20 results about "Sulfotransferase" patented technology

The invention relates to the field of biosynthesis of glycosaminoglycan compounds, and discloses a 2-O-sulfotransferase mutant and application of the 2-O-sulfotransferase mutant in heparin synthesis. On the basis of a chicken source 2-O-sulfotransferase protein sequence, site-directed mutagenesis design and modification are carried out, mutation sites comprise 6I, V60I, R120K, G118H, S157H, L227P, E270D, V258I and A275P, and the 2-O-sulfotransferase mutant is obtained. Compared with a wild type, the 2-O-sulfotransferase mutant provided by the invention has the advantages that the expression level, the thermal stability and the catalytic efficiency are obviously improved, and the 2-O-sulfotransferase mutant shows higher conversion rate and structural modification consistency in a reaction system taking N-sulfoheparin precursor polysaccharide as a substrate. The mutant has a wide application prospect in the structural precise control and industrial synthesis process of biological heparin.

The invention provides an N-deacetylase / N-sulfotransferase and an application of the N-deacetylase / N-sulfotransferase. According to the invention, systematic stability design and surface electrostatic optimization are carried out on the N-deacetylation structural domain of NDST, and multiple engineering strategies such as structural domain fusion and linker peptide optimization are combined to synergistically improve the specific activity, substrate affinity and bifunctional catalytic synergy of enzyme. Experimental results show that the specific enzyme activity of the modified NDST mutant is obviously higher than that of natural enzyme while the difunctional catalytic capability is maintained.

This invention discloses a recombinant 2-O-sulfonyltransferase mutant and its preparation method, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (E28~N356) mutant of hamster-derived 2-O-sulfonyltransferase (L119V-R188E-A240I) with an N-terminal maltose-binding protein (MBP) tag. The recombinant 2-O-sulfonyltransferase mutant exhibits higher activity, 1.9 times that of the recombinant wild type, and greater stability, retaining 86.6% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant 2-O-sulfonyltransferase is increased by 5 times. This mutant, as a novel biocatalyst with high efficiency, stability, and controllable production costs, has significant application value and broad industrialization prospects in the industrial enzymatic synthesis of glycosaminoglycans such as heparin and heparan sulfate.

The invention relates to a heparin 3-O-sulfuric acid transferase mutant with high enzyme activity and high thermal stability as well as a coding gene and application of the heparin 3-O-sulfuric acid transferase mutant. The amino acid sequence of the 3-O-sulfate transferase mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The 3-O-sulfuric acid group transferase mutant has remarkable and superior performance in the enzymatic synthesis of heparin glycosaminoglycans. Performance determination shows that the 3-O-sulfate transferase mutant SM1 realizes the synergistic improvement of the catalytic activity and the thermal stability. The total enzyme activity of the 3-O-sulfotransferase is improved by 1.5 times compared with that of wild chicken source 3-O-sulfotransferase. More prominently, the thermal stability of the 3-O-sulfo-transferase is fundamentally improved: the activity of the wild chicken source 3-O-sulfo-transferase is generally and rapidly attenuated within 24 hours at 37 DEG C, while the 3-O-sulfo-transferase mutant SM1 can still retain about 50% of initial activity after being subjected to heat preservation for 8 days under the same condition, so that the 3-O-sulfo-transferase mutant SM1 shows excellent operation stability.

The present invention relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising a step (I) of modifying the nucleotide sequence of DNA encoding an N-deacetylase / N-sulfotransferase, and a step (II) of introducing DNA containing the nucleotide sequence modified in step (I) into the microorganism in an expressible manner, wherein the step (I) is a step of codon optimization according to the frequency of use of codons of a biological species different from the microorganism. Furthermore, the present invention relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising the step (i) of expressing a specific protein in the microorganism.

