Biocatalysis of Heterogenously-Expressed Chitosanase for the Preparation of Desirable Chitosan Oligosaccharides Applied against Phytopathogenic Fungi

Chitosanase (EC3.2.1.132) can be used to selectively produce partially acetylated chitosan oligosaccharides (COSs), which are functional biopolymers that can widely be utilized as environmentally friendly agents, due to their bioactive, nontoxic, nonallergenic, and biodegradable properties. Different chitosanases can give rise to hydrolysates with diverse functions, making the development of novel chitosanases of great importance in applied biotechnologies. Here, we successfully expressed a novel chitosanase derived from Aspergillus fumigatus CJ22-326 in Pichia pastoris GS115, which could controllably produce chitosan oligosaccharides with desirable polymeric degrees. After glycosylation, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant enzyme Csn75 showed two specific protein bands with sizes of 25.5 and 23.5 kDa individually. The activity of the glycosylated Csn75 was 3.6-fold of that of the deglycosylated Csn75, indicating that glycosylation can greatly increase the activity. K(m )and V-max values of the purified Csn75 were 0.46 mg/naL and 6.03 mu mol.mL(-1).min(-1) separately, revealing a good affinity and high catalytic efficiency to the substrate. Moreover, biochemical tests confirmed that Csn75 had the highest activity from 55 to 65 degrees C when pH ranged from 5.0 to 6.0 and exhibited 4-fold activity in the presence of 1 mM Mn2+. Interestingly, the hydrolysis rate of the crude Csn75 (30 U/mL) can reach up to 90.65% in 4 h when using 2% chitosan as the substrate, producing a mixture of COS with desirable degrees of polymerization (DP), which significantly inhibited the mycelium growth of phytopathogenic fungi. This study proved Csn75 to be a promising chitosanase for the preparation of desirable chitosan oligosaccharides that are green biocides applicable for sustainable agricultural production and environmental protection.