35 results about "Hydroxysteroid dehydrogenase" patented technology

The invention discloses a high-stability hydroxysteroid dehydrogenase mutant, a co-immobilized enzyme construction method and application. According to the invention, a site-directed mutant library is constructed based on computer-aided design, and mutants with significantly improved thermal stability and greatly improved catalytic efficiency compared with wild type mutants are obtained through screening; meanwhile, a co-immobilized enzyme construction method based on electrostatic adsorption and chemical crosslinking is established, the hydroxysteroid dehydrogenase mutant and lactic dehydrogenase are co-immobilized, the co-immobilized enzyme can still keep high activity under high substrate concentration, and the conversion rate is still maintained to be 90% or above after 30 batches of co-immobilized enzyme are repeatedly used. And an important foundation is laid for industrial application of the hydroxysteroid dehydrogenase.

The invention relates to the field of biological catalysis, and discloses a method and a device for generating ursodesoxycholic acid through continuous flow catalysis of chenodeoxycholic acid. Comprising the following steps: (1) in the presence of first immobilized cells, carrying out a first catalytic reaction on a reaction system containing chenodeoxycholic acid to obtain a first product, the first immobilized cells expressing 7 alpha-hydroxysteroid dehydrogenase and lactic dehydrogenase; the concentration of the chenodeoxycholic acid in the reaction system is 40 to 70 mM; (2) adjusting the pH value of the first product to 8-9, and then in the presence of a second immobilized cell, carrying out a second catalytic reaction on the first product after the pH value is adjusted to obtain ursodesoxycholic acid, and the second immobilized cell expresses 7 beta-hydroxysteroid dehydrogenase and glucose dehydrogenase. According to the method, the stable activity of a catalytic system is kept, efficient conversion of high-concentration CDCA is achieved, meanwhile, precipitation of 7-KLCA is thoroughly avoided, and the synthesis efficiency, the product purity and the process continuity of UDCA are remarkably improved.

The application discloses an N-terminal protection sequence for improving stability of 7beta-hydroxysteroid dehydrogenase and a high-efficiency preparation method of the fusion protein, and belongs to the technical field of biology.The N-terminal protection sequence for improving the stability of 7beta-HSDH is designed and synthesized, the protection sequence is composed of 45 amino acids, the N-terminal alpha helix of the original 7beta-HSDH is embedded by a backfolding structure, and the stability of the 7beta-HSDH can be effectively improved.The half-life of the enzyme mutant N45-7beta-HSDH with the protection sequence is prolonged by 2.8 times compared with that of the original 7beta-HSDH under the catalytic condition of pH 8.0 and 30 DEG C.The enzyme consumption for generating UDCA from 7-KLCA by using the above N45-7beta-HSDH is reduced by 45.5%, the molar conversion rate is as high as 99.41%, the production cost is greatly reduced, and the method has actual industrial production value.

Compositions and methods useful for reducing expression of the 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13) gene and treating HSD17B13-associated diseases and conditions are provided. HSD17B13 dsRNA agents, HSD17B13 antisense polynucleotide agents, compositions comprising HSD17B13 dsRNA agents, and compositions comprising HSD17B13 antisense polynucleotide agents are provided, which can be used to reduce HSD17B13 expression in cells and subjects.

The invention relates to the field of biological medicine, and discloses a protein degradation targeting chimera (PROTAC) compound targeting 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) and medical application of the protein degradation targeting chimera (PROTAC) compound. The compound comprises a structural unit shown as a formula I, X is a 11 beta-HSD1 binding ligand, Y is a connecting chain, Z is an E3 ubiquitin ligase ligand, ubiquitination degradation of 11 beta-HSD1 can be induced, and therefore the function of the compound can be regulated and controlled at the protein level. Different from a traditional small-molecule inhibitor, the degradation agent can significantly reduce expression of 11 beta-HSD1 in cells and improve glucocorticoid signal abnormality, and shows a novel action mode at molecular and cellular levels. In diabetes, obesity, lipid metabolism disorder and related complication models, the compound can reduce blood sugar, improve insulin sensitivity, regulate blood fat and relieve inflammation, and shows a good treatment prospect. The half degradation concentration of a representative compound in vitro is about 1000 nM, and feasible degradation efficiency is achieved. The compound can be synthesized by Click chemistry and other methods, and can be prepared into a pharmaceutical composition for prevention and treatment of type II diabetes, metabolic syndrome, polycystic ovarian syndrome and other diseases, and a new strategy is provided for research and development of related drugs.

