Cuitural Condition Optimization Of Rhizopus Nigricansand Cloning Expression Of CPR Gene

All of steroidal compounds have a cyclopentanoperhydrophenanthr-ene structure, such as steroid hormone, progesterone, testosterone, cholesterol and endogenous substances. Steroids have important physiological function on sugar metabolism, fat metabolism, protein synthesis, mineral synthesis, sexual function and neuromodulation etc. Now there are dozens of steroidal drugs in the sale, its therapeutic areas including anti-inflammatory, anti-allergic, step-down, contraception, Enhance immune system, antitumor, etc.This dissertation consist of the optimization of fermentation medium, preparation and regeneration the protoplast of Rhizopus nigericans. Cloning and expression of CPR gene in Rhizopus nigericans.Currently, the optimization for Rhizopus nigricans fermentation medium are most concentrated in the optimum kind of nitrogen source, carbon source, inorganic salts, etc. Using RSM optimization rarely reported. This thesis includes the single factor optimum the Rhizopus nigericans’culture medium, then use the Plackett-Burman testing and ascent procedures testing define optimal response region of these 3 factors, finally the optimal factor concentrations were determined by adopting RSM analysis. The results showed that glucose 39.2 g/L, corn flour 25 g/L, silkworm chrysalis powder 10 g/L, (NH4)2SO40.8 g/L K2HPO4·3H2O 10.8 g/L, pH 4.85 were combined as the most suitable Rhizopus nigericans fermentation medium. The substrate conversion was increased 62.47%, which is increased 7.48% higher than the original fermentation medium.The protoplast with higher regeneration activity was obtained using Yatalase (7.5%) and Lywallzyme (5.0%) to process the spore 4 h, at 30 ℃.The spore was cultivated 14 h. Determine the Minimum inhibitory concentration of hygromycin B on Rhizopus nigericans spore.The MIC is 200μg/mL.Using RT-PCR method obtain CPR gene from Rhizopus oryzae. The sequence was cloned to corresponding site of pCB1004-Pgpd A. Then the expression plasmid pCB 1004-Pgpd A-CPR which promoted by strong promoter PgpdA was obtained. REMI mediated protoplast transformation of Rhizopus nigericans with expression plasmid was performed and CPR overproducing transformat was obtained. The CPR transformat’s conversion of 11α-hydroxylation were increased 4.31% than starting strain.