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KMID : 1094720120170030512
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Biotechnology and Bioprocess Engineering
2012 Volume.17 No. 3 p.512 ~ p.525
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Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme
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Kretza Eirini
Papaneophytou Christos P.
Papi Rigini M.
Karidi Konstantina
Kiparissides Costas
Kyriakidis Dimitrios A.
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Abstract
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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 Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract 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°C and pH 7.0, while the T1/2 was 1 h at 100°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 Ca2+ and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, 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.
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KEYWORD
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Thermus thermophilus HB8, thermostable lipase, organic solvent tolerance, carbon source, oxygen transfer rate, biochemical characterization
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