Enzymes can be defined as nature’s catalyst. Enzymes are proteins that act as a catalyst and increases the rate of a chemical reaction that occur within the living cells, without altered themselves. Chemical reactions generally involve a process that can convert a reactant a (reagents, substrates) into the product. A catalyst (as an enzyme) makes it easier to process a chemical reaction again and again.
Microbial enzymes are obtained from the various microbial sources. Microbial enzymes are generally obtained from microorganisms such as bacteria, fungi and yeasts have been universally studied for biosynthesis of economically viable preparations of various enzymes for commercial applications. Microbial enzymes have several applications, particularly in industries on commercial scales. As compared to enzymes obtained from plants or animals, microbial enzymes are more useful because of high catalytic activity, the high yield possible, ease of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms or inexpensive media (Sharma et al., 2001; Iftikhar et al., 2007; Iftikhar et al., 2008; Helen ; Oliveira, 2009)
Laccases are generally monomeric or multimeric glycoprotein containing copper and posses additional heterogeneity as it contain variable carbohydrate content, differences in copper content or due to their expression as the product of multiple genes ((Dahiya and Singh 1998). Laccase can catalyses both phenolic and non phenolic substrates. Non phenolic substrates can be catalyzed by insertion of mediators (low molecular weight compounds). Most commonly used mediators are 1-hydroxy benzotriazole (HOBT), N-hydroxyphthalimide (NHPI), 2, 2 – azinobis-(3 ethylbenzthiazoline-6-sulfonate) (ABTS), and 3 hydroxyanthranilic acid (Bourbonnais et al 1995 and Gochev et al 2007). Nowadays there has been great interest in studying laccase because of broad specificity and their chemical and catalytic features of wide biotechnological applications (Messner and Srebotnik 1994; Sayadi and Ellouz 1995; Ferraz et al. 2008).
Lignin Peroxidase and Manganese Peroxidase
LiPs and MnPs are a well known glyco protein that contains a heme group that requires hydrogen peroxide as oxidant. By the abstraction of electron and cation radical non phenolic lignin substructure are oxidized by LiPs which will then decomposed chemically (Kirk et al., 1986; Kirk and Farrell, 1987). Decomposition of compounds by MnPs involves Mn (II) oxidation to Mn (III) which will after oxidation generating phenoxyl radicals (Gold et al., 1989).
The mechanistic action and substrate specificities generally varies among different cellulases, they are categorized into exoglucanases (EC 184.108.40.206), endoglucanases (EC 220.127.116.11), cellobiohydrolases (EC 18.104.22.168), and ?-glucosidases (EC 22.214.171.124) 36, 37.
Endo-glucanases (EG, endo-1, 4-D glucanohydrolasase) belongs to endo-type enzyme that hydrolyzes cellulose micro fibrils preferentially in the amorphic part of the plant fibril. The catalytic region of the enzyme is groove-shaped that enables the attachment of the enzyme and the hydrolysis in the middle part of the cellulose fibre (Divine et al. 1994). Endo-glucanase activity in other white rot fungi is a common feature and probably exists in all wood degrading fungi including brown rot fungi (Highley 1998).
Exo glucanase or cellobiohydrolases
Exo glucanase are exo-type enzyme that attack cellulose fibre from both reducing and non-reducing ends. Degradation of pure crystalline cellulose is not shown by fungi belongs to brown rot as they do not posses exo type activity. Few brown rot fungi belong to family Coniphoraceae are exception to this generalization (Nilsson and Ginns 1979).
?- Glucosidase hydrolyzes the product of cellobiose to two glucose units. Cellobiose and glucose can be taken up and assimilated by the hyphae. The cellobiose taken up is probably hydrolyzed to glucose by cell wall bound on intercellular -glucosidase. All cellulolytic enzymes share the same chemical specificity for -1, 4-glycosidic bonds which they cleave by a general acid-catalyzed hydrolysis.
