63 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 discloses a 7alpha-hydroxysteroid dehydrogenase mutant with improved catalytic activity as well as a coding gene and application thereof, and the amino acid sequence of the 7alpha-hydroxysteroid dehydrogenase mutant is obtained by mutation at one or more sites of the 120th site, the 195th site and the 207th site on the basis of the amino acid sequence shown in SEQ ID NO: 1. According to the 7alpha-HSDH mutant disclosed by the invention, the key site of the 7alpha-HSDH enzyme is modified, so that the mutant with higher activity than that of a wild enzyme is obtained, the efficiency bottleneck in UDCA biosynthesis is effectively broken through, and an efficient and green enzyme catalyst is provided for industrial application.

The invention provides a 7beta-hydroxysteroid dehydrogenase mutant and application thereof in synthesis of ursodesoxycholic acid by a biological enzyme method, through a genetic engineering technology and computer-aided design, phosphoric acid coordination residues of protein are modified, the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant with higher activity under an NADH (Nicotinamide Adenine Dinucleotide Hydroxide) system is constructed, and the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant can be used for synthesizing ursodesoxycholic acid. The modification strategy is suitable for other proteins for modifying cofactor preference and improving enzyme activity, and has remarkable industrial value when being used for efficiently catalyzing CDCA to be converted into UDCA through a one-pot one-step method.

The invention discloses a modified 7beta-hydroxysteroid dehydrogenase (7beta-HSDH), which has higher activity and stability in the synthesis process of ursodesoxycholic acid (UDCA), and takes NAD (P) H as a coenzyme. According to the 7beta-hydroxysteroid dehydrogenase mutant, the V104 site in the 7beta-hydroxysteroid dehydrogenase is subjected to saturated mutation modification, and the mutant with enhanced activity and changed coenzyme specificity is screened out. The mutant enzyme can keep high catalytic efficiency under the condition of taking NADH as a coenzyme, so that the synthesis yield of UDCA is remarkably increased, and the production cost is reduced. The enzyme mutant disclosed by the invention has a wide application prospect in industrial production, is particularly suitable for the production process of large-scale biosynthesis of UDCA, can effectively reduce the production cost, improve the product yield and reduce the environmental pollution and the production cost, and provides a technical support for wide application of UDCA.

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 invention discloses an N-terminal protection sequence for improving the stability of 7 beta-hydroxysteroid dehydrogenase and an efficient preparation method of a fusion protein, and belongs to the technical field of biology. An N-terminal protection sequence capable of remarkably improving the stability of the 7beta-HSDH is designed and synthesized, the protection sequence is composed of 45 amino acids, and the stability of the 7beta-HSDH can be effectively improved by spirally embedding an N-terminal alpha exposed to the outside of the original 7beta-HSDH through a section of inflection structure. Compared with the original 7beta-HSDH, the half-life period of the enzyme mutant N45-7beta-HSDH added with the protection sequence is prolonged by 2.8 times under the catalytic conditions that the pH is 8.0 and the temperature is 30 DEG C. When the N45-7beta-HSDH is used for catalyzing 7-KLCA to generate UDCA, the enzyme dosage is reduced by 45.5%, the molar conversion rate is as high as 99.41%, the production cost is greatly reduced, and the N45-7beta-HSDH has actual industrial production value.

The application discloses a 17beta-hydroxysteroid dehydrogenase mutant, an engineering bacterium and application, and uses the 17beta-hydroxysteroid dehydrogenase mutant to biotransform methyl diketone as a substrate to prepare nandrolone. The 17beta-hydroxysteroid dehydrogenase is 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl) from a fungus Cochliobolus lunatus, and the mutant is a single mutant in which histidine His at the 164th position is mutated into valine Val, a single mutant in which threonine Thr at the 151st position is mutated into alanine Ala, or a double mutant in which the 164th position and the 151st position are jointly mutated. The application uses pET28a as an expression plasmid, realizes heterologous expression of the 17beta-HSDcl mutant in E.coli BL21 (DE3), and through 24h whole cell transformation of the engineering bacterium, the nandrolone yield of the single mutant H164V, the single mutant T151A and the double mutant H164V / T151A is 1.27 times, 1.30 times and 1.34 times that of before mutation, respectively. The application provides a basis for industrialized production of nandrolone by microorganisms.

