Abstract
The LIP2 isoenzyme gene from Candida rugosa has been completely synthesised and functionally expressed under the AOX1 promoter control in Pichia pastoris. The on-line monitoring and control of methanol, the key inducer carbon source in fed-batch cultures, has enhanced the yield product/biomass 7.8-fold and the productivity 12.8-fold compared to the best batch cultivation with the Pichia system and, 10-fold compared to the fed-batch cultivation process using the native C. rugosa strain. Nevertheless, the high ionic strength of culture broth favoured aggregation of Lip2, leading to total loss of lipolytic activity. After cultivation, a diaultrafiltration process was implemented to diminish ionic strength, allowing for the recovery of lipolytic activity in the diaultrafiltrate. The developed bioprocess resulted into a reproducible product in terms of quality and productivity.
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