Improved catalytic functionalities of purified pristine and chitosan-immobilized polygalacturonase, and pectin lyase

Abstract

In this study, novel fractions of polygalacturonase (PG), and pectin lyase (PL) were extracted from Neurospora crassa-fermented citrus peel waste. A central composite design from response surface methodology was used to study the in-depth interactions between various fermentation parameters. The active PG and PL fractions were purified to homogeneity and surface immobilized using various concentrations of chitosan (CHI) and glutaraldehyde as a cross-linking agent. A significant increase in the specific activities of PG and PL with 12.3-fold and 6.52-fold purification after Sephadex G-100 column chromatography was achieved. Evidently, the successful immobilization was confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). The catalytic characterization of free parts, i.e., FP–PG and FP–PL and CHI-immobilized parts, i.e., CHI–PG and CHI–PL was investigated using various parameters including pH, thermal behavior, Michaelis–Menten kinetic constants, inhibitors/stimulators, i.e., divalent cations and chelating agents including Ethylenediaminetetraacetic acid and Sodium dodecyl sulfate. The immobilized fractions were highly stable over a broader pH and temperature range from 4-9 and 45–85°C, respectively. The catalytic=constants Km and Vmax were 0.20, 0.13, 0.14, and 0.10mg/mL and 204, 154, 208, and 143U/mL/min for FP–PG, FP–PL, CHI–PG, and CHI–PL, respectively. The negligible difference between the Km and Vmax values of free and immobilized fractions revealed that the conformational flexibility was retained as such. The CHI–PG and CHI–PL fractions retained more than 75% of their operational activities even after seven consecutive cycles.