Response surface methodology based optimization of ß-glucosidase production from Pichia pastoris.

The thermotolerant yeast Pichia etchellsii produces multiple cell bound ß-glucosidases that can be used for synthesis of important alkyl- and aryl-glucosides. Present work focuses on enhancement of ß-glucosidase I (BGLI) production in Pichia pastoris. In the first step, one-factor-at-a-time experimentation was used to investigate the effect of aeration, antifoam addition, casamino acid addition, medium pH, methanol concentration, and mixed feed components on BGLI production. Among these, initial medium pH, methanol concentration, and mixed feed in the induction phase were found to affect BGLI production. A 3.3-fold improvement in ß-glucosidase expression was obtained at pH 7.5 as compared to pH 6.0 on induction with 1 % methanol. Addition of sorbitol, a non-repressing substrate, led to further enhancement in ß-glucosidase production by 1.4-fold at pH 7.5. These factors were optimized with response surface methodology using Box-Behnken design. Empirical model obtained was used to define the optimum "operating space" for fermentation which was a pH of 7.5, methanol concentration of 1.29 %, and sorbitol concentration of 1.28 %. Interaction of pH and sorbitol had maximum effect leading to the production of 4,400 IU/L. The conditions were validated in a 3-L bioreactor with accumulation of 88 g/L biomass and 2,560 IU/L ß-glucosidase activity.