Effect of Sorbitol on Laccase Production in Recombinant Pichia pastoris

Yit, Hui Ying (2022) Effect of Sorbitol on Laccase Production in Recombinant Pichia pastoris. [Final Year Project Report] (Unpublished)

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Abstract

The production of laccases, which are known for their potential in oxidising a wide range of phenolic and nonphenolic substrates into harmless molecules, has been studied for quite some time. Many works reported the production of laccases by recombinant Pichia pastoris yeast using carbon sources such as glycerol and methanol whilst the use of potential other carbon sources like sorbitol is still lacking. Sorbitol is revealed capable of reducing repression of AOX1 promoter which plays an important role in laccases production by recombinant P. pastoris by reducing cytotoxic effects or metabolic burden of the yeast’s growth. This work aims to study the feasibility of sorbitol as an alternative carbon source for recombinant P. pastoris to produce laccases. In this study, recombinant P. pastoris GS115 strains incorporated with laccase enzyme producing gene from Marasmius cladophyllus was used. Buffered methanol complex (BMMY) was used as the basic medium for all fermentations. The concentrations of sorbitol incorporated in the fermentation were 40 g/L, 50 g/L and 60 g/L. The activity of laccases was determined based on the oxidation reactions of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). The results showed that supplementation with 1% methanol + 50 g/L sorbitol yielded the highest laccase activity of 0.5994 x 10-4 U mL-1 recorded on the fourth day of fermentation. The highest laccase activity recorded when sorbitol is incorporated in BMMY was observed to be almost twice the amount of the highest laccase activity when there is no sorbitol included in BMMY with 0.3590 x 10-4 U mL-1 on the seventh day of fermentation. Thus, the presence of sorbitol as an alternative carbon source could be investigated as it plays an important role in laccase production.

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