Multi-analytical profiling of glycoside hydrolase 13 (GH13) α-amylases on potato starch

In this study, 20 α-amylases covering several glycoside hydrolase 13 (GH13) subfamilies were characterized with regard to their impact on the molecular properties of gelatinized potato starch. Using a multi-analytical approach, the enzyme-induced changes in the relative amylose content, molecular weight distribution, chain length distribution, and vibrational properties (FTIR) of the starch were assessed. The results showed that structurally and phylogenetically diverse α-amylases produced distinct starch degradation patterns, resulting in different molecular properties of the starch. Principal component analysis (PCA) of the starch molecular weight and chain length distribution data enabled functional classification of the enzymes into six distinct groups, revealing amylopectin-degrading ability as a central performance parameter. While subfamily classification alone turned out to be a less reliable predictor of the α-amylases' starch degradation patterns, protein structural features - particularly active site openness - were found to correlate well with enzymatic substrate preference and proposed mode-of-action. The study highlights the benefits of a multi-analytical framework for differentiating functional nuances of α-amylases beyond conventional classification schemes, potentially providing a basis for rational selection of these for targeted starch modification in the future.