Abstract
The previously reported functional expression of the γ-isoenzyme of pig liver carboxylesterase (γ-rPLE) in Pichia pastoris is hampered by the small amount of active enzyme formed. Earlier attempts for expression in Escherichia coli failed completely and not even inactive protein was detected. The lack of glycosylation ability of E. coli was ruled out as a possible reason, as it could be shown in this work that deglycosylated PLE also is active. Expression of γ-rPLE was studied using a range of E. coli strains with careful design of the constructs used and control of the cultivation conditions. Indeed, expression in E. coli strains Rosetta, Origami and Rosetta-gami was successful, but the majority of enzymes was present as inclusion bodies and only little soluble but inactive protein was detected. Denaturation and refolding of inclusion bodies failed. However, with the E. coli strain Origami, coexpressing the molecular chaperones GroEL und GroES, a functional expression of γ-rPLE was possible. The recombinant enzyme was released by cell disruption and subjected to His-tag purification. The purified esterase had a specific activity of 92 U mg−1 protein and a V max/K m value of 10.8×10−3 min−1 towards p-nitrophenyl acetate. Activity staining of native polyacrylamide gels gave a single band at 175 kDa with esterolytic activity indicating a trimeric form of γ-rPLE (∼60 kDa per monomer). γ-rPLE was biochemically characterized and its properties were compared to the enzyme previously expressed in P. pastoris. pH and temperature profiles were identical and highest activity was found at pH 8–8.5 and 60 °C, respectively. In the kinetic resolution of (R,S)-1-phenyl-2-butyl acetate with esterase from both expression hosts, similar enantioselectivities (E=50) were found.
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References
Besette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci 96:13703–13708
Bornscheuer UT, Kazlauskas RJ (2005) Hydrolases in organic synthesis—Regio—and stereoselective biotransformations. 2nd edn. Wiley-VCH, Weinheim
Brocca S, Persson M, Wehtje E, Adlercreutz P, Alberghina L, Lotti M (2000) Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa. Protein Sci 9:985–990
Chen CS, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299
Chung CT, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A 86:2172–2175
de Maria PD, Kossmann B, Potgrave N, Buchholz S, Trauthwein H, May O, Gröger H (2005) Improved process for the enantioselective hydrolysis of prochiral diethyl malonates catalyzed by pig liver esterase. Synlett 11:1746–1748
Faber K (2000) Biotransformations in organic chemistry, 3rd edn. Springer, Berlin Heidelberg New York
Heymann E, Junge W (1979) Characterization of the isoenzymes of pig-liver esterase. 1. Chemical studies. Eur J Biochem 95:509–518
Hirschberg HJHB, Simons J-WFA, Dekker N, Egmond MR (2001) Cloning, expression, purification and characterization of patatin, a novel phospholipase A. Eur J Biochem 268:5037–5044
Jones JB (1990) Esterases in organic synthesis: present and future. Pure Appl Chem 62:1445–1448
Jones JB, Hinks RS, Hultin PG (1985) Enzymes in organic synthesis. 33. Stereoselective pig liver esterase-catalyzed hydrolyses of meso cyclopentyl-, tetrahydrofuranyl-, and tetrahydrothiophenyl-1,3-diesters. Can J Chem 63:452–456
Krebsfänger N, Zocher F, Altenbuchner J, Bornscheuer UT (1998) Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens. Enzyme Microb Technol 21:641–646
Lam LKP, Hui RAHF, Jones JB (1986) Enzymes in organic synthesis. 35. Stereoselective pig liver esterase catalyzed hydrolyses of 3-substituted glutarate diesters. Optimization of enantiomeric excess via reaction conditions control. J Org Chem 51:2047–2050
Lange S, Musidlowska A, Schmidt-Dannert C, Schmitt J, Bornscheuer UT (2001) Cloning, functional expression, and characterization of recombinant pig liver esterase. ChemBioChem 2:576–582
Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538
Musidlowska A, Lange S, Bornscheuer UT (2001) By overexpression in the yeast Pichia pastoris to enhanced enantioselectivity: new aspects in the application of pig liver esterase. Angew Chem Int Ed 40:2851–2853
Musidlowska-Persson A, Bornscheuer UT (2002) Substrate specificity of the γ-isoenzyme of recombinant pig liver esterase towards acetates of secondary alcohols. J Mol Catal B Enzym 19–20:129–133
Musidlowska-Persson A, Bornscheuer UT (2003) Site directed mutagenesis of recombinant pig liver esterase yields in mutant with altered enantioselectivity. Tetrahedron Asymmetry 14:1341–1344
Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ and GroEL-GroES in assisting folding of an allergen of Japanese Cedar Pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699
Novy R, Drott D, Yaeger K, Mierendorf R (2001) Overcoming the codon bias of E. coli for enhanced protein expression. Innovations 12:1–3
Prinz WA, Aslund F, Holmgen A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272:14769–14775
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Snehasis J, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298
Thomas JG, Baneyx F (1997) Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli. Protein Expr Purif 11:289–296
Yoshimasu M, Tanaka T, Ahn J-K, Yada RY (2004) Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris. Glycobiology 14:417–429
Acknowledgements
We thank the Deutsche Bundesstiftung Umwelt (DBU, Osnabrück, Germany, Grant No. AZ13071 and AZ13141) for financial support and the BRAIN AG (Zwingenberg, Germany) for helpful discussions. We are also grateful to Dr. H. Trauthwein and Dr. O. May at Degussa’s Service Center Biocatalysis for their support.
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Böttcher, D., Brüsehaber, E., Doderer, K. et al. Functional expression of the γ-isoenzyme of pig liver carboxyl esterase in Escherichia coli . Appl Microbiol Biotechnol 73, 1282–1289 (2007). https://doi.org/10.1007/s00253-006-0585-1
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DOI: https://doi.org/10.1007/s00253-006-0585-1