In silico designing of thermophilic Bacillus circulans xylanase

Abstract

Applications of Xylanase nowadays have become very important and widely used by the textile industry as the enzyme is safe and environmental friendly besides having more advantages compared to the use of chemical reagents. Previously, chlorine was widely used in the paper whitening process in pulp and paper industry. Because of the huge size of this industry, chlorine residues released to the environment became a huge environmental threat. Due to the hazardous effect of chlorine, xylanases have been proposed to decrease the usage of chlorine in pulp and paper industry. However, xylanases have to be functional at 60-70 C which is the temperature of the incoming pulp for the bleaching operation. Generally, xylanases have an optimal activity at 55- 60 C. Bacillus circulans xylanase (BcX) has been proposed to be used in the pulp and paper industry because of its small size (20.4kDa), but due to the high temperature used in the process BcX will not survive. In this research, Molecular Dynamics simulation (MD) was used as an in silica approach to design a thermostable BcX that can survive at high temperatures. Experimentally proven thermostable Bacillus subtilis xylanase (BsX) is used as a reference system to identify the structural and dynamic factors responsible for the thermostability of mutant BsX. Similar structural and dynamic attributes of BsX are incorporated into BcX by suitable mutations to produce similar thermostability behavior in BcX. Molecular Dynamics simulations of BsX and BcX were performed to identify structural and dynamic factors influencing thermostability. The assumption was, if the proposed mutant has the same attributes of structural interactions and dynamic behavior as those of thermostable mutant BsX, then the proposed mutant BcX would be thermostable as well. Both BsX and BcX were examined by MD at 318 Kand 338 K. Thermostability of mutant BsX was found to be contributed by the stability of the overall structure analyzed by root mean square deviation (RMSD), the existence of an important salt bridge within the active site, and an increase in the hydrophobic area. This research reported similar structural and dynamic factors between experimentally proven thermostable BsX and the proposed thermostable mutant BcX. By comparing the trends obtained in MD simulation, mutant BcX is expected to have similar or lesser thermostability compared to mutant BsX. Major factors contributing to the thermostability of BsX were also observed in mutant BcX. However, changes in the number of hydrogen bonds in mutant BcX were slightly different compared to those in BsX. Further in silica mutantions need to be carried out to pinpoint the thermostability factors in BcX.