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

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 degrees C for up to 1 h. Its optimal pH and temperature were observed to be 7.6-7.8 and 60 degrees 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 micromol min(-1) mg(-1) protein and 1,149 micromol 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, beta-mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co(2+) .

Hydrolysis of Amides

“…A very thermostable leucine aminopeptidase has been identified in Geobacillus thermoleovorans (formerly Bacillus thermoleovorans). This enzyme, which was developed as a thermotolerant debittering enzyme for use in food protein processing, retains its full activity after a 1 h incubation up to 90 C [191]. In addition, solvent resistant aminopeptidases have been described in the literature.…”

Characterisation of leucyl aminopeptidase from Solanum tuberosum tuber

“…Since the formation of carcinogens is the most serious problem in the production of protein acid hydrolyzates, the enzymatic production of amino acid mixtures from proteins using peptidases has been paid considerable attention in the food protein processing industry (Deejing, Yoshimune, Lumyong, & Moriguchi, 2005;Tang, Wang, & Yang, 2009). Recently, it has been shown that enzymes have high potential for hydrolysis of chicken breast meat (Chi, Lyu, Lin, & Huang, 2008) and for the proteolysis of enzymemodified Cheddar cheese (Azarnia, Lee, Yaylayan, & Kilcawley, 2010).…”

“…Although many industrial peptidases have been developed, commercial aminopeptidases are limited in number (Deejing et al, 2005). For realisation of this potential, purification and biochemical characterisation of aminopeptidase from various sources is necessary.…”

Identification of an aminopeptidase from the skeletal muscle of grass carp (Ctenopharyngodon idellus)

“…Because of its debittering function in food industry, the production of LAP from microorganism by fermentation has also been paid considerable attention (Chien et al 2002;Deejing et al 2005).…”