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Cloning and characterization of a rhamnose isomerase from Bacillus halodurans

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Abstract

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l -RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k cat of 8,971 min−1 and a k cat/K m of 17 min−1 mM−1 for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far.

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Acknowledgment

This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0070463). This study was also supported by a grant (2008A0080126) from ARPC, Republic of Korea.

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Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, South Korea, 143-701

    Ponnandy Prabhu

  2. Department of Advanced Technology Fusion, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, South Korea, 143-701

    Thanh Thi Ngoc Doan & Lin-Woo Kang

  3. Department of Chemical Engineering, Konkuk University, 1 Hwayang-Dong, Gwangjin-Gu, Seoul, South Korea, 143-701

    Marimuthu Jeya & Jung-Kul Lee

Authors
  1. Ponnandy Prabhu
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  2. Thanh Thi Ngoc Doan
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  3. Marimuthu Jeya
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  4. Lin-Woo Kang
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  5. Jung-Kul Lee
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Correspondence to Lin-Woo Kang or Jung-Kul Lee.

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Ponnandy Prabhu and Thanh Thi Ngoc Doan contributed equally to this work.

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Supplementary Table 1

Salt bridge forming residues in the α-helix of BHRI, ECRI, and YPRI (DOC 38 kb)

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Prabhu, P., Doan, T.T.N., Jeya, M. et al. Cloning and characterization of a rhamnose isomerase from Bacillus halodurans . Appl Microbiol Biotechnol 89, 635–644 (2011). https://doi.org/10.1007/s00253-010-2844-4

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  • DOI: https://doi.org/10.1007/s00253-010-2844-4

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