH can be widely used in detoxification treatment of OTA in feed and food, and the harm of OTA to human beings and animals is reduced.

The invention discloses an amide hydrolase mutant and application thereof. The invention provides a mutant protein of amidase CaADH, which is obtained according to any one of the following modes: the 67th amino acid residue G on the amino acid sequence of the amidase CaADH as shown in SEQ ID NO: 1 is mutated into D, or the 67th amino acid residue G is mutated into E, or the 67th amino acid residue G is mutated into L, and the mutant protein has the same function; experiments prove that the amide hydrolase CaADH and the mutant thereof can be used for efficiently degrading OTA and realizing OTA detoxification.

The invention discloses an amide hydrolase mutant, a biological material, a screening method, a catalyst and application, and compared with wild type amide hydrolase, the amide hydrolase mutant has amino acid residue mutation at the 317th site, or amino acid residue mutation at the 317th site and amino acid residue mutation at the 63rd site, or amino acid residue mutation at the 317th site, amino acid residue mutation at the 63rd site and amino acid residue mutation at the 65th site. The catalytic efficiency of the mutant can reach 16 times as high as that of wild type amide hydrolase, the yield of R-isobutylglutaric acid monoamide generated by catalyzing isobutylglutaric imide reaches 99%, the stereoselectivity ee value is greater than 99%, the reaction condition is mild, the reaction can be carried out only at 20-45 DEG C, and the mutant has high industrial application value.

The invention discloses a double-enzyme sensing system for detecting ochratoxin A and application of the double-enzyme sensing system. The invention provides a method for detecting OTA in a to-be-detected sample, which comprises the following steps: A1) carrying out hydrolysis reaction on the to-be-detected sample by using amide hydrolase to obtain a first-step reaction solution; a2) adding phenylalanine dehydrogenase, coenzyme NAD < + > and Glycine into the first-step reaction solution, and reacting again to obtain a second-step reaction solution; adding a color developing agent into the second-step reaction solution, and carrying out color developing reaction to obtain a reaction product; detecting OTA in the sample to be detected according to the reaction product; according to the invention, a novel OTA degrading enzyme ThADH of an OTA sensing element and a product L-phenylalanine sensing element phenylalanine dehydrogenase are combined, and efficient, visual and quantitative detection of OTA is realized by using a color reaction in a product catalysis process.

The invention discloses a controlled-release fertilizer containing signal substances and capable of promoting transfer of nitrogen to corn kernels and a preparation method of the controlled-release fertilizer. The controlled-release fertilizer comprises the following raw materials in parts by weight: 1-2 parts of an inner-layer signal core, 88-91 parts of a middle-layer nutrient layer and 8-10 parts of an outer-layer controlled-release coating, the inner layer signal core is a compound containing N-acetyl-L-proline; the middle nutrient layer is a compound fertilizer containing nitrogen, phosphorus and potassium; the outer-layer controlled-release coating comprises chitosan, cellulose nanocrystals and nano calcium carbonate. A signal core is prepared through spray granulation, a nutrient layer is covered through roller granulation, and a controlled-release coating is sprayed on a fluidized bed, so that whole-course controlled release of nitrogen and activation and release of a signal substance in a filling stage are realized. The controlled-release fertilizer disclosed by the invention responds to rhizosphere acidification and amide hydrolase in the filling stage of the corn, releases L-proline, promotes the corn to up-regulate nitrogen elements such as ZmGS1-3 and ZmGln1-3, then mobilizes gene expression, promotes nitrogen elements of nutritive organs to be transferred to grains, and greatly improves the utilization rate of fertilizer nutrients.

The present invention provides antisense oligonucleotides (oligomers) complementary to ASAH1 (N-acylsphingosine amidohydrolase 1) pre-mRNA sequences and capable of inhibiting the expression of ASAH1. Such antisense oligonucleotides may be used in in vivo and in vitro methods for reducing the level of ASAH1 transcript in a target cell. The inhibition of ASAH1 expression may be used in the treatment of Krabbe disease and cancer.

The invention discloses a colorimetric biosensing system based on phenylalanine dehydrogenation, which is used for detecting ochratoxin A. The invention provides a method for detecting OTA in a to-be-detected sample, which comprises the following steps: A1) carrying out hydrolysis reaction on the to-be-detected sample by using amide hydrolase VeADH to obtain a first-step reaction solution; a2) adding phenylalanine dehydrogenase, coenzyme NAD < + > and Glycine into the first-step reaction solution, and reacting again to obtain a second-step reaction solution; adding a color developing agent into the second-step reaction solution, and carrying out color developing reaction to obtain a reaction product; detecting OTA in the sample to be detected according to the reaction product; according to the invention, a novel OTA degrading enzyme VeADH of an OTA sensing element and a product L-phenylalanine sensing element phenylalanine dehydrogenase are combined, and efficient, visual and quantitative detection of OTA is realized by using a color reaction in a product catalysis process.

The application discloses a product for degrading ochratoxin and a special protein. The application provides a protein or a mutant thereof, which is any one of the following: a protein as shown in an amino acid sequence of SEQ ID NO: 1; or a protein in which an amino acid residue S at the 66th position in the amino acid sequence shown in SEQ ID NO: 1 is mutated into E and the protein has the same function; or a protein in which an amino acid residue V at the 324th position in the amino acid sequence shown in SEQ ID NO: 1 is mutated into E and the protein has the same function; or a protein in which a label sequence is connected to the N terminal or the C terminal of each of the above proteins. The application finds that an amide hydrolase VeADH and a mutant thereof can efficiently degrade OTA, and OTA detoxification is realized.

The invention relates to the field of synthetic biology and enzyme engineering, in particular to a method for displaying fatty amide hydrolase on the surface of a bacillus subtilis biological membrane. The method disclosed by the invention comprises the following steps: inoculating recombinant engineering bacteria into an LB liquid culture medium containing chloramphenicol, adding sterile fermentation liquor and overnight culture, and carrying out fermentation culture, so as to obtain the fatty amide hydrolase displayed on the surface of bacillus subtilis, the host bacteria of the recombinant engineering bacteria are tasA and sinR gene defect type bacillus subtilis; the recombinant engineering bacterium contains a tapA-sipW-tasA-FAAH (tapA-sipW-tasA-FAAH) recombinant plasmid, and the nucleotide of the recombinant plasmid is as shown in SEQ ID NO: 4. According to the method, the FAAH enzyme is immobilized by using a bacillus subtilis biological membrane surface display system, so that the stability and the catalytic efficiency of the enzyme are remarkably improved. The method is simple and convenient to operate and low in cost, can realize efficient reutilization of the FAAH enzyme, reduces enzyme loss in an industrial catalysis process, and has a wide biological catalysis application prospect.