30 results about "S-Adenosylmethionine Synthetase" patented technology

The invention discloses a method for producing S-adenosylmethionine through fermentation of recombinant pichia pastoris, which comprises that strain seed liquid of the recombinant pichia pastoris of a S-adenosylmethionine synthetase gene integrated with a heterologous expression gene is inoculated into a fermentation medium for fermentation culture, and is characterized in that: the inoculum size of the strain seed liquid of the recombinant pichia pastoris into the fermentation medium is between 5 and 10(v / v)percent; the culture condition in the fermentation culture is that the temperature is between 28 and 32 DEG C, the pH is between 3.0 and 8.0, and the dissolved oxygen level is kept between 30 and 100 percent; and when the strain seed liquid is cultured until the microbial density is between 80 and 120 g / L, the fermentation medium is added with methanol to begin methanol induction, the concentration of the methanol is controlled to be between 0.3 and 1.0(w / v)percent, and the fermentation medium is simultaneously added with L-methionine of which the concentration is controlled to be between 12.5 and 100mmol / L and ammonium sulphate in which the ammonium ion concentration is controlled to be between 300 and 600mmol / L. The method for producing the S-adenosylmethionine through the fermentation can accumulate SAM yield over 11.6g / L in 100h of fermentation time, so the method has short fermentation period, high SAM yield and simple and convenient operation, and can be used for preparing the SAM in mass production.

The invention relates to the method of gene fixed-point mutation which is a modified overlap extension PCR method and the mutated gene. The process includes the fragment composition, the double mixing, the prepared extension, the whole DNA composition and after extension. The method is detected by the synzyme gene sam1 from the SAM of the Saccharomyces cerevisiae and gets the mutated gene modified by the rare coden. The SAM synzyme encoding by the gene can express in the yeast and improves the yield of the intracellular SAM. The invention can reach the many fixed-point mutations in a same reaction process.

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.

A process for preparing refractory S-adenosylmethionine synthetase gene and the polypeptide for coding it and the said gene used for preparing its transgenic microbe or animal and plant and recovering the enzyme coded by it, and in addition, the amino acid sequence of the said polypeptide with the activity of the said refractory S-adenosylmethionine synthetase and its functional analog, and the process based on the whole genom sequencing and analyzing of thermophilic anaerobe for preparing, separating and purifying the said polypeptide are disclosed. It also relates to coding of the separatedDNA with activity or its function identical variant and use of the recombinant DNA technique with the said DNA to produce the above-mentioned polypeptide or its function identical variant.

The invention discloses recombinant streptomyces nodosus capable of producing amphotericin and applications of the recombinant streptomyces nodosus. The recombinant streptomyces nodosus is obtained byimporting vitreoscilla hemoglobin (Vhb), S-adenosylmethionine synthetase (Metk), amphotericin synthetic gene cluster regulating factor (AmphRIV), secondary metabolite global regulation factor (AraC)and erythrocin strong promoter ermE*p sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 into streptomyces nodosus ZJB2016050. Through importing and overexpression of the gene, in recombinant streptomyces nodosus, the yield of amphotericin B is improved by about 45%, the yield of the by-product amphotericin A is reduced by 60%, meanwhile, the growth cycle of the thallus during the fermentation process is shortened, and thus the purpose of improving the production efficiency is achieved.

The invention discloses a high-throughput screening method for catalytic activity modification of S-adenosylmethionine synthetase; an eMAT mutant with significantly improved catalytic activity is obtained by the high-throughput screening method. The specific enzyme activity of the mutant is increased from 1.82 U / mg of wild-type eMAT to 4.15 U / mg. By combined mutation with reported mutation at position 303, the obtained mutant is increased in catalytic activity in comparison to encoded eMAT in SEQ ID NO. 1, so that the time to completely convert a 1 mM substrate is 5 min earlier under the samereaction conditions.

