Fungi are vast group of eukaryotic organisms including yeast and molds that have a significant role in nature by breaking down organic material. These organisms are also producers of important antibodies, enzymes and food.They are the only organisms that are able to completely mineralize lignocelluloses, the most abundant recalcitrant renewable material available in nature. Fungi cells do so by producing several sets of enzymes for breaking down polysaccharides, celluloses and hemicelluloses as well as lignin, a natural aromatic polymer. Ligninases or ligninolytic enzymes constitute a group of oxidoreductases that are specialized in polymerization as well as in the degradation of lignin (Thurston, 1994). These enzymes are mostly produced by so called white-rot fungi and litter decomposing fungi (Assavanig et al., 1992).
Laccase is one of the three main ligninases and differs from the others in its ability to catalyze the oxidation of lignin components using molecular oxygen as the electron acceptor, which in turn is reduced to water (Kiiskinen and Saloheino, 2004). In order to bring about the degradation of complex structures, laccases make use of small molecules as electron mediators both in nature and in the application developed by humans. Laccase is among the oldest known enzymes, laccase activity was first discovered in 1883 in the sap of the Japanese lacquer tree Rhus vernicifera leading to its polymerization and the product is used commonly as Japanese lacquer varnish (Benfield et al., 1964). Since then, the oxidative activity of laccase has been used in a number of industrial and environmental applications. Although laccases originate from different sources like higher plant, bacteria, fungi and insect (Mayer and Staples, 2002) those from fungi origin are used in most of the application.
Laccases from a large number of fungi have been studied. These enzymes offer great variability in terms of induction mechanisms, degree of polymorphism, expression of different isoenzymes and physico–chemical and catalytic properties. This variability determines the application for the isolated laccase and depends on whether the enzyme has a potential to oxidize a substrate or whether it is robust enough to be used in a certain application.
Hence, the search for fungal laccases with different properties and potential application is still on-going. It is expected that new isolated enzymes may catalyze old known process by a better way or may oxidize new substrates for novel application.
1.1 Statement of problem
One of the limitations for large scale application of laccases is the lack of capacity to produce large volume of highly active laccase at affordable cost and identifying inexpensive raw material for this enzyme production could be viewed as a solution to make the entire process cost effective.
In view of this enzyme importance in large scale application, this research is aimed at searching for highly efficient laccase producing microorganism with ability to produce copious amount of this enzyme from agro-waste materials so as to reduce the cost of production.
1.2.1 Objectives of the study
- To isolate and screen fungi for laccase production from agro- waste material (decayed wood, plantain waste and banana waste).
- To determine isolates capable of producing highest quantity of laccase, identify and characterize them.
- To investigate the possible use of agro- industrial waste as organic source of carbon for laccase production.
- To optimize the cultural parameters for laccase production.
1.3 Literature Review
1.3.1 FUNGI: A USEFUL SOURCE OF OXIDATIVE ENZYME
Fungi constitute a kingdom including eukaryotic organisms such as yeast and molds. The principal characteristics include the presence of cell walls, basically containing chitin and glucans, their heterotrophic behavior and the ability to produce extracellular hydrolytic enzymes in order to obtain the nutrients,which can be absorbed through the cell wall and cell membrane.From an evolution point of view, fungi are believed to be closer to animals than plants, since they have chitin rather than cellulose structures, store nutrients such as glycogen and produce spores similar to gametes (Barr, 1992). Their life cycle includes the formation of spores,germination, development of hyphae and mycelia and finally the growing of fruit body.
FIGURE 1.1 LIFE CYCLE OF FUNGI (Carlite et al., 2001).
The above mentioned characteristics of fungi determine their expansion over considerable long distances and even their vast distribution all over the world, including in extreme conditions such as deserts, salty environments and deep sea sediments. Around 70000 species of fungi have been described, however it is estimated that 1.5 million species might exist. Most of the fungal species occur as saprophytes (they use carbon fixed by other organisms) in woods, soils, leaf litter and animals while others are biotrophs, which means that they form symbiotic association with plants(mycorrhizae and endophytes) animal, algae (lichens) and prokaryotes.
As far as is known, fungi have 7 phyla or division: they include, Microsporidia, Chytridiomycota, Neocallimastigomycota, Glomeromycotina (before Zygomycota), Ascomycota and Basidiomycota (Hibbett et al., 2007). Fungi are among the most useful organism for industry and biotechnology. First of all they are the main agent responsible for decomposition of lignocellulosic material and thus for mobilizing the carbon into the ecosystem. They are also involved in bread and alcohol production, cultivation of edible mushroom (Barbado, 2003), production of compounds with biological activity like antibiotics (cephalosporin and penicillin) from Cephalosporium acremonium and Penicillium chrysogenum respectively, production of enzymes (Table 1.1), cholesterol lowering agent(lovastatin from Monascus ruber and mevastatin from Penicillium citrinum) organic acids (itaconic, malic and fumaric acids candida sp) amino acids ( lysine, tryptophan and phenyalanine from Saccharomyces sp, Hansenula sp and Rhodoturula sp respectively), recombinant therapeutics (insulin using Saccharomyces cerevisiae), recombinant enzymes and immunosuppressing drugs (cyclosporine A from Tolypocladium inflatum) (Murphy and Horgan, 2005).
1.3.2 LIGNOCELLULOSE DEGRADING FUNGI
Certain groups of fungi are able to efficiently degrade lignocellulosic material. These are divided into four categories: Stain fungi, Soft- rot fungi, Brown- rot fungi and White fungi (Martinez et al., 2005).Stain fungi and Soft -rot fungi are ascomycetes unable to degrade lignin. Brown- rot fungi are exclusively basidomycetes (Martinez et al., 2005) and produce enzymes that break down cellulose and hemicelluloses and partially breaks lignin and soft wood too (Tanesaka et al.,1993).