Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris

The lipases of the Rhizopus species family are important and versatile enzy mes that are mainly used in fat and oil modification due to their strong 1, 3-regiospecificity. Inexpensive synthetic medium was used for the productio n of Rhizopus oryzae lipase in the methylotrophic yeast Pichia pastoris. Me thanol accumulation inside the bioreactor has previously been shown to nega tively influence the production level. Three different methanol fed-batch s trategies for maintaining the methanol concentration within optimal limits have been assayed in high-density cultures. One methanol feeding strategy, which is based on the monitoring of the methanol concentration by gas chrom atography, resulted in a 2.5-fold higher productivity compared to an initia l cultivation, where the feeding rate was adjusted according to the dissolv ed oxygen concentration (DO) in the supernatant. Finally, productivity coul d be further increased by introducing a transition phase that involved the simultaneous feeding of glycerol and methanol followed by a single methanol feed. This optimized strategy resulted in the highest productivity (12 888 U l(-1) h(-1)), which is 13.6-fold higher than the DO-based strategy. (C) 2001 Elsevier Science B.V. All rights reserved.