Purification and identification of novel alkaline pectinase PNs31 from Bacillus subtilis CBS31 and its immobilization for bioindustrial applications

A robust alkaline pectinase PNs31 was produced from Bacillus subtilis CBS31 that was isolated from kimchi (a traditional Korean fermented food). The isolated strain CBS31 was identified as having 99.93% similarity to Bacillus subtilis subsp. The PNs31 was purified to homogeneity using two-step column purification, with an estimated 13.6% pectinase yield and 29.2-fold purity. The molecular mass (~35 kDa) and the N-terminal sequence residues of PNs31 established it as a novel pectinolytic enzyme. PNs31 was stable in the alkaline pH range (9-14) and up to 70 oC and had an optimum pH and temperature were 12.2 and 60 oC, respectively. PNs31 had definite Vmax and Km values of 220.43 mmol/min and 0.42mg/mL, respectively, when different concentrations (5 to 50mg/mL) of pectin were used as substrate. To develop the functional stability and reusability of the pectinase enzyme, PNs31 was immobilized in calcium alginate beads. The result was that the immobilized enzyme maintained approximately 83% and 65% relative activity in the second and third cycles of reusability experiments, respectively.