Effects Of The Rational Evolution Of Glucosidase From Trichoderma Reesei On Disaccharide Synthetic/Hydrolytic Activity

The important industrial enzyme β-glucosidase is a bifunctional enzyme because it catalyzes not only the hydrolysis of oligosaccharides but also the synthesis of oligosaccharides. Most studies of β-glucosidase focused on the hydrolytic activity, but there were few reports about the synthetic activity. In this study, Cellb, a kind of β-glucosidase derived from Trichoderma reesei, was chosen for improving the synthetic activity of disaccharides by rational design. The three-dimensional structure of Cellb was built by homology modeling. The structure of Cellb and cellobiose was docked though flexible docking. The results showed that the second glucose moiety of cellobiose was surrounded by a generally hydrophilic binding pocket. The hydrophobic residues that were close to cellobiose were W173,1174 and 1177. Therefore, a hypothesis was proposed as the hydrophobicity property of the binding pocket was recalcitrant to the disaccharide formation. We performed the rational engineering of Cellb through site-directed mutagenesis and obtained protein with increasing the hydrophilic property in the binding pocket. They were named I177S, I177S/I174S and I177S/I174S/W173H. Through turning the hydrophobic residues into hydrophilic residues in catalytic site. On the contrary, Cellb was mutated to hydrophobic enhanced of active center of the protein named N240I by turning a hydrophilic amino acid residue into a hydrophobic amino acid residue.The Cellb and its mutants were tested their synthetic and hydrolytic activity. Results were showed that the Cel1b and its mutants could synthesize laminaribiose, cellobiose and sophorose with glucose as substrate. When hydrophilicity of the active center was increased, the disaccharides synthetic activity of mutants was increased, but the hydrolytic activity were decreased; when hydrophobicity of the binding pocket was increased, the disaccharides synthetic activity of mutants was disappeared and the hydrolytic activity remain unchanged. In our study, a high disaccharide synthetic activity of β-glucosidase is engineered via rational design, suggesting our hypothesis is feasible to Cellb protein. The maximum sophorose yield was 25g/L synthesized by I177S/I174S/W173H after 96h at 80% glucose as substrate. The disaccharide synthetic/hydrolytic activities are controlled by the hydrophobicity of the binding pocket of the second glucose in Cellb:increased hydrophobicity improves the hydrolysis activity while hampers disaccharide synthetic activity. We need to decrease the hydrophobicity of the binding pocket to improve synthetic activity of (3-glucosidase. Our analysis of Cellb has important effects for further rational design and shed light to research the catalytic mechanism of β-glucosidase.Innovations are listed as follows:1. The first time to analyze the structure of intracellular β-glucosidase from T. reesei through bioinformatics tools. We proposed a hypothesis related to catalytic activity. And then we validated the applicability of the hypothesis through the rational design, providing new thoughts for further rational design of other β-glucosidase.2. We obtained a high value-added disaccharide synthetic activity β-glucosidase via rational design, which can synthesize laminaribiose, cellobiose and sophorose. The high efficient disaccharides syntheticβ-glucosidase provides the possibility and great prospects for the industrialization of laminaribiose and sophorose.