In this work, three factors, five-level central composite design was used to optimize protease production by Rhizopus oryzae CH4 grown on wheat gluten. Gluten concentration, starch concentration and inoculums sizes significantly affected protease production. The optimal combinations of media constituents for maximum protease production (266.5 UP ml-1) were determined as gluten concentration 22.5 g l-1, starch concentration 30 g l-1 and inoculums size 5x106 spores ml-1. Under the proposed optimized conditions, the protease experimental yield (266.5 UP ml-1) closely matched the yield predicted by the statistical model (254.4 UP ml-1) with R2=0.981. An overall 2.9-fold increase in enzyme production was achieved in the optimized medium. On subsequent scale-up in a 20-l bioreactor using conditions optimized through RSM, 258.2 UP ml-1 of protease was produced in 60-72 h. This clearly indicated that the model remained valid even on a large scale. The relatively higher protease production by Rhizopus oryzae CH4 showed promise of offering great potential as additives in the bread making industry.
