Lignin degradation

 Importance of Lignin Degradation:

 Of all naturally produced organic chemicals, lignin is probably the most recalcitrant. This is consistent with its biological functions, which are to give vascular plants the rigidity they need to stand upright and to protect their structural polysaccharides (cellulose and hemicelluloses) from attack by other organisms. Lignin is the most abundant aromatic compound on earth and is second only to cellulose in its contribution to living terrestrial biomass. When vascular plants die or drop litter, lignified organic carbon is incorporated into the top layer of the soil. This recalcitrant material has to be broken down and recycled by microorganisms to maintain the earth’s carbon cycle. Were this not so, all carbon would eventually be irreversibly sequestered as lignocellulose.

 Lignin biodegradation has diverse effects on soil quality. The microbial degradation of litter results in the formation of humus, and ligninolysis probably facilitates this process by promoting the release of aromatic humus precursors from the litter. These precursors include incompletely degraded lignin, flavanoids, terpenses, lignans, condensed tannins, and uberins. Undegraded lignocellulose, e.g. in the form of straw, has a deleterious effect on soil fertility because decomposing (as opposed to already decomposed) lignocellulose supports high populations of microorganisms that may produce phytotoxic metabolites. High microbial populations in undecomposed litter also compete with crop plants for soil nitrogen and other nutrients. By breaking down the most refractory component of litter, ligninolysis thus contributes to the removal of conditions that inhibit crop productivity. Conditions that disfavour the biological breakdown of lignocellulose lead to soils with pronounced accumulations of litter. Warm temperature, high moisture content, high oxygen availability, and high palatability of the litter to microorganisms all favour decomposition. The more highly lignified litter is, the less digestible it is, and the more its decomposition depends on the unique organisms that can degrade lignocellulose.Lignin is the most abundant source of carbon in the soil after cellulose. Lignin degradation can thus play a major role in improving earth’s biofuel resources and also serve as an alternative to harsh technologies used in the paper and pulp industry.

 Lignin Structure and Its Biosynthesis:

In the plant cell, lignin is biosynthesized by the combination of three basic hydroxycinnamoyl alcohol monomers or monolignols:

1. p-Coumaryl alcohol;

2. Coniferyl alcohol;

3. Sinapyl alcohol.

These monolignols are often referred to as phenylpropanoids, which differ in the substitutions at the 3-C and 5-C positions in the aromatic ring. Lignin synthesis starts with the random self-replicating radical coupling of phenoxy radical to form an oligomeric product. After polymerization, these polymers are referred as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) (from p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, respectively). Consequently, the relative intracellular concentrations of the various monolignols influence the resultant composition and structure of lignin in individual plants.

For example, grasses assemble lignin from G, S, and H monomers, hardwood contains roughly equal parts G and S, and softwood is up to 90% G .

 The linkages formed during radical coupling of lignin monomers varies between species and can be either C-C or C-O ether linkages, though typically more than two-thirds of bonds are ether bonds.

 As the phenolic and β-hydrogen are shared between G, S, and H these sites participate in most of the bonding, with the β-aryl ether (e.g., β-O-4) linkage estimated to represent up to 80% of the bonding motifs found in lignin

biosynthesized, lignin polymers cross-link with the polysaccharides of hemicellulose and cellulose to form the complex matrix, which provides the tissues and cell walls of all vascular plants strength, rigidity and protection from microbial degradation.

Lignin Degrading organisms :

Chemically and morphologically distinct types of decay result from attack of different microorganisms. Wood decay fungi are usually separated into three main groups, causing white, soft, or brown rot. Bacterial degradation of wood has also been 

reported including erosion, tunneling, and cavity formation.

Bacteria:

Certain bacteria can degrade lignified cell walls of wood. Filamentous bacteria belonging to the genus Streptomyces are well-known degraders of lignin and can mineralize up to 15% of labeled lignins but usually much less. Typically, Streptomyces spp. solubilize part of lignin, and the end product is water-soluble acid-precipitable polymeric lignin. Streptomyces cyaneus CECT 3335 was one of the most efficient new isolates in solubilizing 45% of the 14C- (lignin) fraction and mineralizing 3% of the label in 21 d. However, the reference species S. viridosporus solubilized 59% and mineralized 4.5% of the same substrate. The presence of phenol oxidase and peroxidase was found in S. cyaneus, the former activity being 100 times greater as determined by the oxidation of ABTS and 2,4-dichlorophenol, respectively. S. cyaneus degraded a nonphenolic arylglycerol b-aryl ether by a mechanism indicating the cleavage of Ca±Cb bond in lignin.

Streptomyces sp. EC1 produces peroxidase and cell-bound demethylase requiring H2O2 and Mn2, both of which are produced at relatively high levels in the presence of Kraft lignin or wheat straw, while protochatechuate 3,4-dioxygenase and b-carboxymuconate decarboxylase activity were less induced. Lignin model compound studies demonstrated that monomeric compounds, i.e., vanillic acid and protocatechuic acid were formed, indicating the cleavage of Ca±Cb bonds, as well as demethylation and oxidation of Ca in the side chain to carbonyl group.

