Microbial host selection and periplasmic folding in Escherichia coli affect the biochemical characteristics of a cutinase from Fusarium oxysporum.
A cutinase from the mesophilic fungus Fusarium oxysporum (FoCut5a) was functionally expressed in different hosts and their recombinant products were characterized regarding their activity, thermostability and tolerance in organic solvents. The cutinase gene cut5a was expressed in the BL21 and Origami 2 Escherichia coli strains and the resulting protein was folded either in the cytoplasm or in the periplasmic space, with the aim of correct formation of disulfide bonds. Increase of thermostability occurred when the enzyme was expressed in the oxidative cytoplasm of Origami 2. All expression products showed maximum enzyme activity at 40 °C, while thermostability increased by 73% when expressed in the Origami strain compared to the cytoplasmic expression in BL21 cells. The melting temperature of each protein construct was determined by fluorescence spectroscopy showing an additional transition at about 63 °C for enzymes expressed in Origami cells, indicating the co-presence of a different thermostable species. Kinetic studies performed on three p-nitrophenyl synthetic esters of aliphatic acids (C2, C4, C12) indicated that this cutinase shows higher affinity for the hydrolysis of the butyl ester.
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece.
Efstratios
E
Nikolaivits
Physics Laboratory, Department of Biotechnology, Agricultural University of Athens, Athens 11855, Greece.
Areti
A
Kokkinou
Physics Laboratory, Department of Biotechnology, Agricultural University of Athens, Athens 11855, Greece.
Michael
M
Karpusas
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece. Electronic address: vtopakas@chemeng.ntua.gr.
Evangelos
E
Topakas
D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D005670:Fusarium / Q000235:genetics*; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004220:Disulfides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
Cutinase; Escherichia coli; Fusarium oxysporum; Heterologous expression; Origami; Thermostability