Perspectives for biocatalytic lignin utilization: cleaving 4-O-5 and C??-C?? bonds in dimeric lignin model compounds catalyzed by a promiscuous activity of tyrosinase

Background: In the biorefinery utilizing lignocellulosic biomasses, lignin decomposition to value-added phenolic derivatives is a key issue, and recently biocatalytic delignification is emerging owing to its superior selectivity, low energy consumption, and unparalleled sustainability. However, besides heme-containing peroxidases and laccases, information about lignolytic biocatalysts is still limited till date. Results: Herein, we report a promiscuous activity of tyrosinase which is closely associated with delignification requiring high redox potentials (>1.4 V vs. normal hydrogen electrode [NHE]). The promiscuous activity of tyrosinase not only oxidizes veratryl alcohol, a commonly used nonphenolic substrate for assaying ligninolytic activity, to veratraldehyde but also cleaves the 4-O-5 and C??-C?? bonds in 4-phenoxyphenol and guaiacyl glycerol-??-guaiacyl ether (GGE) that are dimeric lignin model compounds. Cyclic voltammograms additionally verified that the promiscuous activity oxidizes lignin-related high redox potential substrates. Conclusion These results might be applicable for extending the versatility of tyrosinase toward biocatalytic delignification as well as suggesting a new perspective for sustainable lignin utilization. Furthermore, the results provide insight for exploring the previously unknown promiscuous activities of biocatalysts much more diverse than ever thought before, thereby innovatively expanding the applicable area of biocatalysis

Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells

It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with L-3,4-dihydroxyphenylalanine (L-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme

The crystal structure of an extracellular catechol oxidase from the ascomycete fungus Aspergillus oryzae

Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled type 3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. The crystal structures of a limited number of bacterial and fungal tyrosinases and plant catechol oxidases have been solved. In this study, we present the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4) at 2.5-Å resolution. AoCO4 belongs to the newly discovered family of short-tyrosinases, which are distinct from other tyrosinases and catechol oxidases because of their lack of the conserved C-terminal domain and differences in the histidine pattern for CuA. The sequence identity of AoCO4 with other structurally known enzymes is low (less than 30 %), and the crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region. A diatomic oxygen moiety was identified as a bridging molecule between the two copper ions CuA and CuB separated by a distance of 4.2–4.3 Å. The UV/vis absorption spectrum of AoCO4 exhibits a distinct maximum of absorbance at 350 nm, which has been reported to be typical of the oxy form of type 3 copper enzymes