Characterization of the gene encoding chitinase enzyme from bacillus isolates insulated from some locations in Southeast Sulawesi

Abstract

This study aimed to determine the isolates of local Bacillus that have potential chitinolytic activity and to know the characteristic of the gene encoding chitinase enzyme from local Bacillus isolates from Southeast Sulawesi that were selected to have chitinolytic activity. Selection of chitinolytic bacteria based on bacterial ability to form clear zone on chitin agar medium which was grew by spread method and incubated for 4 days. From 5 test isolates used, one isolate which had chitinolytic activity was isolate Bacillus sp. Rh 3.8. The amplification of the gene encoding chitinase enzyme selected bacterial isolates was done by PCR (Polymerase Chain Reaction) technique using Chitbac F and Chitbac R primers. Sequence analysis was conducted by BLASTn, mapping of restriction enzyme using Bioedit software, analysis of amino acid using expasy software, analysis of hydrophobicity using Bioedit software, phylogenetic tree construction using MEGA software. The results showed that the characters of the gene encoding chitinase enzyme was a gene measuring 804 bp. Based on BLASTn analysis, the gene has 100% similarity with Bacillus thuringiensis SCG0402 (CP017577). The gene has 9 restriction enzyme cutting sites. Based on hydrophobicity analysis shows that the amino acid sequence of chitinase enzyme is dominant exist on hydrophilic region. The results of phylogenetic tree construction show isolates of the Bacillus sp. Rh 3.8 is a group with Bacillus thuringiensis so this strain is a species of Bacillus thuringiensis.