"Ετερόλογη έκφραση και χαρακτηρισμός μίας λακκάσης του θερμόφιλου μύκητα Myceliopthora thermophila - χρήση της σε εφαρμογές βιοτεχνολογικού ενδιαφέροντος"

Αμαλία Καραγκούνη Καθηγήτρια, Γιώργος Διαλλινάς Καθηγητής, Δημήτρης Χατζινικολάου Επίκ. Καθηγητής (Επιβλέπων)

"Ετερόλογη έκφραση και χαρακτηρισμός μίας λακκάσης του θερμόφιλου μύκητα Myceliopthora thermophila - χρήση της σε εφαρμογές βιοτεχνολογικού ενδιαφέροντος"

"Heterologous expression and characterization of a laccase of the thermophilic fungus Myceliopthora thermophila - use in applications of biotechnological interest"

Laccases (EC 1.10.3.1) are multicopper enzymes that catalyze the oxidation of a variety of phenolic compounds, with concomitant reduction of O2 to H2O. Their unique characteristics such as high chemical stability, the ability to catalyze both anabolic and catabolic procedures and the ecological nature of the reaction make them extremely useful tools for biotechnology applications. Myceliopthora thermophila is a thermophilic fungus capable of degrading cellulosic biomass at high speeds. This fungus produces a variety of enzymes such as endoglucanases, exoglucanases, laccases, xylanases, enzymes that are used in various industrial applications. In the present study, a laccase from Myceliopthora thermophila was heterologously expressed in the methylotrophic yeast Pichia pastoris. The selected gene had a total size of 2093 bp and was organized in 3 exons (258,1498,197 bp) interrupted by 2 introns (58 and 82 bp). The removal of the interfering sequences was achieved using the overlapping PCR technique. The gene was then inserted into a specific vector and was heterologously expressed in Pichia pastoris. The isolated protein from the heterologous expression was tested for the ability to oxidize ABTS and the ability to oxidize ascorbic acid. For the biotechnological use of laccases, an industrially available laccase from Myceliopthora thermophila was used to polymerize a variety of aromatic compounds. A variety of factors affecting the reaction were tested such as the enzyme-substrate ratio, the temperature, pH and time of the reaction. The evaluation of the isolated polymers was examined using ultraviolet-visible (UV-vis) spectrums and infrared (FT-IR) spectrums.

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