Chitin, the second richest polymer in nature, is composed of the monomer N-acetylglucosamine (GlcNAc), which has numerous functions and is widely applied in the medical, food, and chemical industries. However, due to the highly crystalline configuration and low accessibility in water of the chitin resources, such as shrimp and crab shells, the chitin is difficult utilize, and the traditional chemical method causes serious environment pollution and a waste of resources. In the present study, three genes encoding chitinolytic enzymes, including the N-acetylglucosaminidase from Ostrinia furnacalis (OfHex1), endo-chitinase from Trichoderma viride (TvChi1), and multifunctional chitinase from Chitinolyticbacter meiyuanensis (CmChi1), were expressed in the Pichia pastoris system, and the positive transformants with multiple copies were isolated by the PTVA (post-transformational vector amplification) method, respectively. The three recombinants OfHex1, TvChi1, and CmChi1 were induced by methanol and purified by the chitin affinity adsorption method. The purified recombinants OfHex1 and TvChi1 were characterized, and they were further used together for degrading chitin from shrimp and crab shells to produce GlcNAc through liquid-assisted grinding (LAG) under a water-less condition. The substrate chitin concentration reached up to 300 g/L, and the highest yield of the product GlcNAc reached up to 61.3 g/L using the mechano-enzymatic method. A yield rate of up to 102.2 g GlcNAc per 1 g enzyme was obtained.
Keywords: N-acetylglucosamine; chitinase; heterologous expression; mechanoenzymology; shrimp and crab shell.