The application discloses a 7beta-hydroxysteroid dehydrogenase mutant and application thereof, relates to the field of bioengineering technology, and is designed and reformed according to the amino acid sequence of wild-type 7beta-hydroxysteroid dehydrogenase derived from Collinsella aerofaciens, that is, 17 single-mutation sites or 7 combined-mutation sites of 7beta-hydroxysteroid dehydrogenase are provided, the obtained 7beta-hydroxysteroid dehydrogenase mutant has the thermal stability increased by 2-21.5 DEG C and the activity increased by nearly 1.2-2.24 times compared with the wild-type 7beta-hydroxysteroid dehydrogenase. According to the mutation sites with obvious increase in thermal stability and activity, three preferred mutant proteins are further provided, the three preferred mutant proteins have higher yield, activity and thermal stability compared with the wild-type 7beta-hydroxysteroid dehydrogenase, have wider application conditions, are more suitable for the efficient production of ursodeoxycholic acid by the bioconversion method, and are favorable for large-scale production and industrial application.

Devices and methods for measurement of a hydroxysteroid analyte concentration in a host is provided, including a sensor housing, the sensor housing comprising an implantable analyte sensing portion configured to generate a signal associated with a concentration of a hydroxysteroid analyte, the analyte sensing portion comprising at least one (NAD(P)+)- dependent hydroxysteroid dehydrogenase enzyme; and at least one NAD(P)H oxidase. Detecting a signal corresponding to the hydroxysteroid analyte of the implantable portion after implantation of the implantable portion is also provided.

This invention relates to the field of artificial bear bile powder technology, specifically to a highly efficient cloning and temperature-inducible expression vector for the 7αβ-hydroxysteroid dehydrogenase gene and its construction method. The expression vector is formed by integrating the hydroxysteroid dehydrogenase gene into the pBV220 plasmid; the hydroxysteroid dehydrogenase gene is either the 7α-hydroxysteroid dehydrogenase gene or the 7β-hydroxysteroid dehydrogenase gene. The construction of this expression vector makes it possible to obtain the protease through induced fermentation using a temperature-inducible expression system, avoiding the introduction of toxic inducers and simplifying the process. During vector construction, the target gene and the linear plasmid ligate spontaneously within the engineered bacteria, independent of endonucleases and ligases, further reducing costs and process complexity. This technical solution can solve the technical problem of toxic residues in the engineered bacterial expression products of hydroxysteroid dehydrogenase in existing technologies, and has ideal prospects for widespread application.

The application relates to the technical field of biology, and discloses a method for inducing a cell line of immortalized musk deer scent glands to synthesize dihydrotestosterone, wherein the cell line is obtained by transfecting a primary musk deer scent gland epithelial cell with a lentivirus carrying an SV40 Large T antigen and performing resistance screening; the cell line is in an epithelial cell form and stably expresses keratin 18, cholesterol side chain cleavage enzyme, 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. Through lentivirus-mediated SV40 Large T antigen transfection, the primary musk deer scent gland epithelial cell breaks through the limit of replicative senescence and obtains unlimited proliferation capacity, effectively overcomes the defects that the primary cell is prone to fibrosis and rapidly loses metabolic function during in-vitro culture, provides a standardized cell model with a clear genetic background and persistent metabolic activity for biological manufacturing of dihydrotestosterone, and relieves the dependence on endangered animal live resources.

