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Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability
Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
* Author to whom correspondence should be addressed.
Received: 9 January 2012; in revised form: 8 March 2012 / Accepted: 13 April 2012 / Published: 25 April 2012
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.
Keywords: CGTase; thermostable enzyme; site-directed mutagenesis; protein engineering; calcium binding site
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Goh, P.H.; Illias, R.M.; Goh, K.M. Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability. Int. J. Mol. Sci. 2012, 13, 5307-5323.
Goh PH, Illias RM, Goh KM. Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability. International Journal of Molecular Sciences. 2012; 13(5):5307-5323.
Goh, Poh Hong; Illias, Rosli Md.; Goh, Kian Mau. 2012. "Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability." Int. J. Mol. Sci. 13, no. 5: 5307-5323.