The development of methods for the structure elucidation of riverine humic-derived iron- complexes is challenging but an essential task at the present time since they play a pivotal role in maintaining the productivity of marine ecosystems. Low-molecular-weight phenolic compounds represent the most important group of iron-carrying humic substances in peatland-influenced rivers. These compounds may be converted into multidentate Fe-binding ligands via the activity of polyphenol oxidases produced by soil bacteria.[1] In order to develop an accurate model system for the formation of these iron-chelating ligands we focus our studies on the immobilization of a functional mushroom tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4, 65 kDa)[2] on mesoporous Santa Barbara Amorphous type material #15 (SBA-15, pore size: 8.5 nm). The enzyme adsorption isotherm was obtained at 4 °C and pH 7.4, resulting in a maximum amount of 7.2 μmol of immobilized AbPPO4 per gram of non-modified SBA-15. To verify that the tyrosinase preserved its catalytic performance throughout the immobilization process, enzymatic activity tests were developed following the oxidation of L-tyrosine (2 mM) by UV-vis spectroscopy (λ = 475 nm). The influence of the surface charge of the silica support on the loading efficiency of AbPPO4 was evaluated through functionalization of SBA-15 with a positively charged amino-silane, 3-Aminopropyltriethoxysilane (APTS), introduced by a post-grafting strategy.[3] When the specific enzymatic activity of the obtained catalyst (SBA15-NH₂-Tyr) was compared with the free enzyme a considerable decrease in the catalytic performance of the immobilized AbPPO4 was observed. This apparent drop in activity could be attributed to absorption of the reaction product Dopachrome in the mesopores, which was removed after several washings. The oxidation of other phenolic compounds (4-tert-butylcatechol, dopamine and tyramine) was followed by ¹H-NMR, HPLC/MS and UV-Vis using AbPPO4 loaded on non-functionalized SBA-15 (20% w/w), which allowed to elucidate the structure of different oxidized products. This catalyst (SBA15-Tyr-20) exhibited enhanced stability over a wide range of pH and temperatures compared to the free AbPPO4, retaining enzymatic activity after 10 cycles of reuse and up to 66% of its initial biocatalytic performance after 6 months of immobilization. The results obtained point to the possible use of SBA15-Tyr-20 for the preparation of a column-type bioreactor to mimic specific processes in the soil and allow structure elucidation of humic-derived iron- complexes.

References
[1]    H. Kang, M. J. Kwon, S. Kim, S. Lee, T. G. Jones, A. C. Johncock, A. Haraguchi, C. Freeman, Nat. Commun. 2018, 9, DOI 10.1038/s41467-018-06259-1.
[2]    M. Pretzler, A. Bijelic, A. Rompel, Sci. Rep. 2017, 7, 1–10, DOI 10.1038/s41598-017-01813-1.
[3]    C. von Baeckmann, R. Guillet-Nicolas, D. Renfer, H. Kählig, F. Kleitz, ACS Omega 2018, 3, 17496–17510, DOI 10.1021/acsomega.8b02784.