Abstract
The l-rhamnose isomerase gene (L-rhi) encoding for l-rhamnose isomerase (l-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6×His sequence at a C-terminal of the protein. The open reading frame of L-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced l-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant l-RhI exhibited maximum activity at 65°C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60°C for 60 min. The apparent affinity (K m) and catalytic efficiency (k cat/K m) for l-rhamnose (at 65°C) were 4.89 mM and 8.36 × 105 M−1 min−1, respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50°C, for d-allose, l-mannose, d-ribulose, and l-talose from d-psicose, l-fructose, d-ribose and l-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant l-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.
Similar content being viewed by others
References
Altschul F, Gish W, Miller W, Meyers EW, Lipman Stephen DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Badia J, Baldoma L, Aguilar J, Boronat A (1989) Identification of the rhaA, rhaB and rhaD gene products from Escherichia coli K-12. FEMS Microbiol Lett 65:253–258
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 173:5144–5150
Bertelsen H, Jensen BB, Buemann B (1999) d-tagatose—a novel low-calorie bulk sweetener with prebiotic properties. World Rev Nutr Diet 85:98–109
Bhuiyan SH, Itami Y, Izumori K (1997) Isolation of an l-rhamnose isomerase-constitutive mutant of Pseudomonas sp. strain LL172: purification and characterization of the enzyme. J Ferment Bioeng 84:319–323
Bhuiyan SH, Itami Y, Takada G, Izumori K (1999) Preparation of l-talose and d-gulose from l-tagatose and d-sorbose, respectively, using immobilized l-rhamnose isomerase. J Biosci Bioeng 88:567–570
Bradford MM (1979) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Bustard MT, Wright PC (2002) Biodegradation of high concentration isopropanol by a solvent tolerant thermophile, Bacillus pallidus. Extremophiles 6:319–323
Cramp R, Gilmour M, Cowan DA (1997) Novel thermophilic bacteria producing nitrile-degrading enzymes. Microbiology 143:2313–2320
Dishe Z, Borenfreud E (1951) A new spectrophotometric method for the detection of keto sugars and trioses. J Biol Chem 192:583–587
Domagk GF, Zech R (1963) _ber den Abbau der Desoxyzucker durch Bacterienenzyme. I. l-Rhamnose-isomerase aus Lactobacillus plantarum. Biochem Z 339:145–153
Garcia-Junceda E, Shen GJ, Alajarin R, Wong CH (1995) Cloning and overexpression of rhamnose isomerase and fucose isomerase. Bioorg Med Chem 3:1349–1355
Granström TB, Takata G, Tokuda M, Izumori K (2004) Izumoring, A novel and complete strategy for bioproduction of rare sugars. J Biosci Bioeng 97:89–94
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmid. J Mol Biol 166:557–590
Illanes A (1999) Stability of biocatalysts process biotechnology. Electronic J Biotechnol 2:0717–3458
Itoh H, Okaya H, Khan AR, Tajima S, Hayakawa S, Izumori K (1994) Purification and characterization of d-tagatose 3-epimerase from Pseudomonas sp. ST-24. Biosci Biotechnol Biochem 58:2168–2171
Itoh H, Sato T, Izumori K (1995) Preparation of d-psicose from d-fructose by immobilized d-tagatose 3-epimerase. J Ferment Bioeng 80:101–103
Izumori K, Mitchell M, Elbein AD (1976) Evidence that the isomerization of d-ribose and l-rhamnose is catalyzed by the same enzyme in Mycobacterium smegmatis. J Bacteriol 126:553–555
Jeong LS, Yoo SJ (1998) Synthesis and antiviral activity of novel isodideoxy nucleosides with exocyclic methylene. Bioorg Med Chem Lett 8:847–852
Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30
Kim HJ, Oh DK (2005) Purification and characterization of an l-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing d-tagatose. J Biotechnol 120:162–173
Kornderfer 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 300:917–933
Kuroda K, Hanajima D, Fukumoto Y, Suzuki K, Kawamoto S, Shima J, Haga K (2004) Isolation of thermophilic ammonium-tolerant bacterium and its application to reduce ammonia emission during composting of animal wastes. Biosci Biotechnol Biochem 68:286–292
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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 70:3298–3304
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 1674:68–77
Lerner LM, Mennitt G (1994) A new synthesis of l-talose and preparation of its adenine nucleosides. Carbohydr Res 259:191–200
Ly KA, Milgrom P, Rothen M (2006) Xylitol, sweeteners, and dental caries. Pediatr Dent 28:192–198
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 175:5585–5594
Menavuvu BT, Poonperm W, Leang K, Noguchi N, Okada H, Morimoto K, Granstrom TB, Takada G, Izumori K (2006) Efficient biosynthesis of d-allose from d-psicose by cross- linked recombinant l-rhamnose isomerase: separation of product by ethanol crystallization. J Biosci Bioeng 101:340–345
Mozhaev V (1993) Mechanism-based strategies for protein thermostabilization. Trend Biotechnol 11:88–95
Noltmann EA (1972) Aldose–ketose isomerases. In: Boyer PD (ed) The enzymes, 3rd edn, vol 6. Academic, London, pp 271–354
Oudega B, Koningstein G Rodrigues L, de Sales Ramon M, Hilbert H, Dusterhoft TM, Pohl TM, Weitzenegger T (1997) Analysis of the Bacillus subtilis genome: cloning and nucleotide sequence of a 62 kb region between 275 degrees (rrnB) and 284 degrees (pai). Microbiology 143:2769–2774
Poonperm W, Takata G, Morimoto K, Granström TB, Izumori K (2007a) Production of l-xylulose from xylitol by a newly isolated strain of Bacillus pallidus Y25 and characterization of its relevant enzyme xylitol dehydrogenase. Enzyme Microb Technol 40:1206–1212
Poonperm W, Takata G, Sahachaisaree V, Lumyong P, Lumyong S, Izumori K (2007b) Efficient conversion of allitol to d-psicose by Bacillus pallidus Y25. J Biosci Bioeng 103:282–285
Sui L, Dong Y, Watanabe Y, Yamaguchi F, Hatano N, Tsukamoto I, Izumori K, Tokuda M (2005) The inhibitory effect and possible mechanisms of d-allose on cancer cell proliferation. Int J Oncol 27:907–912
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci 74:5463–5467
Takami H, Takaki Y, Uchiyama I (2002) Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environments. Nucleic Acids Res 30:3927–3935
Takeshita K, Shimonishi T, Izumori K (1996) Production of l-psicose from allitol by Gluconobacter frateurii IFO 3254. J Ferment Bioeng 3:212–215
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Wasserman B (1984) Thermostable enzyme production. Food Technol 38:78–88
Yoshida H, Yamada M, Ohyama O, 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 365:1505–1516
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Poonperm, W., Takata, G., Okada, H. et al. Cloning, sequencing, overexpression and characterization of l-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production. Appl Microbiol Biotechnol 76, 1297–1307 (2007). https://doi.org/10.1007/s00253-007-1109-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-007-1109-3