Compositions and methods are disclosed herein for inducing (up-regulating) the expression of sulfotransferases in the hair follicles, e.g., the scalp. Increasing sulfotransferase is beneficial for metabolizing pro-drugs that require sulfonation to be activated, e.g., minoxidil sulfate is the active metabolite of minoxidil. A method for combining the compositions described herein with topical minoxidil to enhance minoxidil treatment for androgenetic alopecia is described. Additional methods and compositions include the use of retinoid X receptor agonists, retinoic acid receptor agonists, and nuclear receptor agonists in an RXR-NR heterodimer. Additional methods and compositions include the use of a topical solution containing an alkalinizing agent or an alkalinizing agent used with a penetration enhancer for up-regulating the sulfonating capacity of hair bearing skin, hair follicles, and / or keratinocyte cells. In addition, compositions and methods for increasing or decreasing the growth rate of hair follicles is disclosed by altering the intracellular pH. Additionally, methods for preparing a stable liposomal containing solution are described.

The application discloses a method for synthesizing chondroitin sulfate with specific molecular weight by metabolic engineering of Pichia pastoris, and belongs to the technical field of metabolic engineering and synthetic biology. The application uses Pichia pastoris GS115 as a host, constructs a chondroitin synthesis pathway by heterologous expression of kfoA, kfoC and tuaD genes, realizes intracellular synthesis of chondroitin, further strengthens the supply of precursors UDP-GlcA and UDP-GalNAc and redirects carbon metabolism, and improves the yield of chondroitin to 564 mg / L, and the yield in a 5-L fermenter reaches 1.15-5.3 g / L. Further expression of chondroitin sulfate sulfotransferase C4ST and an endogenous phosphorylase system realizes synthesis of chondroitin sulfate with a molecular weight of 40-115 kDa. Finally, by integration of an inducible expression chondroitin lyase ABCI, the control of induction time is 1-20 h, and controllable regulation of chondroitin sulfate molecular weight from 500 Da to 115 kDa is realized.

The invention relates to a recombinant chondroitin-6-O-sulfotransferase mutant and application of the recombinant chondroitin-6-O-sulfotransferase mutant in synthesis of chondroitin sulfate C, and belongs to the technical field of bioengineering. According to the invention, a dissolution promoting label is fused at the N end of a chondroitin-6-O-sulfotransferase mutant, so that the efficient soluble expression of the enzyme is successfully realized, and the proportion of soluble components is as high as 90%. The mutant HsC6ST-delta loop has the advantages that by carrying out amino acid sequence truncation on chondroitin-6-O-sulfotransferase, the constructed mutant HsC6ST-delta loop has higher chondroitin-6-O-sulfonation activity compared with HsC6ST chondroitin-6-O-sulfonation, the sulfonation rate reaches 78.4% after reaction is carried out for 12 h under the conditions that the temperature is 40 DEG C and the pH is 8.0, and the Tm and half-life period are increased by 9 DEG C and 1.54 h compared with those of an original enzyme.

The application discloses a chondroitin-4-O-sulfotransferase mutant construction method and belongs to the field of bioengineering technology. The application is characterized in that a wild-type chondroitin-4-O-sulfotransferase (C4ST) is precisely reformed through a calculation-guided semi-rational design, a SUMO label and an AE connecting peptide are fused at an N end to improve soluble expression, and on this basis, a three-point combined mutant V74L / M117T / Y235V is constructed through a site-directed mutagenesis technology, the enzyme activity of the mutant in a 5L fermenter is as high as 76328 U / L, the specific enzyme activity is 327 U / mg, and the enzyme activity and the specific enzyme activity are 16.6 times and 1.3 times of the wild type, respectively. The mutant is used for catalyzing preparation of chondroitin sulfate A, the sulfation degree can reach 97.0% after 48 h of reaction at 37 DEG C. The application significantly improves the catalytic efficiency of C4ST and provides a key technology of efficient and stable enzyme method for preparing CSA.

The invention discloses a parabacteroides gore DZ001 strain with a cholesterol sulfation function and application of the parabacteroides gore DZ001 strain. The preservation number of the parabacteroides gore DZ001 is GDMCC No: 67376, and the preservation number of the parabacteroides gore DZ001 is H. The strain is separated from faeces of healthy people, a genome carries a sequence for coding sulfotransferase, cholesterol can be specifically catalyzed to generate cholesterol sulfate with higher water solubility, intestinal epithelium reabsorption is blocked, and faeces excretion is promoted. In-vitro experiments show that the strain has excellent cholesterol degradation capacity, gastrointestinal tract tolerance and safe gamma-hemolysis characteristics; in-vivo experiments prove that the traditional Chinese medicine composition can remarkably reduce serum total cholesterol and triglyceride levels of high-fat diet mice, increase high-density lipoprotein cholesterol, improve liver fatty degeneration and regulate blood glucose metabolism. The strain and viable bacteria, supernate or cell-free extract thereof can be widely applied to preparation of cholesterol-reducing health-care foods, medicines and animal feeds, and a new way is provided for prevention and treatment of hypercholesterolemia and related cardiovascular and cerebrovascular diseases.

