Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C2 to C12 and the preferred substrate was pNP-pentanoate showing a kcat = 26.2 ± 0.1 s−1, KM = 0.122 ± 0.006 mM and a kcat/KM = 215 ± 11 mM−1 s−1. The enzyme was strongly inhibited by Hg2+, Zn2+, Cu2+, Fe3+, Mn2+ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C50 values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.

The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

TUTINO, MARIA LUISA;PARRILLI, ERMENEGILDA;DEL VECCHIO, POMPEA GIUSEPPINA GRAZIA;
2010

Abstract

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C2 to C12 and the preferred substrate was pNP-pentanoate showing a kcat = 26.2 ± 0.1 s−1, KM = 0.122 ± 0.006 mM and a kcat/KM = 215 ± 11 mM−1 s−1. The enzyme was strongly inhibited by Hg2+, Zn2+, Cu2+, Fe3+, Mn2+ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C50 values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/366788
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