Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli

  • Rui Min Ong
  • Kian Mau Goh
  • Nor Muhammad Mahadi
  • Osman Hassan
  • Raja Noor Zaliha Raja Abdul Rahman
  • Rosli Md Illias
Original Paper

Abstract

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD.

Keywords

Bacillus sp. G1 Cyclodextrin Cyclodextrin glucanotransferase Extracellular expression Predominant β-CGTase Signal peptide 

Abbreviations

CGTase

Cyclodextrin glucanotransferase

CD

Cyclodextrin

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Copyright information

© Society for Industrial Microbiology 2008

Authors and Affiliations

  • Rui Min Ong
    • 1
  • Kian Mau Goh
    • 1
    • 2
  • Nor Muhammad Mahadi
    • 3
  • Osman Hassan
    • 4
  • Raja Noor Zaliha Raja Abdul Rahman
    • 5
  • Rosli Md Illias
    • 1
  1. 1.Department of Bioprocess Engineering, Faculty of Chemical and Natural Resources EngineeringUniversiti Teknologi MalaysiaUTM Skudai, JohorMalaysia
  2. 2.Faculty of Bioscience and BioengineeringUniversiti Teknologi MalaysiaJohorMalaysia
  3. 3.Malaysia Genome Institute, UKM-MTDC Smart Technology CenterBangi, SelangorMalaysia
  4. 4.School of Chemical Science and Food Technology, Faculty of Science and TechnologyUniversiti Kebangsaan MalaysiaSelangorMalaysia
  5. 5.Enzyme and Microbial Technology Research Group, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSelangorMalaysia

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