Abstract
Glucoamylases (GAs) hydrolyse both a-1,4 and a-1,6 glucosidic bonds from the non-reducing end of polysaccharides and releases b-D-glucose with inversion of the anomeric configuration. Fungal GA is widely used in the manufacture of glucose and fructose syrups, however, some fungi are toxic to humans and can not be safely used in the food and wine industries. For the application purpose, we express recombinant GA in yeast, Saccharomyces cerevisiae. We constructed a series of truncated mutants in the starch binding domain and linker region of GA from Rhizopus oryzae and expressed in different hosts performing different degrees of glycosylation, then we assayed their functional activity. We found that when GA is expressed in the yeast that performing hyperglycosylation, its stability increased with slightly decreased in its enzyme activity. It suggested that glycosylation was heavily related to the enzyme’s stability, but unnatural hyperglycosylation in the heteroorganisms may slightly hold back the catalyzed reaction to proceed. We also found that glycosylation increased rGAs’ pH stability at the low pH range but decreased rGAs’ stability at the high (neutral) pH range. The linker was also indicated to be related to the structural stability, full length linker region stabilized the secondary structures of the catalytic domain of GA.