Diversity screening for novel enzymes degrading synthetic polymers

The objective of this PhD study was to evaluate the feasibility of enzymatic degradation of synthetic polymers used as binder materials in marine coatings. Enzymatic modification of synthetic polymers like epoxy resin, polyurethanes and various acrylics is desirable in several industrial processes: waste management, production of biofuels and valueadded biochemicals.

Secondly, the goal was to identify new ways in which relevant enzymatic activities may be identified. Finally, the aim was to contribute to the known diversity of biocatalysts with potential to: oxidize a number of low-molecular phenolics – including some of low-molecular coating binder models, perform the kinetic resolution of selected epoxides and to enzymatically fucosylate various oligosaccharide molecules.

In this work discovery and characterization of a wide diversity of novel enzymes of bacterial and fungal origin is reported. First, a collection of fungal strains was screened for the capability to degrade several compounds of synthetic origin. Strains with the ability to modify colloidal polyester polyurethane, as well as various commercial emulsions of acrylates were identified.

Secondly, we have used metagenomic library from heavily polluted soil – one of the most challenging habitats and source of potent enzymes, potentially degrading a range of recalcitrant xenobiotics from oil and paint industries. It is well-establisehd that ‘you get what you screen for’, therefore the major challenge was the availability and the choice of proper substrates for identification of promising enzyme candidates.

Several genes coding for enzymes with the capability to oxidize various natural and synthetic substrates were identified, expressed heterologously and characterized. Three multicopper oxidases (MCOs) were identified encoded by genes identified in a metagenomic library constructed from soil contaminated with heavy metals and various paint residues.

Mco1 is a three domain CopA-like protein and Mco2 and Mco3 are representatives of bacterial twodomain mutlicopper oxidases (MCOs). The enzymes catalyzed oxidation of 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,6-Dimethylphenol (2,6-DMP) under diverse reaction conditions and catalyzed the conversion of majority of aromatic alcohols tested.

Under conditions tested, the enzymes catalyzed decolourization of common industrial wastewater dye contaminants. Mco2 was identified as being a hyperthermostable enzyme with a remarkable half-life of 21 hours at 80 OC. Cloning and heterologous expression of a potent laccase from the fungus Ganoderma lucidum was also performed.

Interestingly, the recombinant LacGL1 enzyme was able to promote higher yields of glucose during cellulase-catalyzed hydrolysis of pretreated sugarcane bagasse. We have further utilized the constructed metagenomic library for functional identification of epoxide hydrolase activities using a new agar-plate assay.

Using this method, clones with epoxide hydrolase activity were identified. mepox1 gene was re-cloned for heterologous production of recombinant epoxide hydrolase. Despite low enantioselectivity, the purified enzyme exhibited a broad substrate specificity and wide pH range. Finally, the discovery of novel metagenome-originating α-L-fucosidases and evaluation of their potential to catalyze the transglycosylation reaction to produce fucosylated human milk oligosaccharides is reported in this work.

Seven fucosidases were identified, all belonging glycosyl hydrolase family 29, that exhibit different hydrolytic capacity for 2’- fucosyllactose, 3-fucosyllactose and various fucosylated plant cell wall polymers. Several enzymes catalysed transglycosylation either using lactose or pNP-Fuc as acceptor and Mfuc6 exhibited an unusually high transglycosylation/hydrolysis ratio.

Using 25 mM pNP-Fuc as donor and under conditions tested, the maximum yields of 1.6 ± 0.1 mM 2’-fucosyllactose and 0.9 ± 0.01 mM of 3-fucosyllactose were achieved. In conclusion, results included in this work further substantiate that the functional mining of a metagenome can lead to the successful discovery of diverse biocatalysts.

This work paves the way for efficient identification of novel enzymatic catalysts and generates enzymes as well as small molecules with valuable biological properties and market potential.