Simultaneous measurement of alpha-amylase and glucoamylase activities in sake rice koji by capillary electrophoresis of sodium dodecyl sulfate-protein complexes and activity measurement of glucoamylase by in-capillary enzyme reaction method.

Abstract

Capillary electrophoresis (CE) of sodium dodecyl sulfate (SDS)-protein complexes using a nongel sieving matrix (CE-SDS) has been applied to the simultaneous analysis of alpha-amylase and glucoamylase activity in sake rice koji which is employed for the brewing of sake. Alpha-amylase and glucoamylase in sake rice koji extracts were successfully analyzed by CE-SDS. Alpha-amylase and glucoamylase were found to have molecular masses of 53000 and 63000 Da, respectively, as determined by the migration times of eight standard proteins. These values agree with those determined by SDS-polyacrylamide gel electrophoresis (PAGE). The results of CE-SDS method were compared with those achieved by the official method. The relative standard deviations (RSD) of the alpha-amylase and glucoamylase activities by CE-SDS were less than 5.0% in both intra-day and inter-day experiments. An electrophoretic analysis of products of an enzyme reaction of a substrate by in-capillary reaction was also useful for the activity measurement of glucoamylase in sake rice koji. p-Nitrophenyl-beta-D-maltoside (PNP-Mal) was employed as a substrate and p-nitrophenyl-beta-D-glucopyranoside (PNP-Glu) was the product of the enzyme reaction. The glucoamylase activity of sake rice koji samples gave the good linear relationship with the peak area observed in the in-capillary enzyme reaction method. The glucoamylase activity in sake rice koji was measured by either CE-SDS or the in-capillary enzyme reaction more easily than by the official method. Both methods can be applied to the routine quality control of alpha-amylase and glucoamylase activities in sake rice koji.