Rational engineering of a cold-adapted α-amylase from the Antarctic ciliate Euplotes focardii for simultaneous improvement of thermostability and catalytic activity

The α-amylases are endo-acting enzyme which hydrolyze starch by randomly cleaving the 1,4-α-D-glucosidic linkages between the adjacent glucose units in linear amylose chain. It has significant advantages in a wide range of applications, in particular in food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, differently from most of the α-amylases characterized so far. Furthermore, EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants: in one mutant we introduced Pro residues on the A and B domains in surface loops. In the second mutant we changed Val into Thr residues close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered the novel EfAmy with improved thermostability, substrate binding affinity and catalytic efficiency to various extents, without impact on its pH preference. These characteristics can be considered an important property to be used in food, detergents, textiles and other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.