Chitosanase-coated silica-gels were prepared via cross-linking of the chitosanase onto silica-gels for the efficient production of multisize chitooligosaccharides (MCOs) in a continuous process. The kinetic aspects of immobilized chitosanase (IMMCTase) were investigated based on the reaction time, production of MCOs, and MALDI-TOF mass analyses to achieve maximum bioconversion of high molecular weight chitosan (HMWC) to MCOs. IMMCTase revealed a negligible loss of chitosanase activity after multi uses in continuous digestion of HMWC. The optimal temperature of IMMCTase was 37 degrees C, and kinetic parameters toward HMWC were determined to be K-m = 1.45 mM and V-max = 360 mu mole/mu g/min, respectively. Under optimal conditions, the recovery of enzyme activity of IMMCTase was determined to be 82.3%, thus indicating that it can still be reused few more times. In conclusion, use of IMMCTase resulted in rapid and efficient digestions of HMWC with consistent results to produce MCOs. (C) 2014 Elsevier Ltd. All rights reserved.