PRODUCTION PARTIAL PURIFICATION AND CHARACTERIZATION OF CELLULASE BY ASPERGILLUS NIGER FROM SUBMERGED FERMENTATION OF GRAPE BAGASSE

ABSTRACT

The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study the ability of selected cellulosic substrate to induce cellulase production by Aspergillus niger and the ability of the induced enzyme to hydrolyze the cellulosic substrate were assessed. The enzyme was produced by submerged fermentation technique in which grape bagasse was the cellulosic substrate which served as a carbon source. Crude enzyme was harvested after 5 days of growth with activity of 8.2μmole/min for enzyme produced by Aspergillus niger. Cellulase produced from Aspergillus niger was subjected to a three step purification process: 50% ammonium sulphate precipitation, dialysis and gel column chromatography for characterization of the cellulase. The gel column chromatography yielded two peaks. Gel elution fractions were assayed for total cellulase activity and protein concentration. The 2 peaks indicate isoforms of the enzyme produced by Aspergillus niger. The total cellulase activity as well as β-glucosidases activity was characterized using filter paper and cellobiose as substrate. The partially purified enzyme showed that total cellulase activity had an optima pH and temperature of 5.5 and 50oC for isoform A and  5.0 and 55oC for isoform B using filter paper as substrate. Similarly, β-glucosidases activity had an optima pH 5.5 and 6.0 with optima temperature of 50oC for both isoforms using cellobiose as substrate. Kinetic parameter showed a Vmax and Km of 90.9μmole/min and 0.09mM cellobiose and 83.3μmole/min and 0.08mM cellobiose for both isoforms respectively. This kinetic study showed that grape bagasse is a good substrate for cellulase from Aspergillus niger and can be utilized as substrate for cellulase production. These results obtained in this study have established suitable conditions for maximizing the production of cellulase which is used for conversion of high cellulosic waste into wealth as found in bioethanol production.

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

Grape fruit (Citrus paradisi) is a subtropical citrus tree known for its sour to semi-sweet fruit. It has been part of human diet for ages due to its nutritional and medicinal values. The frequent use of grape fruits for production of juices, nectars, concentrates, jams, jelly powders and flakes generates wastes in the form of grape peel and bagasse which could bring about environmental pollution if not properly handled. Agricultural wastes and in fact all lignocellulosics can be converted into products that are of commercial interest such as ethanol, glucose, and single cell protein such as in the conversion of grape bagasse to cellulase. There is great interest in utilising cellulose wastes as feedstocks for fermentation processes, thereby converting low cost starting materials into products of greater value (Ojumu et  al., 2003). Substantial efforts are going  into  investigations on refining biomass to  derive  liquid  fuel,  chemical  feed  stock and  improved  animal  feeds  to  meet  global bioenergy  demand  through  the  biorefinery  concept,  since  agricultural  food  processes  generate millions of tons of waste each year (Xeros and Christakopoulos, 2009) such as grape bagasse, sugar cane bagasse, wheat straw and rice straw. Cellulose, a basic structural component of plant cell wall (Dewey and Mary, 1980) is a polymer of β-D-Glucose which links successfully through a beta- configuration between carbon 1 and carbon 4 of adjacent units to form a long chain 1,4 glucans. Cellulase refers to a class of enzymes produced by fungi, bacteria and protozoans and it causes hydrolysis of cellulose (Bhat, 2000; Sherief et al., 2010). They are widely distributed throughout the biosphere and  are  most  manifest  in  fungal and  microbial organisms (Chinedu et  al.,  2011).  A cellulosic enzyme system consists of three major components: endo-β-glucanase (EC 3.2.1.4), exo-β- glucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) (Knowles et al., 1987). These components act synergistically in the conversion of cellulose to glucose (Chen et al., 1992; Begum and Lemaire,

1996; Chirico and Brown, 1987). Cellulase production has been described for many Aspergillus species (Lockington et al., 2002; Kang et al., 2004; Wang et al., 2006; Gao et al., 2008) under submerged fermentation. The submerged cultivation is carried out by using rotary shaker (Bakare et al., 2005). In enzyme production, purification is important to study the function and expression of the enzyme and to remove any contaminants (other proteins or completely different molecules) that are present in the mixture. The ability to secrete large amounts of extra cellular protein is characteristic of certain fungi and  such strains are  most  studied  for production of higher levels  of extracellular cellulases.  Fungal cellulases  are  preferred  for  industrial application  because  they  are  inducible

enzymes which can produce large quantities of cellulase (Immanuela et al., 2007). This process reflects well the fact that filamentous fungi are naturally excellent protein secretors and can produce industrial enzymes in feasible amounts (Bergquist et al., 2002). Cellulase are used in the textile industry for cotton softening; in laundry detergents for colour care, cleaning, and anti-deposition; in the food industry for mashing; in the pulp and paper industries for deinking, drainage improvement, and fibre modification and they are even used for pharmaceutical applications.

