78 results about "Homosteroids" patented technology

The invention discloses a hydroxysteroid dehydrogenase mutant with improved thermal stability, a screening method and application, key hotspot amino acid residues related to the thermal stability are rapidly identified based on computer-aided design technologies such as hydroxysteroid dehydrogenase structure prediction, PROSS and the like, so that a limited site-directed mutant library is constructed; and further screening to obtain a dominant mutant. The semi-inactivation temperature (T5105) of the obtained mutant is increased by 1.0-7.7 DEG C compared with that before mutation, wherein the semi-inactivation temperatures (T5105) of the mutants A126I and A132H are increased by 7.7 DEG C and 3.7 DEG C respectively. And the relative enzyme activity of the mutant reaches 99.86-136.73% of the relative enzyme activity before mutation. The invention provides a new method for rapidly improving the thermal stability of the enzyme, and lays a foundation for the industrial application of the hydroxysteroid dehydrogenase.

The invention belongs to the field of protein rational design and enzyme engineering, and particularly relates to a 7beta hydroxysteroid dehydrogenase mutant and application thereof in preparation of UDCA. Firstly, ruminococcus torquens 7beta-hydroxysteroid dehydrogenase is used as a template, hydrogen bonds are increased through homologous modeling prediction, a mutant 7beta-HSDH-3M is obtained through site-specific mutagenesis, the 50-DEG C thermal stability of the obtained 7beta-hydroxysteroid dehydrogenase is remarkably improved, the 7beta-hydroxysteroid dehydrogenase is used for catalyzing 80g / L L7-KLCA to generate UDCA, the conversion rate exceeds 99%, and the 7beta-hydroxysteroid dehydrogenase can be used for preparing a mutant 7beta-HSDH-3M mutant 7beta-HSDH-3M mutant 7beta-HSDH-3M mutant 7beta-HSDH-3M mutant 7beta-HSDH-3M. Complete conversion (greater than 99%) of a product substrate is realized through high conversion rate of 7beta-HSDH.

The invention discloses efficient synthesis of ursodesoxycholic acid by improving the positive enzyme activity of 7 beta-hydroxysteroid dehydrogenase through directed evolution, and belongs to the field of biological catalysis engineering. According to the invention, the 7beta-hydroxysteroid dehydrogenase mutant TEAE / F152L / W101N with improved positive reaction activity and improved affinity with a substrate is obtained. Compared with TEAE, the forward enzyme activity of the mutant enzyme is improved by 2.9 times, the reverse activity of the mutant enzyme is reduced by 3.6 times, the catalytic efficiency kcat / Km of the mutant enzyme to 7K-LCA is improved by 4.3 times, and compared with a wild type mutant TEAE / F152L / W101N, the enzyme activity of the mutant enzyme is improved by 206.23 times, and the catalytic efficiency of the mutant enzyme to 7K-LCA is improved by 58.56 times. The invention provides a universal technical platform for directed evolution of hydroxysteroid dehydrogenase, provides an excellent mutant enzyme for biological efficient synthesis of ursodesoxycholic acid, and lays a solid research foundation for efficient and low-cost preparation of ursodesoxycholic acid.

This present disclosure provides various diamine centered PEG-saccharide-lipid conjugates. In various embodiments, the conjugates can safely be used as water solubility or bioavailability enhancers.. In one aspect, the present disclosure provides a PEG-saccharide- lipid conjugate having the structural formula (I) wherein m has a number-average value in the range of 2-10; S is a mono-, di- or trisaccharide group, in which each saccharide unit is a sugar, a sugar alcohol, an amino sugar or a sugar acid; L is -C(O)-R1 in which R1 is an alkanyl or alkenyl group having a number-average number of carbons in the range of 6-22, and / or is a steroid acyl group; P is -(CH2-CH2-O)nR2 in which "n" has a number-average value in the range of 5-50 (e.g., 8-45) and R2 is hydrogen and / or alkanyl and has a number average number of carbons in the range 0-4.

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 present invention provides a recombinant Bacillus subtilis engineering strain and its application in the biological preparation of ursodeoxycholic acid, belonging to the field of bioengineering technology. The present invention provides a recombinant Bacillus subtilis engineering strain that simultaneously and efficiently and stably expresses 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). During biosynthesis, chenodeoxycholic acid is used as a substrate and combined with oxidized coenzyme NADP under the action of 7α-HSDH. + , the 7α-hydroxyl group in chenodeoxycholic acid is oxidized to a carbonyl group, and the resulting intermediate 7-keto-lithocholic acid is reduced to ursodeoxycholic acid under the action of 7β-HSDH and NADPH. The recombinant Bacillus subtilis engineered strain can efficiently and stably biosynthesize ursodeoxycholic acid, and greatly reduces the purification work of the product in industrial applications, greatly reducing production costs.

