Expressed Cellobiohydrolase Enzyme of Thermobifidia fusca in Pichia pastoris as Host Can Act on Cotton Substrate

Cellulose which is extremely produced by plants, can be used for biofuel production but this function needs chemical or enzymatic digestion. Cellulose hydrolysis of plant wastes for ethanol production requires a mixture of three enzyme groups, including endoglucanases, exoglucanases, and beta-glucosidases. The cellobiohydrolase enzyme (Cel۶B) from Thermobifidia fusca has been used for cellulase activity extensively. This research aimed to express recombinant Cel۶B enzyme in Pichia pastoris. For this purpose, cel۶B gene in control of AOX۱ promoter (methanol inducible) was introduced into Pichia pastoris. Amplification of cel۶B gene was performed by PCR technique and then introduced into the Phil-S۱ yeast vector. The recombinant construct contained the cel۶B gene sequence and PHO۱ signal sequence as secretion signal was transferred into Pichia pastoris GS۱۱۵ strain. The transformed yeast cells expressed the recombinant Cel۶B to yield ۲.۱۰۴ U (µmol/min)/ml of culture medium. Purified recombinant enzyme showed the best activity at ۶۰ °C and pH ۴.۵ and this was agreed with optimum conditions for recombinant Cel۶B enzymes which were produced in other systems. The purity of the enzyme was examined by SDS–PAGE technique, and a single band with a molecular weight about ۵۹.۶ kDa was observed. As cel۶B gene sequence was not optimized for expression in the Pichia pastoris yeast, this could be one of the reasons for low level activity of recombinant Cel۶B enzyme. This thermostable enzyme can be used for cellulolytic digestion of biomaterials in biofuel production research and other uses.

Keywords:

Cellulose , Cel۶B , Yeast , AOX۱ promoter , Expression