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

Jing-Jing Chen,Xiao Liang,Ping Zhu,Jin-Ling Yang,Tian-Jiao Chen

doi:10.3390/catal11091042

Jing-Jing Chen, Xiao Liang + Show 3 more

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https://doi.org/10.3390/catal11091042

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Abstract

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.

Highlights

Enzyme-based biocatalysis has been applied in many areas, especially in pharmaceuticals, chemicals, fragrances, cosmetics, and biofuels [1,2,3,4,5]
To further confirm whether the L220G mutation is beneficial for the further improvement of EP2, we introduced the L220G mutation into EP2, and the enzyme activity of corresponding recombinant yeast was measured
The results showed that the introduction of L220G mutation into the EP2 led to the improvement of the β-glycosidase activity and the ability to hydrolyze the substrate XDT of the recombinant yeast GS115-EP2-L220G

Summary

Introduction

Enzyme-based biocatalysis has been applied in many areas, especially in pharmaceuticals, chemicals, fragrances, cosmetics, and biofuels [1,2,3,4,5]. Developments in biotechnology, in the area of protein engineering, have provided important tools for efficiently improving enzyme properties [6,7,8,9], such as increasing catalytic efficiency [10] and/or specific substrate recognition [11,12] or improving thermal stability [13,14,15,16]. The specific amino acid residues of enzyme can be chosen to be precisely designed to improve the enzyme property. This method has the characteristics of simple operation and high success rate, and can obtain mutants with improved properties in a short time [19,20,21]. The results obtained through rational design can, in turn, increase the understanding of the enzyme catalytic mechanism, further increase the successful rate of beneficial enzyme modification, and lay a foundation for the functional elucidation of unknown protein [22]

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Discussion

Conclusion