The present invention relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, comprising the steps of (I) modifying the nucleotide sequence of DNA encoding N-deacetylase / N-sulfotransferase and (II) introducing the DNA containing the modified nucleotide sequence in step (I) into a microorganism so that it can express, wherein step (I) is a step of codon optimization to match the codon usage frequency of a different species of organism than the microorganism. The present invention also relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, comprising the step of (i) expressing a specific protein in the microorganism.

The present disclosure relates to heterocyclic compounds, pharmaceutical compositions comprising said compounds and a method of treating cancer comprising administering the compounds, or compositions thereof to a subject in need thereof. Specifically, the present disclosure provides heterocyclic compounds useful as anti-cancer agents, pharmaceutical compositions comprising said compounds and a method of treating SULT1 positive cancer comprising administering the compounds, or compositions thereof to a subject in need thereof.

This invention belongs to the field of bioengineering and enzyme engineering technology, specifically relating to a method for improving the soluble expression and catalytic activity of chondroitin 4-O-sulfotransferase. Using chondroitin 4-O-sulfotransferase MamC4ST as the research object, this invention constructs various fusion expression systems of soluble tags in prokaryotic hosts, and systematically evaluates its soluble expression behavior and catalytic performance under different host strains and molecular chaperone co-expression conditions. The MBP tag has significant advantages in improving the soluble expression of MamC4ST; under the synergistic effect of the GroEL / GroES molecular chaperone, the recombinant engineered strain MBP-Δ60MamC4ST-Shuttle7-pGro7 exhibits excellent catalytic performance on chondroitin substrates, with conversion and sulfation rates reaching 98.41% and 95.03%, respectively, at a substrate concentration of 2 g / L.

The invention relates to heparin 6-O-sulfate transferase, a high-robustness mutant of heparin 6-O-sulfate transferase, a mutant screening method and an application of heparin 6-O-sulfate transferase. The amino acid sequence of the heparin 6-O-sulfate transferase is as shown in SEQ ID NO.2, and the amino acid sequence of a truncated body of the heparin 6-O-sulfate transferase is as shown in SEQ ID NO.4. The invention also provides a high-robustness heparin 6-O-sulfuryl transferase mutant M10 (SRC), the amino acid sequence of which is as shown in SEQ ID NO.8, and the mutant M10 (SRC) is characterized in that after 22 amino acids are truncated from the N end of heparin 6-O-sulfuryl transferase Om6OST-1, 43 amino acids are mutated. The heparin 6-O-sulfuryl transferase is brand new heparin 6-O-sulfuryl transferase Om6OST-1 which is derived from qiangqiangfish, and has the activity of transferring sulfate radicals to-GlcNS-of a heparin structure. The heparin 6-O-sulfuric acid group transferase truncated body Om6OST-1-delta N22 has the longest half-life period while the activity is maintained. The 6-O-sulfuryl transferase mutant M10 (SRC) has higher activity of transferring sulfate radicals to-GlcNS-of a heparin structure, and the activity of the 6-O-sulfuryl transferase mutant M10 (SRC) is 25.94 times that of a wild type.

The application discloses a compound, a composition and application, and claims the compound shown in formula (I) or a salt thereof. The compound provided by the application is an enzyme responsive fluorescent substrate specially designed for sulfotransferase SULT1A1, and precise recognition and specific catalytic response to SULT1A1 are realized from the molecular structure level. The compound has a low fluorescent background itself, the sulfonated fluorophore in the catalytic reaction product has the characteristics of near-infrared emission and large Stokes shift, can effectively avoid the background interference of excitation light on fluorescent detection, and is excellent in specificity, stability and linearity. Therefore, the compound or the salt thereof and the composition containing the compound or the salt thereof have the prospect of being developed into a reagent, a kit or a microplate for in vitro quantitative or qualitative detection of SULT1A1 activity, in situ imaging and positioning of SULT1A1 in cells or in vivo, and high-throughput screening of SULT1A1 inhibitors or activators.

