Site-Directed Mutagenesis of a β-Glycoside Hydrolase from Lentinula Edodes.

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

doi:10.3390/molecules24010059

Jing-Jing Chen, Tian-Jiao Chen + Show 3 more

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https://doi.org/10.3390/molecules24010059

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Abstract

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.

Highlights

As the biocatalysts, enzymes are widely used in the production of food products, commodities, and pharmaceutical intermediates [1,2]
The key amino acids that may affect the enzyme properties can be chosen for site-directed mutagenesis, which includes the single site-directed mutation, multiple site-directed mutations, and saturation mutation
The thermostability of Geobacillus stearothermophilus xylanase was improved by directed evolution in combination with rational design and up to 13 amino acids were mutated during this process

Summary

Introduction

Enzymes are widely used in the production of food products, commodities, and pharmaceutical intermediates [1,2]. The prompt developments in protein engineering technology have provided the useful tools for improving enzyme critical traits, such as stability and catalytic efficiency [3,4,5,6,7,8]. Directed evolution is a method that mimics the natural evolution in the laboratory It utilizes the error-prone PCR or DNA shuffling technique in combination with the high-throughput screening method to continuously accumulate the dominant mutations with improved characteristics of the enzyme [5,11,12,13,14,15]. The thermostability of Geobacillus stearothermophilus xylanase was improved by directed evolution in combination with rational design and up to 13 amino acids were mutated during this process. The reaction temperature for maximum activity increased from 77 ◦ C to 87 ◦ C, and the catalytic efficiency increased by 90% [13]

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