30 results about "Glutamate kinase" patented technology

The invention relates to a recombinant corynebacterium crenatum to enhance the expression of N-acetyl glutamic acid kinase and the application thereof, which belongs to the technical field of gene engineering. The classification and nomenclature of the recombinant corynebacterium crenatum to enhance the expression of N-acetyl glutamic acid kinase is corynebacterium crenatum SYPA/pJC1-tac-argB with an access number of CCTCC NO: M 208133; The application is as follows: using a key enzyme N-acetyl glutamic acid kinase from corynebacterium crenatum pathway arginine to construct a corynebacterium crenatum expression vector pJC1-tac-argB; introducing the corynebacterium crenatum expression vector pJC1-tac-argB into corynebacterium crenatum SYPA by electrotransformation to obtain the recombinant corynebacterium crenatum SYPA/pJC1-tac-argB; and excessively expressing N-acetyl glutamic acid kinase, which greatly enhances the utilization rate of a precursor glutamic acid and allows the metabolic flow to flow to the arginine synthesis pathway, weakens the synthesis of proline, achieves the purpose of improving the output of arginine and the output of arginine is increased by 23.4 percent than C. crenatum SYPA.

The invention relates to a method for improving the yield of arginine by the mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase. L-arginine is one of semi-essential basic amino acids for a human body, and has various particular physiology and pharmacology functions. High-yield arginine mutant strain C. crenatum SYPA5-5 is compounded into the arginine through a circulating way, and N-acetyl glutamic acid kinase (NAGK) is a key enzyme in the compounding way and is subject to the feedback inhibition of the product arginine. By using an overlapping PCR (Polymerase Chain Reaction) technique, a GAT (glycerol phosphate acyl transferase) codon used for coding aspartic acid is used for substituting a GGA (General Gonadotropic activity) codon used for coding glycine at the site 287 in the coding NAGK albumen, and the NAGK which has high activity and arginine with obviously reduced by the feedback inhibition can be obtained after the mutation. An argBSD gene is brought into the Corynebacterium crenatum of the high-yield arginine through pJCl-tac, and the expression volume of the key enzyme is further improved. The final acid yield is increased to 36.3g/L from original 28g/L, and the yield of the L-arginine is increased by 29.7 percent.

The invention discloses a preparation method of a genetically engineered bacterium for synthesizing glutathione. The preparation method comprises the following steps: (i) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase fusion protein into a host bacterium cell, and highly expressing the gene; (ii) converting a gene for encoding gamma-glutamoyl cysteine synthetase-linker peptide-glutathione synthetase into the host bacterium cell, and highly expressing the gene; (iii) knocking off the gamma-glutamyltranspeptidase of the host bacterium cell; (iv) knocking off an inositol-3-phosphatep synthase gene of the host bacterium cell; and completing the steps (i), (ii), (iii) and (iv) in any sequence, thereby obtaining the genetically engineered bacterium for synthesizing glutathione. The invention further relates to the prepared genetically engineered bacterium. The genetically engineered bacterium for synthesizing glutathione, which is disclosed by the invention, is not only high in efficiency in synthesizing glutathione, but also can greatly improve the conversion efficiency of exogenous cysteine, and can be applied to glutathione production.

The invention provides a metabolic transformation strategy for efficiently improving production capacity of corynebacterium crenatum SYPA5-5 L-arginine, and belongs to the biological technical field. Histidine at a site 268 in N-acetyl glutamatekinase, NAGK, argB of SYPA-5 is mutated into asparaginate, so that feedback inhibition effect of the L-arginine on NAGK can be effectively relived; a mutation site is precisely introduced to a corresponding position of SYPA5-5 genome, so that yield of the obtained feedback inhibition-preventing bacterial strain (H-7)L-arginine is lowered, and metabolic intermediate (L-ornithine and L-citrulline) is largely accumulated. Transcriptional analysis is carried out on an arg gene cluster of h-7 to find that arg GH transcriptional level is lowered. Arg GH reinforced expression is carried out in H-7, and production capacity of the obtained recombinant bacterial strain H-7-GH, L-arginine is improved by 99.0% in comparison with SYPA5-5. Preliminary optimization is carried out on a fermentation culture medium of H-7-GH, so that yield of the L-arginine is 45.1g/L, which is improved by 49.8% in comparison with that before optimization, and improved by 1.13 times in comparison with SYPA5-5. Feedback inhibition of the L-arginine on NAGK is relieved, and metabolic transformation strategy of the arg GH gene is expressed in a strengthened manner at the same time, so that production capacity of the L-arginine of the SYPA 5-5 is effectively improved.

The invention discloses a method for producing trans-4-hydroxyproline from glucose in a fermentation manner by virtue of recombinant Escherichia coli without adding exogenous L-proline. A recombinant plasmid carried by the recombinant Escherichia coli has a mutant gene proBA2 and hyp (4-hydroxyproline), wherein proBA2 is mutated on a glutamyl kinase encoding gene proB, and the inhibition effect of L-proline on glutamyl kinase encoded by the mutated gene is remarkably reduced. proBA2 and hyp are co-expressed, and trans-4-hydroxyproline can be produced directly from glucose in the fermentation manner without adding exogenous L-proline. The recombinant plasmid is obtained by recombining proBA2 and hyp onto the same expression plasmid or by co-transforming recombinant plasmids which contain the two genes respectively and have different resistances. The invention further discloses the application of the Escherichia coli to the production of hydroxyproline.

