Microassay for Rapid Screening of α-Amylase Activity

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Abstract
A microassay was developed for measuring the activity of α-amylases in the nanogram enzyme concentration range, based on the use of dye-labeled cross-linked starch as the substrate, and the release of soluble colored fragments formed in enzyme hydrolysis. Reaction conditions were optimized to generate a linear correlation between the increase in absorbance and a reaction time of 0−10 min, as well as enzyme concentrations in the range of 0−50 ng. A standard curve for the conversion of absorbance to enzyme activity units was constructed. The protocol developed was applied to monitoring the production of ultralow concentrations of recombinant barley α-amylase in yeast cells. Keywords: α-Amylase; barley α-amylase; microassay