The invention provides a method for preparing an ademetionine synthetase in large scale. In the method, an SAM expression level of engineering bacteria in fermentation liquor is improved, and simultaneously the turbidity of the fermentation liquor can be reduced so as to bring more contribution to further extraction and preparation of an enzyme by selectively adding divalent metal ions and a flocculating agent.

The invention belongs to the field of plant genetic engineering, and relates to a soybean GmSAMS1 gene and an application thereof. According to the soybean GmSAMS1 gene and the application thereof, one soybean S-adenosylmethionine synthetase gene GmSAMS1 is cloned from a soybean, and the sequence is SEQ ID NO.1. The protein level of the gene in a soybean leaf is changed after being induced by prodenia litura. Through a plant expression vector, the GmSAMS1 gene is introduced in a tobacco cell, the tobacco with overexpression GmSAMS1 is compared with a comparison tobacco, and the insect resistance to the prodenia litura is improved obviously. The invention discloses a genetic engineering improvement method for conducting plant insect resistance transformation through the gene. By the adoption of the method, a certain function is achieved on cultivation of a plant variety with the insect resistance changed, in particular a soybean variety, and the soybean GmSAMS1 gene and the application thereof can be used for increasing the yield and improving the quality of crops especially the soybean by directly transforming the insect resistance of the crops.

The invention discloses a monoclonal antibody of S-adenosylmethionine synthetase. A heavy chain variable region sequence of the monoclonal antibody contains CDR amino acid sequences shown by SEQ ID NO.2, 4 and 6; a light chain variable region sequence contains CDR amino acid sequences shown by SEQ ID NO.9, 11 and 13. The polypeptide of the enzyme is used as an immunogen for immunizing a mouse to obtain lymphocyte; a hybridoma fusion technology is used for preparing fusion cells; a cell strain capable of detecting and identifying the S-adenosylmethionine synthetase at high affinity and high specificity is obtained. The monoclonal antibody can be used for detecting the content of S-adenosylmethionine synthetase in bodies of animals and plants. The monoclonal antibody provides a targeted protein study tool, so that the deep level study of protein network atlas and the protein expression change on different plant types, different strains or different development periods can be realized.

The invention discloses a method for synthesizing S-adenosylmethionine (SAM) through coupling of escherichia coli and saccharomyces cerevisiae. The method comprises basic processes of firstly, respectively cultivating an escherichia coli engineered strain BL21 / pET-28a-SAMS containing S-adenosylmethionine synthetase gene (SAMS) and saccharomyces cerevisiae at proper conditions, then mixing according to a certain proportion, coupling so as to generate SAM; secondly, allowing the synthesized SAM to secrete to outer side of the cell by taking an organic solvent as a permeability agent; and finally, analyzing and detecting the SAM by a high performance liquid chromatography technology. The invention provides a novel method for producing SAM at low cost, and the method is simple and convenient to operate, and can catalyze ATP and L-Met by the whole cell so as to synthesize SAM.

The invention relates to an S-adenosylmethionine synthetase preparation and a preparation method and application thereof.The preparation method includes the steps that S-adenosylmethionine synthetase genes are constructed into a prokaryotic expression vector through the gene engineering technology, and escherichia coli is converted to construct recombinant host cells; high-yield thalli are obtained through batch fermentation of supplementary materials; then through the affinity chromatography technology, S-adenosylmethionine synthetase is purified, wherein the chromatographic purity of obtained protein is 90% or above.The invention further relates to the application of S-adenosylmethionine synthetase to preparation of a homocysteine diagnostic reagent.

The invention discloses a series of Methanococcus jannaschii S-adenosylmethionine synthetase mutants with improved thermostability and high catalytic activity obtained by using gene mutation technique, characterized in that these mutants refer to an enzyme using Sequence 2 in the Sequence Listing as the reference sequence and contains at least one mutation at position 102, position 93, position 230, and position 357 and has a catalytic activity at least 70% higher than that of the wild-type S-adenosylmethionine synthetase using adenosine triphosphate (ATP) and methionine as substrates. These S-adenosylmethionine synthetase mutants can be used in the production of S-adenosylmethionine.

