Preparation of gentiooligosaccharides using Trichoderma viride β-glucosidase

Area of Science:

• Biotechnology and Industrial Microbiology
• Enzyme Engineering
• Carbohydrate Chemistry

Background:

• Recombinant β-glucosidase (bgl1) from Trichoderma viride holds potential for oligosaccharide synthesis.
• Efficient production and protein folding are critical for industrial enzyme applications.
• Protein disulfide isomerase (PDI) can enhance disulfide bond formation and protein stability.

Purpose of the Study:

• To construct and express the bgl1 gene in Pichia pastoris for enhanced enzyme production.
• To improve β-glucosidase activity through co-expression of PDI and fermentation optimization.
• To investigate the synthesis of gentiooligosaccharides using the recombinant β-glucosidase.

Main Methods:

• Construction of the recombinant plasmid pPIC9K-bgl1 using overlapping PCR.
• Integration of the bgl1 gene into Pichia pastoris KM71, with co-expression of protein disulfide isomerase (PDI).
• Fermentation optimization in a bioreactor and characterization of enzyme activity and gentiooligosaccharide synthesis.

Main Results:

• The engineered Pichia pastoris strain (KM71/pPIC9K-bgl1/pPICZ-A-pdi) achieved a β-glucosidase activity of 143 U/ml in flask fermentation, increasing to 1402 U/ml after optimization in a bioreactor.
• Gentiooligosaccharides were synthesized via reverse hydrolysis of glucose, yielding 130 g/l with a 16.25% conversion rate.
• Transglycosylation using glucose and cellobiose produced gentiooligosaccharides with a yield of 116 g/l and a 19.4% conversion rate.

Conclusions:

• Co-expression of PDI and fermentation optimization significantly enhanced β-glucosidase production in Pichia pastoris.
• The recombinant β-glucosidase is effective for synthesizing gentiooligosaccharides through both reverse hydrolysis and transglycosylation.
• This study provides a robust platform for the biotechnological production of valuable gentiooligosaccharides.