1.1      History of Cellulose

Cellulose, a complex carbohydrate or polysaccharide consisting of 3000 or more glucose units and a basic structural component of plant cell wall (Dewey and Mary, 1980) was discovered in 1838 by the French chemist Anselme Payen, who isolated it from plant matter and determined its chemical formula. Cellulose was used to produce the first successful thermoplastic polymer, celluloid, by Hyatt Manufacturing Company in 1870. Hermann Staudinger determined the polymer structure of cellulose in 1920. The compound was first chemically synthesized (without the use of any biologically derived enzymes) in 1992 (Klemm et al., 2005).

1.1.1   Cellulose

Cellulose is a long chain polymer, made up of repeating units of glucose, a simple sugar joined together with β-1, 4 glycosidic linkages. Cellulose is the most abundant biopolymer on earth (Lynd et al., 2002; Ahmed et al., 2003; Mazeau and Huex, 2003). It is a fibrous, tough, tasteless water-soluble substance, which is found in the protective cell walls of plants, particularly in stalks, stems, trunks and all woody portions of plant tissues (Figure 1). The xylem tissue mainly consists of cellulose which is further protected by hemicelluloses and pectin (Lynd et al., 2002). It is the main structural component of plant cell walls, consisting 505 of the biomass in trees.

Figure 1: Diagram of Plant cell wall (Wikipedia, 2012a)

1.1.2   Structure of Cellulose

Cellulose, a major structural material of plants is a polymer of β-D-Glucose which links successfully through a beta-configuration between carbon 1 and carbon 4 of adjacent units to form a long chain 1, 4 glucans. It consist of linear beta-1, 4-linked D-glucosopyranoside chains (so-called anhydroglucose units) that are condensed into crystalline structure called microfibrils (Mohammed and Obasola, 2007). The molecule adopts an extended and rather stiff rod like conformation, aided by equatorial conformation of glucose from one chain of the hydrogen bonds with oxygen molecule on the same or on a neighbouring chain, holding the chains firmly together side-by-side and forming micro-fibrils with high tensile strength (Montes and Cavaille, 1999; Wei et al., 2009). The cellulose

molecule forms a straight, almost fully extended chain, where glucose residue is rotated about 180o

relative to each other along the main axis. Each chain is stabilised by intra-chain hydrogen bonds formed between the pyranose ring oxygen in one residue and the hydrogen of the –OH group on C3 in the next residue (Gradner and Blackwell, 1974). The functional group in the cellulose chain are the hydroxyl groups, these hydroxyls groups are able to interact with each other or with O-N- and S- group, forming hydrogen bonds. H-bonds also  exist between OH-groups of cellulose and water molecules. These hydroxyl groups make the surface of cellulose largely hydrophilic. The cellulose chains are oriented in parallel, with reducing ends of adjacent glucan chains located at the same end of a micro-fibril.

Figure 2: Structure of cellulose showing Amorphous and crystalline areas (Mohammed and Obasola, 2007).

Cellulose can take on at least four different crystalline forms (I to IV), as determined by X-ray crystallography (Gardner and Blackwell, 1974). Generally, the percentage and crystalline from of cellulose within a plant cell wall varies according to cell type and developmental stage. Cellulose almost never occurs alone in nature but is usually associated with other plant substances. Cellulose fibrils are embedded in a matrix of other polymers, primarily including hemicelluloses, pectin and proteins  (Meiser,  1995).  This  association  may  affect  its  natural degradation.  Native  crystalline cellulose  is  generally  found as  a  mixture of cellulose  1α  and  cellulose  1β  polymorphic forms (Mazeau and Heux, 2003). The proportions of the two allomorphs of crystalline cellulose vary depending on the sources of the cellulose. The mechanical properties of cellulose strongly depend on the  ratio  between  crystalline  and  amorphous  phases;  crystalline  cellulose  has  such  a  strong mechanical strength that it could be used as reinforcement in composite materials (Favier et al., 1995; Igarashi et al., 2009) whereas amorphous cellulose displays important viscosity elastic properties (Montes et al., 1997; Montes and Cavaille, 1999).

