Studies On The C₁₁-α Hydroxylation Process Of Steroid With Metarhizium Anisopliae

In this paper, the C11-αhydroxylation process of 16α, 17α-epoxy-4-pregnene- 3,20-dione with Metarhizium anisopliae including single-step transformation process, dilution process and the factors affecting the formation of clustering mycelium was studied.In single-step transformation process, the composition of medium, feeding time and dissolution method of substrate were examed. The process conditions in shake flask were obtained as follows:inoculum volume 10%(seed age 36~38h), cultivate temperature 28℃, rotation speed of shake flask 220r/min, hypha age 22～24h,(pH down to about 3.6), concentration of substrate 20g/L(dissolved by 0.03% tween), conversion temperature 30℃, conversion period 72h. The conversion rate of substrate reached 70%. However, under 10L stirred-tank bioreactor, the result decreased 20% maybe because of the poor dissolved oxygen or the unstability of hydroxylases under high stirred speed.To improve dissolved oxygen condition and conversion environment, dilution process was investigated. By a series of studies involving the effect of dilution rate, tween and defoamer, pH, metal ion and feeding way of substrate on conversion, feasible process was obtained:After 36~38h cultivation under 28℃, primary seed was inoculated to fermentation medium at the concentration of 10%, after continue cultivation of 20～22h, the culture liquid was diluted by 1:1 ratio and 1/4 substrate was fed first time. 0.4g/L MnSO4 was added to accelerate conversion and temperature was adjusted to 30℃. Afer 3h, the rest substrate was added. The composition of medium is the same as that of single-step process. The substrate was dissolved by 0.5‰tween-80 and 0.5‰defoamer. The dissolved oxygen was bettered under 10L, 20L stirred-tank bioreactor and the conversion rate is closed to 60% after 45h.Thus, it can be further magnified and applied.The factors affecting formation of clusting mycelium involved:lack of nitrogen source and growth factor, low inoculum, change of strain, clustering of spores, low temperature and the control of pH , et al.