Glyoxyl-agarose immobilized Bacillus subtilis chitosanase as a reusable biocatalyst for modulated production of chitooligosaccharides.

Abstract

Enzymatic production of low-molecular-weight chitosan and chitooligosaccharides (COS) with consistent properties is challenging since chitosanases are unstable under acidic, high-temperature conditions. To address this, a recombinant chitosanase was covalently attached to glyoxyl-activated agarose via multipoint attachment to improve stability and influence its hydrolysis pattern. The immobilized enzyme showed increased activity and stability at pH 4.5 and 55 °C, where the free enzyme quickly lost activity. Immobilization also reduced temperature sensitivity and enabled sustained catalysis up to 10 reuse cycles. Size-exclusion HPLC showed similar reductions in average molecular weight for both free and immobilized enzymes, while dynamic light scattering indicated that immobilization accelerated the development of a narrower, more stable particle size. MALDI-TOF spectra showed qualitative differences, with the immobilized enzyme displaying a more persistent, limited low-DP signal pattern mainly associated with DP2-DP4 oligomers. These results suggest that multipoint covalent immobilization not only stabilizes the enzyme but also influences the evolution of chitosan depolymerization products over time. Immobilizing the enzyme on glyoxyl-activated agarose enhanced its stability and produced a more consistent, distinct profile of hydrolysis products, supporting immobilization as a strategy to strengthen biocatalytic performance and tailor polymer breakdown without changing the enzyme's primary structure.