A Continuous Fluorescent Assay for -Amylase Activity

The production and degradation of starch is key for the survival of plants during the day/night cycle. The stored energy is harnessed through the work of many different enzymes including the β‐Amylase (BAM) family of proteins. BAMs break down long chains of starch into the disaccharide maltose (4‐O‐α‐D‐Glucopyranosyl‐D‐glucose). The model organism Arabidopsis thaliana has 9 β‐Amylase proteins with apparently distinct functions, though not all have the ability to hydrolyze starch. Each BAM seems to have its own unique characteristics, although the specific functional differences are not clear. The objective of our research is to create an assay that can show the kinetics of BAM proteins, that is continuous and sensitive while able to work with synthetic and natural substrates of the BAM proteins. We aim to apply this assay to determine the unique biochemical features of the BAMs. To detect BAM activity, we used a maltose binding sensor composed of Maltose Binding Protein fused to Green Fluorescence Protein (MBP‐GFP). When maltose is bound to MBP it causes conformational changes to GFP resulting in fluorescence. We have tested the assay with maltodextrin chains from 4 to 20 residues in length and with soluble starch finding that we can detect maltose production from all of these substrates with the MBP‐GFP biosensor in the single micromolar range. Moreover, we can detect amylase activity in a continuous measurement format using a plate reader allowing for higher throughput sampling with reduced assay time. Support or Funding Information NSF‐RUI MCB‐1616467 to J.M.