Purification and characterization of pectinase from gut-associated Klebsiella oxytoca af-G4 of dwarf honey bee, Apis florea

Abstract

ObjectivesPectinases are enzymes that hydrolyze pectin or pectic compounds. Microorganisms in the gut of bees are involved in the breakdown of these macromolecules. In this study, we aimed to optimize the physicochemical parameters for enhancing enzyme pectinase production by bacteria Klebsiella oxytoca af-G4 isolated from Apis florea gut; in addition, purification and characterization of purified enzyme were carried out. MethodsOptimization of growth conditions for pectinase production using different synthetic and agro-industrial wastes by Klebsiella oxytoca af-G4 were performed. Purification and characterization of the pectinase were carried out. Application of the pectinase in juice extraction was evaluated. ResultsPectinase activity was maximum at 30 °C at pH 5.0, in media supplemented with lactose (1 % w/v) and ammonium sulfate (1 % w/v) as carbon and nitrogen sources, respectively, with a growth period of 72 h. The pectinase production with agro-industrial wastes showed higher production with pomegranate peel (1 % w/v) as substrate at a growth condition of 40 °C, pH 6.0, and a growth period of 92 h. The enzyme was purified to homogeneity (4.8-fold) using DEAE cellulose ion-exchange chromatography. The purified enzyme showed the molecular weight of ∼ 60 kDa on SDS-PAGE with an activity of 23.26 U/ml and specific activity of 14.36 U/mg. The pectinolytic activity was confirmed on the zymogram. The purified enzyme was more active at an optimum pH of 6.0, and stable at a pH range between 5 and 8. The enzyme was stable at temperatures ranging from 30 °C to 80 °C, with 60 °C being the optimum. The enzyme showed Km and Vmax values of 6.04 mg/ml and 4.92 U/ml, respectively. The enzyme showed maximum activity with the addition of metal ion Mn2+ at pH 6 and 60 °C. The enzyme enhanced the extraction of apple juice and clarification significantly. ConclusionThe enzyme characteristics and kinetics as well as the higher production with agro-waste support its pectin-based industrial application. The enzyme properties prove that it is a good candidate for the hydrolysis of pectin and juice clarification for industrial applications.