Development and Evaluation of Rapid and Simple Assay Method for α-Amylase Activity in Rice Koji

A chromogenic substrate assay using 2-chloro-4-nitrophenyl-6⁵-azido-6⁵-deoxy-β-maltopentaoside (G5 method) is one of the official analytical methods for α-amylase activity in rice koji for sake-brewing. Although it is useful because of its rapidity, simplicity, and accuracy, there are concerns about unavailability of the substrate in near future. In this study, an alternative assay using 4,6-O-ethylidene-α-4-nitrophenyl-maltoheptaoside (G7 method) was developed as a method that can be measured using the same procedure as the G5 method. α-Amylase in the sample and the coupled enzymes, glucoamylase and α-glucosidase, hydrolyzes the substrate, and the resulting chromophore, measured at 400 nm, was directly proportional to the α-amylase activity. As the G7 method exhibited good precision, repeatability, and linearity with the G5 method for 41 kinds of rice koji (y = 1.096x, R² = 0.996), it is useful for the determination of α-amylase activity in rice koji. When switching the G5 method to the G7 method, it should be noted that the G7 method tends to exhibit ca. 10% higher values than the G5 method as an average. Moreover, the ratios of the measurement values between the G7 and G5 methods can vary from 1.02 to 1.38 depending on the rice koji samples. Evaluation of K_m values suggested that the catalytic properties of α-amylase for the G7 and G5 methods can vary among rice koji samples. Therefore, if the G7 method is used instead of the G5 method, the comparison of assay results of the two methods for the target koji sample is preferable to ensure data compatibility. PRACTICAL APPLICATION: The previous chromogenic substrate assay, which is one of the official analytical methods for α-amylase activity in rice koji for sake-brewing, may become unavailable in near future. The alternative method was developed while maintaining simplicity and rapidity. It should be noted that the new method tends to exhibit ca. 10% higher values than the previous chromogenic substrate method. Checking the compatibility of two methods for each target koji seems to be desirable if the new method is used as the substitute for the previous method.