Xylanase is a group of enzymes that having properties such as tolerance to high pH and temperature, catalyze the hydrolysis of xylan (Coughlan and Hazelwood, 1993). Typical hemicellulases are of three types endo-1, 4-?-D-xylanases (E.C. 126.96.36.199), endo- 1,4-?-D mannanases (E.C.188.8.131.52), and endo-1, 4-?-D-galactanases (E.C. 184.108.40.206). Endo-?-1, 4-xylanases catalyse the random hydrolysis of ?-1, 4-glycosidic bonds in xylan. Endo-1, 4-?-D mannanases hydrolyse randomly the 1, 4-?-D-mannopyranosyl linkage of gluco- and galacto glucomannans, releasing oligomeric fragments. Endo-1,4-?-D-galactanases have been characterized in Irpex lacteus (Tsumuraya et al., 1990)
Occurrence and distribution
Laccases (benzenediol: oxygen oxidoreductase, EC220.127.116.11) are a family of blue multicopper oxidases that are capable of oxidizing a wide range of phenolic and aromatic compounds, with concomitant reduction of molecular oxygen to water (Hoegger et al 2006). Laccase is a large molecule that can penetrate deep into wood and because it has low redox potential (; 0.8 V) compared to ligninolytic peroxidase (; 1 V), laccase can only oxidize lignin phenolic fragments. However, the number and type of substrate oxidized by laccase can be extended by a mechanism involving participation of redox mediators (Rodgers et al., 2010). Laccases are produced from various sources such as from higher plants (Blingy et al., 1983; Dean et al., 1994 ; Gregory et al,11996), most of the fungi (Thurston et al., 1994); bacteria and also from insects. Laccases isolated from insects generally used during sclerotization processing for epidermal cuticle synthesis (Nakamura and Goa, 2005; Sakurai and Kataoka, 2007). The natural function of these fungal enzymes is degradation of lignin, morphogenesis of fungi, sporulation, pigment production and fruiting body formation (Herman et al., 1983; Langfelder et al., 2003; Fang et al., 2010, Thurston et al.,1994., Kuznetsov et al., 1984; Bourbonnais et al, 1990). In bacteria laccases have several functions such as morphogenesis processes, copper homeostasis, pigment biosynthesis (such as melanin and brown spore pigment), and spore protection against UV light and hydrogen peroxide (Santhanam et al., 2011; Strong and Claus, 2011). In bacteria laccases have several functions such as morphogenesis processes, copper homeostasis, pigment biosynthesis (such as melanin and brown spore pigment), and spore protection against UV light and hydrogen peroxide (Santhanam et al., 2011; Strong and Claus, 2011). Furthermore, fungal laccases are involved in sporulation, pigment production, fruit body formation, and plant pathogenesis (Alcalde, 2007). White rot fungi are also known for their efficient degrading capabilities of various dyes such as azo, heterocyclic, reactive and polymeric dyes by ligninolytic enzymes produced from them (1 Murugesan et al 2009, Murugesan et al 2007). In addition to various industrial applications laccases are also known for their catalytic conversion of phenolic and non phenolic compounds (Bourbonnais et al 1997) and can mineralize various types of dyes produced from different sources. Among white rot fungi Ganoderma lucidium is a representative laccases producing organism able to grow on withered wood and agro-lignocellulosic biomass material and can decay lignin efficiently by secreting laccase enzyme (Zhou et al 2013). Ganoderma lucidium represents a model candidate for processing of lignocellulosics, because of the fact that it can modify lignin or lignin derived components (Sitarz et al 2013) by producing laccases enzymes in liquid cultures (D’Souza et al 1999, D’Souza et al 2005 Sharma et al 2013).
White rot fungi
White rot fungi are generally responsible for litter decomposition as they are the only organisms capable of lignin degradation (Kirk and Cullen, 1998). White rot fungi degrade lignocellulosic substances with two modes of actions namely selective decay and simultaneous decays. (Blanchette., 1995). In selective decay lignin and hemicelluloses fractions are selectively degraded by fungi and cellulose fractions remains unaffected. Selectivity of lignin degradation by white rot fungi depends on lignocellulose sp. In simultaneous decay almost all lignocellulosic fractions are degraded approximately equal (Blanchette 1995; Hatakka 2001). Some of the white rot fungi that show selective degradation are C. subvermispora, Dichomitus squalens, P. chrysosporium and Phelbia radiata and that posse’s non selective degradation are Trametes versicolor and Fomes fomentarius.
Solid state fermentation
Solid state fermentation is defined as a fermentation process that is carried out in without free flowing water; however substrate must provide sufficient moisture to the growing microorganism for their support and metabolism (Pandey et al 1992; Pandey et al 1994; Pandey et al 2000; Pandey et al 2001). Solid state fermentation offers several opportunities over other fermentation techniques in processing of agrochemical residues. This has been possible only in solid state fermentation because of low energy requirement, production of lesser wastewater and also resolve the problem of solid disposal as it is an environmental friendly technique.
