Heterologous expression and characterization of Bacillus coagulans l-arabinose isomerase
- 344 Downloads
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn2+ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K m, V max and k cat/K m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM−1min−1, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L−1 h−1.
Keywordsl-arabinose l-arabinose isomerase Bacillus coagulans l-ribulose Bioconversion
This research was supported by the Science and Engineering Research Council (SERC) of the Agency for Science, Technology and Research (A*STAR) of Singapore (SERC grant no 0921590133). We are grateful to Dr. Keith Carpenter for critical reading of this manuscript.
- Bucke C (1983) Practicality of industrial enzymes. Biochem Soc Trans 11:13–14Google Scholar
- 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
- Heath EC, Horecker BL, Smyrniotis PZ, Takagi Y (1958) Pentose fermentation by Lactobacillus plantarum. II. l-arabinose isomerase. J Biol Chem 231:1031–1037Google Scholar
- Kim P (2004) Current studies on biological tagatose production using l-arabinose isomerase: a review and future perspective. Appl Microbiol Biotechnol 65:243–249Google Scholar
- Kim BC, Lee YH, Lee HS, Lee DW, Choe EA, Pyun YR (2002) Cloning, expression and characterization of l-arabinose isomerase from Thermotoga neapolitana: bioconversion of d-galactose to d-tagatose using the enzyme. FEMS Microbiol Lett 212:121–126Google Scholar
- Lee DW, Choe EA, Kim SB, Eom SH, Hong YH, Lee SJ, Lee HS, Lee DY, Pyun YR (2005a) Distinct metal dependence for catalytic and structural functions in the l-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus. Arch Biochem Biophys 434:333–343CrossRefGoogle Scholar
- Liu SY, Wiegel J, Gherardini FC (1996) Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489. J Bacteriol 178:5938–5945Google Scholar
- Mizanur RM, Takata G, Izumori K (2001) Cloning and characterization of a novel gene encoding l-ribose isomerase from Acinetobacter sp. strain DL-28 in Escherichia coli. Biochim Biophys Acta 1521:141–145Google Scholar
- Zhu W, Manjasetty BA, Chance MR (2007) Crystal structure of Mn2+-bound Escherichia coli l-arabinose Isomerase (ECAI) and Implications in protein catalytic mechanism and thermo-stability. J Young Investigators 17. http://www.jyi.org/research/re.php?id=1255. Accessed 7 Feb 2012