Preparation of gentiooligosaccharides using Trichoderma viride β-glucosidase.

The recombinant plasmid pPIC9K-bgl1 containing β-glucosidase bgl1 from Trichoderma viride was constructed by overlapping PCR and integrated into Pichia pastoris KM71. In order to assist the formation of disulfide bonds and thus improve protein folding efficiency, protein disulfide isomerase pdi was co-expressed in the P. pastoris KM71/pPIC9K-bgl1/pPICZ-A-pdi strain, and fermentation in flasks resulted in enzyme activity of 143 U/ml. The enzyme activity of β-glucosidase reached 1402 U/ml following optimisation of fermentation conditions in a 3.6 l bioreactor. With 80% glucose as substrate, gentiooligosaccharides were synthesised by β-glucosidase-based reverse hydrolysis. A yield of 130 g/l was achieved with a conversion rate of 16.25%. With 20% glucose and 40% cellobiose as substrates, gentiooligosaccharides were synthesised by transglycosylation with a yield of 116 g/l and a conversion rate of 19.4%.