Utilization of Size Exclusion Chromatography for the Recovery of Microbial Pectinases

Size exclusion chromatography (SEC) is an effective analytical technique employed for the purification of biomolecules. In size exclusion chromatography (SEC), biomolecules are sorted according to their size. Objectives: To investigate the purification of pectinases from a microbiological source by size exclusion chromatography (SEC). To evaluate the amount of pectinase and total proteins in the collected fractions through measurement and qualitative analysis. Methods: Utilizing Sephadex G-25 as the stationary phase and a 0.05 M sodium phosphate buffer with increasing concentrations of NaCl as the mobile phase. Using a 3,5-dinitro-salicylic acid (DNS) assay and a pectin-containing agar plate assay, the existence of pectinases in the fractions that were taken was verified quantitatively and subjectively. Results: The increasing order of salt concentration was 0.15, 0.5, 0.8, 1 and 1.6 M NaCl concentration. At 0.15 and 0.5 M salt concentrations, desired proteins were strongly combined to the stationary phase of the Sequential Injection Chromatography (SIC) column and eluted at the last fraction while at 0.8, 1 and 1.6 M sodium chloride concentration pectinases were eluted in the early fractions as compared to the buffers containing a lower concentration of sodium chloride. Conclusions: It was concluded that the suitable NaCl concentration for the purification of pectinase enzyme through SEC was 0.8 M because at these concentrations pectinases can be separated very short time and at a low cost.