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 E. coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterized. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 degrees C at pH 8.0. The enzyme resulted to be active on p-NP-esters of fatty acids with acyl chain length from C(2) to C(12) and the preferred substrate was p-NP-pentanoate showing a k(cat)= 26.2+/-0.1 s(-1), K(M)= 0.122+/-0.006 mM and a k(cat) /K(M)= 215+/-11 mM(-1)s(-1). The enzyme was strongly inhibited by Hg(2+), Zn(2+), Cu(2+), Fe(3+), Mn(2+) ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C(50) 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 characterized 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 characterisation
De Santi C;Mandrich L;de Pascale D
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 E. coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterized. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 degrees C at pH 8.0. The enzyme resulted to be active on p-NP-esters of fatty acids with acyl chain length from C(2) to C(12) and the preferred substrate was p-NP-pentanoate showing a k(cat)= 26.2+/-0.1 s(-1), K(M)= 0.122+/-0.006 mM and a k(cat) /K(M)= 215+/-11 mM(-1)s(-1). The enzyme was strongly inhibited by Hg(2+), Zn(2+), Cu(2+), Fe(3+), Mn(2+) ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C(50) 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 characterized 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
