8 results about "Cytochrome Reductases" patented technology

The invention discloses a heme oxygenase mutant and application of the heme oxygenase mutant in preparation of bilirubin through multienzyme coupling, and belongs to the field of biological catalysis engineering. The forward mutant of the heme oxygenase is obtained through a directed evolution technology, and heme can be efficiently catalyzed to synthesize biliverdin; the bilirubin is synthesized by further introducing biliverdin reductase, coupling cytochrome P450 reductase and formate dehydrogenase, and coupling the two core enzymes with foreign assistant enzymes to construct recombinant escherichia coli to catalyze heme to synthesize bilirubin. In the system, NADPH does not need to be exogenously added, the reaction time is about 4 h, the yield of bilirubin reaches 2.10 g / L, accumulation of intermediate products is not observed in the catalysis process, and the highest yield and the shortest reaction time in bilirubin synthesis reported at home and abroad at present are achieved. Compared with the traditional pig bile extraction, the method disclosed by the invention breaks through the resource limitation, is simple to operate and high in yield, and realizes efficient and green synthesis of bilirubin.

The invention provides a steroid compound 11 alpha-hydroxylase and application of the steroid compound 11 alpha-hydroxylase. The coexpression vector provided by the invention can simultaneously express the steroid compound 11alpha-hydroxylase and cytochrome P450 reductase in escherichia coli or mycobacteria, a genetic engineering strain containing the coexpression vector can be suitable for 11alpha-hydroxyl conversion of various steroid substrates, the specificity is relatively strong, the highest conversion rate can reach 90.39%, and the coexpression vector can be applied to the conversion of 11alpha-hydroxyl of various steroid substrates. And the production technology is simple, efficient and controllable, and has important industrial application value.

The invention belongs to the technical field of gene engineering and microbial fermentation, and particularly discloses a cytochrome P450 reductase coding gene CPR2 from aspergillus nidulans and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO: 1, and the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO: 2. According to the invention, the CPR2 gene (OECPR2) is identified as an optimal electron transfer regulatory element by performing function screening on eight CPR genes annotated in a genome, and the CPR2 gene is over-expressed in an echinocandin B production strain aspergillus nidulans, so that the intracellular electron transfer efficiency is remarkably enhanced, the enzyme catalytic activity of cytochrome P450 is driven, and the cytochrome P450 can be used for preparing an echinocandin B cell. And finally, the yield of echinocandin B is efficiently increased. Experiments show that the engineering strain for over-expression of CPR2 can enable the yield of echinocandin B to reach 1869.97 + / -96.98 mg / L in shake flask fermentation, which is 24.37% higher than that of the original strain. The invention provides a key gene element and an engineering strain for industrial efficient production of echinocandin B, and has important economic value.

This invention discloses a thermophilic recombinant *Yersinia lipolytica* strain producing carotenoids, its construction method, and its applications. The recombinant strain exogenously expresses thermophilic precursor fatty acid desaturase (FAD3) and isocitrate dehydrogenase (IDH2) mutants from *Rhodotorula rubrum*, achieving efficient synthesis at 30°C. Secondly, the metabolic pathway of the recombinant strain is optimized based on a metabolic flux push-pull strategy, overexpressing isopentenyl pyrophosphate isomerase (IDI), farnesyl pyrophosphate synthase (ERG20), phytene dehydrogenase (CarB), and bifunctional phytene synthase (CarRP) to increase precursor throughput. Finally, multiple copies of the genes for astaxanthin synthase (crtS) and cytochrome P450 reductase (crtR) from *Rhodotorula rubrum* are performed to further enhance the carotenoid production capacity of *Yersinia lipolytica*. The thermophilic *Yersinia lipolytica* construction method of this invention is simple, can achieve more efficient carotenoid synthesis at higher temperatures, significantly reduces fermentation costs, and has potential for industrial production.

The application discloses a soluble expression vector of a P450 enzyme for producing indole-3-methylamine and a construction method and application thereof. The construction method is that a nucleotide sequence for coding a P450 enzyme tAMIS protein and a nucleic acid sequence for coding a NADPH-cytochrome reductase CPR protein are expressed in a vector, so that a soluble expression vector of a plant-derived P450 enzyme in Escherichia coli is obtained. The expression vector solves the problem that most plant-derived P450 enzymes cannot exhibit biological activity in the Escherichia coli, and compared with using plants or yeasts as hosts, the time consumed for expressing proteins is reduced. A method for detecting indole-3-methylamine synthesized by the Escherichia coli through an exogenous gene is provided.

The application discloses a kind of heme oxygenase mutant and its multienzyme coupling preparation of application of bilirubin, belong to biological catalysis engineering field.The application obtains the positive mutant of heme oxygenase by directed evolution technology, can efficiently catalyze heme to synthesize chlorophyllin;Further introduce chlorophyllin reductase, coupling cytochrome P450 reductase, formic acid dehydrogenase, and two kinds of core enzyme are coupled with exogenous auxiliary enzyme, constructs recombinant escherichia coli, catalyzes heme to synthesize bilirubin.In the system, without exogenous addition NADPH, reaction time is about 4h, and bilirubin yield is as high as 2.10g / L, and no intermediate accumulation is observed in catalytic process, reaches the highest yield of the synthesis of bilirubin reported at home and abroad, and the shortest reaction time level.Compared with traditional pig bile extraction, the method of the application breaks through the resource limit, and the operation is simple, the yield is high, realizes the efficient, green synthesis of bilirubin.

The present application relates to a kind of CYP11B1-BM3 fusion protein, which is connected by connecting peptide by steroid 11 beta hydroxylase CYP11B1 and cytochrome P450 BM3 reduction domain.The encoding gene of the fusion protein is transformed into escherichia coli for transformation expression, can efficiently synthesize CYP11B1-BM3 fusion protein, and the prepared fusion protein has higher cytochrome P450 reductase activity and in vitro hydroxylation enzyme activity, can efficiently catalyze hydroxylation reaction, can be applied to the synthesis of cortisol drug.

The application provides a steroid compound 11alpha-hydroxylase and its use. The co-expression vector provided by the application can simultaneously express the steroid compound 11alpha-hydroxylase and cytochrome P450 reductase in Escherichia coli or Mycobacterium, and the genetically engineered bacterial strain containing the co-expression vector can be suitable for 11alpha-hydroxylation of various steroid substrates, has high specificity, and the conversion rate can be up to 90.39%, and the production technology is simple, efficient, controllable, and has important industrial application value.