1.1.3   Properties of Cellulose

Two glucose molecules react to form a cellobiose which is the basic chemical unit of a cellulose molecule. The pyranose rings are in the 4 conformation which means that the CH2OH and OH groups as well as glycosidic bonds are all equatorial with respect to the mean planes of the rings. The hydroxyl groups at both end of the cellulose chain show different behaviour. The C-1 end has reducing property, while the glucose end group with a free C4-OH is non-reducing. The absence of side chains allows cellulose molecules to lie close together and form rigid structures. Cellulose has no taste, it is odourless,  hydrophilic with the contact angle of 20–30 (Charles, 2007),  insoluble in water and most organic solvents, chiral and is biodegradable. It can be broken down chemically into its glucose units by treating it with concentrated acids at high temperature.

1.2      Lignocellulosic Substrate

Lignocelluloses are composed mainly of cellulose, hemicelluloses and lignin. Lignocellulosic biomass includes materials such as agricultural residues (Corn straw and wheat straw): forestry residues such as saw dust and various industrial wastes (Howard et al., 2003). Nowadays, lignocellulosic materials are not just used in their old ways but their applications have expanded into the high level as in enzymatic and bio-fuel products.

1.2.1   Methods of lignocellulose substrate pre-treatment

Pre-treatments are designed to open the structure of lignocellulosic biomass prior to enzyme hydrolysis, to allow efficient production of C5  and C6  sugars. Hydrolysis of the major component, cellulose has received the most attention, as it  can be used to produce cellulose and ethanol by fermentation. Cellulose exists in nature as a compact and complex matrix with lignin and hemicelluloses. The cellulose is highly ordered and crystalline with amorphous regions. It is surrounded by lignin which acts as a physical barrier and is associated with the hemicelluloses. Obviously, reduction of crystallinity of cellulose  and  removal of lignin  and  hemicelluloses are important goals for any pre-treatment process. The -1, 4 glucosidic bonds in cellulose are easier to cleave than the – 1, 4-glycosidic bonds of starch (Tsao, 2004). This makes it clear that the main problem in cellulose hydrolysis of biomass cellulose is due to the secondary and tertiary structures, not the primarily linkage structure. A number of pre-treatment methods have been developed to improve cellulose hydrolysis from lignocelluloses. They include mechanical pulverization, pyrolysis, treatment  with  concentrated  acid,  dilute  alkali,  hydrogen  peroxide,  biological  treatment,  and ozonolysis etc. Each method in some way decreases the size of the biomass and opens its physical structure.

1.2.1.1 Mechanical Pulverization

Lignocellulosic biomass can be pulverized by chipping, grinding and shearing. The goal of mechanical pulverization is to reduce the particle sizes of the biomass, as increased surface area leads to improved cellulose hydrolysis. Some of the crystalline structure of cellulose is also destroyed using these methods, though the amount varies according to the type of biomass and power applied in milling and grinding. A Vibration ball mill is the most effective mechanical tool for breaking the crystalline structure of cellulose. Mechanical pulverization methods generally are high cost and do not remove the lignin or hemicellulose.

1.2.1.2 Pyrolysis

Lignocellulosic biomass can be rapidly decomposed to gas, bio-oil, and char when heated to temperatures above 300oC in the absence of oxygen. Biomass pyrolysis is carried out at 400-600oC. At these temperatures, the biomass structure separates randomly. Presence of oxygen, zinc chloride and sodium carbonate can accelerate the decomposition of cellulose, reducing temperature requirements but producing CO2 and H2O. However, pyrolysis does not allow recovery of sugars that can be used to make products such as ethanol.

1.2.1.3 Acid treatment

With  hydrolysis  limited  by  resistance  of  the  crystalline  cellulose  regions,  dilute  acid processing requires time extension and high temperature to increase the reaction rate. Dilute acid has the advantage of a lower abundance of glucose degradation products – furfural and HMF (Lee et al.

2003). There are many methods for acid treatments including use of phosphorus acid, sulphuric acid, hydrochloric acid or peracetic acid (Beldman et al., 1985). Reacting Biomass with dilute sulphuric acid alters the crystalline nature of the cellulose structure by expanding the surface area of the biomass, allowing water penetration into the crystalline structure. Dilute sulphuric acid treatment improves ease of solubilisation of biomass and formation of glucose. Concentrated sulphuric acid pre-treatment solubilises cellulose by breaking down the hydrogen bonds. According to Zhao et al. (2012), peracetic acid can also be used to treat lignocellulosic biomass. Peracetic acid is a powerful oxidizing agent that removes lignin, in a manner similar to ozone treatment. It can also cleave the aromatic molecules in lignin.

1.2.1.4 Alkali treatment

Alkali treatment  reduces the  lignin  and  hemicelluloses content  in  biomass  increases the surface area, allowing penetration of water molecules to the inner layers, and breaks the bonds between hemicelluloses and lignin-carbohydrate. Dilute sodium hydroxide is usually used for alkali treatment. On the other hand, alkaline-based methods are generally more effective at solubilising a greater fraction of lignin while  leaving behind much of the  hemicelluloses in an insoluble and polymeric form (Beldman et al., 1985).