The invention relates to the technical field of biological enzyme engineering, in particular to a 3beta-hydroxysteroid dehydrogenase mutant and application thereof. The 3beta-hydroxysteroid dehydrogenase mutant provided by the invention is obtained by carrying out point mutation on an amino acid sequence shown by wild type 3beta-hydroxysteroid dehydrogenase. Compared with a wild enzyme, the 3beta-hydroxysteroid dehydrogenase mutant constructed by the invention has the advantages that the enzyme activity of the mutant is greatly improved and is 1.68-3.30 times that of a parent, and the use amount of an industrial enzyme can be remarkably reduced. Meanwhile, the mutant can specifically oxidize 3beta-ursodesoxycholic acid to generate 3ketoursodesoxycholic acid, and then the 3ketoursodesoxycholic acid is converted into ursodesoxycholic acid under the action of 3alpha-hydroxysteroid dehydrogenase, so that the defect that 3beta-ursodesoxycholic acid impurities generated in the ursodesoxycholic acid chemical synthesis process are difficult to remove is overcome; and a wide application prospect is provided for industrial production of ursodesoxycholic acid.

The invention discloses a 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) gene. The nucleotide sequence of the 7 alpha-HSDH gene is shown as SEQ ID No: 1. The method comprises the following steps: jointly transforming bacillus subtilis by using a modified 7alpha-HSDH gene, a 7beta-HSDH gene and a GDH gene to obtain recombinant bacillus subtilis; the recombinant bacillus subtilis is used for directional transformation of TCDCA in poultry bile, the recombinant bacillus subtilis has outstanding advantages in the aspect of green manufacturing of artificial bear gall powder, the expression quantity and activity of key enzymes are regulated and controlled through a genetic engineering technology, NADPH coenzyme circulation coupled with the key enzymes is enhanced, efficient and stable directional transformation from TCDCA to TUDCA can be achieved, and the recombinant bacillus subtilis can be used for preparing the artificial bear gall powder. Therefore, comprehensive utilization of resources is achieved, and the requirements of the medicine market are met more effectively.

This invention discloses sterol side-chain cleaving enzymes and 3β-hydroxysteroid dehydrogenases / Δ from different sources. 4,5 Amino acid sequences, coding sequences, and applications of isomerases. This invention, based on synthetic biology and molecular pharmacognosy, explores sterol side-chain cleaving enzymes and 3β-hydroxysteroid dehydrogenases / Δ from different plant sources. 4,5 Isomerases, which will yield sterol side-chain cleavage enzymes and 3β-hydroxysteroid dehydrogenases / Δ 4,5 The coding sequences of the isomerases were constructed into expression cassettes, and using Saccharomyces cerevisiae as the chassis strain, highly efficient pregnenolone or progesterone synthesizing strains were obtained. The pregnenolone yield of the constructed strains reached as high as 14.46±2.16 mg / L, and the progesterone yield reached as high as 32.78±1.84 mg / L. This achieved high-level synthesis of pregnenolone and progesterone by genetic engineering methods, providing an important foundation for the green synthesis of steroid hormones.

[Problem] To provide a novel compound having 17β-hydroxysteroid dehydrogenase 13 (HSD17B13) inhibitory activity. [Solution] A compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof.　

Xanamem, also known as UE2343, is a potent inhibitor of 11 [beta]-hydroxysteroid dehydrogenase type 1 (11 [beta]-HSD1). Due to the inhibitory action of Xanamem, it has been proposed for the treatment of disorders relieved by inhibition of 11 [beta]-HSD1, such as metabolic syndrome, cardiovascular conditions and CNS disorders. The present disclosure relates to formulations of Xanamem and excipients, in particular lactose and microcrystalline cellulose, as immediate release tablet formulations.

This invention discloses the application of red ginseng polysaccharide in the preparation of pharmaceutical compositions for the prevention and treatment of inflammatory bowel disease, belonging to the field of medical formulation technology. It is the first to clearly demonstrate that red ginseng polysaccharide promotes the expression of bile salt hydrolase and 7α-hydroxysteroid dehydrogenase by targeting and upregulating the relative abundance of Bacteroides acidophilus and Clostridium lysate in the intestine, thereby driving the production of 7-ketolithocholic acid in the intestine. Through the TGR5 activation mediated by 7-ketolithocholic acid, it improves intestinal damage caused by inflammatory bowel disease, providing a solid experimental basis and theoretical support for the application of red ginseng polysaccharide in the preparation of drugs or functional foods for the prevention and treatment of inflammatory bowel disease.

