33 results about "Methionine synthetase" patented technology

L-Methionine is produced by culturing a microorganism which is deficient in repressor of L-methionine biosynthesis system and / or enhanced intracellular homoserine transsuccinylase activity is cultured in a medium so that L-methionine should be produced and accumulated in the medium, and collecting the L-methionine from the medium. The microorganism preferably further exhibits reduced intracellular S-adenosylmethionine synthetase activity, L-threonine auxotrophy, enhanced intracellular cystathionine γ-synthase activity and enhanced intracellular aspartokinase-homoserine dehydrogenase II activity. The present invention enables breeding of L-methionine-producing bacteria, and L-methionine production by fermentation.

The invention relates to a method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine. The method has the following technical characteristics of: crosslinking amino resin carrier Seplite LX-1000HA and glutaraldehyde solution; and reacting the amino resin carrier crosslinked with glutaraldehyde with solution of recombinant adenosylmethionine synthetase to obtain the immobilized adenosylmethionine synthetase. The amino resin carrier has high protein fixation rate, the recovery rate of the enzyme activity of the immobilized adenosylmethionine synthetase is high, and the stability and continuous operation performance are good. When the immobilized adenosylmethionine synthetase is used for producing the adenosylmethionine, the reaction operation is simple, the production cycle is short, and products are easy to purify; the immobilized enzyme can be recycled, the production cost is greatly reduced, the production efficiency is improved, and a new way is provided for industrialized production of the adenosylmethionine.

Methods for inhibiting metalloprotease activity; treating a pathological condition influenced by the action of MMP; treating a pathological condition influenced by cyclooxygenases, endonucleases, metallopepitidases and 5-epoxigenase; treating a pathological condition for which the presence of a metal ion is required; inhibiting the lethal factor produced by toxigenic strains of anthrax; inhibiting activities of fungi, bacteria or plants that utilize zinc-dependent methionine synthetase; inhibiting the activity of a zinc-dependent enzyme in prokaryotic systems; and inhibiting the activity of zinc-dependent enzymes; and compositions thereof, are disclosed.

The invention discloses a recombinant escherichia coli capable of high-yielding L-methionine and an application thereof. The recombinant escherichia coli is constructed according to a method as follows: adopting a CRISPR-Cas9 gene editing technology for respectively replacing L-methionine synthase gene promoters with trc promoters in escherichia coli genomes, introducing a host strain genome and screening, thereby acquiring the recombinant escherichia coli capable of high-yielding L-methionine. The host strain is E.coli W3110 Delta metJ Delta metI / pTrc99A / metA* / yjeH which is constructed by knocking out metJ and metI in escherichia coli E.coli W3110 and then introducing a pTrc99A / metA* / yjeH plasmid. The maximal titer of L-methionine can reach up to 2.8g / L and DAP of by-products is obviouslyreduced to above 60% (reduced from 1g / L to below 0.4g / L).

The invention discloses a kit, a PCR (polymerase chain reaction) primer and a nucleic acid hybridization membrane strip for folic acid metabolism genotyping. The nucleic acid hybridization membrane strip comprises a substrate and specific polymorphic detection probes fixed to the substrate, the polymorphic detection probes are in one-to-one correspondence to folic acid metabolism related gene polymorphic sites, and the related gene polymorphic sites include at least one of a c.677 site and a c.1298 site of an MTHFR (methylene tetrahydrofolate reductase) gene, a c.56 site and a c.66 site of an MTRR (methionine synthase reductase) gene, a -186 site, a c.2756 site and a +905 site of an MTR (methionine synthase) gene and a -551 site of a CBS (cystathionine-beta-synthetase) gene. Each polymorphic detection probe comprises 13-25 base sequences. The kit, the PCR primer and the nucleic acid hybridization membrane strip are easy for judgment of polymorphic heterozygotes, polymorphic homozygotes and the like and have advantages of high flux, high efficiency, low cost, convenience in use and the like.

The invention provides a recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299 and the composition method of the recomposed large intestine Escherichia coli. The invention further provides a recomposed expression carrier pMETK of adenosine methionine synthetase. In the recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299, the expression quantity of the adenosine methionine synthetase accounts for 31.36 percent of soluble albumen of the strain, the activity of the adenosine methionine synthetase expressed is as high as 7.38U / ml, far higher than that of wild type strains.

