Functional expression of the gamma-isoenzyme of pig liver carboxyl esterase in Escherichia coli.
The previously reported functional expression of the gamma-isoenzyme of pig liver carboxylesterase (gamma-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 gamma-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 gamma-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 10.8x10(-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 gamma-rPLE ( approximately 60 kDa per monomer). gamma-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 degrees 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.
Böttcher
Dominique
D
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Friedrich-Ludwig-Jahnstr. 18c, 17487, Greifswald, Germany.
Brüsehaber
Elke
E
Doderer
Kai
K
Bornscheuer
Uwe T
UT
D000818:Animals; D043182:Carboxylesterase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013696:Temperature
D007527:Isoenzymes; D011994:Recombinant Proteins; D043182:Carboxylesterase