Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering

Xiaoyu Wang,Xiangming Xie,Tao Tu,Rui Ma,Jianzhong Ge,Bin Yao,Huifang Xie,Weina Liu,Fei Zheng,Shuai You,Huiying Luo

doi:10.1038/s41598-017-12659-y

Abstract

Thermophilic xylanases with high catalytic efficiency are of great interest in the biofuel, food and feed industries. This study identified a GH11 xylanase gene, Tlxyn11B, in Talaromyces leycettanus JCM12802. Recombinant TlXyn11B produced in Pichia pastoris is distinguished by high specific activity (8259 ± 32 U/mg with beechwood xylan as substrate) and excellent pH stability (from 1.0 to 10.5). The beechwood xylan hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose, thus TlXyn11B could be used for the production of prebiotic xylooligosaccharide. By using the structure-based rational approach, the N-terminal sequence of TlXyn11B was modified for thermostability improvement. Mutants S3F and S3F/D35V/I/Q/M had elevated Tm values of 60.01 to 67.84 °C, with S3F/D35I the greatest. Homology modeling and molecular dynamics (MD) simulation analysis revealed that the substituted F3 and I35 formed a sandwich structure with S45 and T47, which may enhance the overall structure rigidity with lowered RMSD values. This study verifies the efficiency of rational approach in thermostability improvement and provides a xylanase candidate of GH11 with great commercialization potential.

Highlights

Xylan accounts for approximately 35% of the dry weight of plant cell wall and represents one of the most important, abundant, and renewable bioresources
It is found that the N-terminal sequence plays a key role in the stability of GH11 xylanases
A highly active GH11 xylanase with a broad pH adaptability range but weak thermostability was identified in Talaromyces leycettanus JCM12802

Summary

Introduction

Xylan accounts for approximately 35% of the dry weight of plant cell wall and represents one of the most important, abundant, and renewable bioresources. The directed evolution is a robust strategy to engineer enzymes by accelerating protein evolution[7] This method involves in construction of large libraries and laborious screening. Computer-aided approaches have been used to analyze the primary sequence, simulate the tertiary structure and de novo predict the protein interaction[9]. Based on these bioinformatic analyses, key amino acids are identified and site-directed mutagenesis is conducted. This in silico rational method dramatically reduces the library size and workload and increases the engineering efficiency as well, and is widely used for protein improvement. A mutant with greater thermostability was obtained, and the underlying mechanism was revealed by the analysis of molecular dynamics simulation

Methods

Results

Conclusion