The present invention relates to a pregestational gene alarm diagnosis kit and its detection method, including DNA extraction liquor for extracting DNA template of testee, PCR mixed liquor for amplifying specific fragment on the genomic DNA of testee, DNA polymerase, PCR product enzyme excision mixed liquor, restriction endonuclease, positive control template of homozygous mutation and coupling pipe for making PCR reaction. The described alarm gene includes N5, N10-methylene tetrahydrofolate reductase and methionine synthetase reductase gene, their mulant sites are respectively 667-cytosine-thymine and 66 adenine-guanine, and their primer sequences respectively are 5'-TGA AGG AGA AGG TGT CTG CGG GA-3', 5'-AGG ACG GTG CGG TGA GAG TG-3', 5'-GCA AAG GCC ATC GCA GAA GAC AT-3' and 5'-GTG AAG ATC TGC AGA AAA TCC ATG TA-3'. It can be used for pregestational diagnosis of that filial generation produces neural tube deform or not.

The invention features a novel gene encoding methionine synthase reductase. The invention also features a method for detecting an increased likelihood of hyperhomocysteinemia and, in turn, an increased or decreased likelihood of neural tube defects, cardiovascular disease, Down's Syndrome or cancer. The invention also features therapeutic methods for treating and / or reducing the risk of cardiovascular disease, Down's Syndrome, cancer, or neural tube defects. Also provided are the sequences of the human methionine synthase reductase gene and protein and compounds and kits for performing the methods of the invention.

The invention belongs to the technical field of enzyme engineering, and relates to a S-adenosyl-L-methionine synzyme mutant and a preparation method using the same. The mutant is mutated into a plurality of amino acid sites in wild S-adenosyl-L-methionine synzyme of which the amino acid sequence is shown as SEQID NO.1, and the mutated amino acid sites comprise C9R and K224R. According to the S-adenosyl-L-methionine synzyme mutant and the preparation method using the same, compared with the wild S-adenosyl-L-methionine synzyme, the mutant has higher specific activity and has higher product yield and lower enzyme activity reduction when catalyzing is performed for preparing the S-adenosyl-L-methionine synzyme.

The invention discloses a raltitrexed related substance C with inhibition activity on dihydrofolate reductase and methionine synthetase, a preparation method and application thereof as an impurity control group.

The invention discloses a methionine synthetase mutant, a mutant gene and an application of the methionine synthetase mutant and the mutant gene in preparation of vitamin B12. Overexpressed genetic engineering strains of a methionine synthetase gene and a mutant gene in Sinorhizobium meliloti have greatly-improved vitamin B12 producing capability, scarcely have influence on biomass during fermentation culture and have relatively high application and popularization values.

The invention discloses a method for producing L-5-methyltetrahydrofolate (L-5-MTHF) by a biological method. The method comprises the following steps of knocking out a methionine synthetase gene (metH) in a catabolism pathway in escherichia coli, and introducing a new metabolic pathway for producing the L-5-MTHF to construct an engineering bacterium BL21-WL strain or BL21 (delta metH)-C1T strain for producing the L-5-MTHF. By optimizing fermentation conditions for producing the L-5-MTHF by the constructed engineering bacteria, the maximum dry cell weight yield of the engineering bacteria can reach 445.94 mu g.g <-1>, is increased by 99.71% compared with that before optimization and reaches 10.0 times of that of an original strain, and the maximum dry cell weight yield of the engineering bacteria can reach 1249.11 mu g after being converted into the yield of each liter of culture and is far greater than the yield (119.84 mu g.L <-1>) of the L-5-MTHF which takes E.coli as host bacteria in the current report. It is indicated that the method and the constructed engineering bacteria have great application value and market prospects.

The invention relates to a method for catalyzing and synthesizing adenosyl methionine through complete cells. The method comprises the following steps of: (1) culturing recombinants capable of greatly expressing adenosyl methionine synthetase through fermentation; (2) carrying out permeability treatment for the cells used for catalyzing and synthesizing adenosyl methionine with an organic solventor surfactant as the perm solution; (3) using the cells having undergone the permeability treatment to synthesize adenosyl methionine with L-methionine and adenosine triphosphate as raw material. After eight hours of reaction, the amount of synthesized adenosyl methionine reaches 10-12g / L, and the substrate conversion ratio reaches above 95 percent with very simple constituents of the reaction fluid, thereby greatly simplifying the abstraction and purification process of adenosyl methionine. The method can realize production of high-quality adenosyl methionine with large scale, low cost and high efficiency, thereby providing a new way of producing adenosyl methionine.

