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

Overview

Journal Biomolecules

Publisher MDPI

Specialties Biochemistry
Molecular Biology

Date 2024 May 24

PMID 38785941

Authors

Nestor David Ortega-de la Rosa

Evelyn Romero-Borbon

Jorge Alberto Rodriguez

Angeles Camacho-Ruiz

Jesus Cordova

Affiliations

Soon will be listed here.

Abstract

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.

References

Pfeifer K, Ergal I, Koller M, Basen M, Schuster B, Rittmann S . Archaea Biotechnology. Biotechnol Adv. 2020; 47:107668. DOI: 10.1016/j.biotechadv.2020.107668. View

Simossis V, Heringa J . PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information. Nucleic Acids Res. 2005; 33(Web Server issue):W289-94. PMC: 1160151. DOI: 10.1093/nar/gki390. View

Suzuki Y, Miyamoto K, Ohta H . A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. FEMS Microbiol Lett. 2004; 236(1):97-102. DOI: 10.1016/j.femsle.2004.05.026. View

Allers T . Overexpression and purification of halophilic proteins in Haloferax volcanii. Bioeng Bugs. 2011; 1(4):288-90. PMC: 3026470. DOI: 10.4161/bbug.1.4.11794. View

Camacho R, Mateos-Diaz J, Diaz-Montano D, Gonzalez-Reynoso O, Cordova J . Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1. Extremophiles. 2009; 14(1):99-106. DOI: 10.1007/s00792-009-0291-x. View

Shao H, Xu L, Yan Y . Biochemical characterization of a carboxylesterase from the archaeon Pyrobaculum sp. 1860 and a rational explanation of its substrate specificity and thermostability. Int J Mol Sci. 2014; 15(9):16885-910. PMC: 4200780. DOI: 10.3390/ijms150916885. View

Camacho R, Mateos J, Gonzalez-Reynoso O, Prado L, Cordova J . Production and characterization of esterase and lipase from Haloarcula marismortui. J Ind Microbiol Biotechnol. 2009; 36(7):901-9. DOI: 10.1007/s10295-009-0568-1. View

Le L, Yoo W, Jeon S, Kim K, Kim T . Characterization and Immobilization of a Novel SGNH Family Esterase (SGNH1) from NCFM. Int J Mol Sci. 2019; 21(1). PMC: 6981805. DOI: 10.3390/ijms21010091. View

Kumar Meghwanshi G, Verma S, Srivastava V, Kumar R . Archaeal lipolytic enzymes: Current developments and further prospects. Biotechnol Adv. 2022; 61:108054. DOI: 10.1016/j.biotechadv.2022.108054. View

10.

Liu Y, Zhang Y, Wen H, Liu X, Fan X . Cloning and rational modification of a cold-adapted esterase for phthalate esters and parabens degradation. Chemosphere. 2023; 325:138393. DOI: 10.1016/j.chemosphere.2023.138393. View

11.

Grajales-Hernandez D, Velasco-Lozano S, Armendariz-Ruiz M, Rodriguez-Gonzalez J, Camacho-Ruiz R, Asaff-Torres A . Carrier-bound and carrier-free immobilization of type A feruloyl esterase from Aspergillus niger: Searching for an operationally stable heterogeneous biocatalyst for the synthesis of butyl hydroxycinnamates. J Biotechnol. 2020; 316:6-16. DOI: 10.1016/j.jbiotec.2020.04.004. View

12.

Martin Del Campo M, Camacho R, Mateos-Diaz J, Muller-Santos M, Cordova J, Rodriguez J . Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea. Extremophiles. 2015; 19(6):1121-32. DOI: 10.1007/s00792-015-0784-8. View

13.

Robert X, Gouet P . Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014; 42(Web Server issue):W320-4. PMC: 4086106. DOI: 10.1093/nar/gku316. View

14.

Johan U, Rahman R, Kamarudin N, Latip W, Mohamad Ali M . A new hyper-thermostable carboxylesterase from Anoxybacillus geothermalis D9. Int J Biol Macromol. 2022; 222(Pt B):2486-2497. DOI: 10.1016/j.ijbiomac.2022.10.033. View

15.

Liu Y, Tang S, Wang X, Wang X, Tang X, Wu Q . A novel thermostable and salt-tolerant carboxylesterase involved in the initial aerobic degradation pathway for pyrethroids in Glycomyces salinus. J Hazard Mater. 2023; 451:131128. DOI: 10.1016/j.jhazmat.2023.131128. View

16.

Liu X, Zhou M, Xing S, Wu T, He H, Bielicki J . Identification and Biochemical Characterization of a Novel Hormone-Sensitive Lipase Family Esterase Est19 from the Antarctic Bacterium sp. E2-15. Biomolecules. 2021; 11(11). PMC: 8615396. DOI: 10.3390/biom11111552. View

17.

Cordova-Garcia G, Esquivel C, Perez-Staples D, Ruiz-May E, Herrera-Cruz M, Reyes-Hernandez M . Characterization of reproductive proteins in the Mexican fruit fly points towards the evolution of novel functions. Proc Biol Sci. 2022; 289(1977):20212806. PMC: 9240691. DOI: 10.1098/rspb.2021.2806. View

18.

Muller-Santos M, de Souza E, de O Pedrosa F, Mitchell D, Longhi S, Carriere F . First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui. Biochim Biophys Acta. 2009; 1791(8):719-29. DOI: 10.1016/j.bbalip.2009.03.006. View

19.

Jaeger K, Dijkstra B, Reetz M . Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol. 1999; 53:315-51. DOI: 10.1146/annurev.micro.53.1.315. View

20.

Kelley L, Mezulis S, Yates C, Wass M, Sternberg M . The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10(6):845-58. PMC: 5298202. DOI: 10.1038/nprot.2015.053. View