One-step immobilization of chitosanase on microcrystalline cellulose using a carbohydrate binding module family 2

Enzyme immobilization technology holds significant value in biocatalysis. Carbohydrate-binding modules (CBMs), with their specific binding to natural polysaccharides, offer a highly promising immobilization method. In the present study, the binding ability with their natural substrates and heterologous expression levels of four CBMs using fluorescent protein tagging were studied, revealing that CBM2r presented the highest immobilization efficiency and expression level. Using the Design of Experiments (DOE), the immobilization conditions for mCherry-CBM2r were optimized, achieving a protein loading of 2.45 wt% on Avicel under optimal conditions: a solid-liquid ratio of 1:30, NaCl concentration of 108 mM, protein concentration of 6 mg/mL, and incubation time of 120 min. Subsequently, CBM2r gene was fused with chitosanase gene from Bacillus subtilis (BsCsn) and expressed in Escherichia coli for establishing a novel one-step immobilization of fusion enzymes mediated by CBM2r on microcrystalline cellulose. The immobilized CBM2r-BsCsn-Avicel was used for batch hydrolysis of high-concentration chitosan to produce chito-oligosaccharides, with the enzyme retaining 96 % substrate degradation efficiency over seven cycles and achieving a space-time yield of 232.8 kg/m 3 /h. This study provides a simple, cost-effective, environment friendly, and competitive biocatalytic immobilization strategy.