Effect of Sorbitol and Methanol Co-feeding on Laccase Production in Recombinant Pichia pastoris

Nur Nadhirah, Wasli (2022) Effect of Sorbitol and Methanol Co-feeding on Laccase Production in Recombinant Pichia pastoris. [Final Year Project Report] (Unpublished)

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Abstract

White-rot fungi, as a natural sources of laccase, are not adequate for commercial usage, however laccase production can be boosted using Pichia pastoris. The AOX1 promoters in P. pastoris are activated by the presence of methanol to express recombinant proteins. Due to methanol constraints, bioprocessing research for P. pastoris is currently focused on the methanol mixed feeding approach. This study evaluated the effects of employing sorbitol as a co-inducer with methanol on laccase synthesis in recombinant P. pastoris. In addition, the fermentation performance of P. pastoris for the laccase production was examined and compared between the use of sorbitol and methanol co-feeding and the use of methanol and sorbitol alone as the feed strategies. During the induction phase, the fermentation medium is supplied for every 24 hours with different induction strategy; 0.5% (v/v) Methanol, 1:1 ratio (0.5% (v/v) Methanol + 0.5% (v/v) Sorbitol), 0.5% (v/v) Sorbitol. Cell growth and laccase activity analysis were performed to assess the fermentation performance. The results showed that the use of sorbitol and methanol co-feeding, 0.5% sorbitol + 0.5% methanol, which is the 1:1 ratio was found to give the highest activity of laccase (6.85×10-4 U mL-1) compared to the approach of using 0.5% (v/v) sorbitol (6.12 ×10-4 U mL-1) and 0.5% (v/v) methanol (4.29×10-4 U mL-1). The enhancement of laccase activity brought using sorbitol and methanol induction was 1.6-fold over that achieved by control fermentation. This study offers valuable insights into the possible use of sorbitol and methanol co-feeding as a viable approach to synthesise heterologous laccase in recombinant P. pastoris since sorbitol has various benefits for P. pastoris fermentation compared to methanol. This study will also contribute to future research on laccase production employing a methanol and sorbitol mixed strategy.

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