Abstract
Cellulases are important in the hydrolysis of lignocellulosic materials and thereby contribute to biomass conversion into fuels and chemicals. A cellulase-producing bacterium was isolated from decayed plant leaf litter in soil of a botanical garden. Based on morphological, biochemical and 16S rRNA gene sequencing, it was identified as Enterobacter cloacae IP8, with gene bank accession number NR118568.1. The bacterial cellulase was purified in a three-step procedure using lyophilization, ion exchange chromatography (QAE Sephadex A-50) and gel filtration (Biogel P-100). Two isoforms of the enzyme were purified 1.21 and 1.23 folds, respectively, with yields of 30 and 29% for isoforms A and B, respectively. Apparent molecular weights of 36.61 ± 1.40 and 14.1 ± 0.10 kDa were obtained for isoforms A and B, respectively, using gel filtration chromatography. Kinetic parameters Km and Vmax were 0.13 ± 0.04 mg/ml and 3.84 ± 0.05 U/ml/min, respectively, for isoform A and 0.58 ± 0.06 mg/ml and 13.8 ± 0.10 U/ml/min, respectively, for isoform B. Optimum pH (7.0) and temperature (60 °C) of cellulase activity were determined for both isoforms A and B. Na+ and Ca2+ enhanced the activities of both isoforms. Mg2+ inhibited the enzyme activity at concentrations 4–15 mM but, while it stimulated the activity of isoform A at concentrations 15–200 mM, it inhibited that of isoform B at same concentration range. The strong inhibition of the enzyme by ethylenediaminetetraacetic acid (EDTA) confirmed the enzyme as a metalloenzyme. These results reveal the purified cellulase from E. cloacae IP8 as a thermostable, acidic to neutral metalloenzyme, suggesting that it has good potential for biotechnological applications.
Acknowledgements
Authors appreciate the Departments of Microbiology and Biochemistry, and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Nigeria, for provision of chemical reagents and instrumental facilities.
Disclosure statement
The authors report no declarations of interest.