Characterization and Low-Resolution Structure of an Extremely Thermostable Esterase of Potential Biotechnological Interest from Pyrococcus furiosus.
Enzymes isolated from extremophiles often exhibit superior performance and potential industrial applications. There are several advantages performing biocatalysis at elevated temperatures, including enhanced reaction rates, increased substrate solubility and decreased risks of contamination. Furthermore, thermophilic enzymes usually exhibit high resistance against many organic solvents and detergents, and are also more resistant to proteolytic attack. In this study, we subcloned and characterized an esterase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Est) that exhibits optimal activity around 80 °C using naphthol-derived substrates and p-nitrophenyl palmitate (pNPP). According to the circular dichroism spectra, the secondary structure of P. furiosus esterase, which is predominantly formed by a β-sheet structure, is very stable, even after incubation at 120°C. We performed SAXS to determine the low-resolution structure of Pf_Est, which is monomeric in solution at 80 °C and has a molecular weight of 28 kDa. The Km and Vmaxvalues for this esterase acting on pNPP were 0.53 mmol/L and 6.5 × 10-3U, respectively. Pf_Est was most active in the immiscible solvents and retained more than 50 % in miscible solvents. Moreover, Pf_Est possesses transesterification capacity, presenting better results when isobutanol was used as an acyl acceptor (2.69 ± 0.14 × 10-2μmol/min mg) and the highest hydrolytic activity toward olive oil among different types of oils testes in this study. Collectively, these biophysical and catalytic properties are of interest for several biotechnological applications that require harsh conditions, including high temperature and the presence of organic solvents.
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe Máximo Scolfaro, nº 10000, Campinas, SP, 13083-970, Brazil.
F
F
Mandelli
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe Máximo Scolfaro, nº 10000, Campinas, SP, 13083-970, Brazil.|Departamento de Bioquímica, Instituto de Biologia (IB), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
T A
TA
Gonçalves
Departamento de Física e Biofísica, Instituto de Biociências de Botucatu, UNESP Univ Estadual Paulista, Botucatu, SP, Brazil.
C A
CA
Gandin
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe Máximo Scolfaro, nº 10000, Campinas, SP, 13083-970, Brazil.|Departamento de Bioquímica, Instituto de Biologia (IB), Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
A C P
AC
Oliveira
Departamento de Física e Biofísica, Instituto de Biociências de Botucatu, UNESP Univ Estadual Paulista, Botucatu, SP, Brazil.
M
M
Oliveira Neto
Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe Máximo Scolfaro, nº 10000, Campinas, SP, 13083-970, Brazil. fabio.squina@bioetanol.org.br.
F M
FM
Squina
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D008970:Molecular Weight; D009284:Naphthols / Q000378:metabolism; D017433:Protein Structure, Secondary; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D019843:Archaeal Proteins; D009284:Naphthols; D004950:Esterases
Circular dichroism; Hyperthermophilic; SAXS; Transesterification; kinetics parameters
Protein Expr Purif. 2015 Dec;116:90-7
26276473
Appl Environ Microbiol. 2006 Apr;72(4):3021-5
16598011
Biochem J. 1999 Oct 1;343 Pt 1:177-83
10493927
Nat Protoc. 2006;1(6):2876-90
17406547
PLoS One. 2013 Jul 29;8(7):e70014
23922891
PLoS One. 2010 Oct 01;5(10):null
20957207
FEMS Microbiol Lett. 1999 Oct 15;179(2):241-6
10518722
Biochem Soc Symp. 1992;58:149-69
1445404
Microb Biotechnol. 2010 Nov;3(6):677-90
21255363
Appl Microbiol Biotechnol. 2013 Aug;97(15):6759-67
23229566
Biochemistry. 2015 Mar 10;54(9):1841-8
25711934
Lett Appl Microbiol. 2013 Jul;57(1):40-6
23530753
Biotechnol Lett. 2011 Sep;33(9):1841-6
21544610
Biochemistry. 2001 Oct 23;40(42):12524-32
11601976
Appl Microbiol Biotechnol. 2005 Apr;67(2):160-9
15630579
Protein Expr Purif. 2011 Aug;78(2):120-30
21513802
Biochimie. 2000 Nov;82(11):1063-85
11099803
Trends Biotechnol. 1998 Sep;16(9):396-403
9744114
FEBS J. 2007 Jun;274(11):2832-42
17466017
Enzyme Microb Technol. 1993 Feb;15(2):133-9
7763454
Protein Expr Purif. 2008 Jun;59(2):327-33
18400514
Appl Biochem Biotechnol. 2015 May;176(1):1-12
25869294
Protein Pept Lett. 2012 Feb;19(2):132-43
21933125
FEMS Microbiol Rev. 2002 Mar;26(1):73-81
12007643
Braz J Microbiol. 2014 Mar 10;44(4):1113-9
24688500
Eur J Biochem. 1992 Jan 15;203(1-2):25-32
1730231
J Biol Chem. 2014 Jul 4;289(27):19031-41
24867954
Biotechnol Bioeng. 1998 Mar 5;57(5):624-9
10099242
Curr Protein Pept Sci. 2000 Dec;1(4):349-84
12369905
FEBS J. 2013 Dec;280(24):6658-71
24127688
Acta Biochim Biophys Sin (Shanghai). 2010 Apr;42(4):288-95
20383468