Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase |
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Authors: | Libby Carol Baker; Cornett Catherine AG; Reilly Peter J; Ford Clark |
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Affiliation: | Department of Chemistry, Colby College Waterville, ME 04901, USA
1Department of Zoology and Genetics, Iowa State University Ames, IA 50011, USA
2Departments of Chemical Engineering, Iowa State University Ames, IA 50011, USA
3Departments of Food Science and Human Nutrition, Iowa State University Ames, IA 50011, USA |
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Abstract: | Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1471 and 509616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441471) in the crystal structurewraps around an / -barrel formed by residues 1440. Thesecond (residues 472508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466512 (GA 1), 485512 (GA 2) and 466483 (GA 3).GA 2 and GA 3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA 1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA 3 was critical for secretion, while the region deletedin GA 2 contributed to the production of a stable enzyme structure.The activities of purified GA 2 and GA 3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA 2 and GA 3thermos tabilities. |
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Keywords: | deletions/ glucoamylase/ linker/ O-glycosylation/ secretion/ thermostability |
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