Original Paper

Journal of Industrial Microbiology & Biotechnology

, Volume 35, Issue 12, pp 1705-1714

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

  • Rui Min OngAffiliated withDepartment of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia
  • , Kian Mau GohAffiliated withDepartment of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi MalaysiaFaculty of Bioscience and Bioengineering, Universiti Teknologi Malaysia
  • , Nor Muhammad MahadiAffiliated withMalaysia Genome Institute, UKM-MTDC Smart Technology Center
  • , Osman HassanAffiliated withSchool of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia
  • , Raja Noor Zaliha Raja Abdul RahmanAffiliated withEnzyme and Microbial Technology Research Group, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia
  • , Rosli Md IlliasAffiliated withDepartment of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia Email author 

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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