Versatile fungal polyphenol oxidase with chlorophenol bioremediation potential : Characterization and protein engineering

• Polyphenol oxidases (PPOs) have been mostly associated with the undesirable post-harvest browning in fruits and vegetables and with implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome of Thermothelomyces thermophila and expressed in Pichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/L and it appeared to be a glycosylated and N-terminally processed protein. TtPPO showed broad substrate specificity as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols and methoxyphenols. Its optimum temperature was 65 °C with a half-life of 18.3 h at 50 °C, while its optimum pH was 7.5. The homology model of TtPPO was constructed and site-directed mutagenesis was performed in order to increase its activity on mono- and di-chlorophenols (CPs). TtPPO variant G292N/Y296V had a 5.3-fold increased activity on 3,5-diCP compared to wild-type.Importance A novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)

Nyckelord

Biokemisk processteknik

Biochemical Process Engineering

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