During development of bioprocess in SSF several aspects should be considered such as micro organism and substrate, various parameters that has to be optimized and also the isolation and purification of products obtained after extraction (Pandey et al 1992; Pandey et al 2000; Nampoothiri et al 1996; Selvakumar et al 1999; Pandey et al 1998). Solid substrate or material in solid state fermentation provides physical support and as source of nutrient. Earlier it has been concluded that the SSF has advantage over SmF in terms of product yield, but there is no established scale or method to compare the product yield in SSF and SmF in exact term. The logic behind this might be that in SSF microbial cultures are closer to their natural environment and probably their activity increased (Pandey et al 1992; Pandey et al 2000;Pandey et al 2001; Pandey et al 2000; Pandey et al 2000; Pandey et al 2000;Pandey et al 2000;Pandey et al 2001; Peralta et al 2001; Hoogschagen et al 2001).
It is also very important to screen potential micro-organisms and select the suitable one. It has also been reported that use of inert material such as polyurethane foam will make it easier to isolate product in a simpler way as compare to natural substrate such as wheat bran because during extraction after fermentation along with the product several water soluble components leech out and make purification process difficult. Nowadays enzyme production is an increasing field of biotechnology. Earlier most of the enzyme production was carried out under sub – merged fermentation (SmF) technique. It has been reported that during last decade’s thers has been increasing interest towards using solid substrates for several enzyme production under SSF technique. SSF is found to be more suitable for filamentous fungi (Pandey et al 1999; Young et al 1983), since they will get the natural conditions to reproduce (Pandey et al 1999).
Due to the low amount of laccase production from selected strains in native form, several compounds have been widely used to elicit enhanced laccase production (Kumar, 2010). To meet the current requirement of fungal laccase in various biotechnological applications efforts were made to enhance the laccase production in economic and efficient manners by addition of various inducers or enhancers. Compounds (inducers) that are generally used for enhancing enzyme production are natural substrate or substrate analogue for enzyme (Kumar, 2010), nature of the compound that induces laccase activity differs greatly with the species. Inducers that are reported to be responsible for enhancing laccase production are copper sulfate (Revankar and Lele., 2006; Baldrian., 2003), ferulic acid and veratryl alcohol (Couto et al., 2002; Dela Rubia et al., 2002), 4-hydroxy-benzoic acid, 2, 5-xylidine or vanillin (Couto et al., 2002), alcohol, heavy metals, vitamins, amino acids, antibiotics (Baldrian et al., 2004), syringic acid, cinnamic acid, gallic acid and guaicol (Yang et al 2013), copper sulphate and tryptophan (Sharma et al., 2014), aromatic and phenolic compounds have been widely used to elicit enhanced laccase production by different organisms (D’Souza et al., 2004; Revankar and Lele, 2006). In recent years efforts have been made to produce enzyme on lignocellulosic wastes produced from agriculture to reduce the cost.
Lignocellulose is comprised of three main components, that is, cellulose, hemicellulose, and lignin 21
Table Typical chemical composition of various lignocellulosic materials (Betts et al. 1991) from lignocellulose biodegradation by S malherb
Raw material Lignin (%) Cellulose (%) Hemicellulose (%)
Hardwoods 18-25 45-55 24-40
Softwoods 25-35 45-50 25-35
Grasses 10-30 25-40 25-50
Mode of action of white rot fungi on lignocellulosic composition
For the degradation of cellulose, basidiomycetes utilizes a set of hydrolytic enzymes typically composed of – Endoglucanse (EG, endo-1, 4-D glucanohydrolasase) to randomly cleave the intermonomer bonds, Exoglucanase to remove mono and dimmers from the end of glucose chain, – glucosidase to hydrolyze glucose dimer (Deobald and Crawford 1997; Tomme et al. 1995). The enzymatic degradation of hemicelluloses requires a complex set of enzymes. Endo type librates shorter fragment of substituted oligosaccharide, which are further degraded by side group cleaving enzyme and exotype enzyme.
Fig. A schematic view of the cellulose structure and action of the CBHs, EGs, and ß glucosidases (ß-gluc) in Trichoderma reesei. C defines the highly ordered crystalline region, R the reducing ends (filled circles), and NR the non reducing ends (open circles). EGs attack the more disordered structures of the cellulose. ß glucosidase action produces glucose. Modified from Teeri (1997).
Lignin does not contain hydrolysable linkages, which means that the enzymes must be oxidative. Lignin is stereo irregular, which also points to more nonspecific attack compared to many other natural polymers. Laccase is a copper-containing oxidase that utilizes molecular oxygen as oxidant and also oxidizes phenolic rings to phenoxyl radicals (Thurston, 1994 LiP oxidizes nonphenolic lignin substructures by abstracting one electron and generating cation radicals that are then decomposed chemically (Kirk et al., 1986; Kirk and Farrell, 1987). MnP oxidizes Mn (II) to Mn (III) which then oxidizes phenolic rings to phenoxyl radicals which lead to decomposition of compounds (Gold et al., 1989) from biodegradation of lignin.
