Applied Microbiology and Biotechnology

, Volume 89, Issue 3, pp 635–644 | Cite as

Cloning and characterization of a rhamnose isomerase from Bacillus halodurans

Biotechnologically Relevant Enzymes and Proteins


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.


Bacillus halodurans Characterization Rhamnose isomerase Thermostability 



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.

Supplementary material

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


  1. Badia J, Gimenez R, Baldoma L, Barnes E, Fessner WD, Aguilar J (1991) l-Lyxose metabolism employs the l-rhamnose pathway in mutant cells of Escherichia coli adapted to grow on l-lyxose. J Bacteriol 173:5144–5150Google Scholar
  2. Barlow DJ, Thornton JM (1983) Ion-pairs in proteins. J Mol Biol 168:867–885CrossRefGoogle Scholar
  3. Bautista DA, Pegg RB, Shand PJ (2000) Effect of l-glucose and d-tagatose on bacterial growth in media and a cooked cured ham product. J Food Prot 63:71–77Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  5. Cai G, Zhu S, Yang S, Zhao G, Jiang W (2004) Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability. Appl Environ Microbiol 70:2764–2770CrossRefGoogle Scholar
  6. Chakravarty S, Varadarajan R (2000) Elucidation of determinants of protein stability through genome sequence analysis. FEBS Lett 470:65–69CrossRefGoogle Scholar
  7. Costantini S, Colonna G, Facchiano AM (2008) ESBRI: a web server for evaluating salt bridges in proteins. Bioinformation 3:137–138Google Scholar
  8. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C (2000) Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol 301:1041–1057CrossRefGoogle Scholar
  9. Dische Z, Borenfreund E (1951) A new spectrophotometric method for the detection and determination of keto sugars and trioses. J Biol Chem 192:583–587Google Scholar
  10. Dragan AI, Potekhin SA, Sivolob A, Lu M, Privalov PL (2004) Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56. Biochemistry 43:14891–14900CrossRefGoogle Scholar
  11. Fersht AR (1972) Conformational equilibria and the salt bridge in chymotrypsin. Cold Spring Harb Symp Quant Biol 36:71–73Google Scholar
  12. Fu Y, Ding Y, Chen Z, Sun J, Fang W, Xu W (2010) Study on the relationship between cyclodextrin glycosyltransferase’s thermostability and salt bridge by molecular dynamics simulation. Protein Pept Lett (in press)Google Scholar
  13. Gottschalk G (1986) Bacterial metabolism. Springer, New York, pp 79–81Google Scholar
  14. Izumori K (2002) Bioproduction strategies for rare hexose sugars. Naturwissenschaften 89:120–124CrossRefGoogle Scholar
  15. Kabsch W, Sander C (1983) How good are predictions of protein secondary structure? FEBS Lett 155:179–82CrossRefGoogle Scholar
  16. Karshikoff A, Ladenstein R (2001) Ion pairs and the thermotolerance of proteins from hyperthermophiles: a “traffic rule” for hot roads. Trends Biochem Sci 26:550–55CrossRefGoogle Scholar
  17. Korndorfer IP, Fessner WD, Matthews BW (2000) The structure of rhamnose isomerase from Escherichia coli and its relation with xylose isomerase illustrates a change between inter and intra-subunit complementation during evolution. J Mol Biol 300:917–933CrossRefGoogle Scholar
  18. Kumar S, Nussinov R (1999) Salt bridge stability in monomeric proteins. J Mol Biol 293:1241–1255CrossRefGoogle Scholar
  19. Kumar S, Nussinov R (2004) Different roles of electrostatics in heat and in cold: adaptation by citrate synthase. Chembiochem 5:280–290CrossRefGoogle Scholar
  20. Lawson CJ, Homewood J, Taylor AJ (2002) The effects of l-glucose on memory in mice are modulated by peripherally acting cholinergic drugs. Neurobiol Learn Mem 77:17–28CrossRefGoogle Scholar
  21. Leang K, Takada G, Ishimura A, Okita M, Izumori K (2004a) Cloning, nucleotide sequence, and overexpression of the l-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli. Appl Environ Microbiol 70:3298–3304CrossRefGoogle Scholar
  22. Leang K, Takada G, Fukai Y, Morimoto K, Granstrom TB, Izumori K (2004b) Novel reactions of l-rhamnose isomerase from Pseudomonas stutzeri and its relation with d-xylose isomerase via substrate specificity. Biochim Biophys Acta 1674:68–77Google Scholar
