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

Abstract

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_α-C_β bonds in 4-phenoxyphenol and guaiacyl glycerol-β-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.

Keywords: 4-Phenoxyphenol; Guaiacyl glycerol-β-guaiacyl ether (GGE); Promiscuous activity; Sustainable lignin utilization; Tyrosinase.

Fig. 1

The catalytic promiscuity in tyrosinase oxidizes veratryl alcohol to veratraldehyde. Gas chromatographic (GC) profile and mass spectra of the catalytic product using veratryl alcohol as the substrate in the absence (control, blue line) and presence of 100 unit tyrosinase (reactant, red line). The analyte was extracted with ethyl acetate. The chromatographic peaks were identified by the retention time and mass spectra library (NIST02). Authentic standard chemicals were used to double check the identified substrates and products (standards, black line). Inset: (i) mass spectrum and (ii) magnification of the peak at 10.95 min, and (iii) mass spectrum of the peak at 14.63 min

Fig. 2

The cyclic voltammetry. a The cyclic voltammograms of phosphate buffer (50 mM, pH 6.5) and tyrosinase with and without veratryl alcohol in phosphate buffer (50 mM, pH 6.5). Only in tyrosinase in the coexistence of veratryl alcohol, the oxidation peak was shown at +1.22 V vs. Ag/AgCl, which might be corresponding to +1.42 V vs. NHE. b The cyclic voltammogram of veratryl alcohol (1.7 mM) and veratraldehyde (1.7 mM) in phosphate buffer (pH 6.5). NO oxidation and reduction peaks were observed in each of veratryl alcohol and veratraldehyde. In all the cyclic voltammetries, glassy carbon, coiled Pt wire, and Ag/AgCl electrode were used as the working, counter, and reference electrode, respectively. Cyclic voltammetry was carried out using potentiostat/galvanostat controlled by commercial WMPG software. The scan rate was 50 mVs⁻¹

Fig. 3

Cleavage of 4-O-5 bond in 4-phenoxyphenol by the catalytic promiscuity of tyrosinase. a GC profile and mass spectra (MS) of the catalytic product using 4-phenoxyphenol in the absence (control, blue line) and presence of 100 unit tyrosinase (reactant, red line). The analyte was extracted with ethyl acetate. The chromatographic peaks were identified by the retention time and mass spectra library (NIST02). The authentic standard chemicals were used to confirm the identified (standard, black line). Inset: (i) mass spectrum and (ii) magnification of the peak at 10.15 min, and (iii) mass spectrum of the peak at 39.7 min. b Reaction scheme illustrates the catalytic promiscuity cleaves the 4-O-5 bond in 4-phenoxyphenol. The catalytic product phenol is a primary substrate of tyrosinase (cresolase activity)

Fig. 4

Decomposition of guaiacyl glycerol-β-guaiacyl ether (GGE) by the catalytic promiscuity of tyrosinase. a HPLC profile of GGE and vanillin in the control without tyrosinase and in the reaction sample which includes tyrosinase. The chromatographic peaks were identified by the retention time and authentic standard chemicals. b GC–MS of the catalytic product when using GGE as the substrate in the presence of 500 unit tyrosinase (red line). Black line represents authentic vanillin standard. Inset: mass spectrum of the peak at 10.31 min c Reaction scheme illustrates that the catalytic promiscuity cleaves the C_α–C_β bond in GGE