Purification and biochemical characterization of halophilic, alkalithermophilic protease AbCP from Alkalibacillus sp. NM-Fa4

• Protease AbCP was purified and determined to be monomeric with molecular mass 20 kDa.
• The enzyme exhibited maximal activity at pH 9.5, 1 M sodium chloride and 52 °C.
• The enzyme is stimulated with SDS, hydrogen peroxide, urea, EDTA, DTT and β-mercaptoethanol.
• The enzyme is stimulated in the presence of MgCl2, CaCl2 and FeSO4.
• The enzyme has broad substrate specificity hydrolyzing natural and synthetic substrates.

An extracellular alkaline, halo- and thermostable protease (AbCP) produced by a novel Alkalibacillus sp. NM-Fa4, isolated from the alkaline, hypersaline lakes of the Wadi An Natrun, was purified to homogeneity by precipitation with ethanol and anion-exchange chromatography. The molecular weight of the purified protease was 19.7 kDa. AbCP retains proteolytic activity over broad sodium chloride, pH and temperature ranges, with maximal activity at 1 M NaCl, pH45 °C 9.5 and 50–52 °C. AbCP was resistant to phenylmethylsulfonyl fluoride (2 mM) and ethylene diamine tetra-acetic acid (2 mM), stimulated by the reducing agents dithiothreitol (2 mM) and β-mercaptoethanol (1% v/v) and inhibited with iodoacetic acid (5 mM), suggesting that AbCP is a cysteine protease. AbCP showed stability toward anionic surfactants (sodium dodecyl sulfate), oxidizing agents (H2O2), chemical denaturants (urea), and retained most of its activity in the presence of 1% v/v of the non-ionic surfactant Tween 80. The protease was stable in 50% mixtures of ethanol and, to a lesser extent, methanol, and was stimulated by Mg2+, Ca2+, and Fe2+. AbCP shows a broad substrate specificity and hydrolyzes both natural and synthetic substrates. Based on the Lineweaver–Burk plot, the Km with casein as substrate was 1.3 ± 0.007 mg/mL and Vmax was 1111 mg/ml/min. The stability of the enzyme under the combined extreme conditions of high salt concentration, alkaline pH and high temperature, in addition to being resistant to chemical denaturants, oxidizing agents, surfactants as well as exhibiting broad substrate specificity makes this enzyme a promising candidate for a variety of biotechnological applications.

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