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/mL
and 6.03 μmol·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 °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.
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