| Insufficient coenzyme is a main limiting factor in biocatalyst an-d biosynthesis in relation to oxidoreductase, products of synthesis cat-alyzed by oxidoreductase are widely employed in the areas of pharm-aceutical industry, foodstuff industry and pesticide field. Therefore, th-e study of coenzymes regeneration has important academic meaning and economic value. Researches about steroidal C11α-hydroxylation by biotransformation using Rhizopus nigricans have been conducted for decades, and technologies of production are relatively mature, hence, there is considerable difficulty to improve conversion rate from existi-ng basises. Cytochrome P450 enzymes is a major catalysator of ster-oidal C11α-hydroxylation by Rhizopus nigricans, the catalytic process require enough coenzyme NADPH to ensure continual reaction, accor- ding to current theoretical researches, hard to gain additional convers-ion rate in production mainly results from restriction of NADPH qua-ntity. Therefore, based on reinforcing NADPH regulation, this paper researched the impacts of the clone of G6PDH gene on steroidal C11-a-hydroxylation by rhizopus nigricans.Firstly, we isolated a fragment of G6PDH gene from Rhizopus oryzae. In this study, total RNA of Rhizopus oryzae is isolated by tr-izol method, then we obtained a G6PDH gene by using RT-PCR, a-nd agarose electrophoresis shows the G6PDH fragment band is betw-een 1200-1400 bp, which according with 1346 bp reported in BRO-AD datebase. Positive clones were selected by the transformation of E. coli competent cells experiment, colony PCR, restriction enzyme reaction confirmation, and the sequencing result is exactly consistent with the reported sequence. The eukaryotic expression vector PCB10-04-G6PDH was constructed successfully by ligation and transformatio nof G6PDH and PCB1004 with same cohesive end obtained from Ba-mH I and Apa I restriction enzyme reaction.Secondly, this study performed the preparation of Rhizopus nigric ans protoplasts and transformation mediated by PEG. The optimal co-nditions of protoplasts preparation:the concentration of enzyme mixt-ure is 6% lywallzyme and 8% yatalase, the reaction temperature is 30℃, the reaction time is 4 hour. The concentration of homomycin resistance screening is 200μg/ml, which is built by a hygromycin s-ensitivity test. In the protoplasts transformation mediated by PEG and stains screening,2 strains named RG3 and RG12 are found, and are proved at the level of genomic DNA, The results showed that both RG3 and RG12 strains genomic DNA contain HPH gene encoding h-ygromycin B phosphotransferase and G6PDH gene.Finally, this study investigated the strains growth and conversion-n rate of 16,17a-epoxyprogesterone by original strain and RG3, RG12 strains in different carbon and nitrogen source through the singlefact-or experiments, results indicated that glucose are the best organic ca-rbon source and soybean are the best nitrogen source, olive oil has an obvious effect on RG12 strain conversion rate of 16,17-a-epoxypr-ogesterone, the conversion rate of 16,17a-epoxyprogesterone is 14.3± 0.70% using glucose and is 28.5±0.50% using olive oil, this sugges-ted that olive oil has a promoting effect on catalytic process, this pr-ovided a basis for further optimizing RG12 stain conversion abilities. Experiments about the effect of inorganic nitrogensources on convers-ion rate showed that inorganic nitrogen sources have negative effects on the orginal strain conversion of 16,17a-epoxyprogesterone, for RG-3, conversion rate reached 48.75±0.70% after adding sodium nitrate. The RG12 has relatively high conversion rate 45.55%±0.42% by add-ing ammonium dihydrogen phosphate, compared with the highest co- nversion rate 41.69±0.90% of the original strain, RG3 and RG12 h-ave advantages in application of 16,17α-epoxyprogesterone C11α-hydr-oxylation, therefore, this study proved that G6PDH has a great poten-tial and application value in improving the capability of steroidal C11-a-hydroxylation by Rhizopus nigricans. |
PDF Full Text Request