The invention provides a peanut S-adenosylmethionine synthetase gene AhSAMS and protein and application thereof, and the sequence of the gene is shown in the first sequence in a sequence table. Peanut S-adenosylmethionine synthetase which is interactive with calmodulin protein (CaM) is screened out by screening a protein library, and the genetic sequence and the protein sequence of the peanut S-adenosylmethionine synthetase are obtained. The functions of the peanut S-adenosylmethionine synthetase are determined through further experiments, the different expression conditions of the peanut S-adenosylmethionine synthetase in adversity stress are obtained through draught, high salt, ABA and high temperature highlight stress treatment, and a factual basis is supplied to following further utilization of the peanut S-adenosylmethionine synthetase gene, so that the regulation and control capacity of peanuts to adversity is conveniently improved by further utilizing Ca<2+> and the gene AhSAMS.

The invention provides a combination product for detecting enzyme activity of thiopurine S-methyltransferase. The combination product contains S-adenosyl-L-methionine or an enzymatic reaction system producing the S-adenosyl-L-methionine, a methyl receptor and S-adenosyl-L-homocysteine hydrolase, wherein the enzymatic reaction system producing the S-adenosyl-L-methionine comprises adenosine triphosphate, methionine and S-adenosyl methionine synthetase. Preferably, the combination product is in the form of kit, wherein each ingredient contained in the combination product is in a separately self-existent reagent state. The combination product can be used on semi-automatic or fully-automatic analyzing detection equipment, the detection sensitivity is high, the specificity is high, and the operation is simple and convenient, so that the combination product can be practically popularized.

The invention discloses a Raltitrexed-related substance F with raltitrexed with dihydrofolate reductase and methionine synthetase inhibitory activity, a preparation method thereof, and an applicationof the Raltitrexed-related substance F as an impurity reference substance.

The invention provides a recombined strain capable of simultaneously compressing glutathione synthetase system and adenosyl methionine synthetase and a method for simultaneously producing glutathione and S-adenosyl methionine at high yield. The recombined strain comprises: fragment capable of exogenously expressing gama-glutamyl cysteine synthetase or glutathione synthetase and fragment capable of exogenously expressing adenosyl methionine synthetase and the strain is capable of expressing glutathione synthetase system and / or adenosyl methionine synthetase, thereby fermenting glutathione and adenosyl methionine. The recombined strain is capable of simultaneously producing glutathione and adenosyl methionine by fermentation at high yield and independently producing glutathione or adenosyl methionine by fermentation.

The invention provides a recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299 and the composition method of the recomposed large intestine Escherichia coli. The invention further provides a recomposed expression carrier pMETK of adenosine methionine synthetase. In the recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299, the expression quantity of the adenosine methionine synthetase accounts for 31.36 percent of soluble albumen of the strain, the activity of the adenosine methionine synthetase expressed is as high as 7.38U / ml, far higher than that of wild type strains.

This application relates to S-adenosylmethionine synthetase preparation, its preparation method and use. The preparation method of the present disclosure uses genetic engineering technology to construct the S-adenosylmethionine synthetase gene into a prokaryotic expression vector, and transforms Escherichia coli to construct a recombinant host cell; obtains high-yield bacterial cells through fed-batch fermentation; Afterwards, the S-adenosylmethionine synthetase is purified by affinity chromatography, and the chromatographic purity of the obtained protein is above 90%. The present application also relates to the use of S-adenosylmethionine synthetase in the preparation of homocysteine ​​diagnostic reagents.

The invention discloses a methionine synthetase mutant, a mutant gene and its use in the preparation of vitamin B 12 in the application. Genetically engineered bacteria of the methionine synthase gene and mutant gene overexpressed in Sinorhizobium meliloti, which produce vitamin B 12 The ability of the method has been greatly improved, and it has almost no impact on the biomass during fermentation, which has great application and promotion value.

The invention relates to a genetically engineered bacterium for producing N-acetyl-5-methoxytryptamine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium for producing the N-acetyl-5-methoxytryptamine comprises a caffeic acid O-methyltransferase gene and a methionine adenosine transferase gene or / and a methionine synthetase gene, and can be used for producing a large amount of caffeic acid O-methyltransferase with biological activity and methionine adenosine transferase or / and methionine synthetase in cells. According to the method, through overexpression of the caffeic acid-O methyltransferase (COMT) gene and the methionine adenosine transferase gene or / and the methionine synthetase gene, an N-acetyl-5-methoxytryptamine biosynthesis pathway is reconstructed, and the synthesis yield of N-acetyl-5-methoxytryptamine is obviously increased.

