Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1

• Published in: Biomolecules (Basel, Switzerland) Switzerland), 2024-04, Vol.14 (5), p.534
• Main Authors: Ortega-de la Rosa, Nestor David, Romero-Borbón, Evelyn, Rodríguez, Jorge Alberto, Camacho-Ruiz, Angeles, Córdova, Jesús
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Biochemical characteristics; biochemical characterization; Carboxylesterase; Carboxylesterase - chemistry; Carboxylesterase - genetics; Carboxylesterase - metabolism; Cloning; Cloning, Molecular; Diatomaceous earth; E coli; enzyme immobilization; Enzyme Stability; Enzymes; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - genetics; Enzymes, Immobilized - metabolism; Genomes; Halobacterium salinarum; Halobacterium salinarum - enzymology; Halobacterium salinarum - genetics; Halophilic archaea; Hydrogen-Ion Concentration; Immobilization; Industrial applications; Kinetics; Metal ions; Molecular weight; n-Hexane; Proteins; recombinant carboxylesterase; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sodium chloride; Substrate Specificity; Temperature; Thermal stability
• ISSN: 2218-273X; 2218-273X
• DOI: 10.3390/biom14050534

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 NRC-1 was cloned and successfully expressed in . 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 -nitrophenyl valerate as substrate (K = 78 µM, k = 0.67 s ). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, -hexane, -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.