Industrially produced pullulanases with thermostability: Discovery, engineering, and heterologous expression.

Pullulanases (EC 3.2.1.41) are well-known starch-debranching enzymes widely used to hydrolyze α-1,6-glucosidic linkages in starch, pullulan, amylopectin, and other oligosaccharides, with application potentials in food, brewing, and pharmaceutical industries. Although extensive studies are done to discover and express pullulanases, only few are available with desirable characteristics for industrial applications. This raises the challenge to mine new enzyme sources, engineer proteins based on sequence/structure, and regulate expressions. We review here the identification of extremophilic and mesophilic microbes as sources of industrial pullulanases with desirable characteristics, including acid-resistance, thermostability, and psychrotrophism. We present current advances in site-directed mutagenesis and sequence/structure-guided protein engineering of pullulanases. In addition, we discuss heterologous expression of pullulanases in prokaryotic and eukaryotic microbial systems, and address the effectiveness of the expression elements and their regulation of enzyme production. Finally, we indicate future research needs to develop desired industrial pullulanases.