ABSTRACT
The establishment of a fermentation process for the production of pig liver esterase (PLE) in high yields is necessary for industrial applications. In our previous studies, we reported the recombinant expression of PLE in Escherichia coli Origami (DE3) in shake flask. Only a coexpression with chaperones GroEL/ES allowed the production of soluble and active enzyme. The optimization of the cultivation conditions, such as temperature, inducer concentrations, or media compositions to increase enzyme yield in a fermentation process is described here. Using fed-batch fermentation cell densities up to OD = 50 were obtained, but almost no active enzyme was expressed. Only batch fermentation was found suitable for production of active pig liver esterase and cell densities between OD = 7-13 and activities of 300-400 U L(-1) for isoenzyme PLE-1 (gammaPLE) and 1,400 U L(-1) for PLE-5 were obtained after 22 h total cultivation time or 18 h after induction of PLE expression, respectively.
Subject(s)
Cloning, Molecular/methods , Escherichia coli/genetics , Esterases/biosynthesis , Liver/enzymology , Affinity Labels , Animals , Esterases/genetics , Fermentation , Industrial Microbiology , Recombinant Fusion Proteins , Swine , Temperature , Thioredoxin-Disulfide Reductase/geneticsABSTRACT
The possible physiological role of PLE (E.C. 3.1.1.1) located in the endoplasmic reticulum (ER) of pig liver cells in the conversion of endogenous compounds was investigated as it was reported, that PLE acts as prenylated methylated protein methyl esterase (PMPMEase) hydrolysing methylesters of prenylated proteins. Using the specific PMPMEase substrate benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM), six different PLE isoenzymes expressed recombinantly in the yeast Pichia pastoris were found active. Activities ranged from 1.6-15.6mU per mg protein and it is suggested that Pro285 has a major influence on high activity. In addition, the role of the C-terminal HAEL retention signal for translocation of pig liver esterase (PLE) in the endoplasmic reticulum (ER) of eukaryotic cells was studied using the gamma-isoenzyme of PLE expressed in Pichia pastoris. Using truncated versions (HAE, HA, H and without retention signal) of the enzyme it was found that in contrast to earlier reports no influence of the signal peptide on the expression rate of PLE was found. However, higher enzyme activities were obtained in the periplasmatic fraction compared to the supernatant irrespective of the presence or absence of HAEL and the trimeric formation seems to occur in the supernatant of P. pastoris X33 enabling an easier transition of monomeric forms through cell membranes.
Subject(s)
Endoplasmic Reticulum/enzymology , Esterases/metabolism , Liver/enzymology , Swine/metabolism , Amino Acid Motifs , Animals , Electrophoresis, Polyacrylamide Gel , Isoenzymes/metabolism , Methylation , Models, Molecular , Mutagenesis, Site-Directed , Protein Prenylation , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
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.
