Cloning and characterization of a novel GH75 family chitosanase from Penicillium oxalicum M2

Abstract

In this study, we aimed to clone and further investigate the novel chitosanase PoCSN75A from Penicillium oxalicum M2 that we have previously identified and characterized. Sequence alignment and bioinformatics analysis revealed the conserved regions, enzyme classification, and other properties of PoCSN75A. Furthermore, the gene encoding PoCSN75A was heterologously expressed in Pichia pastoris GS115 and successfully purified via Ni-NTA chromatography. The purified recombinant PoCSN75A had an optimum pH and temperature of 5.5 and 60 °C, respectively. The enzyme was stable within a pH range of 3.0–6.0 and thermostable at 45 °C and below. Additionally, treatments with cations (Ca2+, Mn2+), non-ionic surfactants (Tween 20/40/60/80, Triton X-100), and common reducing agents (dithiothreitol [DTT], β-mercaptoethanol [β-ME]) were observed to significantly enhance the catalytic ability of the recombinant protein, which exhibited an obvious substrate preference for chitosan. For the enzyme kinetic parameters, the Vmax and Km values against colloidal chitosan were determined to be 4.36 U/mL and 0.27 mg/mL, respectively. Molecular docking (MD) for the overall model of enzymes and substrates identified several potentially important amino acid residues (D148, D150, T149 and E159). The recombinant protein was also found to exhibit a typical endohydrolytic pattern, with a chitooligosaccharide dimer and trimer as the final hydrolytic products.