Construction of a laccase chimerical gene: recombinant protein characterization and gene expression via yeast surface display.
The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.
CNR - Istituto di Scienze delle Produzioni Alimentari (ISPA), via Lecce-Monteroni, Lecce, 73100, Italy.
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G
Bleve
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Lezzi
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Spagnolo
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Rampino
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Perrotta
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Mita
Francesco
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Grieco
D061505:Cell Surface Display Techniques; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000235:genetics* / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000378:metabolism; D020076:Pleurotus / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
C087578:FLO1 protein, S cerevisiae; D005656:Fungal Proteins; D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D006360:Heat-Shock Proteins; D037241:Mannose-Binding Lectins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D042845:Laccase