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Characterization of a novel protease from Anoxybacillus kamchatkensis strain M1V with biotechnological interest
* 1 , 2 , 3 , 3 , 3 , 3 , 2 , 3 , 2 , * 3
1  Laboratory of Microbial Biotechnology, Enzymatic, and Biomolecules (LMBEB), Centre of Biotechnology of Sfax (CBS), University of Sfax, P.O. Box 1177, Sfax 3018, Tunisia
2  Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of Biological Sciences, University of Sciences and Technology of Houari Boumediene (USTHB), PO Box 32, El Alia, Bab Ezzouar, 16111 Algiers, Algeria
3  Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia


Over the last few decades, researchers have been intrigued by the fascinating polyextremophiles microrganisms that inhabit extreme environments. Such organisms are being looked upon as stars for industrial biotechnology that function under acute conditions in which usually the enzymes of their non-extremophilic counterparts could not. The application of hydrolases in industrial processes sometimes fails due to the lack of robustness, stability and undesirable properties. Thanks to their biochemical properties, they are relevant for specific industrial applications that determine the demand for tailor-made enzymes and shift the industrial interest towards biocatalysts from extremophiles including proteases. A new extracellular thermostable serine alkaline protease (designated SAPA) was produced, purified, and characterized from Anoxybacillus kamchatkensis M1V. The bacterial strain was found to hyper-produce extracellular protease when grown at 45 °C in optimized media (4,600 U/mL). The purification to homogeneity of the SAPA enzyme was achieved, simultaneously, by precipitation with ammonium sulfate fractionation-dialysis, anion exchange (FPLC), and gel filtration (HPLC) chromatographies. It had a relative molecular mass of 28 kDa as estimated by SDS-PAGE. The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. It showed optimal activity at pH 11 and 70 °C. The thermoactivity and thermostability of SAPA were enhanced in the presence of 2 mM Ca2+. Irreversible inhibition of the enzyme activity by DFP and PMSF confirmed its belonging to the serine proteases family. Interestingly, SAPA displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than proteases viz. SPVP from Aeribacillus pallidus strain VP3, SAPB from Bacillus pumilus strain CBS, Subtilisin A from B. licheniformis, and Subtilisin 309 from B. clausii. More interestingly, SAPA showed a high detergent stability and compatibility and an outstanding stain removal compared to commercial enzymes viz. Alcalase™ and Savinase™. The sapA gene was cloned, sequenced, and expressed in the extracellular fraction of E. coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPA) were similar to those of native SAPA. The deduced amino-acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from B. subtilis, with 16 amino-acids of difference. Above all, SAPA exhibited remarkable biochemical proprieties which may be considered as a potential candidate for biotechnological applications such as a cleaning bio-additive in laundry detergent formulations.

Keywords: Protease; Anoxybacillus kamchatkensis; thermophilic; hot spring; detergent; expression.