Please use this identifier to cite or link to this item:
|Title:||Experimental and computational studies of a fungal chitinase||Authors:||Khan, Faez Iqbal||Issue Date:||2015||Abstract:||Chitin, the second most abundant natural biopolymer, is composed of repeating units of N-acetyl-β-D-glucosamine and primarily forms the structural component of protective biological matrices such as fungal cell walls and exoskeletons of insects. Chitinases are a ubiquitous class of extracellular enzymes that have gained attention in the past few years due to their wide range of biotechnological applications, especially in the field of agriculture for bio-control of fungal phytopathogens. They play an important role in the defense of organisms against chitin-containing parasites by hydrolyzing the β-1,4-linkages in chitin and hence act as anti-fungal as well as anti-biofouling agents. Moreover, the effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance and thus, chitinases offer a potential alternative to the use of chemical fungicides. In recent years, thermostable enzymes isolated from thermophilic microorganisms have gained widespread attention in industrial, medical, environmental and biotechnological applications due to their inherent stability at high temperatures and a wide range of pH optima. Determination of the three- dimensional structure of a protein can provide important details about its biological functions and its mode of action. However, despite their significance, the precise three-dimensional structures of most of the chitinases, including those isolated from Thermomyces lanuginosus is not fully characterized so far. Hence, the main focus of the present study was to gain a better understanding of the structural features of chitinases obtained from this thermostable fungus using both experimental and computational techniques, and their relationship with their activity profiles. The genes encoding thermostable chitinase II from T. lanuginosus were isolated and cloned in both E. coli as well as the Pichia pastoris expression system. Analysis of the nucleotide sequences revealed that the chitinase II gene (1196 bp) encodes a 343 amino acid protein of molecular weight 36.65 kDa whereas the chitinase I gene (1538 bp) encodes a 400 amino acid protein of molecular weight 44.14 kDa. In silico protein modeling was helpful in predicting the 3D models of the novel chitinase II enzyme, followed by the prediction of its active sites. The presence of Glu176 was found to be essential for the activity of chitinase II. Similarly, analysis of chitinase I revealed several active sites in its structural framework. A 10 ns Molecular dynamics (MD) simulations was implemented to assess the conformational preferences of chitinases. The MD trajectories at different temperatures clearly revealed that the stability of the enzymes were maintained at higher temperatures. Additionally, a constant pH molecular dynamics simulations at a pH range 2-6 was performed to establish the optimum activity and stability profiles of chitinase I and chitinase II. For this purpose, the Molecular Dynamics simulations were carried out at fixed protonation states in an explicit water environment to evaluate the effect of the physiological pH on chitinase I and II enzymes obtained from T. lanuginosus. The results suggest a strong conformational pH dependence of chitinases. These enzymes retained their characteristic TIM Barrel fold at the respective protonated conditions, thus validated the experimental outcomes. Further, the different stability and flexibility predictions were used to assess the relation of point mutations and enzyme stabilities. Our results pave the way to engineer new and better thermostable enzymes.||Description:||Submitted in fulfillment of the requirements of the degree of Doctor of Philosophy: Chemistry, Durban University of Technology, 2015.||URI:||http://hdl.handle.net/10321/1267|
|Appears in Collections:||Theses and dissertations (Applied Sciences)|
Show full item record
Page view(s) 50269
checked on Nov 19, 2018
checked on Nov 19, 2018
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.