The invention discloses a method for improving the acetylglucosamine yield of recombinant bacillus subtilis by knocking out argCJBD, and belongs to the field of genetic engineering. Recombinant bacillus subtilis BSGN6-PxylA-glmS-P43-GNA1 serves as the original strain, acetyl glutamate glutamate-5-semialdehyde dehydrogenase coding gene (argC), ornithine acetyltransferase coding gene (argJ), acetyl glutamate kinase coding gene (argB) and acetyl ornithine aminotransferase coding gene (argD) are knocked out through homologous recombination, and therefore the approach of host bacteria for generating arginine from glutamic acid is stopped, and the reaction of converting glutamic acid into glutamine is promoted; glutamine serves as amino donors synthesized through acetylglucosamine, and therefore the yield of acetylglucosamine is improved. In the bottle shaking fermentation process through a semi-synthesized culture medium, the acetylglucosamine yield of recombinant bacillus subtilis reaches 6.4 g/L by knocking out argCJBD and is improved by 16.3% compared with the before-knockout yield. A foundation is laid for further producing glucosamine through bacillus subtilis transformed through metabolic engineering.

The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, and/or alleviating a limitation such that yield is increased, or a combination of these and that have been transformed using a novel vector construct including a synthetic N-acetyl glutamate kinase (NAGK) gene that modulates nitrogen use in plants. The invention also includes the overexpression and enzymatic characterization of an arginine-insensitive NAGK isolated from a bacterial strain that improves stress tolerance and nitrogen uptake, metabolism or both. In various embodiments, the vector construct includes one or more nucleic acid sequences including SEQ ID NO: 1. The invention also relates to isolated vectors for transforming plants and to antibodies used for detecting transformed plants. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants or are modulated by nitrogen conditions.

The present invention relates to transgenic plants that have increased nitrogen use efficiency, stress tolerance, and/or alleviating a limitation such that yield is increased, or a combination of these and that have been transformed using a novel vector construct including a synthetic isocitrate dehydrogenase (icdh) gene that modulates nitrogen use in plants. The invention also relates to stacking the icdh gene with other exogenous or heterologous genes that modulate nitrogen use in the plant, including a N-acetylglutamate kinase gene. The invention also relates to methods of expressing in plants the nucleic acid molecules corresponding to the nucleic acid sequences that modulate nitrogen use in plants or are modulated by nitrogen conditions.

The invention discloses an N-acetylgutamate kinase mutant with heat stability improved remarkably and belongs to the field of bioengineering .The gene of N-acetylgutamate kinase from Corynebacterium crenatum SYPA5-5 is subjected to site-specific mutagenesis, so that lysine K at the site 234 of the encoded amino acid sequence is mutated into threonine T .the half-life period of the N-acetylgutamate kinase mutant at 37 DEG C is prolonged to be 112 h from 36 h of a wild type N-acetylgutamate kinase mutant, which is 3.11 times of the length before mutation .The N-acetylgutamate kinase mutant can better meet the production requirement of arginine, and lays a foundation for efficient synthesis of L-arginine.

The invention provides corynebacterium glutamicum for producing L-proline and a method for producing the L-proline by using the strain. According to the corynebacterium glutamicum for producing the L-proline, proline dehydrogenase/pyrrole-5-carboxylate dehydrogenase PutA is inactivated, activity of glutamate kinase ProB, glutamic acid-5-semialdehyde dehydrogenase ProA, pyrrole-5-carboxylate dehydrogenase ProC, pyruvate carboxylase Pyc, glyceraldehyde-3-phosphate dehydrogenase GapN, L-proline efflux protein ThrE or SerE is enhanced, and activity of L-proline is enhanced. The L-glutamic acid efflux protein MscCG is inactivated, the L-proline yield, the conversion rate and the production intensity of the obtained strain are remarkably improved compared with those of an original strain, and the production cost of the L-proline can be reduced.

The invention relates to an ammonia (ammonia ion) content determination method by using an enzyme colorimetric method and an enzyme coupled reaction method, a reagent composition and components. The technical principle of the determination method is that a series of catalytic reactions can be carried out according to ammonia kinases, glutamine kinases and glutamine synthetase. The invention also relates to an ammonia (ammonia ion) diagnosis/determination kit. The determination method of invention has the advantages of high sensitivity and small error. The determination method and the kit of the present invention can be widely used for clinical medicine/food examination.

The invention relates to a determination method for determining ammonia (ammonium ion) content by utilizing an enzymatic cycling amplification method, an enzymatic colorimetric method and enzyme couple reaction technology, and composition and components of a reagent. The technology principle for the determination is that the determination is finished according to series catalytic reactions of ammonia kinase, glutamic kinase and glutamic dehydrogenase. The invention further relates to a diagnosis/determination reagent kit of ammonia (an ammonium ion). The determination method is high in sensitivity and small in errors, and accordingly the determination method and the reagent kit can be widely applied to clinical medicine/food inspection.