The invention relates to the technical field of microbial fermentation related to production of bear gall powder, and discloses a method for increasing the fermentation expression quantity of 7 alpha / beta-hydroxysteroid dehydrogenase and application of the method in production and transformation of bear gall powder, the method comprises a fermentation induction stage, in the fermentation induction stage, lactose is used as an inducer for primary induction and secondary induction, and the fermentation expression quantity of 7 alpha / beta-hydroxysteroid dehydrogenase is increased. The primary induction is to add lactose into the fermentation liquor at one time, and the secondary induction is to add lactose into the fermentation liquor in a fed-batch manner. According to the scheme, a fermentation culture medium and an induction mode are synergistically optimized, the OD600 value and the protein expression quantity of thalli are increased, the method for producing the hydroxysteroid dehydrogenase by fermenting the escherichia coli is provided aiming at the requirements of products produced by the company, the process is simple, the yield is high, the protein expression quantity is considerable, the method is suitable for industrialization, and the method is suitable for industrial production. The method is suitable for producing and transforming bear gall powder.

The present invention belongs to the field of bioengineering technologies, and in particular, to a production method of recombinant Escherichia coli (E. coli) and high-purity ursodeoxycholic acid (UDCA). The present invention constructs novel double-enzyme co-expression gene engineered bacteria, that is, recombinant E. coli. The bacteria simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH). The bacteria is applicable to the production of high-purity UDCA. The yield of a target product is increased through the joint expression and application of 7β-HSDH and GDH. The production method of high-purity UDCA in the present invention is simple, generates a small amount of impurities in a production process, is a green process that satisfies environmental protection requirements, and has significant industrial application value.

The present disclosure relates to RNAi agents, e.g., double stranded RNAi agents, able to inhibit 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13 or 17β-HSD13) gene expression. Also disclosed are pharmaceutical compositions that include HSD17B13 RNAi agents and methods of use thereof. The HSD17B13 RNAi agents disclosed herein may be conjugated to targeting ligands to facilitate the delivery to cells, including to hepatocytes. Delivery of the HSD17B13 RNAi agents in vivo provides for inhibition of HSD17B13 gene expression. The RNAi agents can be used in methods of treatment of HSD17B13-related diseases and disorders, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic fibrosis, and alcoholic or non-alcoholic liver diseases, including cirrhosis.

The invention discloses a preparation method of high-purity ursodesoxycholic acid, and belongs to the technical field of biological medicines. The method provided by the invention specifically comprises the following steps: carrying out a catalytic oxidation reaction on chenodeoxycholic acid by using 7 alpha-hydroxysteroid dehydrogenase to generate a 7-ketolithocholic acid intermediate, and then carrying out a catalytic reduction reaction on 7 beta-hydroxysteroid dehydrogenase to generate a crude ursodeoxycholic acid product. Further, the crude ursodesoxycholic acid reacts with a triethylamine reagent to generate ursodesoxycholic acid triethylamine salt, and a part of cell impurities are removed; dropwise adding an acid reagent to hydrolyze ursodesoxycholic acid triethylamine salt; and finally, recrystallizing and refining by using ethyl acetate and ethanol to obtain high-purity ursodesoxycholic acid. The method is simple in process and low in cost, the obtained product is high in purity, few organic reagents are used, environmental pollution is little, and industrial production can be achieved.

[Problem] To provide a new compound having 17β-hydroxysteroid dehydrogenase-13 (HSD17B13) inhibitory activity. [Solution] A compound represented by general formula (1) or a pharmaceutically acceptable salt thereof.　

The present invention relates to compounds of formula (I)to salts, solvates and solvates of salts thereof, and to pharmaceutical compositions comprising these compounds as active ingredients. The invention further relates to their use as aldo-keto reductase family 1 C3 (AKR1C3), also known as 17β-hydroxysteroid dehydrogenase type 5 (17β-HSD5, HSD17B5) and prostaglandin (PG) F2α synthase, inhibitors. The invention further relates to methods for their preparation, and to uses of said compounds.

