Afrah Shafique (2012-VA-577)
Production, Purification And Characterization Of Laccase From White Rot Fungus - 2014. - 90p.;
Laccase (oxidoreductase, EC 1.10.3.2) are blue copper dependent oxidases and the mainligninolytic enzyme produced by white rot fungus. Laccase catalyze the oxidation of large snumbers of phenolic compounds (Kunamneni et al. 2007; Poonkuzhali et al. 2011). These enzymes have a molecular weight 60-90 kDa and consist of 15–30% carbohydrate. Laccases are the earliest and maximum investigated enzymatic systems. Laccase was initially found by Yoshilda in 1883 in the sap of Japanese laquer tree named as Rhusvernicifera. After a while in 1896, Bertrand and Laborde determined that laccase is a fungal enzyme.(Shraddha et al. 2007; Giardina et al. 2010).
Laccases are present extensively in nature, originating from plants, bacteria and fungi (Poonkuzhali and Palvannan 2011). In fungi, laccases are widely distributed in ascomycetes, deuteromycetes and basidiomycetes. The laccase producing fungus include Trametes versicolor, Pleurotus ostreatus, Polyporus, Trametespubescens, Cerrenaunicolour,PhanerochaetechrysosporiumandFunaliatrogiietc (Dwivediet al. 2011). Laccases occur morein fungi, than in the higher plants. Laccases are also present in few bacteria such as S.lavendulae, S.cyaneus, and M.mediterranea(Viswanath et al. 2008; Arias et al. 2003). In vegetables laccases have been recognized in turnips, apples, pears, cabbages, potatoes, beets, asparagus and various other vegetables (Jhadav et al. 2007).
Enzymes are produced by every living organism, however enzyme produced by microbes have various benefits over the enzyme originated from plants and animal origins.Laccases by nature
are important because of its huge diversity of catalytic activities, economical in production and comparatively more stable than other enzymes.The field of biotechnology proposes expanding possibilities for the production of several enzymes from microorganisms. New methods and techniques have been advanced by using enzyme as biocatalysts to produce big added value products like growing food requirements,good quality chemicals and medicines. Moreover enzymes are also utilized for environmental actions and for diagnostic and analytical motives. (Buchholz et al. 2005). Microbial enzymes are used as cost effective and environmentally sensitive substitutes for chemical processing in several industries and bioremediation. Therefore the commercial demand for microbial enzymes is increasing (Radhika et al. 2013).
Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolicselimination from wines, in preparation of biosensors in detergents blockindye transfer- functions (Yaver et al. 2001).It is also used in the formation of anticancer drugs, and included in few cosmetics to lessen their toxicity (Couto and Herrera 2006).In recent years, laccase have been skillfully practiced to the field of nanobiotechnology due to its capacity to mobilize electron transfer reactions without further addition of cofactor(Shraddha et al. 2007).
Laccase is ample in several white- rot fungi that are involved in lignin metabolism (Bourbonnais et al. 1997, Leontievskyet al. 1997). Fungal laccases have immense redox potential (up to +800 mV) than bacteria or plant laccases. The action of these laccases seems to be appropriate in nature and also has significant applications in the field biotechnology. These laccases are associated with the deterioration of lignin and also in the elimination of conceivably lethal phenols appear during the breakdown of lignin (Thurston et al. 1994).
The white rot fungus is corporeal in preference to morphological and composes of those fungi that are adequate of degrading lignin, which is a heterogenous polyphenolic compound in huge amount within the lignocellulose wastes(Eaton and Hale. 1993).Theircapability to deteriorate cellulose, hemicellulose, these are the polysaccharides forming the essential part of lingo cellulose is the basic metabolic processbetween the fungi and happen under the span of environmental conditions.The degeneration of lignindoesn’tprovide net energy so it is degraded during the secondary metabolism in order to gain polysaccharides present in lignin and carbohydrate complexes, supplying energy to which the organisms don’t have access(Jeffrics. 1990).The white rot fungi varyingly secrete one or more three extracellular enzyme namely manganese peroxidase, lignin peroxidase and laccase that are fundamental for degradation of lignin, ant they are generally mentioned as lignin modifying enzymes LMEs (Pickard et al. 1999).
Laccase is the subjects of demanding research in the recent years, because of their several properties like extensive substrate relevance, doesn’t required the inclusion of cofactors because they use oxygen as cofactor which is frequently present in the environment (Eugenio et al. 2009). Maximum number of laccases produced by various organisms is excreted as extracellular enzymes and this makes the purification process quite accessible. Laccase commonly display appreciable extent of stability. Due to these properties laccases are ideally applicable in diverse biological processes such as the treatment of industrial effluent, biopulping and biobleaching (Eggert et al. 2006).