Application of laccase enzyme
Textile industries are one of the greatest generators of liquid effluent containing synthetic dyes and are responsible for the deep color of the effluents. Dyes are usually resistant to fading on exposure to light, water and also various chemicals due to their complex structure, and are most of them are difficult to decolorize due to their synthetic origin (Lin et al. 2010a, b) Currently many researchers have optimized dye decolorization using laccase (Fazli et al. 2010; Sharma et al. 2009). White-rot fungi are a class of microorganisms that produce efficient enzymes capable of decomposing dyes under aerobic conditions. They produce various oxido reductases that degrade lignin and related aromatic compounds (Ali 2010; Khan et al. 2013). Hence, fungal decolorization of dye by using laccase can provide an attractive solution due to their potential in degrading dyes of diverse chemical structure including synthetic dyes.
One of the major problem that are facing today is contamination of soil and water through environmentally hazardous xenobiotic compounds like polycyclic aromatic hydrocarbons (PAHs), phenols and organophosphorus insecticides that are known for their teratogenic and carcinogenic effects. Their removal from the environment is a priority for most of the environmental agencies (Alcalde et al., 2006; Viswanath et al., 2014).
Nanobiotechnology and biosensors
In biosensor technology laccase is mainly used for the broad range detection of phenolics, oxygen or azides, which disallow the detection of specific constituents (Fogel and Limson 2013). Laccase that is used in biosensor consisting of an electrode that can be used for detection of catechol in tea (a phenolic compound) according to Palmore and Kim 1999 and real water sample (Li et al.2014), in detection of polyphenolic compound in wine (Lanzellotto et al. 2014) and also in detection of lignins and phenols in wastewaters (Giovanelli and Ravasini 1993).
Medical and personal care application
Many antimicrobial, detoxifying active personal care agents or polymerizing agents are generated by laccases. Laccase enzyme polymerizes urushiol and reduces the effect of poison dermatitis and makes it non toxic (Cheong et al. 2010). Erb-Downward et al. (2008) reported synthesis of immune modulatory prostaglandrins. Laccase isolated from Pleurotus cornucopiae was found to have inhibitory effect on proliferation of murine leukemia cell line L1210 and human hepatoma cell line HepG2 and also in reducing the activity of of HIV-1 reverse transcriptase with an IC50 of 22 lM
Application of laccase in food processing
Laccases (benzenediol: oxygen oxidoreductase, (EC:18.104.22.168) are a diverse group of multi-copper proteins that oxidize a surprisingly wide variety of organic and inorganic compounds, including diphenol, polyphenols, substituted phenols, diamines and aromatic amines, with concomitant a reduction of molecular oxygen to water (Thurston, 1994). Due to the low specificity laccases have attracted interest in industrial and environmental biotechnology including as pulp delignification, dye decolorization, environmental pollutant detoxification, biopolymer modification, and biotransformation (Hou et al., 2004; Leonowicz et al., 2001; Yaropolov et al., 1994). Currently one of the most interesting subjects for researcher in environmental biotechnology and food microbiology consisting of Laccases can be used in detection of catecholamine neurotransmitters such as dopamine, norepinephrine, 6, laccase-oxidized form of morphine in the presence of codeine by coupling its oxidation with glucose dehydrogenase has been studied 7. Some fungal laccases degrade toxic fungal metabolites, such as aflatoxin B1 8, and are also useful in the field of food microbiology. From suresh s
The initial aim of this study was to produce lignocellulolytic enzymes from white rot basidiomycetous fungi collected from Arunachal Pradesh. After screening and optimization by OFAT for their lignocellulolytic enzyme production two isolates were found to be potent laccase producers. During the course of the work emphasis moved to laccase and RSM using box behnken was set to know the interactive effect of Cu2+, Tween 20 and inoculum amount on laccase production. Aim of the present study focuses on screening of white rot basidiomycetous fungi (collected from Arunachal Pradesh) for their laccase producing potential. Out of the six species collected (Ganoderma sp, Lentinus sp, Trametes sp, Pleurotus sp, Peniophora sp and Lepiota sp), Ganoderma sp and Lentinus sp were found to be potent laccase producers. In addition, the production of laccase by the positive strains was confirmed by qualitative test and also quantitative assay for laccase using guaicol as substrate. The isolated hypersecretory strain was identified by ITS sequencing. Due to the wide biotechnological applications of laccase. There is a need for screening the wild diversity of white rot fungi for selecting new efficient strains. In this context, the present investigation was designed to isolate wild basidiomycetous fungi with high laccase activity. Optimization was done by OFAT and RSM for maximizing laccase production and laboratory scale up for laccase production was performed.