Bioconversion of lignin by oxidative enzymes for lignin depolymerization from tropical bacteria isolates

Abstract

The conversion of lignocellulosic biomass into bioethanol or biochemical products requires a crucial pre-treatment process to break down the recalcitrant lignin structure. Biological pre-treatment using microbial enzymes appears to be the most promising alternative to depolymerize the lignin fragment, which can simultaneously facilitate conversion into valuable chemical products. Thus, this research focuses on bioconversion of Alkali Lignin (AL) for lignin depolymerization, using several enzymes from bacterial isolates. Two bacteria isolates, Streptomyces sp. strain S6 and Bacillus subtilis strain S11y, were selected as the potential strains for the source of candidate enzymes responsible for lignin depolymerization. Sequencing of the genomic DNA of these strains revealed four successful candidate genes with lignin depolymerizing ability, in which two genes were identified as dye-decolorizing peroxidase (DyP2, ~41 kDa) and multicopper oxidase (CuO1, ~44 kDa) from Streptomyces sp. strain S6, and also two genes identified as Cu/Zn superoxide dismutase (SOD2, ~22 kDa) and monofunctional heme catalase (Kat2, ~55 kDa) from Bacillus subtilis strain S11y. These genes were successfully expressed as recombinant enzymes and confirmed to have the ability to degrade AL polymer. Differential UV-vis spectrum (Δε-spectrum) of AL treated with the candidate enzymes demonstrated increased absorbance at ~295 nm and 350 nm after treatment, indicating increased free and conjugated phenol structure due to depolymerization. These enzymes also showed activity for oxidation of AL, reducing ~100-240 Da of the high-molecular-weight fraction of AL within 24 h treatment. Analysis of reaction components of all enzymes with AL by ultra-high-pressure liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry showed that the enzymes generated various low-molecular-weight products of diverse groups, such as vanillyl alcohol, vanillin, dihydro-ferulic acid, salicylic acid, benzoic acid, 2,4-dimethyl-benzaldehyde, and oxalic acid. Based on the depolymerization products, the reaction mechanisms performed by each enzyme were also successfully elucidated, which involved several types of reactions, including β−O−4, Cα-Cβ, Cβ-Cγ, Aryl-Cα bond cleavages, O-demethylation, polymerization, decarboxylation, benzylic oxidation, and aromatic ring oxidative cleavage. Each enzyme appeared to generate radicals formed on the lignin surface, leading to several bond cleavages and structural modification in AL after enzymatic treatment, proving their ability to depolymerize polymer lignin. Successful evaluation of lignin depolymerizing enzymes can be applicable for lignin pre-treatment process in green energy production as well as generation of valuable chemicals in bio-refinery.