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

• Main Authors: Deejing S., Yoshimune K., Lumyong S., Moriguchi M.
• Format: Article
• Language: English
• Published: 2014
• Online Access: http://www.scopus.com/inward/record.url?eid=2-s2.0-22144459435&partnerID=40&md5=4ac5000328c807fac53345a5d8dd44bb; http://www.ncbi.nlm.nih.gov/pubmed/15937698; http://cmuir.cmu.ac.th/handle/6653943832/4972
• Institution: Chiang Mai University

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 μmol min-1 mg-1 protein and 1,149 μmol 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+. © Society for Industrial Microbiology 2005.