Purifying and Characterizing Cellulase from Bacteria Isolated from Gut of Oryctes rhinoceros

<b>Background and Objective:</b> Cellulose degradation is essential in biomass conversion, but limited access to efficient cellulase-producing microbes constrains industrial applications. Herbivorous insect guts are potential sources of novel cellulolytic bacteria. This study aimed to purify and characterize cellulase enzymes from bacteria isolated from the gut of <i>Oryctes rhinoceros</i>, to evaluate their potential for biotechnological use. <b>Materials and Methods:</b> Bacterial strains were isolated from the gut of <i>O. rhinoceros</i> and cultured in carboxymethyl cellulose (CMC) medium. Cellulase enzymes were purified using Sephadex G-100 gel filtration followed by Diethylaminoethyl (DEAE)-cellulose ion-exchange chromatography. Protein concentrations and enzyme activities were measured to calculate specific activities. The SDS-PAGE was used to determine the molecular weight. Enzymatic activity was assessed under different pH, temperature and metal ion conditions. <b>Results:</b> Two strains, <i>Bacillus tequilensis</i> B01L and <i>Bacillus cereus</i> B19L, were identified as potent cellulase producers. The cellulase from B01L was purified 11.46-fold (specific activity: 8.529 U/mg), while the enzyme from B19L showed a 12.28-fold increase (specific activity: 9.221 U/mg). Both enzymes had a molecular weight of approximately 52.23 kDa. Optimal activity occurred at pH 6.0, with temperature of 50°C for B01L and 60°C for B19L. Enzyme activity was unaffected by Ca<sup>2+</sup> and Mg<sup>2+</sup>, but inhibited by 35-40% in the presence of K<sup>+</sup> and Fe<sup>2+</sup>. <b>Conclusion:</b> The gut of <i>O. rhinoceros</i> harbors cellulolytic bacteria with significant enzymatic potential. These findings highlight a promising source of industrially relevant cellulases. Further studies are warranted to optimize production conditions and evaluate their application in biomass degradation.