title: Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability.
abstract: Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
authors: Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors: Poh Hong
authors: PH
authors: Goh
authors: Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors: Rosli Md
authors: RM
authors: Illias
authors: Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors: Kian Mau
authors: KM
authors: Goh
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D002118:Calcium