title: Characterization of a novel organic solvent tolerant protease from a moderately halophilic bacterium and its behavior in ionic liquids.
abstract: An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40-80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V(max) and K(m) of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba(2+) and Ca(2+) (2 mM) strongly enhanced the enzyme activity, while Fe(2+) and Mg(2+) activated moderately and Zn(2+), Ni(2+), and Hg(2+) decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C(6)MIM][PF(6)]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.
authors.affiliation: Molecular Biotechnology Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz 71454, Iran. karbalaei@shirazu.ac.ir
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Karbalaei-Heidari
authors.forename: Mahnaz
authors.initials: M
authors.lastname: Shahbazi
authors.forename: Ghodratollah
authors.initials: G
authors.lastname: Absalan
mesh_terms: D002364:Caseins / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D052578:Ionic Liquids / Q000494:pharmacology; D007700:Kinetics; D045726:Metalloproteases / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D011489:Protein Denaturation; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology*; D014737:Vibrionaceae / Q000201:enzymology
chemical_list: D002364:Caseins; D007093:Imidazoles; D052578:Ionic Liquids; D008670:Metals; D015842:Serine Proteinase Inhibitors; D012997:Solvents; D045726:Metalloproteases; D057057:Serine Proteases