CHAPTER 2 REVIEW OF LITERATURE

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1 CHAPTER 2 REVIEW OF LITERATURE 2.1 Lignin Lignin is a complex aromatic amorphous polymer, most commonly derived from wood and an integral part of plant cell walls. The term was introduced in 1819 by De Candolle and is derived from the Latin word lignum, meaning wood and is the second most abundant biopolymer after cellulose, employing 30% of non-fossil organic carbon and constituting from a quarter to a third of dry mass of wood (Boerjan et al., 2003). It is also seen as a natural, complex, heterogenous, phenylpropanoid polymer comprising 25-30% of plant biomass. Lignin plays a crucial part in conducting water in plants stem. It is a natural polymer of the cell wall that gives strength to wood. These are abundant plant biopolymers accounting for approximately 30% of the organic carbon in the biosphere (Arora et al., 2002). The ability to synthesize lignin has been essential in the evolutionary adaptation of plants from an aquatic environment to land. Lignocellulosic materials are composed primarily of cellulose, hemicelluloses and lignin due to photosynthetic activity of plants pathogens Structure of lignin Lignin is a structurally complex aromatic biopolymer. It is a highly branched and three dimensional amorphous heteropolymer, non-water soluble and optically inactive. It is derived from the random coupling of phenylpropanoid precursors; coniferyl, sinapyl and p-coumaryl alcohols (Eriksson et al., 1990). It is relatively hydrophobic and aromatic in nature and is known for its heterogeneity in lacking a defined primary structure. It is synthesized from phenyl propanoid precursors by polymerization in higher plants. There are three different types of lignin; hardwood lignin, softwood lignin and grass lignin. Hardwood contains about 15-35% lignin and has a much more complex lignin structure than softwood, which consists of about 20% lignin. Lignin fills the space in the cell wall between cellulose, hemicellulose and pectin components, especially in tracheids, sclereids and xylem. It is covalently linked to hemicellulose and there by crosslinks different plants polysaccharides, conferring mechanical strength to the cell wall and plant as a whole (Chabannes et al., 2001). Lignin is formed by removal of water from sugars to create aromatic structures through the phenylpropanoid pathway. This reaction is

2 irreversible. Lignin polymers are cross-connected structures with molecular weights on the order of 15,000 or more. 2.2 Lignininolytic fungus Fungi are recognized for their superior aptitudes to produce a large variety of extracellular enzymes. These are principally responsible for lignocellulose degradation. Wood-rotting basidiomycetous fungi are usually divided into white-rot, brown-rot and litter- decomposing fungi (Steffen et al., 2000). The only organisms capable of mineralising lignin efficiently are basidiomycetous white rot fungi (WRF) and related litter-decomposing fungi (LDF) (Hatakka, 2001). WRF belonging to the basidiomycetes produce various isoforms of extracellular ligninolytic enzymes: laccases and different peroxidases, including lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP), the latter sharing LiP and MnP catalytic properties (Martinez, 2002). These enzymes are directly involved not only in the degradation of lignin in their natural lignocellulosic substrates but also in the degradation of various xenobiotic compounds including dyes (Wesenberg et al., 2003). Lignin modifying enzymes are produced by WRF during their secondary metabolism. Mushrooms are not plants and do not undergo photosynthesis. These are non-timber forest products which are often found as saprophytes on soil, open field, farm lands and wood. Mushrooms with their delicate flavour and texture are recognised as a nutritious food and are important source of biologically active compounds with medicinal values (Emilia et al., 2006), therefore have been used as a part of regular diet mostly by the ethnic group of Asian people from time immemorial. The higher basidiomycetes include about 10,000 species from 550 genera and 80 families in the basidiomycetes class with macroscopic fruiting bodies; approximately 700 species of higher basidiomycetes have been found to possess significant pharmacological activities. In developing countries like India mushrooms are a boon for progress in the field of food, medicine and unemployment. Mushrooms in the twentieth century are well known to people all over the Asian countries as an important bio-source of novel secondary metabolites. In India, particularly the alternative systems of medicine utilize the curative properties of mushrooms (Mizuno et al., 1995).

