46 results about "TNase activity" patented technology

Disclosed are the uses of specific genes of the mevalonate and isoprenoid biosynthetic pathways, and of inactive gene sites (the pseudogene) to (1) enhance biosynthesis of isopentenyl diphosphate, dimethylallyl diphosphate and isoprenoid pathway derived products in the plastids of transgenic plants and microalgae, (2) create novel antibiotic resistant transgenic plants and microalgae, and (3) create a novel selection system and/or targeting sites for mediating the insertion of genetic material into plant and microalgae plastids. The specific polynucleotides to be used, solely or in any combination thereof, are publicly available from GeneBank and contain open reading frames having sequences that upon expression will produce active proteins with the following enzyme activities: (a) acetoacetyl CoA thiolase (EC 2.3.1.9), (b) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5), (c) HMG-CoA reductase (EC 1.1.1.34), (d) mevalonate kinase (EC 2.7.1.36), (e) phosphomevalonate kinase (EC 2.7.4.2), (f) mevalonate diphosphate decarboxylase (EC 4.1.1.33), (g) isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2), and (h) phytoene synthase (EC 2.5.1.32).

The present invention provides methods of identifying polypeptides that have enzymatic activity associated with nutrient and/or energy homeostasis, and thus, are involved in the development of one or more cardiovascular and metabolic disorders, e.g., cardiovascular disease, obesity, insulin resistance, type 2 diabetes, dyslipidemia, nonalcoholic fatty liver disease, and metabolic syndrome. One such method comprises identifying a polypeptide as a member of the adiponutrin family of proteins. As such, the invention is related to the polynucleotides and polypeptides belonging to the adiponutrin family, and provides novel isolated and purified polynucleotides and polypeptides of a novel member of the adiponutrin family, patatin-like phospholipase domain 1 (PNPLA1). Also provided are methods of using the polynucleotides and polypeptides related to or provided by the invention for screening a test compound, e.g., a small molecule, antibody, etc., for the ability of the test compound to detect and/or modulate the activity of one or more members of the adiponutrin family of proteins. The present invention also is directed to novel methods for diagnosing, prognosing, and monitoring the progress of at least one cardiovascular and metabolic disorder using polynucleotides or polypeptides belonging to the adiponutrin family, and/or modulators of one or more members of the adiponutrin family. The present invention is further directed to novel therapeutics and therapeutic targets for the intervention (treatment) and prevention of cardiovascular and metabolic disorders arising from dysregulated energy homeostasis, as related to one or more members of the adiponutrin family of proteins.

A method for selectively obtaining a natural variant of an enzyme having activity includes (1) a step of detecting an ORF sequence of a protein having enzyme activity from a genome database including base sequences of metagenomic DNA of environmental microbiota; (2) a step of obtaining at least one PCR clone including the ORF sequence having a full length, a partial sequence of the ORF sequence, or a base sequence encoding an amino acid sequence which is formed by deletion, substitution, or addition of at least one amino acid residue in an amino acid sequence encoded by the ORF sequence, by performing PCR cloning on at least one metagenomic DNA of the environmental microbiota by using a primer designed based on the ORF sequence; (3) a step of determining a base sequence and an amino acid sequence which is encoded by the base sequence for each PCR clone obtained in the step (2); and (4) a step of selecting a natural variant of an enzyme having activity by measuring enzyme activity of proteins encoded by each PCR clone obtained in the step (2).

Disclosed is a stabilized enzyme composition for use in clinical examination, comprising: (a) an enzyme component comprising at least two enzymes selected from the group consisting of alkaline phosphatase, creatine kinase and alanine aminotransferase; (b) a stabilizer component comprising effective stabilizing amounts of an albumin, and at least one saccharide selected from the group consisting of trehalose and sorbitol; and (c) an aqueous medium having dissolved therein the components (a) and (b). The enzyme composition of the present invention is stable for a prolonged period of time not only under non-freeze refrigeration conditions, but also under freezing conditions or under conditions for non-freeze refrigeration after thawing of the frozen composition, as compared to the conventional enzymatic compositions. The enzyme composition of the present invention can be advantageously used for the purpose of checking the precision in measurement, correcting measured values and calibrating the amount and activity of an enzyme, in a clinical examination for measuring the enzymatic activity in a sample, such as serum or the like.

