Purification and characterization of cellulase produced by Anoxybacillus sp. UniMAP KB-01 from tropical mangrove soil

Abstract

Cellulase production from bacteria can be an advantage as the enzyme production rate is normally higher due to the higher bacterial growth rate as compared to fungi. This study involved purification and characterization of cellulase produced by Anoxybacillus sp. UniMAP KB-01, the newly isolated bacteria from tropical mangrove forest in peninsular Malaysia. Partial purification was done using ammonium sulphate precipitation method at 80% of saturation and the results show a purification fold of 3.9 and total enzyme activity of 0.1534 U/mL. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was conducted to determine the enzyme molecular weight showed two distinct band on 22.1 kDa and 117.9 kDa. The biochemical properties of the enzymes was determined by using carboxymethylcellulose (CMC) assay at 50°C and pH 4.8. The enzymes possessed highest activity of 0.1132 U/mL at the temperature of 50°C and was stable for 1 hour at the range of 40°C - 60°C. Besides, the enzymes showed maximum enzyme activity of 0.0677 U/mL at pH 6.0, and able to retained the enzyme activity up to 50% at a range of pH 5.0 - pH 8.0 for the duration of 2 hours. The enzyme activity and the stability test were performed by using 1 mM and 5 mM concentrations of 3 types of metal ions which were CaCl2, MgSO4 and FeSO4. Among the metal ions tested, the enzymes demonstrated a highest relative activity of 141.77% (0.0689 U/mL) when incubated at 5 mM of MgSO4 as compared to the control, 0.0486 U/mL. The presence of 5 mM of FeSO4 minimized the relative enzyme activity by 49.37%, giving an enzyme activity of 0.0246 U/mL. Similarly, the enzymes exerted the best stability at 5 mM of MgSO4 where the relative activity remain 60.63% (0.0474 U/mL) at the end of 3 hour incubation. Kinetic analysis was done by constructing a Lineweaver-Burk Plot where the Km and Vmax value obtained were 0.9669 mg/mL and 0.0133 mg/(mL·min) respectively.