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 degrees C, respectively. The catalytic=constants K-m and V-max were 0.20, 0.13, 0.14, and 0.10 mg/mL and 204, 154, 208, and 143 U/mL/min for FP-PG, FP-PL, CHI-PG, and CHI-PL, respectively. The negligible difference between the K-m and V-max 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. (C) 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.