  23. Levin GV, Zehner LR, Sanders JP, Beadle JR (1964) Sugar substitues: their energy values, bulk characteristic, and potential health benefits. Am J Clin Nutr 62:1161–1168Google Scholar
  24. Livesey G, Brown JC (1995) Whole body metabolism is not restricted to d-sugars because energy metabolism of l-sugars fits a computational model in rats. J Nutr 125:3020–3029Google Scholar
  25. Moralejo P, Egan SM, Hidalgo E, Aguilar J (1993) Sequencing and characterization of a gene cluster encoding the enzymes for l-rhamnose metabolism in Escherichia coli. J Bacteriol 175:5585–5594Google Scholar
  26. Mozhaev VV (1993) Mechanism-based strategies for protein thermostabilization. Trends Biotechnol 11:88–95Google Scholar
  27. Musafia B, Buchner V, Arad D (1995) Complex salt bridges in proteins: statistical analysis of structure and function. J Mol Biol 254:761–770CrossRefGoogle Scholar
  28. Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726–739CrossRefGoogle Scholar
  29. Poonperm W, Takata G, Okada H, Morimoto K, Granstrom TB, Izumori K (2007) Cloning, sequencing, overexpression and characterization of l-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production. Appl Microbiol Biotechnol 76:1297–1307CrossRefGoogle Scholar
  30. Power J (1967) The l-rhamnose genetic system in Escherichia coli K-12. Genetics 55:557–568Google Scholar
  31. Richardson JS, Hynes MF, Oresnik IJ (2004) A genetic locus necessary for rhamnose uptake and catabolism in Rhizobium leguminosarum bv. trifolii. J Bacteriol 186:8433–8442CrossRefGoogle Scholar
  32. Richardson JS, Carpena X, Switala J, Perez-Luque R, Donald LJ, Loewen PC, Oresnik IJ (2008) RhaU of Rhizobium leguminosarum is a rhamnose mutarotase. J Bacteriol 190:2903–2910CrossRefGoogle Scholar
  33. Ryu KS, Kim JI, Cho SJ, Park D, Park C, Cheong HK, Lee JO, Choi BS (2005) Structural insights into the monosaccharide specificity of Escherichia coli rhamnose mutarotase. J Mol Biol 349:153–162CrossRefGoogle Scholar
  34. Spek EJ, Bui AH, Lu M, Kallenbach NR (1998) Surface salt bridges stabilize the GCN4 leucine zipper. Protein Sci 7:2431–2437CrossRefGoogle Scholar
  35. Sterner R, Liebl W (2001) Thermophilic adaptation of proteins. Crit Rev Biochem Mol Biol 36:39–106CrossRefGoogle Scholar
  36. Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F, Hirama C, Nakamura Y, Ogasawara N, Kuhara S, Horikoshi K (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317–4331CrossRefGoogle Scholar
  37. Tomazic SJ, Klibanov AM (1988) Mechanisms of irreversible thermal inactivation of Bacillus alpha-amylases. J Biol Chem 263:3086–3091Google Scholar
  38. Veith B, Herzberg C, Steckel S, Feesche J, Maurer KH, Ehrenreich P, Baumer S, Henne A, Liesegang H, Merkl R, Ehrenreich A, Gottschalk G (2004) The complete genome sequence of Bacillus licheniformis DS13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7:204–211CrossRefGoogle Scholar
  39. Vieille C, Epting KL, Kelly RM, Zeikus JG (2001) Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase. Eur J Biochem 268:6291–6301CrossRefGoogle Scholar
  40. Wasserman B (1984) Thermostable enzyme production. Food Technol 38:78–88Google Scholar
  41. Whitlow M, Howard AJ, Finzel BC, Poulos TL, Winborne E, Gilliland GL (1991) A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and d-xylose. Proteins 9:153–173CrossRefGoogle Scholar
  42. Xiao L, Honig B (1999) Electrostatic contributions to the stability of hyperthermophilic proteins. J Mol Biol 289:1435–1444CrossRefGoogle Scholar
  43. Yip KS, Britton KL, Stillman TJ, Lebbink J, de Vos WM, Robb FT, Vetriani C, Maeder D, Rice DW (1998) Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family. Eur J Biochem 255:336–346CrossRefGoogle Scholar
  44. Yoshida H, Yamada M, Ohyama Y, Takada G, Izumori K, Kamitori S (2007) The structures of l-rhamnose isomerase from Pseudomonas stutzeri in complexes with l-rhamnose and d-allose provide insights into broad substrate specificity. J Mol Biol 365:1505–1516CrossRefGoogle Scholar
  45. You DJ, Fukuchi S, Nishikawa K, Koga Y, Takano K, Kanaya S (2007) Protein thermostabilization requires a fine-tuned placement of surface-charged residues. J Biochem 142:507–516CrossRefGoogle Scholar

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

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