The β-glycoside hydrolases (LXYL-P1-1 and LXYL-P1-2) from Lentinula edodes (strain M95.33) can specifically hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol for the semi-synthesis of Taxol. Our previous study showed that both the I368T mutation in LXYL-P1-1 and the T368E mutation in LXYL-P1-2 could increase the enzyme activity, which prompted us to investigate the effect of the I368E mutation on LXYL-P1-1 activity. In this study, the β-xylosidase and β-glucosidase activities of LXYL-P1-1I368E were 1.5 and 2.2 times higher than those of LXYL-P1-1. Most importantly, combination of I368E and V91S exerted the cumulative effects on the improvement of the enzyme activities and catalytic efficiency. The β-xylosidase and β-glucosidase activities of the double mutant LXYL-P1-1V91S/I368E were 3.2 and 1.7-fold higher than those of LXYL-P1-1I368E. Similarly, the catalytic efficiency of LXYL-P1-1V91S/I368E on 7-β-xylosyl-10-deacetyltaxol was 1.8-fold higher than that of LXYL-P1-1I368E due to the dramatic increase in the substrate affinity. Molecular docking results suggest that the V91S and I368E mutation might positively promote the interaction between enzyme and substrate through altering the loop conformation near XDT and increasing the hydrogen bonds among Ser91, Trp301, and XDT. This study lays the foundation for exploring the relationship between the structure and function of the β-glycoside hydrolases.
Submitter: Shu-Ching Ou
Submission Date: March 22, 2019, 11:06 a.m.
|Number of data points||28|
|Assays/Quantities/Protocols||Experimental Assay: Vmax ; Experimental Assay: Km ; Experimental Assay: kcat ; Experimental Assay: kcat/Km ; Derived Quantity: SD of kcat ; Derived Quantity: SD of Km ; Derived Quantity: SD of Vmax|
|Libraries||Variants for LXYL-P1-1_XDT|