The invention discloses a DNA (deoxyribonucleic acid) molecule for expressing a hairpin type RNA (ribonucleic acid) for suppressing sitobion avenae carboxylesterase and application of the DNA molecule. The structure of the DNA molecule disclosed by the invention is SEQ (forward)-X-SEQ (reverse); the sequence of the SEQ (forward) is from a locus 1 to a locus 345 of a sequence 1; the sequence of the SEQ (reverse) is reversely complementary to that of the SEQ (forward); the X is an interval sequence between the SEQ (forward) and the SEQ (reverse); according to the sequence, the X is not complementary to the SEQ (forward) and the SEQ (reverse). An experiment shows that the DNA molecule is converted into an immature embryo callus of wheat to obtain a transgenic line; the transgenic line is subjected to an aphid feeding test; after sitobion avenae is fed on transgenic leaves, the carboxylesterase gene expression quantity and the enzymatic activity are obviously alleviated, and the rate of reproduction is reduced; furthermore, the tolerance of the sitobion avenae to a phoxim solvent is reduced by 20-30 percent. Therefore, the DNA molecule has a great significance for prevention and treatment of insects of agricultural production.

The invention provides a recombinant protein structural domain for enhancing TET enzyme activity. The recombinant protein structural domain is a DNA methylation binding structural domain (MBD), and the amino acid sequence of MBD1-4 is as shown in SEQ ID 1-4. The recombinant protein MBD-TET formed by fusing the MBD and the TET enzyme can be used for remarkably enhancing the oxidation activity of the TET enzymes, including NgTET1, mTET1CD, mTET2CDT, hTET1CD and hTET2CDT, on 5mC. In addition, the invention further provides a simple, convenient and rapid high-throughput DNA methylation detection process, and the sensitivity and accuracy of a TET enzymatic oxidation reaction-based DNA methylation detection technology are improved.

The invention relates to a 7 beta-HSDH enzyme and DPS fusion protein. The 159th glycine of the 7 beta-HSDH enzyme of which an amino acid sequence is as shown in SEQUENCE NO.1 is mutated to form histidine, the 165th lysine is mutated to form histidine, a 7 beta-HSDH enzyme mutant of which an amino acid sequence is as shown in SEQUENCE NO.2 is obtained, the 7 beta-HSDH enzyme mutant and DPS proteinwhich is derived from thermoanaerobacter pseudethanolicus are subjected to fusion expression so as to obtain the 7 beta-HSDH enzyme and DPS fusion protein. Compared with a wild 7 beta-HSDH enzyme, the7 beta-HSDH enzyme and DPS fusion protein obtained by the invention has the advantages that the heat stability is significantly improved, and the enzymic activity is improved by 70%.

The invention relates to construction of a recombinant bacterium for efficiently expressing chitinase and screening of a mutant with high enzyme activity. According to the invention, efficient secretory expression of BcchiA1 in bacillus subtilis is realized by means of a recombinant protein technology, and the problem of insufficient chitinase secretion amount of most microorganisms is solved. Meanwhile, a novel chitinase high-throughput screening method is created, and the screening throughput and efficiency are greatly improved. In addition, the invention relates to several strains of chitinase mutants with high enzyme activity, the enzyme activities of BcchiA1 (Y10A), BcchiA1 (R301A), BcchiA1 (E327A), BcchiA1 (Y10A/R301A), BcchiA1 (Y10A/E327A), BcchiA1 (R301A/E327A) and BcchiA1 (Y10A/R301A/E327A) are respectively improved by 2.49 times, 0.67 times, 3.61 times, 2.39 times, 3.46 times, 4.75 times and 16.89 times compared with the enzyme activities of the wild type BcchiA1, and the enzyme activity of BcchiA1 (Y10A/R301A/E327A) is optimal. Finally, after the chitinase BcchiA1 and the optimal mutant BcchiA1 (Y10A/R301A/E327A) of the chitinase BcchiA1 and the monooxygenase BatLPMO10 from bacillus atrophaeus are subjected to a synergistic effect respectively, the hydrolysis efficiency of the chitinase BcchiA1 can be improved by 50.00%, and the hydrolysis efficiency of the chitinase BcchiA1 (Y10A/R301A/E327A) can be improved by 46.71%.

