Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

Young-Wan Kim; Doo-Kyoung Kim; Myo-Jeong Kim; Hyunju Cha; Cheon-Seok Park; Tae-Wha Moon; Kwan-Hwa Park

doi:10.5458/jag.52.7

Regular Papers

Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

Young-Wan Kim, Doo-Kyoung Kim, Myo-Jeong Kim, Hyunju Cha, Cheon-Seok Park, Tae-Wha Moon, Kwan-Hwa Park

著者情報

Young-Wan Kim
Center for Agricultural Biomaterials, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University
Doo-Kyoung Kim
Center for Agricultural Biomaterials, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University
Myo-Jeong Kim
Department of Food Science and Biotechnology, University of Inje
Hyunju Cha
Center for Agricultural Biomaterials, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University
Cheon-Seok Park
Department of Food Science and Technology, Kyunghee University
Tae-Wha Moon
Center for Agricultural Biomaterials, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University
Kwan-Hwa Park
Center for Agricultural Biomaterials, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University

キーワード: calcium binding site, cyclodextrin-degrading enzymes, mutagenesis, thermostability, Thermus maltogenic amylase

ジャーナルフリー

2005 年 52 巻 1 号 p. 7-13

DOI https://doi.org/10.5458/jag.52.7

詳細

抄録

Thermus maltogenic amylase (ThMA), one of the cyclodextrin (CD)-degrading enzymes, is expected to have a calcium-binding site (Ca2 site) based on multiple sequence alignments. In spite of the presumption of the Ca2 site in ThMA, however, its thermostability is independent of calcium ions. In order to investigate the effect of calcium ions on thermostability, two mutations (Ile152Asn and Ser153Asn) were introduced into Ca2 sites of a thermostabilized ThMA mutant (ThMA-DM2) using site-directed mutagenesis. The resultant mutant (ThMA-DM2-Ca) showed highly improved thermostability in the presence of calcium ions. The relative hydrolysis activity of ThMA-DM2-Ca also increased in comparison with that of ThMA-DM2 whereas transglycosylation activity was not affected by substitutions. The result confirmed that the residues located in the Ca2 site (Ile152 and Ser153) played critical role in stabilizing CD-degrading enzymes.

責任著者(Corresponding author)

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