Green and Efficient Preparation of Nano-chitin for Enzymatic Preparation of N-Acetylchitooligosaccharides

An eco-friendly pretreatment method, ball milling, was reported to efficiently prepare nano-chitin for the enzymatic production of N-acetylchitooligosaccharides (NCOS). In this process, α-chitin and β-chitin were milled into nano-chitin with an average degree of polymerization of 20–25 without any chemical additives. The milling process damaged the intersheet and intrasheet hydrogen bonding of chitin, resulting in a decrease in chitin crystallinity and average molecular weight and making the obtained nano-chitin dispersible in aqueous solution, confirmed by transmission electron microscopy (TEM), X-ray diffraction (XRD), gel permeation chromatography (GPC), and transmittance spectra. The ball milling pretreatment enhances the affinity between chitinase and milled chitin confirmed by the decrease of Km value from 12.66 to 3.75 mg mL–1. Moreover, using milled nano-chitin as the substrate, the catalytic efficiency of chitinase could achieve 4.49 mg mL–1 s–1, which was comparable to that employed colloidal chitin (4.84 mg mL–1 s–1), and the conversion rate of milled α-chitin was 80.5% of that of colloidal chitin. The reported ball milling pretreatment could produce nano-chitin substrate without the requirement of chemical reagents, showing the potential to replace acid treatment for the preparation of chitin substrate in NCOS enzymatic production.