Abstract:To overcome the limitation of the insolubility of chitosan substrates to chitosanase and achieve the green preparation of chitooligo saccharides with a single degree of polymerization（DP），this study conducted steam explosion pretreatment on chitosan and performed enzymatic hydrolysis by using chitosanase Csn-But，which was efficiently expressed in Bacillus subtilis WB800.Scanning electron microscopy （SEM）results showed that the chitosan treated with the steam explosion at 2.4 MPa exhibited a multi-spiked fibrous arrangement and obvious tearing on the surface，with multiple cavities present in the separated and exposed fibers.The results of attenuated total reflectance Fourier transform infrared（ATR-FTIR）spectroscopy showed that the chemical bonds of chitosan were not destroyed by steam explosion treatment，but the hydrogen bonding network was disrupted.After using chitosanase Csn-But to hydrolyze the chitosan treated with the steam explosion（2.4 MPa）for 48 h，the content of chitooligosaccharides was 5.4 times higher than that of the control group without pretreatment，with a product concentration of 2.0 mg/mL.Furthermore，by regulating the substrate concentration，high-purity chitotriose was obtained from the substrate with a concentration of 6%.