Nonfilamentous bacteria usually mineralize less than 10% of lignin preparations and can degrade only the low-molecular weight part of lignin as well as degradation products of lignin. Thus, they may play some role in final mineralization of lignin. Among these eubacteria, Pseudomonas spp. are the most efficient degraders. However, since these bacteria do not produce extracellular oxidoreductases, and large molecules apparently cannot be taken up into the cell, they are obviously unable to attack polymeric lignin.

Fungi:

Ascomycetes and deuteromycetes generally cause soft-rot decay of wood. The decayed wood has a brown, soft appearance that is cracked and checked when dry. Two forms of soft rot have been described, type I consisting of biconical or cylindrical cavities that are formed within secondary walls while type II refers to an erosion form of degradation. In contrast to nonselective white-rot fungi, the middle lamella is not attacked by type II softrot fungi. Xylariaceous ascomycetes from genera such as Daldinia, Hypoxylon, and Xylaria have earlier often been regarded as white-rot fungi, but nowadays these fungi are grouped to soft-rot fungi since they cause typical type II soft rot. They primarily occur on hardwood, and weight losses up to 53% of birch wood were found within 2 months by the most efficient fungus of this group, Daldinia concentrica. The highest lignin loss observed was 44% at the stage when weight loss was 77% after 4 months incubation. Pine wood was degraded, however, very little, only showing 2.5% weight loss.

Ligninolytic peroxidases or laccases of softrot fungi may not have the oxidative potential to attack the recalcitrant guaiacyl lignin. On the other hand, syringyl lignin apparently is readily oxidized and mineralized by the enzymes of soft-rot fungi.The largest group of fungi that degrades wood is the basidiomycetes. It has been calculated that in North America there are 1600±1700 species of wood-degrading basidiomycetes Wood-rotting basidiomycetous fungi are usually divided into white-rot and brown-rot fungi. They are taxonomically closely related, and white-rot and brown-rot fungi can be foundin the same genera. Most wood rotters belong to the orders Agaricales and Aphyllophorales. Brown-rot fungi mainly decompose the cellulose and hemicellulose components in wood, but they can also modify the lignin to a limited extent.

Brown-rotted wood is dark, shrink, and typically broken into brick-shaped or cubical fragments that easily break down into brown powder. The brown color indicates the presence of modified lignin in wood. Many brown-rot fungi such as Serpula lacrymans, Coniophora puteana, Meruliporia incrassata, and Gloeophyllum trabeum are destructive to wood used in buildings and other structures.

The only organisms capable of mineralizing lignin efficiently are basidiomycetous white rot fungi and related litter-decomposing fungi. White-rot fungi are a heterogeneous group of fungi classified in the Basidiomycota. Different white-rot fungi vary considerably in the relative rates at which they attack lignin and carbohydrates in woody tissues.

Enzymes involved:

Extracellular enzymes involved in lignin degradation are lignin peroxidases (LiPs,ligninases, EC 1.11.1.14) and manganese peroxidases (MnPs, Mn-dependent peroxidases, EC 1.11.1.13), as well as laccases (benzenediol:oxygen

oxidoreductase, EC 1.10.3.2). In addition, some accessory enzymes are involved in hydrogen peroxide production. Glyoxal oxidase (GLOX ) and aryl alcohol oxidase (AAO ) (EC 1.1.3.7) belong to this group. Glyoxal oxidase generates extracellular hydrogen peroxide by the oxidation of a variety of simple dicarbonyl and hydroxycarbonyl compounds, especially glyoxal and methylglyoxal. AAO produces hydrogen peroxide by oxidative dehydrogenation of phenolic and non-phenolic aryl-alcohols, polyunsaturated (aliphatic) primary alcohols or aromatic secondary alcohols to their corresponding aldehydes.

LiPs and MnPs are heme-containing glycoproteins which require hydrogen peroxideas an oxidant.  LiP oxidizes nonphenolic lignin substructures by abstracting one electron and generating cation radicals that are then decomposed chemically.

MnP oxidizes Mn( II ) to Mn( III ) which then oxidizes phenolic rings to phenoxyl radicals which lead to decomposition of compounds.Laccase is a copper-containing oxidase that utilizes molecular oxygen as oxidant and also oxidizes phenolic rings to phenoxyl radicals.

Lignin peroxidase:

Lignin peroxidases (LiPs) were the first ligninolytic enzymes to be discovered. Lignin peroxidase (LiP, EC 1.11.1.14) is a glycosylated enzyme containing heme protein with an iron protoporphyrin prosthetic group that requires hydrogen peroxide (H2O2) to catalyze the oxidation of non-phenolic lignin units and mineralize the recalcitrant aromatic compounds.

