Cascade enzymatic cleavage of the β-O-4 linkage in a lignin model compound

The β-O-4 aryl ether linkages represent about 50% of all ethers in various lignins. At least three enzymatic steps are required to break them down: a NAD+-dependent C-α dehydrogenase (such as LigD and L), a glutathione lyase that releases guaiacol (i.e., a β-etherase such as LigE and F), and a glutathione-dependent lyase (i.e., LigG). In this work the LigD, L, E, F, and G fromSphingobium sp. SYK-6 were overexpressed in E. coli and purified with high yields. After characterizing the stability and kinetic properties of LigD and L on the lignin model compound GGE (1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3- diol) and the thermostability of all five recombinant Lig enzymes, the experimental conditions for GGE bioconversion could be optimized (i.e., pH 9.0, 25 °C, ≈0.1 mg mL−1 of each enzyme, and 0.5 mM racemic substrate). Under optimal conditions, and by recycling NADH using the L-lactate dehydrogenase–pyruvate system, GGE was fully converted into the final products 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan- 1-one and guaiacol in <2 hours. Differently from what was previously reported, this result and chiral HPLC analyses demonstrated that LigG catalyzes the glutathione-dependent thioether cleavage of both β(R)- and β(S)-isomer intermediates produced by LigE and LigF β-etherases: this allowed, for the first time, reaching 100% conversion of GGE. Altogether, the recombinant five-enzyme Lig system represents a component well suited for a multienzymatic process, comprising well-known ligninolytic activities (such as peroxidases and laccases), devoted to transforming selected lignins into aromatic compounds as an alternative to the oil source. 1. Introduction Lignocellulose refers to plant dry matter (biomass or the socalled lignocellulosic biomass). It is composed of carbohydrate polymers (cellulose, hemicellulose) and an aromatic polymer (lignin) and represents the most promising feedstock. Although burning lignin still represents a valuable contribution for saving fossil sources, lignin also offers perspectives in terms of higher value-added applications. In fact, after the