1 Bioinformatics analyses of a Thermophilic Fungal Recombinant Chitinase Muthu Prasad Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India prasadmku@gmail.com Peramachi Palanivelu* 1 *Former Professor and Head, Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, India 1 Professor, CARE, Chettinad University, Kelambakkam, Chennai – 603 103, India. ppmkupp@gmail.com * 1 Corresponding author Abstract: A thermophilic fungal chitinase was cloned, sequenced and overexpressed in Saccharomyces cerevisiae. Multiple sequence alignment of the recombinant chitinase revealed that the catalytic domain is 100% conserved with other family GH18 chitinases. Phylogenetic analysis revealed that the recombinant chitinase exhibited a very close proximity to the chitinases of other thermophilic fungi and higher fungi. Glycosylation of the recombinant chitinase was analyzed by in silico methods and confirmed by experimental data. Homology modeling of the chitinase showed the typical (α/β) 8 TIM barrel structure. Molecular docking of the enzyme revealed the involvement of a glutamic acid of the catalytic domain in the catalytic process. Keywords – Chitin, fungal recombinant chitinase, GH18 family, glycosylation, homology modeling, molecular docking. I. INTRODUCTION Chitin is the most abundant, natural, cationic biopolymer present in the world. It is a homopolymer of β, 1-4 linked N-acetyl D- glucosamine residues. The chitinases, which hydrolyze the chitin polymer are broadly classified into exo- and endo-chitinases. The endochitinases are further classified into two different glycosyl hydrolase families, viz., GH18 and GH19, based on their catalytic domain, catalytic mechanism, three dimensional structures, and sensitivity to inhibitors. Chitinases are ubiquitous enzymes and perform several functions in different species. They hydrolyze the chitin and contribute to the recycling and effective utilization of the most abundant marine waste for various applications [1]. The GH18 family chitinases contains (α/β) 8 TIM barrel structures composing of α-helices and β strands. The chitinases have two highly conserved motifs viz., SxGG and DxxDxDxE, corresponding to substrate binding and catalytic domains, respectively [2]. The GH18 family chitinases are predominantly characterized for degradation of chitins to produce industrially and pharmaceutically important, chito-oligosaccharides and N-acetyl D-glucosamine (NAG). Chitinases are produced by fungi during all phases of their growth and these enzymes also contribute to morphogenetic and pathogenic processes such as spore germination, hyphal growth and branching and mycoparasitism [3]. In this study, the chitinase gene was isolated from the thermophilic fungus, Thermomyces lanuginosus RM-B, cloned and overexpressed in S. cerevisiae and characterized further [4-5]. This thermophilic fungal chitinase is identified as GH18 family type. Most reliable method on the prediction utilized bioinformatics analysis, based on the sequence and three dimensional structure of the chitinase. Experimental data were used to validate the