The invention discloses a construction method for pichia pastoris bacterial strain of high-yield S-ademetionine. The construction method is characterized by comprising the following steps of: carrying out PCR (Polymerase Chain Reaction) augmentation on saccharomyces cerevisiae genome to obtain S-ademetionine synthetase gene sam2; connecting the S-ademetionine synthetase gene sam2 to plasmid pPIC9K to obtain an eukaryon expression vector pPIC9K-sam2; transforming the eukaryon expression vector pPIC9K-sam2 to pichia pastoris; carrying out PCR augmentation on the pichia pastoris genome to obtain cystathionine beta synthetase gene cbs; sub-cloning the cystathionine beta synthetase gene cbs to a vector pMD19T to obtain a recombinant vector T-cbs; introducing bleomycin resistance gene Zeocin to the cystathionine beta synthetase gene of the recombinant vector T-cbs to obtain cbs gene, knocking out plasmid T-C-Z, and obtaining cbs gene knockout segment C-Z after enzyme digestion; transforming the segment C-Z to the pichia pastoris containing pPIC9K-sam2 obtained in the step one to obtain pichia pastoris bacterial strain of high-yield S-ademetionine with cbs gene kicked out and sam2 gene reinforced.

The invention relates to a method for preparing S-adenosylmethionine. According to the method, a pet8 gene is transferred into an S-adenosylmethionine production strain so that secretory expression of S-adenosylmethionine by the S-adenosylmethionine production strain is obviously promoted; and an S-adenosylmethionine synthetase gene and the pet8 gene are transferred into the S-adenosylmethionine production strain so that a S-adenosylmethionine yield can be further improved. The method solves the technical problem that based on the prior art, an S-adenosylmethionine production strain carries out secretory expression of S-adenosylmethionine difficultly, and promotes separation or purification of a downstream S-adenosylmethionine.

The invention provides a preparation method of adenosylmethionine butanedisulfonate with health-care functions, and belongs to the technical field of pharmacy. According to the preparation method of the adenosylmethionine butanedisulfonate with the health-care functions, proline, fumaric acid and Na(2)HPO(4)-12H(2)O are added into afermentation medium at a content ratio of 2-4 to 1-2 to 1-2 duringthe implementation process so as to have expression level of s-adenosylmethionine synthetase improved with catalytic activity of the s-adenosylmethionine synthetase in a living body increased; and thus, improvement of L-methionine conversion rate can be guaranteed with addition of adenosine triphosphate at a relatively low content in a fermentation system. Reaction efficiency of the obtained adenosylmethionine with butanedisulfonic acid is high, so that the finally prepared adenosylmethionine butanedisulfonate product has obviously improved purity.

The invention discloses a series of Methanococcus jannaschii S-adenosylmethionine synthetase mutants with improved thermostability and high catalytic activity obtained by using gene mutation technique, characterized in that these mutants refer to an enzyme using Sequence 2 in the Sequence Listing as the reference sequence and contains at least one mutation at position 102, position 93, position 230, and position 357 and has a catalytic activity at least 70% higher than that of the wild-type S-adenosylmethionine synthetase using adenosine triphosphate (ATP) and methionine as substrates. These S-adenosylmethionine synthetase mutants can be used in the production of S-adenosylmethionine.

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.

This invention relates to an isolated nucleic acid fragment encoding a plant enzyme that catalyze steps in the biosynthesis of lysine, threonine, methionine, cysteine and isoleucine from aspartate, the enzyme a member selected from the group consisting of: dihydrodipicolinate reductase, diaminopimelate epimerase, threonine synthase, threonine deaminase and S-adenosylmethionine synthetase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the enzyme, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the enzyme in a transformed host cell.

