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Isolation and Characterization of a Novel Thermophilic-Organic Solvent Stable Lipase From Acinetobacter baylyi

Uttatree, Sasithorn, Winayanuwattikun, Pakorn, Charoenpanich, Jittima
Applied biochemistry and biotechnology 2010 v.162 no.5 pp. 1362-1376
Acinetobacter, EDTA (chelating agent), ammonium sulfate, barium, benzene, bioenergy industry, calcium, coconut oil, corn oil, esters, fatty acids, hexane, hydrolysis, iron, isoamyl alcohol, lithium, magnesium, manganese, mercury, molecular weight, octoxynol, pH, palm oils, pharmaceutical industry, rice bran oil, sesame oil, silver, sodium, temperature, transesterification, triacylglycerol lipase, zinc
The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative molecular mass as 30 kDa. The enzyme expressed maximum activity at 60°C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80°C, respectively. A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely inhibited in the presence of Fe²⁺, Mn²⁺, EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl fluoride (PMSF), Na⁺, and Mg²⁺ and no significant effect was found in the presence of Ca²⁺ and Li⁺. Half of an activity was retained by Ba²⁺, Ag⁺, Hg⁺, Ni²⁺, Zn²⁺, and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C₈-C₁₂). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl esters (FAMEs) revealed 31.64 ± 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs, indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis and pharmaceutical industry.