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Development of a biosensing platform based on a laccase-hydrophobin chimera

Sorrentino, Ilaria, Giardina, Paola, Piscitelli, Alessandra
Applied microbiology and biotechnology 2019 v.103 no.7 pp. 3061-3071
L-dopa, Pichia pastoris, Pleurotus ostreatus, biosensors, caffeic acid, chemical species, coatings, culture media, gene fusion, hydrophobins, laccase, oxidation, polystyrenes, propionic acid, recombinant fusion proteins
A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.