Applied Microbiology and Biotechnology

, Volume 89, Issue 3, pp 635–644

Cloning and characterization of a rhamnose isomerase from Bacillus halodurans

Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-010-2844-4

Cite this article as:
Prabhu, P., Doan, T.T.N., Jeya, M. et al. Appl Microbiol Biotechnol (2011) 89: 635. doi:10.1007/s00253-010-2844-4
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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 kcat of 8,971 min−1 and a kcat/Km of 17 min−1 mM−1 for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. haloduransl-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. haloduransl-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far.

Keywords

Bacillus haloduransCharacterizationRhamnose isomeraseThermostability

Supplementary material

253_2010_2844_MOESM1_ESM.doc (38 kb)
Supplementary Table 1Salt bridge forming residues in the α-helix of BHRI, ECRI, and YPRI (DOC 38 kb)

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Department of Bioscience and BiotechnologyKonkuk UniversitySeoulSouth Korea
  2. 2.Department of Advanced Technology FusionKonkuk UniversitySeoulSouth Korea
  3. 3.Department of Chemical EngineeringKonkuk UniversitySeoulSouth Korea143-701