13 results about "Alanine dehydrogenase" patented technology

Provided are a theanine-producing strain and use thereof in tea fermentation production. A corynebacterium glutamicum is proposed, which includes an alanine decarboxylase CsAlaDC mutant. The theanine-producing strain is obtained by taking the corynebacterium glutamicum as a starting strain, knocking out in sequence an α-ketoglutarate dehydrogenase E1 subunit gene odhA, a glutamate external transporter gene Ncg11221 and a lactate dehydrogenase gene ldh; and / or expressesing a citrate synthase gene gltA, a pyruvate kinase gene pyk and a glutamate dehydrogenase gene gdh; and / or overexpressing an alanine dehydrogenase alaA and integrating a γ-glutamine synthetase GMAS into a cg1960 pseudogene locus of the corynebacterium glutamicum.

The invention discloses a genetically engineered bacterium for high yield of L-alanine as well as a construction method and application of the genetically engineered bacterium, a dominant strain is obtained by knocking out an alanine racemase gene dadX and integrating coding sequences of alanine dehydrogenase from different sources, and the shake flask yield is 10.8 g / L; the activity of a corresponding mutant M18L / E75A / A78S / I266V is 4.3 times that of a wild type; 6 by-product related genes are knocked out, meanwhile, a multi-copy strain integrated with alanine dehydrogenase is constructed, and the yield of E.coli A-LB6 in a 5L fermentation tank is increased to 133.3 g / L. Through a dynamic regulation and control strategy, an oxygen-sensitive promoter Pomp * is adopted to replace a phosphoenolpyruvate carboxylase promoter, so that succinic acid byproducts are reduced by 85.8%, and the yield reaches 143.5 g / L after 48 hours of aerobic and anaerobic two-stage fermentation. And finally, by regulating and controlling the ATP synthetase gene cluster atp promoter, the yield of L-alanine in a 50 L fermentation tank reaches 151.3 g / L, and the saccharic acid conversion rate reaches 95.2%. The combination of polygene knockout and dynamic metabolism regulation effectively enhances the biosynthesis ability of alanine, and provides an efficient engineering strain for industrial production.

The invention belongs to the technical field of microorganism application, and particularly relates to a genetically engineered bacterium for producing L-alanine and application of the genetically engineered bacterium in two-stage fermentation of the L-alanine, and the genetically engineered bacterium takes vibrio natrievibrio as a chassis bacterium and can co-express an alanine dehydrogenase gene and an alanine transporter gene. An alanine dehydrogenase gene is heterologously introduced into a vibrio natriticus genome, and an inactivated gene combination with the optimal L-alanine yield is found out by randomly inactivating a key enzyme in a byproduct production pathway, so that the vibrio natriticus genetically engineered bacterium capable of efficiently producing the L-alanine through a fermentation method is constructed. According to the present invention, the L-alanine is produced through the two-stage fermentation by using the gene engineering bacteria through the temperature-sensitive control strategy so as to reduce the influence of the accumulation of the L-alanine on the cell growth, and the high production of the Xiabanmate is provided. The genetically engineered bacterium is short in growth cycle, glucose can be efficiently utilized to convert the genetically engineered bacterium into L-alanine, the L-alanine is transferred to the outside of cells, separation and purification are simple, cell disruption is not needed, and the yield and conversion rate of the L-alanine can both reach a high level.

According to the alanine dehydrogenase mutant and the application thereof, compared with wild alanine dehydrogenase, the enzyme activity of the alanine dehydrogenase mutant is improved, the alanine dehydrogenase mutant can be used for fermentation production of L-alanine, and the production efficiency of the L-alanine is improved.

The present invention discloses an L-alanine-producing genetically engineered strain, as well as a method of construction and use thereof, and pertains to the field of bioengineering. According to the present invention, through enhancing the glycolysis pathway or / and introducing a gene for thermostable alanine dehydrogenase, a genetically engineered strain capable of high-yield production of alanine under a high temperature condition of 42° C. to 55° C. can be constructed. Moreover, through knocking out alanine racemase genes, optical purity of L-alanine can be significantly increased. When the original strain possesses a lactate synthesis pathway, blocking this lactate synthesis pathway can augment the proportion of a pyruvate synthesis pathway, resulting in an additionally increased yield of L-alanine. The present invention overcomes the problems of fermentation at a low temperature, high cost and the like, which arise from the use of conventional L-alanine production techniques, enables production of L-alanine by fermentation at a high temperature of 42° C. to 55° C. with a yield of 95 g / L or higher, and is of high value to industrial application.

