Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

• Published in: Journal of industrial microbiology & biotechnology 2005-07, Vol.32 (7), p.269-276
• Main Authors: Deejing, S, Yoshimune, K, Lumyong, S, Moriguchi, M
• Format: Article
• Language: English
• Subjects: Ammonium; Bacillaceae - enzymology; Bacillus thermoleovorans; bacterial proteins; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnology; enzyme activity; Enzyme Stability; Enzymes; Fractionation; Fundamental and applied biological sciences. Psychology; Geobacillus thermoleovorans; Hot springs; Hot Temperature; Hydrolysis; Hydroxyapatite; Ionization; Ions; leucyl aminopeptidase; Leucyl Aminopeptidase - chemistry; Leucyl Aminopeptidase - isolation & purification; Leucyl Aminopeptidase - metabolism; Mass spectrometry; Peptides; thermal stability
• ISSN: 1367-5435; 1476-5535
• DOI: 10.1007/s10295-005-0236-z

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 K(m) and V(max) values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min-1 mg-1 protein and 1,149 mol min-1 mg-1 protein, respectively. The turnover rate (k(cat)) and catalytic efficiency (k(cat)/ K(m)) 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+ .