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 invention provides a short-tube lycoris S-adenosylmethionine synthetase and its coding gene LrSAMS and use. After salt treatment, as time went on, gene LrSAMS expression is improved. The coding gene LrSAMS or a DNA sequence which is homologous with the coding gene LrSAMS and can code the same functional protein is introduced into escherichia coli and compared with the original strain, the recombinant strain has higher high-concentration NaCl and KCl resistance. The short-tube lycoris S-adenosylmethionine synthetase and its coding gene have important theoretical and practical significances for research on a plant salt-resistant mechanism, improvement of plant salt and alkali resistance, and improvement of relative characters, have important effects in improvement of plant salt and alkali-resistant gene engineering, and have a wide application prospect.

This invention relates to a method for gene directed evolution, specifically, a method for gene recombination and application. The method comprises the steps of: dividing the reaction into two groups, performing recombination, mixing the two reactants, and performing a series of PCR reactions to realize gene recombination. The sequencing results show that the method is better than previous gene recombination methods, and is especially suitable for recombination of genes with low sequence identity. The effectivity of the method is testified by using adenosylmethionine synthetase genes from Escherichia coli and yeast as the starting genes, and can obtain high activity recombined enzyme.

The invention discloses a method for preparing S-ademetionine and D-methionine. The method comprises the following steps: fermenting D / L-methionine which serves as a raw material to obtain the S-ademetionine; separating the S-ademetionine from bacteria cells; separating the D-methionine from fermentation liquor. According to the method, the low-price D / L-methionine serves as the raw material, strains containing S-adenosylmethionine synthetase are adopted for fermentation, the L-methionine reacts with ATP in the bacteria cells to generate the S-ademetionine, the S-ademetionine is separated from the D-methionine through a cell membrane, and the S-ademetionine and the D-methionine are separated in separation manners such as a centrifugal separation manner and an ion exchange manner respectively; according to the method, the S-ademetionine and the D-methionine can be synchronously produced, and the D-methionine can be recycled.

The invention provides a method for preparing an ademetionine synthetase in large scale. In the method, an SAM expression level of engineering bacteria in fermentation liquor is improved, and simultaneously the turbidity of the fermentation liquor can be reduced so as to bring more contribution to further extraction and preparation of an enzyme by selectively adding divalent metal ions and a flocculating agent.

The invention relates to a method for preparing S-adenosylmethionine. According to the method, a pet8 gene is transferred into an S-adenosylmethionine production strain so that secretory expression of S-adenosylmethionine by the S-adenosylmethionine production strain is obviously promoted; and an S-adenosylmethionine synthetase gene and the pet8 gene are transferred into the S-adenosylmethionine production strain so that a S-adenosylmethionine yield can be further improved. The method solves the technical problem that based on the prior art, an S-adenosylmethionine production strain carries out secretory expression of S-adenosylmethionine difficultly, and promotes separation or purification of a downstream S-adenosylmethionine.

The invention discloses a monoclonal antibody of S-adenosyl methionine synthetase. A heavy-chain variable region sequence of the monoclonal antibody includes a CDR1 amino acid sequence shown as SEQ ID NO.2 and a CDR2 amino acid sequence shown as SEQ ID NO.4, and a light-chain variable region sequence of the monoclonal antibody includes a CDR3 amino acid sequence shown as SEQ ID NO.6. Polypeptide of the S-adenosyl methionine synthetase serves as an immunogen to immunize mice to obtain lymphocytes, and fusion cells are obtained through a hybridoma cell fusion technology, so that a cell strain capable of detecting the S-adenosyl methionine synthetase in a high-affinity and high-specificity manner can be generated. The monoclonal antibody of the S-adenosyl methionine synthetase has the advantages that the monoclonal antibody can be used for detecting the content of the S-adenosyl methionine synthetase in animal and plant bodies; the monoclonal antibody provides a targeting protein study tool and makes protein expression change of different plant types or different strains or in different development periods and deeper studies on protein network maps possible.