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

Kheng Oon Low, Nor Muhammad Mahadi, Raha Abdul Rahim, Amir Rabu, Farah Diba Abu Bakar, Abdul Munir Abd. Murad, Rosli Md. Illias

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 (HlyAs61). 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 1×104 Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs61 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.

Original languageEnglish
Pages (from-to)453-459
Number of pages7
JournalJournal of Biotechnology
Volume150
Issue number4
DOIs
Publication statusPublished - Dec 2010

Fingerprint

Mutagenesis
Hemolysin Proteins
ATP-Binding Cassette Transporters
Cyclodextrins
Escherichia coli
Starch
Amino acids
Screening
Proteins
Amino Acids
Polymerase Chain Reaction
Mutation
cyclomaltodextrin glucanotransferase

Keywords

  • Alpha-hemolysin transport system
  • Cyclodextrin glucanotransferase
  • Directed evolution
  • Escherichia coli
  • Extracellular secretion

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

Enhanced secretory production of hemolysin-mediated cyclodextrin glucanotransferase in Escherichia coli by random mutagenesis of the ABC transporter system. / Low, Kheng Oon; Mahadi, Nor Muhammad; Abdul Rahim, Raha; Rabu, Amir; Abu Bakar, Farah Diba; Abd. Murad, Abdul Munir; Md. Illias, Rosli.

In: Journal of Biotechnology, Vol. 150, No. 4, 12.2010, p. 453-459.

Research output: Contribution to journalArticle

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AU - Abu Bakar, Farah Diba

AU - Abd. Murad, Abdul Munir

AU - Md. Illias, Rosli

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