50 results about "Homosteroids" patented technology

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 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 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 provides a mutant R41D / S193N of 7beta-hydroxysteroid oxidative dehydrogenase, a gene sequence of the mutant enzyme, a recombinant expression vector containing the gene sequence of the enzyme and an engineering strain containing the vector. The invention also provides a construction method of a recombinant expression vector and an engineering strain, a method for preparing the mutant enzyme by using the engineering strain, and application of the mutant enzyme in in-vitro enzyme catalysis of 7-ketolithocholic acid to produce ursodeoxycholic acid. Compared with a wild type, the R41D / S193N mutant provided by the invention shows stronger preference on a cofactor NADH, is more economical than the traditional NADPH utilization, is simple and convenient in preparation process, high in yield and simple in downstream treatment process, does not involve other solvents except water in the preparation process, belongs to a green biosynthesis method, and is suitable for industrial production. Meanwhile, the method has relatively high atom economy and step economy.

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 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.

[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.　

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.

[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.

Polyunsaturated ecdysteroids for use in treatment The present invention relates to polyunsaturated ecdysteroid compounds for use in hormonal, metabolic, inflammatory and oxidative stress related conditions, as well as pharmaceutical compositions comprising said compounds.

To provide a method for determining a subject's susceptibility to developing a liver disease or for diagnosing a subject with liver disease.SOLUTION: Provided is a method for detecting a variant HSD17B13 gene in a human subject. The method comprises or consists of performing an assay on a biological sample collected from the human subject, wherein the assay determines whether a thymine is inserted between positions corresponding to positions 12665 and 12666 of a specific sequence of a wild-type HSD17B13 gene or whether a thymine is present at a position corresponding to position 12666 of another specific sequence of a variant HSD17B13 gene, and wherein the presence of the thymine indicates the variant HSD17B13 gene.SELECTED DRAWING: Figure 1A-1

The present invention belongs to the field of protein rational design and enzyme engineering, and specifically relates to a 7β-hydroxysteroid dehydrogenase mutant and its use in the preparation of UDCA. First, the present invention uses Ruminococcus torques 7β-hydroxysteroid dehydrogenase as a template, and obtains the mutant 7β-HSDH-3M through homology modeling prediction to increase hydrogen bonds and site-directed mutagenesis. The 7β-hydroxysteroid dehydrogenase obtained by the present invention has significantly improved thermal stability at 50°C. The enzyme is used to catalyze 80g / L of L7-KLCA to produce UDCA with a conversion rate of over 99%. The high conversion rate of 7β-HSDH achieves complete conversion of the product substrate (>99%).

Disclosed in the present invention are a steroid compound, and a preparation method therefor and the use thereof. The structure of the steroid compound is as shown in formula I, and the definition of each substituent in the formula are as described in the description and claims. The steroid compound of the present invention is a steroid compound having the dual functions of excellent AR antagonistic activity and AR degradative activity, has the characteristics of good druggability and safety, and can be used for treating diseases related to an AR signaling pathway.

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.

To provide a method for treating a disease.SOLUTION: Provided are potent soft corticosteroids, a pharmaceutical composition comprising the same, and a method for using the same as an anti-inflammatory agent. Also provided is a method for softening fluticasone propionate and similar corticosteroid and attaining a potent but safer alternative. Compounds which are equally as potent as fluticasone but safer than fluticasone, in particular S-fluoromethyl 17α-dichloroacetoxy-6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-dien-17β-carbothioate is provided. Another compound of particular interest is 2-hydroxyethyl 17α-dichloroacetoxy-6α,9α-difluoro-11β-hydroxy-16β-methyl-3-oxoandrosta-1,4-diene-17β-carboxylate.SELECTED DRAWING: None

To provide 17β-hydroxysteroid dehydrogenase type 13 (HSD17B13) iRNA compositions and methods of use thereof.SOLUTION: The present 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 present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a HSD17B13 gene and / or for treating a subject who would benefit from inhibiting or reducing the expression of a HSD17B13 gene, e.g., a subject suffering or prone to suffering from an HSD17B13-associated disease, for example, a chronic fibro-inflammatory liver disease.SELECTED DRAWING: None