Immobilization of XynB from the hyperthermophilic Thermotoga maritima on nickel-chelate IDA (iminodiacetic acid disodium salt monohydrate)-Eupergit C 250L (Ni-E, nickel-chelate Eupergit C 250L) resulted in high immobilization yield (76.3%), catalytic efficiency (77.9%) and bond protein (96.6%). Thermostability of Ni-E XynB was significantly improved by 1 mol l(-1) phosphate buffer (pH 7.0) treatment. Chernical surface modifications were followed by Fr-NIR (Fourier transform near infrared) spectroscopy, strongly indicated that a chemical reaction between the amines or some other groups and epoxy groups took place in Eupergit C 250L during immobilization. Characterization of the immobilized XynB was further evaluated. The optimum pH was not affected by immobilization, but the optimum pH range of the immobilized XynB was pH 4.6-6.6 while that of the free XynB was pH 5.6-7.5. The free XynB had an optimum temperature of 90 degrees C, whereas that of the immobilized XynB was shifted to 100 degrees C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Autohydrolysis liquor of corncob (ALC) was used as the substrate for enzymatic hydrolysis by the immobilized XynB in a packed-bed enzyme reactor. The major hydrolysis product is xylobiose. The immobilization procedure developed provides a promising solution for application of xylanases in continuous hydrolysis of ALC. (c) 2007 Elsevier Inc. All rights reserved.