Directed evolution of Aspergillus niger glucoamylase to increase thermostability.
Bottom Line: Irreversible inactivation tests indicated that CR2-1 increases the free energy of thermoinactivation at 80°C by 10 kJ mol(-1) compared with that of wild-type GA.Thus, CR2-1 is more thermostable (by 5 kJ mol(-1) at 80°C) than the most thermostable A. niger GA variant previously described, THS8.In addition, Val59Ala and Glu408Lys were shown to individually increase the thermostability in GA variants by 1 and 2 kJ mol(-1), respectively, at 80°C.
Affiliation: Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA.Show MeSH
Mentions: Glucoamylase from A. niger has been characterized both biochemically and genetically (Aleshin et al., 1992; 1994; 1996). The enzyme has two domains and a linker: an N‐terminal catalytic domain of 440 amino acids with an (α,α)6‐barrel structure that has been structurally characterized by X‐ray crystallography (Fig. 1); a C‐terminal starch‐binding domain of 108 amino acids, necessary for hydrolysis of insoluble starch granules (but not necessary for hydrolysis of soluble substrates); and a heavily O‐glycosylated linking region of 68 amino acids. The GA gene from A. niger has been cloned and can be expressed conveniently in the yeast Saccharomyces cerevisiae (Cole et al., 1988). Numerous approaches have successfully been taken to improve the thermostability of A. niger GA by site‐directed mutagenesis (Ford, 1999; Reilly, 1999; Liu and Wang, 2003). Recently, Wang and colleagues (2006) have used directed evolution in conjunction with site‐directed mutagenesis to construct the most thermostable A. niger GA previously described (THS8). THS8 contains eight mutations (Asp20Cys, Ala27Cys, Ser30Pro, Thr62Ala, Ser119Pro, Gly137Ala, Thr290Ala and His391Tyr) with a cumulative increase in thermostability (ΔG) over wild‐type GA of 5 kJ mol−1 at 80°C without a reduction in catalytic activity.
Affiliation: Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, USA.