3 Mushrooms are recognized as important food items since ancient times. Their usage is being increased day by day for their significant role in human health, nutrition and disease (Moore and Chiu, 2001) recorded that; fungi are ideal food because they have a fairly high content of protein which contains all of the essential amino acids. Fungal biomass is also a source of dietary fiber and is virtually free of cholesterol. Many of mushrooms produce a range of metabolites of intense interest to the pharmaceutical and food industries. (Chang, 2007). Most mushroom species, if not all, contain biologically active polysaccharides. Fruiting bodies, mycelia, submerged broth are sources of the bioactive compounds. Also, much attention has been drawn to various immunological and anti-cancer properties of the mushroom extracts. They also offer other potentially important therapeutic properties including antioxidants, antihypertensive, cholesterol-lowering and liver protection; antifibrotic, anti-inflammatory, antidiabetic, anti-viral and anti-microbial activities wood decayed by WRF is pale in colour because of oxidative bleaching and loss of lignin and often retains a fibrous texture. Some WRF preferentially attack lignin more readily than hemicellulose and cellulose in the wood tissue (Blanchette et al., 1991) Brown rots fungi (BRF) Brown rot fungi comprise a relatively small group of basidiomycetes that decay cellulose in wood preferentially. They do not degrade the lignin extensively, although they modify it by demethylating it. Brown rot fungi make a large contribution to wood decay, especially in coniferous forests (Dix and Webster, 1995). They deserve much more research attention, but have been difficult to study because they do not exhibit full degradative activity on defined media in vitro (Eriksson et al., 1990). Now-a-days the cultivation of mushroom is disseminated all over the world because of its revenue activity (Ponmurugon et al., 2007) Leaf litter Fungi Leaf litter fungi degrade leaf litter, as opposed to woody litter, are poorly understood. In some cases, leaves are colonized shortly after they fall by basidiomycetes. For example, Marasmius and rosaceus an early colonizer and degrader of pine needles, a relatively recalcitrant and long-lived form of leaf litter. In most cases, leaf litter decomposition is more complex, involving a succession of biodegradative activities that precede attack by lignocellulose degraders (Dix and Webster, 1995).

4 2.2.3 White rots fungi (WRF) These are the most abundant degraders of wood in nature. Their strategy is to decompose the lignin in wood so that they can gain access to the cellulose and hemicelluloses that are embedded in the lignin matrix. Under optimal conditions, the rates at which WRF mineralize lignin rival their rates of polysaccharide degradation. Basidiomycetes, Xylariaceous and Ascomycetes that cause WRF are principally responsible for wood decay in hardwood forests; tropical forest and coniferous forest play a prominent role in ecosysytem (Eriksson et al., 1990, Blanchette, 1991; Dix and Webster, 1995). WRF is known to degrade polyaromatic hydrocarbons (PAHs), chlorinated aromatic hydrocarbons (CAHs), polycyclic aromatics, polychlorinated biphenyls, polychlorinated dibenzo (p)dioxins, the pesticides DDT and lindane and some azo dyes Degradation of PAHs, which include benzo(a) pyrene, pyrene, fluorene, and phenanthrene, is favoured at nitrogen-limiting conditions and low ph It has been documented that WRF is able to mineralize tri-, tetra and pentachlorophenol (PCP) and a group of microbes in soil can completely mineralize PCP Degradation of cyclodiene insecticides, including chlordane, by WRF has been demonstrated. WRF has been observed to degrade Tri nitro toluene (TNT) in laboratory-scale studies (using pure cultures), however, factors that limit their effectiveness may retard wide-spread use in the field Auricularia polytricha Auricularia polytricha (wood ears fungus) is one of the most famous Chinese medicinal fungi that invades and lives in the woodcut or fallen logs of several types of trees. A. polytricha resemble ears in shape and are gelatinous to leathery in texture and brownish-purple in color. Protein, vitamin and carbohydrate content of A. polytricha are reported to be higher than those of many vegetables and fruits. A. polytricha, therefore, it is an ideal food for dietetic prevention of hyperlipidaemia (Chang and Hayes, 1978). The basidiocarp when fresh is rubbery, gelatinous and ear-like in structure but when dried, it is shapeless and brittle. Its edible fruitbodies could be easily identified by pilose upper surface which is strongly capitate with dark brown smooth hymenium (Jonathan, 2002). It is highly valued as medicinal herbs, owing to their various biological and pharmacological activities, such as immuno-stimulating and antitumour activities (Yang et al., 2000). In a dose dependent study, the an exo-biopolymer produced from a submerged mycelial culture of A. polytricha proved that rats fed at mg kg-1 body weight,

