An alkalophilic and thermostable polygalacturonase (PGase) from Pseudomonas sp. 13159349: purification, biochemical characterization and its efficacy in olive oil extraction

The present study deliberates on the biochemical characterization of a highly alkalophilic and thermostable polygalacturonase (PGase) from Pseudomonas sp. 13,159,349. Further, its efficacy in olive-oil extraction was also assessed. The PGase enzyme was produced using wheat bran solid substrate fermentation (SSF) media that was statistically optimised using Placket-Burman and RSM. The extracellularly secreted PGase was purified by gel permeation chromatography with a fold purification of 6.42 and a specific activity of 192.97 U/mg. 12% SDS-PAGE and LC-MS spectra confirmed that the enzyme has a molecular weight of 44.5 kDa. PGase exhibited optimum activity at pH 9 and 45 − 50 °C and the enzyme retained > 50% stability at alkaline pH 9, 10, as well as 45 and 50 °C even after 8 h. The enzyme’s kinetic parameters (Km and Vmax) with pectin substrate were found to be 0.0663 mg/ml and 209.60 mol. min-1, respectively. Metal ions Co 2+ and Mn 2+ enhanced enzyme activity while Ag 2+ , Hg 2+ , and ethylenediaminetetraacetic acid (EDTA) reduced it. Activity could be restored by removing EDTA by dialysis, demonstrating PGase as a metalloenzyme. In addition, the use of non-ionic surfactants displayed considerable stability, while Triton-X 100 increased the activity significantly, indicating a greater interaction between enzyme and substrate. Furthermore, treatment of olive paste with 300 U and 600 U of PGase remarkedly ( P