Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris.
BACKGROUND
The 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.


RESULTS
Recently, 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.


CONCLUSIONS
P. 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.
Centro de Investigaciones Biológicas (CIB), Spanish National Research Council (CSIC), Ramiro de Maeztu 9, Madrid 28040, Spain.
Víctor Barba
VB
Cedillo
Francisco J
FJ
Plou
María Jesús
MJ
Martínez
D055162:Biocatalysis; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D055156:Ophiostoma / Q000201:enzymology*; D010084:Oxidation-Reduction; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008715:Methionine; D002787:Sterol Esterase