The huge potential of laccase requires advancement in its production and, with huge activities and low cost (Herrera et al. 2007). The use of lignocellulosic agricultural waste as substrates is a tradition for the production of enzyme like laccase because it is ligninolytic in nature (Niladeviet al.2011). It is highly crucial to optimize the fermentation parameters for the adequate production of laccase (Revankaret al. 2007). .
The advantages of agro-industrial leftovers for cultivation media is of immense concern as agriculture waste cut down the expenditure of enzyme production and enhance the understanding on energy protection and recycling (Mansuret al. 2003).These agriculture wastes are comparatively economical and also contain ample nutrients such as lignin, cellulose andhemicellulose. These nutrients serve as inducer to energize the production of enzyme (Vassil et al. 2000).Due to these properties these agricultural waste can be used as substrate for the production of ligninolytic enzymes during the process of fermentation.
Laccase can be produced at varying rates by using a wide range of organisms grown on different substrates and by using several methods of fermentation, such as solid state, semisolid state, and submerged (Rodriguez et al. 1999; Boran et al. 2011). However, for effective laccase production, it is very important to use efficient laccase-producing organisms, suitable fermentation methods, and cheap and widespread sources. Accordingly, one of the most suitable approaches for the production of this enzyme is to use the most efficient agricultural wastes for increasing the production of the ligninolytic enzymes (Elisashviliet al. 2008).
Pakistan is an agricultural country and each year manufactures tons of agricultural by products. These agricultural wastes are accessible in markets at a very reasonable price and can be utilized as substrates in fermentation technique (Minussi et al. 2007). Agricultural waste products like rice husk, wheat bran, corn cob, millet husk and cereal huskhave been utilized by various scientists for laccase production (Osma et al. 2011; jhadav et al. 2009).The chemical properties of these agricultural wastes make them important and economical fermentation medium for biotechnological purposes(Giardina et al. 2010).The cellulose and hemicellulose constituents of lignocellulose wastes are widely used by several organisms but lignin, which is the maximum contrary material to microbial degradation, is transformed conveniently by only few organism of thw white rot fungus (Dwivedi et al. 2011).Lignin serves as a barrier that protects cellulose and hemicellulose from enzymatic attack, however, white rot fungi can attack this barrier in order to obtain energy from cellulose. These fungi produce different extracellular ligninolytic enzymes such as laccase, manganese peroxidase, and lignin peroxidase (Couto et al. 2006).
Fermentation is a biological approach that is used for the transformation of complicated substrates into basic composites by different microorganisms like bacteria and fungi. In the procedure of this metabolic breakdown the microorganisms also release various added compounds like carbon dioxide and alcohol asidefrom the conventional products of fermentation. These added compounds are known as secondary metabolites (Pandey et al. 1999). These Secondary metabolites span from enzymes, antibiotics, peptides and growth factors (Balakrishnan and Pandey. 1996; Machado et al. 2004; Robinson et al. 2001). They are also known as bioactive compounds becausethey carry biological activity(Demain et al. 1999).
Submerged fermentation is a type of fermentation in which components are present in a liquid media like broths and syrup. The co-active composites are poured into the fermentation broth. In this media the substrates are employed quiet immediately, due to this reason the nutrients in the media are either fortified or regained continuously. This type of fermentation approach is optimum for microorganisms such as bacteria, fungi because they depend upon on immense moisture content. The increased benefit of this approach is that the purification of the desired products or enzymes is quiet effortless. Submerged fermentation is especially used in the abstraction of secondary metabolites that are utilized in liquid form (Subramaniyam et al. 2012).
Furthermore 75% of the commercial enzymes are made by using submerged fermentation, it also supports the usage of genetically modified organisms to a large expanse then solid state fermentation. Submerged fermentation is also used on large extent because it doesn’t require equipment concerning solid state. On the contrary solid state fermentation is a mechanism operated in absence of free flowing water by utilizing solid support in form of natural substance ( Poonkuzhali et al . 2011). .
The major purpose of conducting this research is to design optimized fermentation process which produces effective amount of enzyme by using agricultural wastes. The use of agricultural wastes as substrates is economical and increase awareness on energy conservation .The enzyme can be used further for bioremediation because it not substrate specific and can act on broad range of substrates.
