Biotechnology and Bioprocess Engineering 17: 512-525 (2012) DOI 10.1007/s12257-011-0481-0 Lipase Activity in Thermus thermophilus HB8: Purification and Characterization of the Extracellular Enzyme Eirini Kretza, Christos P. Papaneophytou, Rigini M. Papi, Konstantina Karidi, Costas Kiparissides, and Dimitrios A. Kyriakidis Received: 30 September 2011 / Revised: 6 December 2011 / Accepted: 7 December 2011 © The Korean Society for Biotechnology and Bioengineering and Springer 2012 Abstract In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca 2+ -oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular ex- tract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80 o C and pH 7.0, while the T 1/2 was 1 h at 100 o C. The K m of the partial purified enzyme was 1 mM and the V max was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca 2+ and Hg 2+ stimulated lipase activity, whereas Zn 2+ , Co 2+ , or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications. Keywords: Thermus thermophilus HB8, thermostable lipase, organic solvent tolerance, carbon source, oxygen transfer rate, biochemical characterization 1. Introduction Lipases (E.C. 3.1.1.3) are very prominent biocatalysts due to their novel and multifold applications in oleochemistry, organic synthesis, detergent formulation and nutrition [1]. These enzymes catalyze the hydrolysis of carboxyl esters and are present in various organisms, including animals, plants, fungi, and bacteria [2]. Moreover, lipases are unique in catalyzing the hydrolysis of fats into fatty acids and glycerol at the water-lipid interface and reversing the reaction in non-aqueous media [3]. They also possess the ability to catalyze several other types of biotransformations, such as esterification and transesterification. Various micro- bial lipases of industrial importance have been purified and characterized, and their biotechnological applications have been evaluated in food, dairy, detergents, pharmaceuticals, textile, cosmetics, paper industry and in preparation of various flavor and fragrances [4]. Despite this potential use of such lipases, biocatalysis with lipases cannot be easily scaled up to large-scale fo industrial applications because of the instability of these enzymes in harsh reaction Eirini Kretza, Costas Kiparissides Chemical Process Engineering Research Institute/Centre for Research & Technology, Hellas C.P.E.R.I./CE.R.T.H., Thessaloniki 57001, Greece Eirini Kretza, Konstantina Karidi, Costas Kiparissides Department of Chemical Engineering, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece Christos P. Papaneophytou, Rigini M. Papi, Dimitrios A. Kyriakidis * Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece Tel: +30-231-099-7771; Fax: +30-231-099-7689 E-mail : kyr@chem.auth.gr RESEARCH PAPER