The invention discloses an application of gallic acid in preparation of a salmonella typhimurium CDSH inhibitor, and belongs to the technical field of biological medicine. CDSH enzyme activity inhibition experiment, H2S detection experiment, antibiotic stimulation experiment and greater wax moth infection model experiment analysis finds that the gallic acid can reduce the stress tolerance of S.Typhimurium by inhibiting the activity of CDSH enzyme, reduce the stress tolerance of S.Typhimurium, reduce the stress tolerance of S.Typhimurium, reduce the stress tolerance of S.Typhimurium, and reduce the stress tolerance of S.Typhimurium by inhibiting the activity of CDSH enzyme. The cysteine thiotransferase has the advantages that the cysteine thiotransferase can be used as a transferase inhibitor, the antibiotic sensitivity and the in-vivo removal effect of the cysteine thiotransferase are enhanced, the gallic acid is used for inhibiting the enzyme activity of CDSH and reducing the salmonella tolerance, and the cysteine thiotransferase inhibitor and the medicine for treating the caused infectious diseases are prepared and are used for preventing or treating salmonella typhimurium infection.

Compositions and methods are disclosed herein for inducing (up-regulating) the expression of sulfotransferases in the hair follicles, e.g., the scalp. Increasing sulfotransferase is beneficial for metabolizing pro-drugs that require sulfonation to be activated, e.g., minoxidil sulfate is the active metabolite of minoxidil. A method for combining the compositions described herein with topical minoxidil to enhance minoxidil treatment for androgenetic alopecia is described. Additional methods and compositions include the use of retinoid X receptor agonists, retinoic acid receptor agonists, and nuclear receptor agonists in an RXR-NR heterodimer. Additional methods and compositions include the use of a topical solution containing an alkalinizing agent or an alkalinizing agent used with a penetration enhancer for up-regulating the sulfonating capacity of hair bearing skin, hair follicles, and / or keratinocyte cells. In addition, compositions and methods for increasing or decreasing the growth rate of hair follicles is disclosed by altering the intracellular pH.

The purpose of the present invention is to provide a 2-O-sulfotransferase having higher 2-O-sulfation activity than the conventional art and exhibiting excellent selectivity for iduronic acid residue in heparosan. The protein comprises a catalytic domain of a protein comprising an amino acid sequence represented by SEQ ID NO: 1 with the 111th amino acid residue substituted with an L-glutamic acid residue or an L-asparagine residue, and has 2-O-sulfation activity. The selectivity of the 2-O-sulfation activity for iduronic acid residues is higher than that of a protein comprising a catalytic domain of a protein comprising an amino acid sequence represented by SEQ ID NO: 1.

The present application relates to the field of biotechnology and the field of heparin biosynthesis, and discloses a heparin modification enzyme gene, a high-efficiency expression engineering strain and application. Specifically, the present application relates to a bifunctional N-deacetylase / N-sulfotransferase NDST gene, a glucuronate C5-epimerase C5epi gene, a sulfated heparan 2-sulfotransferase (2-OST) gene, a sulfated heparan 6-sulfotransferase 6-OST gene derived from a Limosa lapponica and a sulfated heparan 3-sulfotransferase 3-OST gene derived from a Hirundo rustica. The heparin modification enzyme genes obtained by mining are derived from various bird genomes that have not been annotated, and the high-efficiency expression and purification of the genes in Pichia pastoris or Escherichia coli are realized. Meanwhile, it is verified that the genes have biological catalytic activity and can be used for in vitro multi-enzyme catalytic synthesis of heparin. Therefore, the present application has potential application value.

Heparin compounds and synthetic heparin analogues having short acting anticoagulant activity are provided. Methods of synthesizing such heparin compounds, including chemoenzymatic pathways using sulfotransferase enzymes are provided. Methods of treating subjects in need of anticoagulant activity are provided.