Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

• Published in: Applied and Environmental Microbiology
• Main Authors: Yang, Guang, Yao, Hua, Mozzicafreddo, Matteo, Ballarini, Patrizia, Pucciarelli, Sandra, Miceli, Cristina
• Other Authors: Kelly, Robert M.
• Format: Article in Journal/Newspaper
• Language: English
• Published: American Society for Microbiology 2017
• Subjects: Ecology; Applied Microbiology and Biotechnology; Food Science; Biotechnology; Antarctic; The Antarctic; Antarc*
• Online Access: http://dx.doi.org/10.1128/aem.00449-17; https://journals.asm.org/doi/pdf/10.1128/AEM.00449-17

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α- d -glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii ( Ef Amy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, Ef Amy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a 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 Ef Amy with improved thermostability and catalytic efficiency at low temperatures. We engineered two Ef Amy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr 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 Ef Amy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the 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 ...