The application discloses an alanine dehydrogenase mutant, an L-alanine high-yield strain containing the same and application of the L-alanine high-yield strain. The amino acid sequence of the alanine dehydrogenase mutant of the application comprises one or more amino acid mutations of E48T, T180I, H295A, A304E and N344I compared with SEQ ID NO:1. The L-alanine high-yield strain expresses the above alanine dehydrogenase mutant, can produce high-concentration L-alanine through fermentation culture, has the ability of short fermentation time, reaches 136.5 g / kg in 38 hours of fermentation, has a sugar-acid conversion rate as high as 97%, has improved fermentation level and greatly shortened fermentation time, and has the potential of industrial application.

The invention relates to a bactericidal adjuvant, which comprises a preparation capable of knocking out, knocking down or silencing a coding gene of alanine dehydrogenase of bacteria, or a preparation capable of destroying the alanine dehydrogenase function of the bacteria. By knocking out the ald gene, the sensitivity of aeromonas veronii to antibiotics is remarkably improved, and a brand-new technical method is provided for treatment of drug-resistant bacteria.

The application discloses an alanine dehydrogenase mutant, wherein 1-3 amino acids in positions 165, 177, 178, 180, 199, 200, 217, 218, 219, 225, 237 and 238 of the wild-type alanine dehydrogenase amino acid sequence shown in SEQ ID NO. 1 are respectively mutated into one of lysine, arginine, glutamic acid, proline, valine, glycine, alanine and serine. The alanine dehydrogenase mutant has significant preference for NAD(H) analogues, can produce L-alanine or pyruvic acid by using NAD analogues and NADH analogues, and does not cause interference problems. The application further discloses application of the alanine dehydrogenase mutant.

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 method for distinguishing a vibrio alginolyticus wild type virulent strain and a crp gene deletion type attenuated strain based on proteomics. According to the invention, proteomics is utilized to determine the content ratio of glyceraldehyde-3-phosphate dehydrogenase to alanine dehydrogenase in vibrio alginolyticus, and a coding gene crp deleted attenuated strain and a wild virulent strain of cAMP receptor protein are distinguished; the vibrio alginolyticus crp gene deletion type attenuated strain with the result of 3-phosphoglyceraldehyde dehydrogenase / alanine dehydrogenase being lower than 0.9 in the sample to be detected, and the vibrio alginolyticus wild type virulent strain with the result of 3-phosphoglyceraldehyde dehydrogenase / alanine dehydrogenase being higher than 1.2 in the sample to be detected. The method is simple to operate, high in detection speed and reliable in result.

Provided are a theanine-producing strain and use thereof in tea fermentation production. A Corynebacterium glutamicum is proposed, which includes an alanine decarboxylase CsAlaDC mutant. The theanine-producing strain is obtained by taking the Corynebacterium glutamicum as a starting strain, knocking out in sequence an α-ketoglutarate dehydrogenase E1 subunit gene odhA, a glutamate external transporter gene Ncg11221 and a lactate dehydrogenase gene ldh; and / or expressing a citrate synthase gene gltA, a pyruvate kinase gene pyk and a glutamate dehydrogenase gene gdh; and / or overexpressing an alanine dehydrogenase alaA and integrating a γ-glutamine synthetase GMAS into a cg1960 pseudogene locus of the Corynebacterium glutamicum.

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 invention relates to an AlaDH mutant and an application of the AlaDH mutant in production of L-serine. Specifically, the invention provides an alanine dehydrogenase AlaDH mutant, and the alanine dehydrogenase AlaDH mutant has amino acid substitutions of L107A, K161V and E205D or a combination of any two of the amino acid substitutions of L107A, K161V and E205D relative to wild alanine dehydrogenase AlaDH. The present invention also provides a method of producing L-serine or an L-serine derivative, the method comprising culturing a bacterium in a culture medium wherein the bacterium comprises a nucleotide sequence encoding the AlaDH mutant. According to the AlaDH mutant disclosed by the invention, 3-PHP can be specifically catalyzed to generate 3Per, so that the generation of byproducts is reduced, and the production cost is reduced.