The invention belongs to the technical field of enzyme engineering, and relates to a S-adenosylmethionine synthetase mutant and a preparation method using the same. The mutant mutates multiple amino acid positions in the wild-type S-adenosylmethionine synthetase of the amino acid sequence shown in SEQ ID NO.1, and the mutated multiple amino acid positions include C9R and K224R. Utilizing the S-adenosylmethionine synthetase mutant of the present invention and its preparation method can make the mutant have higher specific activity than wild-type S-adenosylmethionine synthetase, and can be used in When catalyzing the preparation of S-adenosylmethionine, it has higher product yield and lower enzyme activity decline.

The invention belongs to the technical field of biology, and relates to a high-selectivity probe of non-cobalt-dependent methionine synthase in higher plants and application thereof. The highly selective probe contains sulfonyl fluoride groups and is capable of specifically and covalently labeling proteins at lysine residues. The probe can be used for detecting the activity and expression quantityof non-cobalt-dependent methionine synthase in higher plants and positioning tissues and subcells. The probe provided by the invention provides a targeted protein research tool, so that deeper research on protein expression changes and protein network maps of different plant types, different strains or different development stages can be realized.

The invention belongs to the technical field of enzyme engineering, and relates to recombinant escherichia coli engineering bacteria and a method for preparing S-adenosylmethionine using the same. Therecombinant escherichia coli engineering bacteria contains a recombinant expression vector, and the recombinant expression vector contains a gene expressing S-adenosylmethionine synthetase and a geneexpressing ATP synthetase. Compared with a traditional fermentation method and an in-vitro enzyme-catalyzed synthesis method, S-adenosylmethionine can be prepared with more simplicity, lowest cost and high yield by using the recombinant escherichia coli engineering bacteria and the method for preparing the S-adenosylmethionine using the same.

The invention discloses a method for preparing S-adenosylmethionine by an enzyme method capable of eliminating key impurities. The method comprises the following steps: step 1, constructing gene-deleted escherichia coli: knocking out a speD gene in escherichia coli BL21 (DE3) to obtain gene-deleted escherichia coli which does not generate S-adenosylmethionine decarboxylase; and 2, transforming the escherichia coli by using a recombinant plasmid containing a target gene: expressing the S-adenosylmethionine synthetase gene metK by using a gene deletion bacterium, culturing and inducing by using an inducer, and directly applying a crude enzyme liquid extracted after expression to enzyme reaction to produce S-adenosylmethionine. The method has the effect of greatly reducing the subsequent SAM purification cost.

The invention discloses an S-adenosylmethionine synthetase mutant and a preparation method of the mutant. The mutant is prepared by single-point, double-point or three-point mutation of a site 65, a site 104 and a site 186 in an amino acid sequence shown as SEQ ID NO. (sequence identifier number) 1. Compared with wild S-adenosylmethionine synthetase, the mutant increases an accumulation amount ofSAM (S-adenosylmethionine) to be increased by 1.06mM in a 10mM substrate reaction system. At the same time, enzyme activity of the mutant is 3.3 times that of wild eMAT (escherichia coli S-adenosylmethionine synthetase), and is 6.02+ / -0.22U / mg. The possibility is further provided for enzyme method preparation of SAM, and a clue is provided for improving a product inhibition phenomenon of an enzymeby molecular modification.

The invention belongs to the technical field of enzyme engineering, and relates to a recombinant Escherichia coli engineering bacterium and a method for preparing S-adenosylmethionine by using the same. The recombinant Escherichia coli engineering bacterium contains a recombinant expression vector, and the recombinant expression vector contains a gene expressing S-adenosylmethionine synthetase and a gene expressing ATP synthetase. Compared with the traditional fermentation method and in vitro enzyme-catalyzed synthesis method, the recombinant Escherichia coli engineering bacteria of the present invention and the method for preparing S-adenosylmethionine can be prepared with simpler, lower cost and high yield. ‑adenosylmethionine.

The invention provides a recombined strain capable of simultaneously compressing glutathione synthetase system and adenosyl methionine synthetase and a method for simultaneously producing glutathione and S-adenosyl methionine at high yield. The recombined strain comprises: fragment capable of exogenously expressing gama-glutamyl cysteine synthetase or glutathione synthetase and fragment capable of exogenously expressing adenosyl methionine synthetase and the strain is capable of expressing glutathione synthetase system and / or adenosyl methionine synthetase, thereby fermenting glutathione and adenosyl methionine. The recombined strain is capable of simultaneously producing glutathione and adenosyl methionine by fermentation at high yield and independently producing glutathione or adenosyl methionine by fermentation.