Abstract
The araA gene encoding an L-arabinose isomerase (L-AI) from the acido-thermophilic bacterium Acidothermus cellulolytics ATCC 43068 was cloned and overexpressed in Escherichia coli. The open reading frame of the L-AI consisted of 1,503 nucleotides encoding 501 amino acid residues. The recombinant L-AI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be approximately 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 75°C and pH 7.5. It required divalent metal ions, either Mn2+ or Co2+, for both enzymatic activity and thermostability improvement at higher temperatures. The enzyme showed relatively high activity and stability at acidic pH. It exhibited over 90% of its maximal activity at pH 6.0 and retained 80% of activity after 12 h incubation at pH 6.0. Catalytic property study showed that the enzyme had an interesting catalytic efficiency. Its apparent K m, V max, and catalytic efficiency (k cat/K m) for D-galactose was 28.9 mM, 4.9 U/mg, and 9.3 mM−1 min−1, respectively. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield over 50% after 12 h under optimal conditions, suggesting its potential in D-tagatose production.
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Acknowledgements
This work was supported by the grants from the National High Technology “863” Program of China, Project No. 2006AA10Z334, the Natural Science Foundation of China, Project No. 20906040, the Natural Science Foundation of Jiangsu Province, Project No. BK2008099 and BK2008003, the Research Program of State Key Laboratory of Food Science and Technology, Jiangnan University, Project No. SKLF-MB-200804 and SKLF-TS-200805, the Specialized Research Fund for the Doctoral Program of Higher Education, Project No. 200802951024, and the Program for Innovative Research Team of Jiangnan University, Project No. 2008CXTD01.
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Cheng, L., Mu, W., Zhang, T. et al. An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization. Appl Microbiol Biotechnol 86, 1089–1097 (2010). https://doi.org/10.1007/s00253-009-2322-z
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DOI: https://doi.org/10.1007/s00253-009-2322-z