Improvement of GH10 family xylanase thermostability by introducing of an extra a-helix at the C-terminal Guangqi Li a, b , Xiaojuan Chen a , Xuan Zhou c , Rong Huang a , Lingbo Li a , Youzhi Miao a , Dongyang Liu a , Ruifu Zhang a, b, * a Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing, 210095, PR China b Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, 100081, PR China c National Agricultural Technology Extension and Service Center, Beijing, 100125, PR China article info Article history: Received 21 May 2019 Accepted 27 May 2019 Available online xxx Keywords: GH10 family xylanase Thermostability Poly-threonine Enzyme engineering abstract Xylanase is an important enzyme in industrial applications, which usually require the enzyme to maintain activity in high-temperature condition. In this study, a GH10 family xylanase XynAF0 from a thermophilic composting fungus, Aspergillus fumigatus Z5, was investigated to determine its thermo- stable mechanism. XynAF0 showed excellent thermostability, which could maintain 50% relative activity after incubation for 1 h at 70  C. The homologous modeling structure of XynAF0 was constructed and an a-helix composed of poly-threonine has been found in the linker region between the catalytic domain and the carbohydrate-binding module domain. Both the molecular dynamics simulation and the biochemical experiments proved that the a-helix plays an important role in the thermostability of XynAF0. Introducing of this poly-threonine region to the C-terminus of another GH10 family xylanase improved its thermostability. Our results indicated that the poly-threonine a-helix at the C-terminus of the catalytic domain was important for improving the thermophilic of GH10 family xylanases, which provides a new strategy for the thermostability modification of xylanases. © 2019 Elsevier Inc. All rights reserved. 1. Introduction Xylan is abundant renewable biomass, with the backbone mainly consisted of b-1,4-linked D-xylopyranosyl residues, and various substituent groups on the side chain, including acetyl, arabinose or 4-O-methyl glucuronic acid [1]. The degradation of xylan relied on the cleavage of b-1,4-glycosidic bond and the release of xylose oligosaccharide products by endo-xylanase. Xylanase became an important enzyme in bioenergy conversion, pulp bleaching, and animal feed [2], these industrial applying en- vironments are usually in high temperature and require the xyla- nase to be thermostable. For example, the pulp bleaching process needs the enzyme to keep hydrolysis in 60  Ce70  C conditions, and the granulation process of the animal feed production requires the protein to withstand the temperature of 75  Ce90  C[3]. Therefore, screening of thermophilic proteins is of great significance for in- dustrial applications. Currently, the widely used xylanases in in- dustry were mainly belong to the GH10 and GH11 families. GH11 xylanases are usually lower in molecular weight and higher in ac- tivity than GH10 xylanases. However, the GH10 xylanases are more resistant to high temperature, and most of the reported thermo- philic xylanases are belong to GH10 family [4]. Numerous researches of thermophilic xylanases provided many high quality models for investigating the mechanisms of their thermostability. These thermostable proteins usually have some commonalities guiding the modification of mesophilic proteins and improvement of their thermostability. For example, the number of hydrogen bonds and salt bridges between amino acid residues could affect the optimal reaction temperature and thermostability of these enzymes [5e7]. Increasing of hydrophobic amino acids, such as the aromatic residues on the protein surface helps to form hydrophobic sticky patches and aromatic clusters, thereby improving the thermostability through increase intermolecular hydrophobic interactions and the rigid regions [8,9]. Some muta- tions with increased number of charged amino acids on the surface * Corresponding author. College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, 210095, PR China. E-mail address: rfzhang@njau.edu.cn (R. Zhang). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc https://doi.org/10.1016/j.bbrc.2019.05.163 0006-291X/© 2019 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (xxxx) xxx Please cite this article as: G. Li et al., Improvement of GH10 family xylanase thermostability by introducing of an extra a-helix at the C-terminal, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.05.163