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Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

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Abstract

The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM®-T easy vector to transform into Escherichia coli DH5 α for sequencing. The obtained gene encoding an amylomaltase consisted of 1,503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21(DE3). The maximum expression was observed when the cloned cells were cultured at 37°C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70°C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the largering cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions.

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Authors and Affiliations

  1. Biochemistry and Molecular Biology Program, Faculty of Medicine, Thammasat University, Patumtani, 12121, Thailand

    K. Sawasdee

  2. Department of Biochemistry, Phramongkutklao College of Medicine, Phramongkutklao Hospital, Bangkok, 10400, Thailand

    P. Rudeekulthamrong

  3. Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Leipzig, 04103, Germany

    W. Zimmermann

  4. Department of Agricultural Chemistry, Faculty of Agriculture, Meiji University, Higashimita, Tama-ku, Kawasaki, 214-0034, Japan

    S. Murakami

  5. Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    P. Pongsawasdi

  6. Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Patumtani, 12121, Thailand

    J. Kaulpiboon

Authors
  1. K. Sawasdee
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  2. P. Rudeekulthamrong
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  3. W. Zimmermann
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  4. S. Murakami
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  5. P. Pongsawasdi
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  6. J. Kaulpiboon
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Corresponding author

Correspondence to J. Kaulpiboon.

Additional information

Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2014, Vol. 50, No. 1, pp. 25–33.

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Sawasdee, K., Rudeekulthamrong, P., Zimmermann, W. et al. Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production. Appl Biochem Microbiol 50, 17–24 (2014). https://doi.org/10.1134/S000368381306015X

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  • DOI: https://doi.org/10.1134/S000368381306015X

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