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Enhanced secretory production of hemolysin-mediated cyclodextrin-glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system.


Citation

Kheng, Oon Low and Mahadi, Nor Muhammad and Abdul Rahim, Raha and Rabu, Amir and Abu Bakar, Farah Diba and Abdul Murad, Abdul Munir and Md. Illias, Rosli (2010) Enhanced secretory production of hemolysin-mediated cyclodextrin-glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system. Journal of Biotechnology, 150 (4). pp. 453-459. ISSN 0168-1656

Abstract

The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.


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Additional Metadata

Item Type: Article
Divisions: Faculty of Biotechnology and Biomolecular Sciences
DOI Number: https://doi.org/10.1016/j.jbiotec.2010.10.001
Publisher: Elsevier
Keywords: Alpha-hemolysin transport system; Cyclodextrin glucanotransferase; Directed evolution; Extracellular secretion; Escherichia coli.
Depositing User: Nida Hidayati Ghazali
Date Deposited: 14 May 2014 03:38
Last Modified: 22 Sep 2015 06:07
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.1016/j.jbiotec.2010.10.001
URI: http://psasir.upm.edu.my/id/eprint/14512
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