Compounds of Formula (I), pharmaceutical compositions or kits thereof are disclosed. Wherein A1 is C (O) or CHRz1, Rz1 is OH, amino, imino, halogen, (C1-C5) alkoxy, OC (O) (C1-C5) alkyl, and OSO2-amino; a2 is CH2 or O; r1 is hydrogen, a (C1-C5) alkyl group optionally substituted by one or more Z substituents, a (C1-C5) alkoxy group optionally substituted by one or more Z substituents, a (C2-C5) alkenyl group optionally substituted by one or more Z substituents, a (C2-C5) alkynyl group optionally substituted by one or more Z substituents; (C3-C8) cycloalkyl, aryl or heteroaryl groups; z is halogen, OH, (C1 to C5) alkyl, (C1 to C5) haloalkyl, (C1 to C5) alkoxy, (C1 to C5) haloalkoxy, or amino; r2 is a heterocyclic aromatic ring having 5, 6 or 7 members and having at least one N, S and / or O; r3 is a (C1-C5) alkyl group, which is substituted by one or more Br, Br76; i, I123, I124 or I131 substitutions; m is an integer selected from 0 to 2; n is an integer selected from 0 to 2; and p is an integer selected from 0 to 5. The compounds of formula I are inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) for use in the treatment or prevention of steroid hormone dependent diseases such as breast cancer, lung cancer, prostate cancer, ovarian cancer, uterine cancer, endometrial cancer and endometrial hyperplasia. (I).

This invention proposes an ancestral 7β-hydroxysteroid dehydrogenase and its application. The amino acid sequence of the ancestral 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO.2. This invention obtains an ancestral 7β-hydroxysteroid dehydrogenase with significantly improved stability by reconstructing the ancestral enzyme sequence. This method is based on the theory that ancestral enzymes have higher stability in early evolutionary stages. Through homology alignment and phylogenetic analysis of the amino acid sequences of modern enzymes 7β-HSDH-T189V & V207M, ancestral enzyme sequences at several key evolutionary nodes were identified and synthesized. After activity screening and stability evaluation, ancestral enzyme No. 81 was finally obtained. Experimental results show that the residual enzyme activity of this ancestral enzyme after incubation at 55℃ for 60 minutes increased from 14% of the original enzyme to over 80%, significantly improving the enzyme's thermostability and providing important support for its application in the industrial production of ursodeoxycholic acid.

The application provides an enzyme inhibiting supramolecular complex and application in cosmetics, and relates to the field of cosmetic raw materials.The supramolecular complex is a supramolecular structure formed by hydrogen bond connection of coriander glycoside and sodium phosphatide through high-pressure homogenization treatment and gradient freeze-drying treatment, and can inhibit key enzymes of sebaceous gland secretion, including aminoacylase N, 3beta-hydroxysteroid dehydrogenase and fatty acid synthase.The supramolecular complex can be added into different types of cosmetics, especially cosmetics with oil control and pore shrinking functions, so that the supramolecular complex can not only play a role of the composition itself, but also improve the transdermal rate by using the lipophilicity of phospholipid, and maximizes the cosmetic effect.

This invention relates to the field of artificial bear bile powder preparation technology, specifically to a novel 7α-hydroxysteroid dehydrogenase gene R-α-1 and its application in the production and conversion of bear bile powder. This method isolates a novel gene catalyzing asymmetric carbonyl reduction reactions from the feces of Sichuan brown bears. This gene encodes a 7α-hydroxysteroid dehydrogenase, which catalyzes the formation of the intermediate tauroursodeoxycholic acid from tauroursodeoxycholic acid, which is further catalyzed by a 7β-hydroxysteroid dehydrogenase to produce tauroursodeoxycholic acid, the main component of bear bile powder. The nucleotide sequence of this gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. This technical solution can solve the technical problem that existing types of 7α-hydroxysteroid dehydrogenases cannot meet the needs of large-scale production due to unsatisfactory enzyme activity and thermostability, and has ideal prospects for widespread application.

