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

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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Abbreviations

CGTase:

Cyclodextrin glucanotransferase

CD:

Cyclodextrin

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Acknowledgments

This work was financially supported by the Malaysia Ministry of Science, Technology and Innovation (MOSTI) under project number 09-02-05-006 BTK/ER/34. The authors also would like to thank Dr. Madihah Salleh for making the HPLC available for product analysis.

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Authors and Affiliations

  1. Department of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia, 81310, UTM Skudai, Johor, Malaysia

    Rui Min Ong, Kian Mau Goh & Rosli Md Illias

  2. Faculty of Bioscience and Bioengineering, Universiti Teknologi Malaysia, 81310, Johor, Malaysia

    Kian Mau Goh

  3. Malaysia Genome Institute, UKM-MTDC Smart Technology Center, 43600, Bangi, Selangor, Malaysia

    Nor Muhammad Mahadi

  4. School of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor, Malaysia

    Osman Hassan

  5. Enzyme and Microbial Technology Research Group, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Selangor, Malaysia

    Raja Noor Zaliha Raja Abdul Rahman

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  1. Rui Min Ong
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  2. Kian Mau Goh
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  6. Rosli Md Illias
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Correspondence to Rosli Md Illias.

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Ong, R.M., Goh, K.M., Mahadi, N.M. et al. Cloning, extracellular expression and characterization of a predominant β-CGTase from Bacillus sp. G1 in E. coli . J Ind Microbiol Biotechnol 35, 1705–1714 (2008). https://doi.org/10.1007/s10295-008-0462-2

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  • DOI: https://doi.org/10.1007/s10295-008-0462-2

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