Highly efficient production of N,N'-diacetylchitobiose according to substrate modification and changes in enzyme kinetics

Abstract

The purpose of this study is to improve the method for characterizing the kinetics of enzymes on chitin and to improve the inconvenience of colloidal chitin production and use. Therefore, we prepared a low molecular weight (MW) substrate by partially hydrolyzing and acetylating high molecular weight chitosan (HMWC) with chitosanase. The activity of chitinase was confirmed under optimal reaction conditions using acetylated low molecular weight chitin (AcCOS). In the hydrolysates of AcCOS, N, N′-diacetylchitobiose (GlcNAc)2 was identified as the main product under optimal reaction conditions using TLC. Residual N-acetylglucosamine (GlcNAc), which could not be sufficiently identified by the sensitivity of TLC, was confirmed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. However, from a quantitative point of view, the TLC results suggest that the content of (GlcNAc)2 is much higher than that of GlcNAc. These results are significant for use as a specific substrate capable of evaluating the reaction rate of a chitinase substrate. Taken together, our results provide novel results for the highly efficient production of (GlcNAc)2 and the evaluation of its biological activity by chemical modification of the substrate.