Biochemical properties and application of a novel pectinase from a mutant strain of Bacillus subtilis

Pectinases have broad applications in food industries attributed to their role as a biocatalyst in degrading pectic compounds. The present work reports the purification and characterization of a pectinase from a mutant strain of Bacillus subtilis . A three-step enzyme purification involving ammonium sulfate precipitation, followed by ion-exchange chromatography (IEC) and size-exclusion chromatography (SEC), was evaluated to recover the enzyme effectively. The pectinase was purified up to 7.98-fold with 65.63% total recovery, resulting in maximum specific activity of 362.04 U/mg after three purification steps. The eluted fractions of IEC and SEC showed pectinase activities of 9520 U and 8288 U, respectively. The molecular weight of this extracellular pectinase was found to be 30 kDa after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimal conditions of temperature and pH for maximum pectinase activity were 37 °C and 4–5, respectively. The enzyme also exhibited high stability when incubated at optimal conditions for 60 min. The addition of calcium and magnesium ions elicited pectinase activity at 4 mM concentrations, while copper and zinc ions inhibited the pectinase activity. The V max and K m values were 362 U and 0.62 mg/ml, respectively, for the pectin substrate obtained from citrus peel. Further analysis on the efficiency of clarification in sweet lime, pineapple, and lime juice showed 79.25, 72.76, and 61.24% reductions in turbidity within 1-h incubation. Therefore, characteristics of pectinase suggest that it can be potentially used for industrial applications as a biocatalyst.