Department of Biochemistry
2213,T
Production, Purification And Characterization Of Laccase From White Rot Fungus - 2014. - 90p.;
Laccase (oxidoreductase, EC 1.10.3.2) are blue copper dependent oxidases and the mainligninolytic enzyme produced by white rot fungus. Laccase catalyze the oxidation of large snumbers of phenolic compounds (Kunamneni et al. 2007; Poonkuzhali et al. 2011). These enzymes have a molecular weight 60-90 kDa and consist of 15–30% carbohydrate. Laccases are the earliest and maximum investigated enzymatic systems. Laccase was initially found by Yoshilda in 1883 in the sap of Japanese laquer tree named as Rhusvernicifera. After a while in 1896, Bertrand and Laborde determined that laccase is a fungal enzyme.(Shraddha et al. 2007; Giardina et al. 2010).
Laccases are present extensively in nature, originating from plants, bacteria and fungi (Poonkuzhali and Palvannan 2011). In fungi, laccases are widely distributed in ascomycetes, deuteromycetes and basidiomycetes. The laccase producing fungus include Trametes versicolor, Pleurotus ostreatus, Polyporus, Trametespubescens, Cerrenaunicolour,PhanerochaetechrysosporiumandFunaliatrogiietc (Dwivediet al. 2011). Laccases occur morein fungi, than in the higher plants. Laccases are also present in few bacteria such as S.lavendulae, S.cyaneus, and M.mediterranea(Viswanath et al. 2008; Arias et al. 2003). In vegetables laccases have been recognized in turnips, apples, pears, cabbages, potatoes, beets, asparagus and various other vegetables (Jhadav et al. 2007).
Enzymes are produced by every living organism, however enzyme produced by microbes have various benefits over the enzyme originated from plants and animal origins.Laccases by nature
are important because of its huge diversity of catalytic activities, economical in production and comparatively more stable than other enzymes.The field of biotechnology proposes expanding possibilities for the production of several enzymes from microorganisms. New methods and techniques have been advanced by using enzyme as biocatalysts to produce big added value products like growing food requirements,good quality chemicals and medicines. Moreover enzymes are also utilized for environmental actions and for diagnostic and analytical motives. (Buchholz et al. 2005). Microbial enzymes are used as cost effective and environmentally sensitive substitutes for chemical processing in several industries and bioremediation. Therefore the commercial demand for microbial enzymes is increasing (Radhika et al. 2013).
Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolicselimination from wines, in preparation of biosensors in detergents blockindye transfer- functions (Yaver et al. 2001).It is also used in the formation of anticancer drugs, and included in few cosmetics to lessen their toxicity (Couto and Herrera 2006).In recent years, laccase have been skillfully practiced to the field of nanobiotechnology due to its capacity to mobilize electron transfer reactions without further addition of cofactor(Shraddha et al. 2007).
Laccase is ample in several white- rot fungi that are involved in lignin metabolism (Bourbonnais et al. 1997, Leontievskyet al. 1997). Fungal laccases have immense redox potential (up to +800 mV) than bacteria or plant laccases. The action of these laccases seems to be appropriate in nature and also has significant applications in the field biotechnology. These laccases are associated with the deterioration of lignin and also in the elimination of conceivably lethal phenols appear during the breakdown of lignin (Thurston et al. 1994).
The white rot fungus is corporeal in preference to morphological and composes of those fungi that are adequate of degrading lignin, which is a heterogenous polyphenolic compound in huge amount within the lignocellulose wastes(Eaton and Hale. 1993).Theircapability to deteriorate cellulose, hemicellulose, these are the polysaccharides forming the essential part of lingo cellulose is the basic metabolic processbetween the fungi and happen under the span of environmental conditions.The degeneration of lignindoesn’tprovide net energy so it is degraded during the secondary metabolism in order to gain polysaccharides present in lignin and carbohydrate complexes, supplying energy to which the organisms don’t have access(Jeffrics. 1990).The white rot fungi varyingly secrete one or more three extracellular enzyme namely manganese peroxidase, lignin peroxidase and laccase that are fundamental for degradation of lignin, ant they are generally mentioned as lignin modifying enzymes LMEs (Pickard et al. 1999).
Laccase is the subjects of demanding research in the recent years, because of their several properties like extensive substrate relevance, doesn’t required the inclusion of cofactors because they use oxygen as cofactor which is frequently present in the environment (Eugenio et al. 2009). Maximum number of laccases produced by various organisms is excreted as extracellular enzymes and this makes the purification process quite accessible. Laccase commonly display appreciable extent of stability. Due to these properties laccases are ideally applicable in diverse biological processes such as the treatment of industrial effluent, biopulping and biobleaching (Eggert et al. 2006).
