Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

• Published in: Catalysts Vol. 11; no. 9; p. 1042
• Main Authors: Chen, Jing-Jing, Liang, Xiao, Chen, Tian-Jiao, Yang, Jin-Ling, Zhu, Ping
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 01.09.2021
• Subjects: Amino acids; biocatalysis; Catalysts; Chemical reactions; Crystal structure; Enzyme activity; Enzymes; Glycosidases; Hydrogen bonds; Molecular docking; Molecular structure; Mutagenesis; Mutation; Proteins; Residues; site-directed mutagenesis; Substrates; Synthesis; Taxol; β-glycosidase
• ISSN: 2073-4344; 2073-4344
• DOI: 10.3390/catal11091042

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.