Visualization of polypeptides including fragmented α-amylase in rice koji grains using mass spectrometry imaging

Abstract

The quality of rice koji greatly affects the quality of sake. To accurately evaluate the quality of rice koji, various approaches for the evaluation of rice koji are required. In this study, we directly and simultaneously visualized the distribution of polypeptides in rice koji using mass spectrometry imaging. We demonstrated four koji-specific polypeptides at m/z 4660, 6140, 8170, and 11,840 and one rice-derived polypeptide at m/z 5330. To identify the koji-specific polypeptides, extracts from rice koji were separated using tricine SDS-PAGE, and the band appeared to coincide with the polypeptide at m/z 11,840 was identified to be the N-terminal fragment of α-amylase. The polypeptide seemed to have no hydrolytic activity based on the primary structure of α-amylase. The polypeptide at m/z 11,840 seemed to coincide with the fragmented α-amylase was detected at the later stage of koji making (after 42h). At the same period during koji making, the increasing rate of α-amylase activity decreased compared to that of glucoamylase activity, suggesting that α-amylase fragmentation possibly leads to the deceleration of the increase in α-amylase activity at the later stage of koji making. This is the first study to directly and simultaneously demonstrate the distribution of polypeptides in rice koji using mass spectrometry imaging and imply the relationship between α-amylase fragmentation and activity in rice koji.