Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

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Abstract
Aspergillus awamori glucoamylase (GA) contains globular catalytic and starch-binding domains (residues 1–471 and 509–616, respectively). A heavily O-glycosylated sequence comprises two parts. The first (residues 441–471) in the crystal structure wraps around an α/α-barrel formed by residues 1–440. The second (residues 472–508) is an extended, semi-rigid linker between the two domains. To investigate the functional role of this linker, we made internal deletions to remove residues 466–512 (GAΔ1), 485–512 (GAΔ2) and 466–483 (GAΔ3). GAΔ2 and GAΔ3 were expressed in Saccharomyces cerevisiae culture supernatants at ∼ 60 and 20% the wild-type level, respectively, while GAΔ1 was almost undetectable. Western blots comparing extracellular and intracellular fractions indicated that the region deleted in GAΔ3 was critical for secretion, while the region deleted in GAΔ2 contributed to the production of a stable enzyme structure. The activities of purified GAΔ2 and GAΔ3 on soluble and insoluble starch were similar to those of wild-type GA, indicating that for soluble starch their deletions did not affect the catalytic domain and for insoluble starch the linker does not coordinate the activities of the catalytic and starch-binding domains. The deletions had a significant negative effect on GAΔ2 and GAΔ3 thermos tabilities.

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