P450 enzyme of anisodus acutangulus and application of P450 enzyme to preparation of tropinone
A technology of reducing enzymes and amino acids, which is applied in the fields of biosynthesis and enzyme catalysis, and can solve the problems of large environmental pollution caused by chemical methods
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Embodiment 1 3
[0071] Example 1 Cloning of three-thirds cytochrome P450 and cytochrome P450 reductase genes
[0072] 1.1 Extraction and detection of total RNA
[0073] Turn on the ultra-clean bench and ultraviolet lamp, burn the mortar, keys, and scissors with ethanol. Cut off 100mg of three-thirds hairy roots, grind the tissue into powder in liquid nitrogen, and distribute it into Ep tubes. After the liquid nitrogen evaporates, add 1ml Trizol-x-100, shake vigorously immediately or use a pipette to blow 5- 8 times (until there are no lumps), let stand at room temperature for 5 minutes; extract twice with an equal volume of chloroform, and centrifuge at 7500g for 15 minutes; Centrifuge at 10,000g for 10min at ℃; add 1ml of 75% ethanol to the precipitate for washing, and centrifuge at 10,000g at 4°C for 10min; dry the precipitate at room temperature for 10min and dissolve in 25μL of DEPC-treated water, and use 1.0% agarose gel electrophoresis to detect the integrity of the RNA. The ratio and...
Embodiment 2
[0083] The construction of embodiment 2 eukaryotic expression vector
[0084] 2.1 Cloning of AaCYP82M3 and AaCPR1-3 genes: Use Axygen’s plasmid extraction kit to extract and sequence the correct T vector, use the AaCYP82M3 and AaCPR1-3 genes in the T vector as templates, and use the primers in Table 1 to perform PCR amplification. The following system [5×phusion buffer 4.0μL, dNTP (2.5mM) 1.6μL, each primer (2μM) 2μL, DMSO 0.6μL, cDNA 0.5μL, Phusion DNA polymerase 0.2μL, add ddH 2 O to a final volume of 20.0 μL] to amplify the gene, the reaction program: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 2min, 30 cycles, final extension at 72°C for 5min, gel recovery using Axygen Reagents are recovered to obtain the corresponding fragments.
[0085] 2.2 Gene double digestion: Digest the PCR product of AaCYP82M3 with restriction enzymes BamHI and KpnI at 37°C for 2h; digest the PCR product of AaCPR1 with restriction...
Embodiment 3
[0089] Example 3 Construction of AaCYP82M3 and AaCPR1-3 Expression Strains
[0090] Saccharomyces cerevisiae By4742 was streaked on a YPD plate and cultured at 30°C for 2 days; a single colony of yeast was picked and placed in 5mL YPAD liquid medium, cultured overnight at 30°C and 200rpm; the yeast liquid was diluted 10 times, and the OD was measured 600 , at this time OD 600 Should be 0.4-0.5 (indicating good growth). Transfer to 2*YPAD (fast growth) at a ratio of 1:20 to 1:25, and culture in 50mL to make the initial OD 600 Less than or equal to 0.2; cultivate to OD at 30°C, 200rpm 600 0.5, about 4-5 hours; the carrier DNA (protist DNA) frozen at -20°C was denatured at 100°C for 5 minutes, and immediately inserted into the ice (keep single strand). Centrifuge at 1,000g for 5 minutes at room temperature to collect bacteria, resuspend with 1 / 2 volume of sterile water (for example, resuspend 50mL of bacterial liquid with 25mL of sterile water) and centrifuge; wash again with ...
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