Abstract
BackgroundThe ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture.ResultsRecently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steady-state kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme.ConclusionP. pastoris resulted to be an optimum biofactory for the heterologous production of recombinant sterol esterase from O. piceae, yielding higher activity levels than those obtained with the saprophytic fungus. The enzyme showed improved kinetic parameters because of its modified N-terminus, which allowed changes in its aggregation behaviour, suggesting that its hydrophobicity has been modified.
Highlights
The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards pnitrophenol, glycerol and sterol esters
An additional reason to choose this system to express the O. piceae sterol esterase was that other fungal lipases have been successfully expressed in this yeast, such as the different isoenzymes (Lip1-Lip5) from C. rugosa [8,10,40,41,42,43,44] and G. candidum [45,46], as well as the sterol esterase from M. albomyces [15]
The screening to select the clones of P. pastoris with sterol esterase activity was carried out by using a simple plate activity assay [47] based on the hydrolysis of tributyrin in MM medium, which showed clear halos in the positive transformants
Summary
The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards pnitrophenol, glycerol and sterol esters. Most of them share a common structural backbone belonging to the structural superfamily of α/β-hydrolases, like esterases and lipases, where residues responsible for its catalytic activity are highly conserved and form the socalled catalytic triad Ser-Asp/Glu-His [17], being the serine residue the nucleophile responsible for the beginning of catalysis. For this reason, all these enzymes are known as serine hydrolases. These proteins show tertiary structure but dimeric, tetrameric, and even hexameric forms or more can be found because of aggregation phenomena giving these pseudo-quaternary structures [6,19,20,21]
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