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 present invention relates to methods of treating an individual who would benefit from reduced expression of HSD17B13, such as an individual with a HSD17B13-related disease, disorder or condition, such as non-alcoholic steatohepatitis (NASH), using a double stranded ribonucleic acid (dsRNA) composition targeting the HSD17B13 gene. The invention also provides methods for preventing at least one symptom in an individual suffering from a HSD17B13-related disease, disorder or condition (e.g., NASH).

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.

Disclosed herein are compounds that inhibit the function of 3β-hydroxysteroid dehydrogenase (3βHSD1), pharmaceutical compositions comprising the compounds, and methods of using the compounds, e.g., for the treatment of prostate cancer, breast cancer, and other diseases dependent on the activity of 3βHSD1.

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 discloses a method for improving heterologous synthesis of pregnenolone and progesterone by using yarrowia lipolytica. The invention provides a method for improving the heterologous synthesis of pregnenolone by using yarrowia lipolytica, which comprises the following steps: knocking out an MHY1 gene on the basis of a recombinant yarrowia lipolytica strain SyBEY12090002, and constructing a strain Y1001; the invention relates to a method for improving heterologous synthesis of progesterone by using yarrowia lipolytica, which is characterized in that on the basis of a recombinant yarrowia lipolytica strain SyBEY12090002, 3beta-hydroxysteroid dehydrogenase is introduced, an MHY1 gene is knocked out, and a strain Y1102 is constructed. The MHY1 gene is knocked out from the strain for synthesizing the pregnenolone or the progesterone from the beginning, so that the yield of the pregnenolone or the progesterone synthesized by the recombinant lipolytic yeast strain is improved, the influence of regulating the morphological change of the yarrowia lipolytica on a heterologous synthesis pathway is explored, and a research direction is provided for subsequently improving the yield of the pregnenolone or the progesterone.

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 present invention discloses a 7α-hydroxysteroid dehydrogenase mutant and its application in the synthesis of 7-oxolithocholic acid, belonging to the fields of genetic engineering and biocatalysis technology. By performing site-directed mutagenesis on specific amino acids in the pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase (Bm7α-HSDH) and combining some advantageous mutations, the present invention obtains mutants with significantly improved thermal stability and catalytic efficiency. In the reaction of catalyzing the synthesis of 7-oxolithocholic acid from the substrate chenodeoxycholic acid, the enzyme activity of the mutant is increased by up to about 45 times at most, the catalytic efficiency is increased by about 122 times, and the T m value can be increased by about 12 °C at most, providing high-quality biocatalysts and precursor compounds for the biocatalytic synthesis of ursodeoxycholic acid and laying a solid research foundation for its industrial production.

To provide 19-nor C3,3-disubstituted C21-N-pyrazolyl steroids, and methods for use thereof.SOLUTION: Provided herein are 19-nor C3,3-disubstituted C21-pyrazolyl steroids of Formula (I), and pharmaceutically acceptable salts thereof. Such compounds are contemplated useful for the prevention and treatment of a variety of CNS-related conditions, for example, treatment of sleep disorders, mood disorders, schizophrenia spectrum disorders, convulsive disorders, disorders of memory and / or cognition, movement disorders, personality disorders, autism spectrum disorders, pain, traumatic brain injury, vascular diseases, substance abuse disorders and / or withdrawal syndromes, and tinnitus.SELECTED DRAWING: None

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 present invention discloses a method for improving the forward enzymatic activity of 7β-hydroxysteroid dehydrogenase by directed evolution to efficiently synthesize ursodeoxycholic acid, belonging to the field of biocatalytic engineering. The present invention obtains a 7β-hydroxysteroid dehydrogenase mutant TEAE / F152L / W101N with improved forward reaction activity and substrate affinity. Compared with TEAE, the mutant enzyme has a 2.9-fold increase in forward enzymatic activity and a 3.6-fold decrease in reverse activity. The catalytic efficiency k of 7K-LCA is 1.3 times higher than that of TEAE. cat / K m The enzyme activity of the mutant TEAE / F152L / W101N increased by 4.3 times compared to the wild type. The catalytic efficiency for 7K-LCA increased by 58.56 times. This invention provides a universal technical platform for the directed evolution of hydroxysteroid dehydrogenases, provides excellent mutant enzymes for the efficient biosynthesis of ursodeoxycholic acid, and lays a solid research foundation for the efficient and low-cost preparation of ursodeoxycholic acid.

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.

This invention relates to compositions and methods for modifying Brassinosteroid Insensitive-1 (BRI1) genes in plants by introducing at least one mutation in a region of the BRI1 gene encoding the kinase domain, optionally to improve yield traits. The invention further relates to a mutated BRI1 gene, a modified BRI1 polypeptide, and plants having increased improved yield traits produced using the methods and compositions of the invention.

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.