Abstract
A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The Km and Vmax values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 μmol min−1 mg−1 protein and 1,149 μmol min−1 mg−1 protein, respectively. The turnover rate (kcat) and catalytic efficiency (kcat/ Km) for Leu-p-NA and LeuGlyGly were 10,179 s−1 and 49,543 s−1 and 15,470 mM−1 s−1 and 1981.7 mM−1 s−1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, β-mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co2+ .
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The authors are very thankful to the Ministry of Education, Culture, Sports, Science and Technology of Japan for a research student grant.
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Deejing, S., Yoshimune, K., Lumyong, S. et al. Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b. J IND MICROBIOL BIOTECHNOL 32, 269–276 (2005). https://doi.org/10.1007/s10295-005-0236-z
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DOI: https://doi.org/10.1007/s10295-005-0236-z