5 significantly decreased the concentrations of the plasma triacylglycerols, total cholesterol and low-density lipoprotein (LDL) cholesterol Morchella spp. Morchella is a true morel. It is called as Guchhi economically important edible wild type mushroom. It belongs to class Ascomycetes. The morels are amogest most highly prized fungi in world (Pegler, 2003). There harvesting represent a seasonal employement. Morchella is used as food products and source of income from sale. All the true morels are known to be edible (Arora et al., 2002). These are good source of protein for human consumption contain % of protein (Kurtzman and Zadrazil, 1989) Helvella spp. Helvella spp. is a genus of ascomycete fungus of the Helvellaceae family. The mushrooms, commonly known as elfin saddles, are identified by their irregularly shaped caps, fluted stems (Ammirati et al., 1985). A lot of different factors can affect the development of macro fungus. Glucose, the most abundant monosaccharide in nature, is the principal and preferred carbon and energy source for cells (Fron, 1996). In addition, glucose can act as a "growth hormone" to regulate aspects of cell growth, metabolism, and development (Ozcan and Johnston, 1999). 2.4 Ligninolytic Enzymes Lignin biodegradation is a process involving the action of oxidative enzymes and in subsequent chemical reactions (Hatakka, 1994). Reactions catalysed by enzymes play a significant role in the complete degradation of lignocellulose biomass. Since the lignin polymer is large and highly branched, lignin-degrading mechanisms must be extracellular and unspecific. WRF were cultivated for purification of extracellular enzyme. WRF are able to biodegrade lignin as well as broad spectrum of organic chemicals containing carbon (Tien and Kirk, 1983). WRF produced isoforms of extracellular oxidase including laccase, mangnease peroxide and lignin peroxidase which are involved in degradation of lignin. These lignolytic enzymes are involved in degradation of various xenobiotics compounds and dyes. All extracellular peroxidises and laccases have the ability to catalyze one-electron oxidation resulting in the formation of radicals, which undergo several spontaneous reactions. These, in turn lead to various bond cleavages

6 including aromatic ring fission (Hatakka, 2001). Apparently, these enzymes act using lowmolecular mass mediators to carry out lignin degradation Laccase Laccase (EC ) are copper-containing enzymes catalysing the oxidation of a broad number of phenolic compounds and aromatic amines by using molecular oxygen as the electron acceptor, which is reduced to water. Laccase is widely distributed in higher plants and fungi. In fungi laccase is present in Ascomycetes, Deuteromycetes, Basidiomycetes and is particularly abundant in WRF that degrade lignin. Ligninolytic enzymes production depends on cultivation conditions: submerged or solid-state fermentation; carbon and nitrogen sources; presence of inducers and other small molecules; and presence of different microelements and their concentration. Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in last decades due to their ability to oxidize both environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes, molecular cloning and applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area (Madhavi, 2009) Aryl alcohol oxidase (AAO) AAO (EC ) is an extracellular flavoenzyme involved in lignin biodegradation by some WRF. These enzymes catalyze the extracellular oxidation of aromatic alcohol to the corresponding aldehydes. AAO activity was described for the first time in P. versicolor in WRF are responsible for lignin degradation and AAO participates in this process by generating hydrogen peroxide in the redox cycling of aromatic fungal metabolites, also involving mycelial dehydrogenases. The most prominent enzymes associated with lignin degradation are lignin peroxidase (LiP) and manganese peroxidase (MnP), as well as laccases and H 2 O 2 producing oxidases (Ferreira et al., 2005) Lignin peroxidise (LiP) LiP (EC ) appears to be the key enzyme in the oxidation of nonphenolic phenylpropanoid units which leads to polymer fragmentation. Lignin is found to be degraded by an enzyme lignin peroxidase produced by some fungi like Phanerochaete chrysosporium. The

7 mechanism by which LiP interects with the lignin polymer which involves versatile alcohol, a secondry metabolite of white rots fungi, act as a cofactor for enzyme. LiP is a hemeprotein from the white rot fungus. LiP is able to oxidize non-phenolic aromatic substrates and does not require the participation of mediators due to its unusually high redox potential (Glenn and Gold, 1985) Manganese peroxidas (MnP) MnP (EC ) belongs to the family of oxidoreductases, to be specific those acting on peroxide as acceptor (peroxidases). The first extracellular MnP was purified from P. chrysosporium, with its expression and production shown to be regulated by the presence of Mn 2+ in the culture medium (Bonnarme and Jeffries, 1990). MnPs are extracellular glycoproteins and are secreted in multiple isoforms which contain one molecule of heme as iron protoporphyrin IX (Ashger et al., 2008). Laccase have been purified from cultures of P. eryngii in glucose-ammonium medium. High level of Mn-oxidizing peroxidase activity was observed in this fungus when peptone was used as Nitrogen source. MnP Compound II oxidizes another Mn (II) ion to Mn (III) and is reduced by the reaction of two H + ions and the iron bound oxygen (Glenn and Gold, 1985).