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Domain replacement to elucidate the role of B domain in cgtase thermostability and activity

Poh, Hong Goh and Md. Illias, Rosli and Goh, Kian Mau (2012) Domain replacement to elucidate the role of B domain in cgtase thermostability and activity. Process Biochemistry, 47 (12). pp. 2123-2130. ISSN 1359-5113

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Official URL: http://dx.doi.org/10.1016/j.procbio.2012.07.033


The B domain of CGTase has been generally accepted as a domain involved in thermostability. However, limited work has been performed in which entire B domain is substituted with the thermostable counterpart. Using overlap extension PCR, we replaced the B domain of a variant of CGTase Bacillus sp. G1 by six other B domains from thermostable CGTases. Likely due to distortion in the substrate-binding cleft adjacent to the active site, variants with the domain replacements from Thermoanaerobacter, Thermococcus, Thermococcus kodakarensis, Anaerobranca gottschalkii and Pyrococcus furiosus completely lost their catalytic function. A mutant designated Cgt_ET1 with a domain replacement from a Bacillus stearopthermophilus ET1 CGTase was the only variant that retained activity after domain exchange. Both the parental enzyme and the mutant Cgt_ET1 had an identical optimum temperature at 60 °C. The activity half-life was 22 min for the parental CGTase, whereas a marked increase to 57 min was observed for the mutant. Further mutagenesis on Cgt_ET1 was performed at residue 188 by replacing a Phe residue with Tyr. The mutant Cgt_ET1_F188Y displayed a decreased activity half-life of 28 min. Both mutants exhibited a better cyclodextrin-forming ability and a faster turnover rate (kcat) than the parental CGTase.

Item Type:Article
Uncontrolled Keywords:Mutagenesis, Thermostability
Subjects:Q Science
Divisions:Biosciences and Bioengineering
ID Code:33450
Deposited By: Fazli Masari
Deposited On:28 Aug 2013 04:07
Last Modified:30 Nov 2018 06:35

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