RESUMO
Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.
Assuntos
Carboxilesterase , Clonagem Molecular , Halobacterium salinarum , Proteínas Recombinantes , Carboxilesterase/genética , Carboxilesterase/metabolismo , Carboxilesterase/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Halobacterium salinarum/enzimologia , Halobacterium salinarum/genética , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Concentração de Íons de Hidrogênio , Cinética , Estabilidade Enzimática , Proteínas Arqueais/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , TemperaturaRESUMO
The tRNA-dependent transamidation pathway is the essential route for Asn-tRNA(Asn) formation in organisms that lack an asparaginyl-tRNA synthetase. This pathway relies on a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS encoded by aspS), an enzyme with relaxed tRNA specificity, to form Asp-tRNA(Asn). The misacylated tRNA is then converted to Asn-tRNA(Asn) by the action of an Asp-tRNA(Asn) amidotransferase. Here we show that Asn-tRNA(Asn) formation in the extreme halophile Halobacterium salinarum also occurs by this transamidation mechanism, and we explore the property of the haloarchaeal AspRS to aspartylate tRNA(Asn) in vivo and in vitro. Transformation of the E. coli trpA34 strain with the H. salinarum aspS and tRNA(Asn) genes led to restoration of tryptophan prototrophy by missense suppression of the trpA34 mutant with heterologously in vivo formed Asp-tRNA(Asn). The haloarchaeal AspRS works well at low and high (0.1-3 M) salt concentrations but it is unable to use Escherichia coli tRNA as substrate. We show that mutations of two amino acids (H26 and P84) located in the AspRS anticodon binding domain limit the specificity of this nondiscriminating enzyme towards tRNA(Asn). Thus, as was observed in an archaeal discriminating AspRS and a bacterial ND-AspRS, amino acids in these positions influence the enzyme's tRNA selection.