Engineering Specificity and Stability in Glucoamylase from AspergiUus nip-
INTRODUCTION Glucoamylases (GAs) 1 (1 ,4-a-D-glucan glucohydrolase, EC 184.108.40.206) constitute glycoside hydrolase family 15 (Henrissat, 1991; Henrissat and Bairoch, 1993) and catalyze the release of jJ-o-glucose from nonreducing ends of starch and related oligo-and polysaccharides. Fungal GAs are applied in the commercial production of high glucose and fructose syrups (Saba and Zeikus, 1989), and the widely used AspergiUus nigtr GA is an obvious target for improvement of industrial properties through protein engineering. GA, at the same active site, cleaves the ~1,4linked with about 500 times higher specificity than ~1,6linked substrates (Hiromi et aL, 1966; Sierks and Svensson, 1994; Frandsen et aL, 1995; Fierobe et aL, 1996). One major goal is to raise the current yield of approximately 96% glucose in industrial saccharification by eliminating the GA catalyzed condensation reactions that lead to accummulation of ~1,61inked oligosaccharides (Nikolov et aL, 1989). Other goals include elevated thermostability or high activity in the neutral pH-range, which would advance application of immobilized GA and enable a single-step liquefaction, saccharification, and isomerization process (Bhosale et aL, 1996).