Lac Man

Water is the basis of all life on our planet. Whether plants, animals, humans or microorganisms, we all depend on it for our survival. Water scarcity and purity cause conflicts worldwide. Above all, water pollution from drugs, such as antibiotics or estrogen derivatives and other non-medical pollutants is proving to be an emerging issue in industrialized countries. For this reason, in 2013 the EU Parliament adopted a directive on the constant analysis of water within the Union [1]. This so-called “watch list” was first evaluated in 2018 by the Joint Research Center [2]. The frequent exceedance of the Predicted No-Effect Concentration (PNEC) for 17-alpha-ethinylestradiol (EE2) was particularly noticeable. In addition to EE2, the watch list includes other potentially dangerous substances for humans and the environment, such as antibiotics and diclofenac (pain relievers). Many of these molecules are characterized by common properties. Examples include aromatic ring structure and their effect as endocrine disruptors (e.g. EE2) and / or the toxicity of these compounds (e.g. diclofenac, carbamazepine), which justify the hazard potential for humans and animals [3].

Why laccases?

Aromatic ring structures serve as potential targets for enzymes, the so-called laccases, that have been identified in various fungi and bacteria. Laccases oxidize phenol groups, creating phenoxyl radicals. Four oxidized electrons are stored and transferred via the copper cluster onto free molecular oxygen, which is reduced to water. The degradation products produced here have no negative effects on people and the environment [4]. Our goal was to improve the stability of these enzymes and by using our selected laccases of T. versicolor and S. cyaneus to neutralize a large number of pollutants due to their wide range of substrates.

How are laccases synthesized?

The laccases are produced using a classic biotechnological approach, in which the insert DNA T. versicolor or S.cyaneus and the expression vectors pPICZalpha or PET28 are amplified via PCR using primers for a subsequent gibson assembly and then assembled using the NEBuilder HiFi DNA Assembly Cloning Kit #E5520S. This is used to transform competent E.coli cells. The expression vector pPICZalpha is integrated into the genome in order to generate a stable expression strain [6]. Because the above Laccase Trametes versicolor has several disulfide bridges, production in eukaryotic cells is necessary. The Pichia pastoris X33 strain has been successfully used for the synthesis of laccases.

Why immobilization?

Immobilization using a mesoporous silica foam is suitable to make the laccases sustainable on the long run [7, 8]. Silicon-based materials are well suited because they are environmentally friendly, biocompatible and, above all, resistant to organic solvents and microbial attacks. Furthermore, the immobilization makes the laccases pH- and thermostable. In addition, immobilized enzymes have a halflife up to 18 times longer [9]. At the same time, the enzymes used are firmly bound to the matrix and therefore do not have to be filtered out of the wastewater again. This process also has the advantage that no genetically modified organisms can be released into the environment.

Innovation

Immobilizing enzymes on a mesoporous silica matrix via amino groups on their surface and glutaraldehyde as a cross-linking agent was reported to significantly decrease enzyme activity [10]. Because the active site could be sterically blocked by the immobilization process, we came up with the idea of adding a poly-Lysine-tag at the c-terminus of the laccase. In doing so, the immobilization rate near the active site is reduced and the structure of the laccase would remain almost unchanged, resembling the free enzyme.

How did we estimate the amount of immobilized enzyme needed?

In order to be able to analyze the properties of our foam and the immobilized laccases in regard to substrate conversion, we established a kinetic model according to Michaelis-Menten. In doing so, the foam is simulated by aligned compartments which represent a substrate concentration gradient inside the foam. This substrate conversion kinetic is further complemented by equations modelling temperature-dependence and other important parameters.

Applications

Could be applied in wastewater treatment plants, efflux of pharma companies, swimming pools and hospitals.

References

„RICHTLINIE 2013/39/EU DES EUROPÄISCHEN PARLAMENTS UND DES RATES,“ 2013.
D. M. I. S. D. N. a. T. L. Robert Loos, „Review of the 1st Watch List under the Water Framework Directivend and recommendations for the 2 Watch List,“ Publications Office of the European Union, Luxembourg, 2018.
P. G. P. G. Benoıt Ferrari, “Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac”, Ecotoxicology and Environmental Safety, vol. 55, 2003.
M. H. J. S. K. P. P. R. S. R. M. Y. Palanivel Sathishkumar, „Laccase mediated diclofenac trans- formation and cytotoxicity assessment on mouse fibroblast 3T3-L1 preadipocytes,“ RSC Adv., vol.4, 2014.
Guex N, Peitsch MC, Schwede T. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis. 2009;30:S162–S173.

Team:Stuttgart/Description