The invention belongs to the technical field of biology, and particularly relates to a Sardinia clostridium 7alpha-hydroxysteroid dehydrogenase mutant S161G. The amino acid sequence of the mutant S161G is as shown in SEQ ID NO: 2, and the amino acid sequence of the mutant S161G is obtained by mutating Ser at the 161st site of a wild type sequence SEQ ID NO: 1 into Gly. The catalytic efficiency of the mutant S161G on substrates NADP < + > and TCDCA is 3.82 times that of a wild type. The mutant S161G provided by the invention has huge application potential in the industrial production process of obtaining TUDCA or UDCA through biotransformation.

In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the wildtype 7β-HSDH of SEQ ID NO:2, a decreased substrate inhibition and / or an altered cofactor usage, and the mutant has, in comparison with the wildtype 7β-HSDH of SEQ ID NO:2, 1 to 15 amino acid additions, substitutions, deletions and / or inversions in the sequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ ID NO:2.

The invention belongs to the field of biology, discloses a variant of 7 alpha-hydroxysteroid dehydrogenase, and aims to improve the catalytic efficiency and stability of catalyzing chenodeoxycholic acid (CDCA) to be converted into UDCA precursor 7-ketolithocholic acid, and particularly, the variant is obtained by carrying out single mutation or multipoint combined mutation on the 93th site, the 94th site, the 96th site, the 146th site, the 150th site, the 155th site, the 188th site, the 189th site, the 190th site, the 195 site, the 195th site, the 196th site, the 196th site, the 195th site, the 195th site, the 195th site, the 196th site, the 199th site, the 200th site, the 204th site and the 249th site

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 discloses a 7alpha-hydroxysteroid dehydrogenase mutant and application thereof in preparation of ursodesoxycholic acid, the enzyme mutant is obtained by mutating a wild type amino acid sequence as shown in SEQ ID NO: 1 through Q31A, H72Q, W111R or A178R, the mutated enzyme activity is high, 7-ketolithocholic acid can be prepared based on catalysis of a high-concentration substrate, and the 7alpha-hydroxysteroid dehydrogenase mutant can be applied to preparation of ursodesoxycholic acid. And the method has a significant meaning for industrial preparation of ursodesoxycholic acid under catalysis of the propulsor.

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.

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the HSD17B13 gene, as well as methods of inhibiting expression of HSD17B13, and methods of treating subjects that would benefit from reduction in expression of HSD17B13, such as subjects having a HSD17B13-associated disease, disorder, or condition, using such dsRNA compositions.

The invention relates to methods of treating subjects that would benefit from reduction in expression of HSD17B13, such as subjects having a HSD17B13-associated disease, disorder, or condition, e.g., nonalcoholic steatohepatitis (NASH), using double-stranded ribonucleic acid (dsRNA) compositions targeting the HSD17B13 gene. The invention also provides methods for preventing at least one symptom in a subject having a HSD17B13-associated disease, disorder, or condition, e.g., NASH.

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.

According to the invention, the 7alpha-HSDH gene derived from Brevundimonas sp. Is artificially synthesized, and the expression efficiency of the 7alpha-HSDH gene in a host is improved through optimization. The nucleotide sequence of the optimized 7alpha-HSDH gene is as shown in SEQ ID NO: 1. The corresponding amino acid sequence is as shown in SEQ ID NO: 2. The 7alpha-hydroxysteroid dehydrogenase gene and the expression system thereof provided by the invention can efficiently convert CDCA into 7K-LCA. The engineering bacterium can realize efficient specific oxidation of a substrate CDCA, a reaction product is analyzed and confirmed as a target product 7K-LCA through high performance liquid chromatography (HPLC), the conversion rate can reach 92%, and the engineering bacterium shows excellent catalytic performance and application potential. The engineering strain construction and catalytic conversion process adopted by the invention is based on an escherichia coli expression system, the operation is simple and convenient, the repeatability is high, and related culture devices and reagents are wide in source, low in price and convenient for large-scale application. The method meets the requirements of green chemistry and sustainable development, and has good environmental friendliness and safety.

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.　

This invention discloses a hydroxysteroid dehydrogenase mutant and its application in the preparation of steroidal drugs. The mutant is obtained by saturation mutation and combination mutation at positions 89, 185, or 190 of the amino acid sequence shown in SEQ ID NO. 1. The hydroxysteroid dehydrogenase mutant of this invention exhibits improved activity and stability. Using a DMSO-aqueous phase system further improves substrate conversion, reducing production costs and increasing production efficiency.

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.