Cloning and characterization of a novel GH75 family chitosanase from Penicillium oxalicum M2. (September 2022)

• Record Type: Journal Article
• Authors: Cao, Shining; Gao, Pei; Xia, Wenshui; Liu, Shaoquan; Liu, Xiaoli
• Is Part Of: Process biochemistry. Volume 120(2022)
• Page Start: 41
• Page End: 52
• Publication Date: 2022-09
• Subjects: Chitooligosaccharides -- GH75 family -- Chitosanase -- Heterologous expression -- Molecular docking; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2022.05.023
• Languages: English
• ISSNs: 1359-5113

Abstract

Abstract: In this study, we aimed to clone and further investigate the novel chitosanase Po CSN75A 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 Po CSN75A. Furthermore, the gene encoding Po CSN75A was heterologously expressed in Pichia pastoris GS115 and successfully purified via Ni-NTA chromatography. The purified recombinant Po CSN75A 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 (Ca 2+, Mn 2+ ), 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 V max and K m 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. Graphical Abstract: ga1 Highlights: ● A novel chitosanase gene from Penicillium oxalicum M2 was cloned. ● Po CSN75A expression in P. pastoris was 4.62 folds that of the original strain ● MD expanded the understanding of the catalytic mechanism of Po CSN75A.