The Laccase‐Lig Multienzymatic Multistep System in Lignin Valorization

Abstract

A laccase‐Lig multienzymatic multistep system for lignin depolymerization was designed and developed. Studies were performed on pristine and fractionated lignins (Kraft and Organosolv) using a specific cascade of enzymes, that is, laccases from Bacillus licheniformis and from Funalia trogii, respectively for Kraft and Organosolv lignin, followed by the Lig system from Sphingobium sp. SYK‐6 (β‐etherases Lig E and Lig F, glutathione lyase Lig G). Careful elucidation of the structural modifications occurring in the residual lignins associated with the identification and quantification of the generated low‐molecular‐weight compounds showed that (i) the laccase‐Lig system cleaves non‐phenolic aryl glycerol β‐O‐4 aryl ether bonds, and (ii) the overall reactivity is heavily dependent on the individual lignin structure. More specifically, samples with low phenolic/aliphatic OH groups ratio undergo net depolymerization, while an increased phenolic/aliphatic OH ratio results in the polymerization of the residual lignin irrespective of its botanical origin and isolation process.

Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid

“…Vanillin was isolated from Kraft lignin following the procedure described by Vignali et al . In detail, a 100 mL mixture containing 0.5 g of Kraft lignin and 2 mM TEMPO was added to the recombinant laccase from Bacillus licheniformis (BALL) (100 U, 0.2 U enzyme per mg of lignin). ,, After 2 h of incubation at 30 °C under shaking (100 rpm) in 0.5 L baffled flasks, 1 N HCl was added until a final pH of 1.0 was reached.…”

“…Vanillin was isolated from Kraft lignin following the procedure described by Vignali et al . In detail, a 100 mL mixture containing 0.5 g of Kraft lignin and 2 mM TEMPO was added to the recombinant laccase from Bacillus licheniformis (BALL) (100 U, 0.2 U enzyme per mg of lignin). ,, After 2 h of incubation at 30 °C under shaking (100 rpm) in 0.5 L baffled flasks, 1 N HCl was added until a final pH of 1.0 was reached. The sample was extracted using 3:1 (v/v) ethyl acetate as the organic solvent; the dewatered organic phase was then filtered using Whatman filter paper, dried in a rotary evaporator, and resuspended in 200 mM Tris–HCl buffer, pH 8.0 for HPLC analyses (to evaluate the recovery yield) and as the substrate for the whole-cell biotransformation.…”

“…The sample was extracted using 3:1 (v/v) ethyl acetate as the organic solvent; the dewatered organic phase was then filtered using Whatman filter paper, dried in a rotary evaporator, and resuspended in 200 mM Tris–HCl buffer, pH 8.0 for HPLC analyses (to evaluate the recovery yield) and as the substrate for the whole-cell biotransformation. Following this procedure, vanillin was obtained with a recovery yield of ≈4.5 mg per gram of lignin, as evaluated by GC–MS analyses …”

“…Here, we focused on the generation of an engineered E. coli strain able to convert in one-pot both vanillin arising from lignin and ferulic acid isolated from wheat bran into ccMA (Scheme 1). The previous aromatic compound was isolated from Kraft lignin using the enzymatic procedure described by Vignali et al, 13 the latter from wheat bran using an optimized thermo-enzymatic process. The engineered cell system efficiently generated ccMA, a useful metabolite of main industrial relevance: the global market of the ccMA derivatives is estimated at $22 billion, 14 with a market for ccMA alone at more than $100 million.…”

“…Following this procedure, vanillin was obtained with a recovery yield of ≈4.5 mg per gram of lignin, as evaluated by GC−MS analyses. 13 Construction of Plasmids and Whole-Cell Biocatalysts. The synthetic genes encoding the enzymes involved in the biosynthetic pathway (Scheme 1) were designed by in silico back translation of the amino acid sequence reported in the GenBank database (Table 1), and the codon usage was optimized for expression in E. coli.…”