Functional Analysis and Characterization of Chitinase Gene/s From Trichoderma Isolates

Abstract

Chitinases have been reported to be capable of hydrolyzing chitin by splitting their ß-1, 4-glucosidic bonds. The aim of this study was to isolate and characterize an endochitinase gene from isolate of Trichoderma spp. that was isolated from the western Uttar Pradesh soil samples. In total, twelve highly antagonistic Trichoderma isolates were screened for chitinolytic activity via dual plate method. Trichoderma isolate TBT6 was identified as a target for isolation of an endochitinase gene due to its high chitinolytic enzyme activity by observing the degradation of chitin substrates. The genomic DNA of Trichoderma isolate TBT6 was extracted amplified using a pair of specific primers designed for ech42 gene. The resulting amplicon (1.3 kb) from them were separately cloned into pJET1.2/blunt cloning vector and sequenced commercially. The cloned DNA sequence was 1,376 base pairs. The endochitinase gene was characterized using bioinformatics tools. It showed 99% homology with Hypocrea lixii YZ2203 endochitinase (ech42) gene. The putative endochitinase gene, then subjected to in silico analysis to obtain physico-chemical, evolutionary and structural information of this protein. The Chitinase protein sequence had 352 amino acids and showed 99% homology to Trichoderma harzianum endochitinase. The maximum parsimony analysis showed that ChitTBT6 protein was clustered into Group III with other fungi. The 3D model showed the β sheets are located within the barrel structure of the protein. Ultimately, the in-silico analysis of ChitTBT6 implicated the involvement of this gene/protein in chitin catabolism.