Enzymatic cleavage of lignin β-O-4 aryl ether bonds via net internal hydrogen transfer

Abstract

The current greening of chemical production processes going along with a rising interest for the utilization of biogenic feedstocks recently revived the research to find new ways for the degradation of the complex lignin-backbone by means of biocatalysis and combined chemo-enzymatic catalysis. Lignin, which accumulates in 50 million t/a, is regarded as a potential substitute for phenolic and other aromatic, oil-based chemicals in the upcoming post oil age. The cleavage of the β-O-4-aryl ether linkage is the most favoured, since it accounts for approximately 50% of all ether linkages in lignin. This enzymatic cleavage was proposed to be a part of the lignin catabolism in the proteobacterium Sphingobium sp. SYK6.Three enzymes, LigD, a Cα-dehydrogenase, LigF, a β-etherase and LigG, a glutathione lyase, are supposed to be involved in lignin degradation. We cloned and recombinantly expressed these genes in E. coli and determined their pH and temperature optima on the lignin model substrate 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol 1. Using an NAD + dependent glutathione reductase from Allochromatium vinosum (AVR) we established an efficient way to regenerate the co-substrates NAD + and glutathione allowing for a self-sufficient balanced enzymatic cascade with net internal hydrogen transfer (hydrogen borrowing). We showed the capability of this enzyme system to release lignin monomers from complex lignin structures coming from differently prepared real lignin substrates. This novel enzyme system could become a useful tool to release lignin monomers from complex lignin structures. † Electronic supplementary information (ESI) available. See

From Gene Towards Selective Biomass Valorization: Bacterial β‐Etherases with Catalytic Activity on Lignin‐Like Polymers

“…Similar to LigF, LigF-NS and LigF-NA exclusively attack the S enantiomer of GVG (b(S)GVG), whereas LigE-NS and LigE-NA are highly selective for the R enantiomer (b(R)GVG), as reported previously for LigE and LigP. [24] Substrate specificity of the novel b-etherases towards dimeric model compounds As a result, all active b-etherases in our study exhibited the highest activity at temperatures between 20 and 30 8C ( Figure S5 in the Supporting Information) and at alkaline pH in the range from 8.5 to 10 ( Figure S6 in the Supporting Information).…”

“…These enzymes are members of the glutathione transferase superfamily (GSTs; EC 2.5.1.18) and cleave the b-aryl ether bond upon glutathione (GSH) consumption. [22] The b-aryl ether degradation pathway of S. paucimobilis SYK-6 has been studied by using lignin model compounds, [23][24][25][26] indicating the necessary additional presence of at least four nicotinamide adenine dinucleotide (NADH)-dependent stereospecific alcohol dehydrogenases (LigD, LigL, LigN, and LigO), which catalyze the initial oxidation of the a-hydroxyl group in guaiacyl-a-veratrylglycerol (GVL) to the corresponding ketone ( Figure 1). [20,21] GST genes present widely divergent sequences grouped into several classes, but the roles of these enzymes are not yet completely understood.…”

“…Subsequently, an unusual reductive ether cleavage through a b-etherase reaction takes place. [26] So far, a more detailed description of the enzymatic properties of b-etherases has only been reported for LigF, [24,25] for which preliminary catalytic studies on polymer-like lignin substrates have been conducted with modest success. The reported b-etherases are stereoselective and attack the two enantiomers of racemic bGVG selectively.…”

“…LigE, LigF, and LigP catalyze nucleophilic attack by the tripeptide GSH on the carbon atom at the b position of substrates containing b-aryl ether linkages, such as bGVG, to produce guaiacol and a GSH-conjugated aromatic compound ( Figure 1). [24] Interestingly, many genes in the National Center for Biotechnology Information (NCBI) database have been annotated as putative betherases, according to sequence homology to ligF, ligE and Figure 1. Whereas LigF produces GS-b(R)VG from b(S)GVG, LigE and LigP are selective for b(R)GVG to provide GS-b(S)VG.…”

Solvent free depolymerization of Kraft lignin to alkyl-phenolics using supported NiMo and CoMo catalysts

“…2,3 Several approaches have been reported for the valorization of lignin. These include enzymatic methods, 4,5 thermal treatment in the absence of air (pyrolysis), 6,7 base catalysed depolymerizations [8][9][10][11] and various chemo-catalytic methods like catalytic hydrotreatment in which lignin is reacted with gaseous hydrogen or a hydrogen donor solvent. 2,[12][13][14] Oxidative approaches are also known and have also recently been established.…”