ORIGINAL PAPER Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25 Maryam Siroosi • Mohammad Ali Amoozegar • Khosro Khajeh • Mostafa Fazeli • Mehran Habibi Rezaei Received: 1 February 2013 / Accepted: 26 September 2013 Ó Springer Japan 2013 Abstract A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent- tolerant amylopullulanase. The maximum enzyme pro- duction was at high salt concentration, 3–4 M NaCl. Optimum pH and temperature for enzyme production were 7.0 and 40 °C, respectively. Molecular mass of purified enzyme was estimated to be about 140 kDa by SDS– PAGE. This enzyme was active on pullulan and starch as substrates. The apparent K m for the enzyme activity on pullulan was 4 mg/ml and for soluble starch was 1.8 mg/ml. Optimum temperature for amylolytic and pullulytic activ- ities was 50 °C. Optimum pH for amylolytic activity was 7 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0–4.5 M) of NaCl. The effect of organic solvents on the enzyme activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. This study is the first report on amylopullulanase production in halo- philic bacteria and archaea. Keywords Extreme halophilic archaea Á Halophilic amylopullulanase Á Halorubrum Á Organic solvent tolerance Á Protein purification Introduction Haloarchaea in order Halobacteriales and family Halo- bacteriaceae are extreme halophiles requiring at least 1.5 M NaCl for growth (Grant et al. 2001). Halophiles are extremophilic microorganisms that live, grow and multiply in highly saline environments and can be a source of hal- ophilic enzymes (Margesin and Schinner 2001). Most industrial processes are carried out in very harsh physico- chemical conditions which may not be definitively adjusted to the optimal points required for the activity of the available enzymes. Thus, it would be of great importance to have extremozymes that expose optimal activities at high salinity, temperature and pH values. The interest in enzymes produced by extremophiles, which are expected to show optimal activities in extreme conditions, has recently greatly increased (Adams and Kelly 1995). Haloarchaeal hydrolytic enzymes seem to be very good candidate for industrial application which not only act at high salt con- centrations, but also may be excellent activity at high temperature, low water activity and high pH value (Mak- hdoumi Kakhki et al. 2011). Proteins from haloarchaea face extremely saline conditions both inside and outside the cell, and are adapted to these conditions because their of unique amino acid composition. The surface of halophilic proteins is highly acidic and halophilic proteins have Communicated by H. Atomi. Electronic supplementary material The online version of this article (doi:10.1007/s00792-013-0589-6) contains supplementary material, which is available to authorized users. M. Siroosi Á M. A. Amoozegar (&) Á M. Fazeli Extremophiles Laboratory, Department of Microbiology, Faculty of Biology, College of Science, School of Biology and Center of Excellence in Phylogeny of Living Organisms, University of Tehran, P. O. Box 14155-6455, Tehran, Iran e-mail: amoozegar@ut.ac.ir K. Khajeh Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran M. Habibi Rezaei Protein Biotechnology Laboratory, Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran 123 Extremophiles DOI 10.1007/s00792-013-0589-6