The application relates to the field of genetic engineering, and particularly relates to an application of a pseudomonas protein. The application comprises: the pseudomonas protein with an amino acid sequence as shown in SEQ ID NO. 1 is used for preparing 7beta-hydroxysteroid dehydrogenase. The application is based on the previous research finding that a kind of pseudomonas protein belonging to the superfamily of short-chain dehydrogenase and reductase can be screened and optimized from the pseudomonas protein gene library. The pseudomonas protein has a homologous spatial structure arrangement with the reported 7beta-hydroxysteroid dehydrogenase, has the core catalytic function of the 7beta-hydroxysteroid dehydrogenase, and can catalyze the reversible oxidation-reduction reaction of 7-ketolithocholic acid and ursodeoxycholic acid. In addition, after the amino acid sequence and structure of the pseudomonas protein are optimized, the intrinsic activity of the pseudomonas protein in catalyzing 7-ketolithocholic acid and the application scene adaptability are improved, and the catalytic efficiency of the 7beta-hydroxysteroid dehydrogenase can be effectively improved.

The present disclosure relates to RNAi agents, such as double-stranded RNAi agents, capable of inhibiting 17beta-hydroxysteroid dehydrogenase type 13 (HSD17B13 or 17beta-HSD13) gene expression. Also disclosed are pharmaceutical compositions comprising HSD17B13 RNAi agents and methods of using the same. The HSD17B13 RNAi agents disclosed herein can be conjugated to a targeting ligand to facilitate delivery to cells, including hepatocytes. Delivery of the HSD17B13 RNAi agents in vivo provides for inhibition of HSD17B13 gene expression. The RNAi agents can be used in methods of treating diseases and disorders associated with HSD17B13, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, and alcoholic or nonalcoholic liver disease, including cirrhosis.

The invention relates to the technical field of microorganisms and enzyme engineering, in particular to a 7beta-hydroxysteroid dehydrogenase mutant and a preparation method thereof, and particularly relates to semi-rational design of the 7beta-hydroxysteroid dehydrogenase mutant. The preparation method comprises the following steps: taking a ruminococcus torqus 7beta-hydroxysteroid dehydrogenase mutant T189Vamp as a template, and taking the mutant T189Vamp as a template for preparing the ruminococcus torqus 7beta-hydroxysteroid dehydrogenase; mutants T189V / V207M / S70P and T189V / V207M / Q133R are obtained through a semi-rational design method of ancestral enzyme reconstruction assisted enzyme directed evolution by taking V207M as a template, the high-temperature stability of the former is remarkably improved, and the specific enzyme activity of the latter is remarkably improved.

The invention provides a directionally modified 7beta-hydroxysteroid dehydrogenase iteration mutant as well as a coding gene and application thereof. According to the invention, the expression vector or recombinant bacterium containing the 7beta-hydroxysteroid dehydrogenase iterative mutant gene is successfully constructed. According to the invention, the 7beta-hydroxysteroid dehydrogenase double mutants are directionally modified by an enzyme modification technology, and alanine at the 203th site of the 7beta-hydroxysteroid dehydrogenase double mutants is mutated into glutamine, so that the pH tolerance and catalytic activity of the enzyme are remarkably improved. The 7beta-hydroxysteroid dehydrogenase iteration mutant disclosed by the invention can be used for preparing UCDA (Urban Cyclin Dehydrogenase).

This invention discloses a 7α-hydroxysteroid dehydrogenase derived from animal fecal metagenomics, its preparation method, and its applications, belonging to the field of genetic engineering technology. The amino acid sequence of the enzyme is shown in SEQ ID NO.2, and the nucleotide sequence encoding the enzyme is shown in SEQ ID NO.1. The enzyme has a specific activity of 444.15 ± 41.38 U / mg, an optimal temperature of 50℃, an optimal pH of 10.0, and retains over 90% of its activity after 12 hours of incubation at pH 10.0. It exhibits good alkali tolerance. EDTA enhances enzyme activity, while Ag+ and SDS completely inactivate the enzyme. This enzyme has broad substrate specificity, catalyzing the conversion of chenodeoxycholic acid to 7-keto-lithocholic acid, providing a highly efficient candidate enzyme for the biosynthesis of ursodeoxycholic acid, and has significant application value in the medical field.