The invention discloses a tannase AfTan2.0 gene mutant and application thereof. The mutants are G475E, T437K and A222V. According to the invention, 20 single-point mutants are designed on the basis of molecular dynamics simulation by applying a calculation program FoldX; activity identification shows that the mutant R212L loses the tannase activity; and the mutants G475E, T437K and A222V which have an obvious improvement effect on the thermal stability of tannase are screened out by measuring the residual enzyme activity of the residual 19 mutants under the treatment conditions of 50 DEG C and 60 DEG C.

The invention is applicable to the technical field of gene engineering, and provides a single enzyme mutant prepared by replacing a higher plant ACS double enzyme activity key structural domain, which comprises a BOX2 conserved domain with a conserved sequence of F-Q-D-Y-H-G-[LM]-[PSKL], a BOX6 conserved domain with a conserved sequence of M-S-S-F-[GT]-L-[VI]-S-S-Q-T-Q, and a BOX4 conserved domain with a conserved sequence of T-N-P-S-N-P-L-G-[TA]-[TAMVIF]. After the conserved domain sequences are subjected to recombination replacement, the ACS recombinant protein only has ACS or C-S lyase single enzyme activity. The key structural domain influencing the ACS and C-S lyase activity of higher plant ACS protein is identified, a key target spot is provided for controlling multiple life processes in which ethylene participates by utilizing a genetic engineering method, and wide application prospects and important economic values are achieved.

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to an activity-enhanced leucine-5-hydroxylase mutant and application thereof. A leucine-5-hydroxylase encoding gene derived from Nostoc punctiforme NIES-2108 is obtained by a molecular biological means, and reverse PCR is adopted to perform site-directed mutagenesis to obtain a geneencoding a mutant, and then the gene encoding the mutant is heterologously expressed to obtain the leucine-5-hydroxylase mutant V77A. When the mutant uses leucine and methionine as substrates, the relative enzymatic activity of the mutant is increased by 69.53% and 23.26% respectively compared with a wild type. The mutant with enhanced activity selected by the invention can hydroxylate the carbonl No. 5 at the distal end of leucine more efficiently, and can catalyze methionine to produce methionine sulfoxide, thereby providing broad prospects for industrial enzymatic catalytic production of pharmaceutical intermediates.

The invention belongs to the technical field of biological engineering, and particularly relates to a mutant of short-chain dehydrogenase, and the mutant is a mutant L104A, a mutant A150Y, a mutant Y155A, a mutant I158A, a mutant I202A or a mutant T205A. The amino acid sequence of the short-chain dehydrogenase is as shown in SEQ ID NO. 1. Compared with wild type short-chain dehydrogenase, the short-chain dehydrogenase mutant has higher enzyme activity, prednisolone can be prepared from prednisone or prednisone acetate matched with hydrolase as a substrate, the conversion rate of the substrate is high, the yield of the product is high, no by-product is generated, the reaction can be carried out at a higher substrate concentration level, and the method is suitable for industrial production. The method is suitable for large-scale production.

The invention belongs to the field of pharmaceutical chemicals, and particularly relates to a BTK inhibitor. According to the invention, the structural characteristics of the listed drug ibrutinib are analyzed,and on the basis of the latest reported structure-function relationship that the difference of the N<2> site substituent R1 of piperidine-2,6-dione has little influence on the activity of Btk enzyme and the R1 faces a hydrophilic pocket of the enzyme, the physicochemical properties of the BTK inhibitor can be adjusted by introducing different hydrophilic groups. The space orientation of the N9 site substituent R2 faces the gatekeeper regions of kinases, the N9 site substituent R2 and specific residues interact in a Tec family, and interacted amino acid residues generated in the gatekeeper regions of different kinases have differences. Thus, a high-cytotoxicity compound is expected to be designed and synthesized so as to provide anti-cancer effect, and a druggability potential is provided. A cytotoxicity experiment shows that the compound has a relatively strong in-vitro inhibition effect on various cancer cells.