Comparative Potential of Chitinase and Chitosanase from the Strain Bacillus thuringiensis B-387 for the Production of Antifungal Chitosan Oligomers.

Abstract

The depolymerization of chitosan using chitinolytic enzymes is one of the most promising approaches for the production of bioactive soluble chitooligosaccharides (COS) due to its high specificity, environmental safety, mild reaction conditions, and potential for development. However, the comparative efficacy of bacterial chitinases and chitosanases in terms of yield, solubility, and antimicrobial activity of produced COS remains understudied. In this work, chitinase (73 kDa) and chitosanase (40 kDa) from the strain Bacillus thuringiensis B-387 (Bt-387) were purified using various chromatographic techniques and compared by their action on chitosan (DD 85%). The molecular mass and structure of generated COS was determined using TLC, LC-ESI-MS, HP-SEC, and C¹³-NMR techniques. Chitosanase converted the polymer more rapidly to short COS (GlcN₂-GlcN₄), than chitinase, and was more specific in its action on mixed bonds between GlcN and GlcNAc. Chitosanase needed a noticeably shorter incubation time and enzyme-substrate ratio than chitinase for production of larger oligomeric molecules (Mw 2.4-66.5 and 15.4-77.7 kDa, respectively) during controlled depolymerization of chitosan. Moreover, chitosanase-generated oligomers demonstrate better solubility and a higher antifungal activity in vitro against the tested plant pathogenic fungi. These features, as well as the high enzyme production and its simplified purification protocol, make chitosanase B-387 more suitable for the production of antifungal chitooligomers than chitinase.