The huge potential of laccase requires advancement in its production and, with huge activities and low cost (Herrera et al. 2007). The use of lignocellulosic agricultural waste as substrates is a tradition for the production of enzyme like laccase because it is ligninolytic in nature (Niladeviet al.2011). It is highly crucial to optimize the fermentation parameters for the adequate production of laccase (Revankaret al. 2007). .
The advantages of agro-industrial leftovers for cultivation media is of immense concern as agriculture waste cut down the expenditure of enzyme production and enhance the understanding on energy protection and recycling (Mansuret al. 2003).These agriculture wastes are comparatively economical and also contain ample nutrients such as lignin, cellulose andhemicellulose. These nutrients serve as inducer to energize the production of enzyme (Vassil et al. 2000).Due to these properties these agricultural waste can be used as substrate for the production of ligninolytic enzymes during the process of fermentation.
Laccase can be produced at varying rates by using a wide range of organisms grown on different substrates and by using several methods of fermentation, such as solid state, semisolid state, and submerged (Rodriguez et al. 1999; Boran et al. 2011). However, for effective laccase production, it is very important to use efficient laccase-producing organisms, suitable fermentation methods, and cheap and widespread sources. Accordingly, one of the most suitable approaches for the production of this enzyme is to use the most efficient agricultural wastes for increasing the production of the ligninolytic enzymes (Elisashviliet al. 2008).
Pakistan is an agricultural country and each year manufactures tons of agricultural by products. These agricultural wastes are accessible in markets at a very reasonable price and can be utilized as substrates in fermentation technique (Minussi et al. 2007). Agricultural waste products like rice husk, wheat bran, corn cob, millet husk and cereal huskhave been utilized by various scientists for laccase production (Osma et al. 2011; jhadav et al. 2009).The chemical properties of these agricultural wastes make them important and economical fermentation medium for biotechnological purposes(Giardina et al. 2010).The cellulose and hemicellulose constituents of lignocellulose wastes are widely used by several organisms but lignin, which is the maximum contrary material to microbial degradation, is transformed conveniently by only few organism of thw white rot fungus (Dwivedi et al. 2011).Lignin serves as a barrier that protects cellulose and hemicellulose from enzymatic attack, however, white rot fungi can attack this barrier in order to obtain energy from cellulose. These fungi produce different extracellular ligninolytic enzymes such as laccase, manganese peroxidase, and lignin peroxidase (Couto et al. 2006).
Fermentation is a biological approach that is used for the transformation of complicated substrates into basic composites by different microorganisms like bacteria and fungi. In the procedure of this metabolic breakdown the microorganisms also release various added compounds like carbon dioxide and alcohol asidefrom the conventional products of fermentation. These added compounds are known as secondary metabolites (Pandey et al. 1999). These Secondary metabolites span from enzymes, antibiotics, peptides and growth factors (Balakrishnan and Pandey. 1996; Machado et al. 2004; Robinson et al. 2001). They are also known as bioactive compounds becausethey carry biological activity(Demain et al. 1999).
Submerged fermentation is a type of fermentation in which components are present in a liquid media like broths and syrup. The co-active composites are poured into the fermentation broth. In this media the substrates are employed quiet immediately, due to this reason the nutrients in the media are either fortified or regained continuously. This type of fermentation approach is optimum for microorganisms such as bacteria, fungi because they depend upon on immense moisture content. The increased benefit of this approach is that the purification of the desired products or enzymes is quiet effortless. Submerged fermentation is especially used in the abstraction of secondary metabolites that are utilized in liquid form (Subramaniyam et al. 2012).
Furthermore 75% of the commercial enzymes are made by using submerged fermentation, it also supports the usage of genetically modified organisms to a large expanse then solid state fermentation. Submerged fermentation is also used on large extent because it doesn’t require equipment concerning solid state. On the contrary solid state fermentation is a mechanism operated in absence of free flowing water by utilizing solid support in form of natural substance ( Poonkuzhali et al . 2011). .
The major purpose of conducting this research is to design optimized fermentation process which produces effective amount of enzyme by using agricultural wastes. The use of agricultural wastes as substrates is economical and increase awareness on energy conservation .The enzyme can be used further for bioremediation because it not substrate specific and can act on broad range of substrates.
Department of Biochemistry
2213,T