Enzymatic Processes to Unlock the Lignin Value

Abstract

Main hurdles of lignin valorization are its diverse chemical composition, recalcitrance, and poor solubility due to high-molecular weight and branched structure. Controlled fragmentation of lignin could lead to its use in higher value products such as binders, coatings, fillers, etc. Oxidative enzymes (i.e., laccases and peroxidases) have long been proposed as a potentially promising tool in lignin depolymerization. However, their application was limited to ambient pH, where lignin is poorly soluble in water. A Finnish biotechnology company, MetGen Oy, that designs and supplies industrial enzymes, has developed and brought to market several lignin oxidizing enzymes, including an extremely alkaline lignin oxidase MetZyme^® LIGNO™, a genetically engineered laccase of bacterial origin. This enzyme can function at pH values as high as 10-11 and at elevated temperatures, addressing lignin at its soluble state. In this article, main characteristics of this enzyme as well as its action on bulk lignin coming from an industrial process are demonstrated. Lignin modification by MetZyme^® LIGNO™ was characterized by size exclusion chromatography, UV spectroscopy, and dynamic light scattering for monitoring particle size of solubilized lignin. Under highly alkaline conditions, laccase treatment not only decreased molecular weight of lignin but also increased its solubility in water and altered its dispersion properties. Importantly, organic solvent-free soluble lignin fragmentation allowed for robust industrially relevant membrane separation technologies to be applicable for product fractionation. These enzyme-based solutions open new opportunities for biorefinery lignin valorization thus paving the way for economically viable biorefinery business.

Keywords: biomass; enzyme; laccase; lignin; lignocellulosic; oxidoreductase.

Figure 1

Standard activity and stability of MetZyme^® LIGNO™. (A) pH dependence of MetZyme^® LIGNO™ activity toward standard low-molecular weight substrates: 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), and 2,6-dimethylphenol (DMP). (B–D) Stability of MetZyme^® LIGNO™ at various pH (9–11) and temperatures (50, 60, and 70°C); pre-incubation time at indicated conditions is plotted against residual activity measured after pre-incubation. Pre-incubation at 50°C was carried out over a span of 24 h, at 60 and 70°C—over 1 h; temperatures and pH are indicated over the graphs. The curves do not represent a model fit, they are line interpolations that Microsoft excel provides as a default and they are added only for convenience.

Figure 2

Molecular weight distribution of enzyme-treated lignin sample and control sample (the same process run without the enzyme). (A) Elution profiles of size exclusion chromatography, profiles were area-normalized to reflect the same total mass; (B) molecular weight distribution calculated based on HPLC profile using molecular weight standards.

Figure 3

Properties of enzyme-treated lignin revealed from acid titration. (A) Acid titration curve of enzyme-treated and control lignin; highlighted areas indicate pH ranges of deprotonation of phenolic hydroxyls (pH 9–11) and phenolic hydroxyls of lignin units with keto group at alpha-carbon (see Discussion), deprotonating hydroxyls in the molecules are outlined with solid line, substituent at alpha-carbon is outlined with dotted line; (B) solubility of enzyme-treated and control lignin at different pH. The results are average of three independent titrations.

Figure 4

Colloidal particle size distribution of enzyme-treated and control lignin in a buffer at pH 10.5.

Figure 5

Fractionation of enzyme-treated lignin. (A) Experiment flow chart and lignin fractions (dried); (B) analysis of lignin fractions by size exclusion chromatography. Calculated average molecular weights are given in the table. Since claimed column resolution range is 100–100,000 Da, and molecular weight standards range starts from 600 Da, smaller molecular weights may not be calculated correctly.

Figure 6

Schematic representation of laccase driven oxidation of a lignin unit. Oxidation of alpha-carbon to a ketone lowers pKa of the corresponding C1 hydroxyl, resulting in increased solubility in neutral-to-acidic pH and facilitated lignin chain brake.