Efstratios Nikolaivits

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential.

BACKGROUND Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers. METHODS A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues. RESULTS The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16°C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40°C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C(2), C(4), C(12)), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers. CONCLUSIONS The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. GENERAL SIGNIFICANCE FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

Marine-Derived Biocatalysts: Importance, Accessing, and Application in Aromatic Pollutant Bioremediation

The aim of the present review is to highlight the potential use of marine biocatalysts (whole cells or enzymes) as an alternative bioprocess for the degradation of aromatic pollutants. Firstly, information about the characteristics of the still underexplored marine environment and the available scientific tools used to access novel marine-derived biocatalysts is provided. Marine-derived enzymes, such as dioxygenases and dehalogenases, and the involved catalytic mechanisms for the degradation of aromatic and halogenated compounds, are presented, with the purpose of underpinning their potential use in bioremediation. Emphasis is given on persistent organic pollutants (POPs) that are organic compounds with significant impact on health and environment due to their resistance in degradation. POPs bioaccumulate mainly in the fatty tissue of living organisms, therefore current efforts are mostly focused on the restriction of their use and production, since their removal is still unclear. A brief description of the guidelines and criteria that render a pollutant POP is given, as well as their potential biodegradation by marine microorganisms by surveying recent developments in this rather unexplored field.

Enzymatic tailoring of oleuropein from Olea europaea leaves and product identification by HRMS/MS spectrometry

Oleuropein, a bioactive compound found in all parts of olive tree, especially in leaves and branches, presents numerous health promoting properties that increase research and market interest the last few years. In addition, oleuropein degradation products, such as hydroxytyrosol, elenolic acid, and the aglycones also exhibit biological activities with different properties compared to the starting compound. Under this view, a commercial lipase preparation Lipolase 100L and a thermophilic β-glucosidase from Myceliophthora thermophila were used for the regioselective hydrolysis of oleuropein towards the production of the corresponding biologically active compounds. The enzymatic degradation products of oleuropein, such as hydroxytyrosol, elenolic acid and its glucoside, and oleuropein aglycones were identified by LC-HRMS/MS and NMR spectroscopy. The latter, was found as a mix of diastereomers of the monoaldehydic form of oleuropein aglycone, identified as (5S, 8R, 9S)-, (5S, 8S, 9S)- and (5S, 8R, 9R). The high substrate specificity exhibited by both lipase and β-glucosidase allows the successful tailoring of oleuropein towards the production of different biologically active compounds with significant potential in the cosmeceutical and food industry.