Lignin oxidation takes place via electron transfer, non-catalytic cleavages of various bonds, and aromatic ring opening. The catalytic cycle of LiP consists of one oxidation and two reduction steps as follows:

Step 1 Two-electron oxidation of the resting (native) ferric enzyme ([LiP]-Fe(III)) by H2O2 to formthe Compound I oxo-ferryl intermediate [Fe(IV)];

Step 2 Reduction of Compound I by the non-phenolic aromatic reducing substrate (A) to formCompound II by gaining one electron;

Step 3 Finally, the oxidation cycle ends when Compound II is returned to the resting ferric state with a gain of one more electron from the reducing substrate A

LiPs have a high redox potential (1.2 V at pH 3.0) as compared with other peroxidases and can oxidize phenolic and nonphenolic structures of lignin directly without a mediator.

 

 

 Manganese Peroxidase:

Manganese (Mn) is essential for the formation of MnP. The enzyme MnP plays an important role during the initial stages of lignin degradation. Compared to laccase, MnP causes greater degradation of phenolic lignin due to its higher redox potential with the eventual release of carbon dioxide. MnP is mainly produced by a broad species of white-rot basidiomycetes such as Phanerochaete chrysosporium. The catalytic cycle of MnP is similar to that of LiP. Like LiPs, MnPs are also heme containing glycoproteins which require H2O2 as an oxidant. Manganese acts as a mediator during MnP enzymatic activity. To begin with, MnP oxidizes Mn2+ to Mn3+. The enzymatically generated Mn3+ oxidant is freely diffusible and participates in the oxidation reaction as a redox couple.

In addition, organic acids such as lactate and malonate can chelate Mn3+ ion. The chelated Mn3+—organic acid complex oxidizes the phenolic compounds in lignin to phenoxy radicals. High levels of Mn can stimulate MnP enzymatic activity and enhance the degradation process of lignin in soils.

Laccase:

Lac (EC 1.10.3.2, p-diphenol oxidase) is a copper-containing enzyme belonging to the oxidoreductase group which oxidizes a wide variety of organic and inorganic substances Mediators are low molecular weight compounds that are easily oxidized by Lacs and subsequently reduced by the substrate. Due to its large size, the substrate cannot reach the active site of the enzyme. A mediator, due to its small size, acts as a conveyer of an electron from the enzyme to the substrate. The mediator reaches the enzyme active site easily and gets oxidized to a more stable intermediate with a high redox potential. The oxidized mediator diffuses away from the enzyme and oxidizes more complex substrates before returning to its original state. The electrons taken by Lacs are finally transferred back to oxygen to form hydrogen peroxide. Most of the enzymes are substrate specific, in contrast to Lac activity which oxidizes a variety of substrates like polyphenols, diphenols, benzenethiol, and aromatic amines

.

Versatile Peroxidase:

Versatile peroxidase, as the name suggests, has catalytic properties of both LiP and MnP. VP was first purified from the genera of fungi Bjerkandera  and was found to transform lignin even without an external mediator.The VP enzyme possesses a hybrid molecular architecture with several binding sites including Mn2+ and is able to oxidize Mn2+ like MnP and LiP. However, unlike MnP, VP has the dual ability to oxidize Mn2+ in the independent oxidation of simple amines and phenolic monomers. VP can also oxidize a variety of substrates (with high and low redox potentials) including Mn2+, phenolic and non-phenolic lignin dimers, and aromatic alcohols.

Dye-Decolorizing Peroxidase:

The DyP enzyme is also a heme-based peroxidase that can cause lignin breakdown through a radical-mediated oxidation process. The DyPs are phylogenetically distinct  from other peroxidases as they possess an α + β ferredoxin-like fold. However, their oxidation mechanism is similar to VP and MnP. They are widely found in microorganisms  and classified into four types: A, B, C, and D. Bacterial enzymes are predominantly found in type A to C, while type D is mostly clustered to fungal DyPs. All kinds of DyPs have peroxidase activities; however, they differ in substrate specificity values. In addition to lignin, DyPs can also oxidize synthetic dyes, non-phenolic methoxylated aromatics, Mn2 , and high redox synthetic dyes such as anthraquinone and azodyes.

REFERENCES:

v  Adam B Fisher and Stephen S Fong. (2014) Lignin biodegradation and industrial implications. AIMS Bioengineering vol1, issue 2,92-112.

v  Prof.Dr.Annele Hatakka. Biodegradation of lignin.

v  K.E. Hammel. Fungal degradation of lignin.

v  Rahul Datta, Aditi Kelkar, et.al.,(2017) Review of enzymatic degradation of lignin in soil, sustainability 2017,9,1163.

v  Thomas WJeffries. Biodegradation of Lignin and hemicellulose.

v  Gonzalo de Gonzalo, Dana I Colpa. (2016) review of Bacterial enzymes involved in lignin degradation, Journal of Biotechnology.236(2016),110-119.

v  Grzegorz Janusz, Anna Pawlik,. et.al. (2017) review article on Lignin degradation: microorganisms, enzymes involved, genome analysis and evolution. FEMS microbiology, vol 4, No 6, 941-962.