Vietnam ginsenoside R7 hydrolase gene cloning and application thereof
A Vietnamese ginseng, hydrolase technology, applied in hydrolase, application, genetic engineering and other directions
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example 1
[0044] Example 1, Vietnamese ginsenoside R 7 Cloning of hydrolase and its coding gene
[0045] Genomic DNA of Herpetosiphonaurantiacus DSM785 was extracted as template, and 5′-CGCGGATCCATGACCGCGAGCGATCAAC-3′(Forward) and 5′-CCCAAGCTTCTAGCCCTGATTGACCTTGGC-3′(Reverse) were used as primers for polymerase chain reaction PCR amplification. PCR system: 2×TaqMixture 25 μL, upstream and downstream primers 1 μL, genome 1 μL and ddH 2 O22 μL.
[0046] The PCR conditions were as follows: pre-denaturation at 95°C for 3 minutes, followed by 30 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min; finally, extension at 72°C for 10 min.
[0047] The above PCR products were analyzed by agarose gel electrophoresis, the gel was tapped and the target band was recovered with a kit.
[0048] Use restriction endonucleases BamHI and HindIII to double digest the recovered product with the pET28a vector (37°C, 6h); use the kit to purify and recover the digested product, and use T4 ligase to l...
example 2
[0050] Example 2 Recombinant Vietnamese Ginsenoside R 7 Expression and Purification of Hydrolase HaGH03
[0051] Inoculate the single colony of BL21(DE3) / pET28a-Ha3 obtained above into LB liquid medium containing kanamycin (final concentration: 50 μg / mL), culture at 37°C for 10 h, and take 1 mL of bacterial liquid (1%, Volume percentage content) was added to 100mL fresh LB liquid medium, and kanamycin (final concentration was 50μg / mL) was added at the same time, and cultured to OD at 37°C 600 When it reached 0.5, IPTG (final concentration: 0.1 mM) was added to the culture medium, and induced at 16° C. for 24 hours. The fermentation broth was collected, and the fermentation broth was centrifuged at 8000 rpm for 5 min at 4°C to collect the bacteria. Wash twice with 50 mL of normal saline, centrifuge, and collect the bacteria.
[0052] Resuspend the bacteria with 10mL of LA solution (sodium phosphate buffer at pH 7.4, containing 20mM imidazole, 500mM NaCl). In an ice-water ba...
example 3
[0054] Example 3 Recombinant Vietnamese Ginsenoside R 7 Optimum Temperature of Hydrolase HaGH03
[0055] In 0.5 mL of pH 6.550 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, add p-nitrophenyl-β-D-glucoside and a certain amount of recombinant Vietnamese ginsenoside R at a final concentration of 2 mM 7 Hydrolase HaGH03, 20-65 ℃ water bath reaction for 5min, add 0.5mL1mM Na 2 CO 3 The solution terminated the reaction, and the absorbance at 405nm was measured. The relative activity results are shown in Table 2. The enzyme has the highest reactivity at 50°C.
[0056] temperature(℃)
[0057] Table 2 Vietnamese ginsenoside R 7 Optimum temperature for hydrolytic enzymes;
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