Abstract Chitinases (CHI) are a series of glycoside hydrolases (GHs) that break chitin into chitooligosaccharides ((GlcNAc)n, 10≥n≥2) or N-acetylglucosamine (GlcNAc). In this study, a chitinase gene derived from Bacillus thuringensis, BtCHI1, was cloned and heterologously expressed in Escherichia coli BL21(DE3)plysS. The enzymatic properties, kinetic parameters, and substrate hydrolytic patterns recombinant enzyme BtCHI1 were characterized. The results showed that the molecular mass of BtCHI1 was approximate 75 kD. Its optimum temperature and pH were 40 ℃ and 7.0, respectively. The enzyme was unstable after heat-challenge at 50 ℃ for 1 h, retaining only 20.11% residual activity. However, the enzyme was considerably stable in broad pH buffers ranging from 3.0 to 10.0, retained over 80% residual activities after incubation for 1 h, respectively. Substrate kinetics assays indicated the recombinant BtCHI1 was active against chitin, colloidal chitin and chitosan, with Vmax values of (4.62±0.46), (0.52±0.03) and (0.22±0.02) μmol/(min·mg), respectively. Nevertheless, the enzyme was inactive towards hydroxypropyl chitosan or glycol chitosan. Meanwhile, the enzyme was considerably resilient to 0.5~10 mmol/L Na+, Al3+ or EDTA (P>0.05). However, after treatment with 0.5~10 mmol/L Ca2+ or 5~10 mmol/L SDS for 1 h, the activities of BtCHI1 decline significantly (P<0.01). Thin-layer chromatography and high-performance liquid chromatography analysis revealed that BtCHI1 releases chitobiose from chitin and colloidal chitin, and further converted it into GlcNAc. Additionally, BtCHI1 (0.28 U) was observed to liberate (1.00±0.04) mg/mL and (1.71±0.11) mg/mL from natural shrimp and crab shell substrates. These results provide new insights into chitooligosaccharides/GlcNAc development and natural chitin substrate utilization.
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