1.2.1.5 Biological treatment

Cellulose degrading microorganism play an important role in the biosphere by recycling cellulose and are common in fields such as forest soils, in manure and on decaying plant tissues.

Among the cellulose utilizing species there are aerobic, anaerobic, mesophilic and thermophilic bacteria,  filamentous fungi,  basidiomycetes and  actinomycetes. A diverse group of fungi utilize cellulose for their carbon and energy sources. Strongly cellulose degrading fungi are represented by species of the genera Aspergillus, Chaetomium, Curvularia, Fusarium, Memoniella Phomo, Thielavia and Trichoderma. These strains produce extracellular cellulose degrading enzymes namely endoglucanase, exoglucanase and cellobiase which act synergistically on the conversion of cellulose to glucose (Anita et al., 2013).Micro-organism, including the brown, white, and soft-rot fungi, attack lignocellulosic biomass, breaking down both lignin and hemicellulose. White and soft rot fungi attack cellulose and lignin. On the other hand, brown rot fungi usually attack only cellulose. The white rot fungi produce powerful lignin degrading enzymes. Phanerochaete chorysosporium, a species of white rot fungi produces both lignin peroxidises and manganese-dependent peroxidises for lignin degradation. Biological treatment requires low energy and normal environmental conditions but the hydrolysis yield is low and requires long treatment times.

1.2.1.6 Ozonolysis

Ozone is a powerful oxidizing agent. Ozonolysis has been previously proposed as a treatment process to  popular wood stocks for  cattle  feed.  The  appeal of using  ozone (O3)  for  feedstock enhancement is due to the accessibility of the nutritional content from an inedible carbohydrate. Whereas other treatments would produce unwanted lignin derivatives, ozonolysis produces mainly carboxylic acid groups, such as formic acid, oxalic acid, acetic acid and other dicarboxylic acid groups, all of which can be metabolised by ruminants. It is used in the paper pulping/bleaching industries; ozonolysis causes the delignification of the paper leaving a whiter finish without compromising the paper strength by breaking the cellulose fibers.

1.3      Cellulase

Cellulases (EC 3.2.1.4) are modular enzymes that break down cellulose, the carbohydrate that is the main part of the cell walls of plants. They are composed of independently folding, structurally and functionally discrete units, referred to as either domains or modules (Henrissat, 1998). They are members of the glycoside hydrolase families of enzymes, which hydrolyze the β-1,4-glucosidic bonds of cellulose to produce smaller β-glucose oligomers and β-D-glucose. A complete cellulase system consists of three classes of enzymes: endo-1,4-β-glucanases (EC 3.2.1.4), which cleave  internal glucosidic bonds; exo-1,4-β-glucan cellobiohydrolase (EC 3.2.1.91), which cleave cellobiosyl units

from the ends of cellulose chains; β-D-glucosidase (EC 3.2.1.21), which cleave glucose units from cellulo-oligosaccharides (Figure 3) (Watanabe and Tokuda, 2001).

The enzymes are classified into families, according to the amino acid sequence similar to their catalytic domains. Such a classification system is based on the concept that similarities in sequence reflect  the  conservation  of  both  the  structural  fold  and  catalytic  mechanism.  Most  effective cellulolytic enzymes are made of at least two constitutive discrete globular domains, catalytic domain and a non-catalytic cellulose-binding domain linked by a flexible peptide. The catalytic domain is responsible for the hydrolysis reaction itself, while cellulose-binding domain has no catalytic activity, but promotes the absorption of the enzyme onto insoluble macromolecular cellulose (Henrissat, 1991; Henrissat and Bairoch, 1993; Beguin and Aubert, 1994). The three types of reaction catalyzed by cellulases includes: Breakage of the non-covalent interactions present in the amorphous structure of cellulose  (Endocellulase); Hydrolysis  of  chains  ends  to  break  the  polymer  into  smaller  sugars (Exocellulase) and the hydrolysis of disaccharides into glucose (β-glucosidase).

1.3.1   Molecular Structure of Cellulases

Most cellulases share a common molecular organisation where a larger catalytic domain (CD) is linked by a highly glycosylated linker-peptide to a small carbohydrate-binding module (CBM) (Hilden and Johansson, 2004). The molecular structure of cellulases can be of two types; single domain or multi-domain (Bhikhbhai et al., 1984; Ong et al., 1989). Most cellulases however, consist of multiple domains which are joined by characteristic linker sequences. The majority of these

sequences are rich in either serine residues or a combination of proline and threonine residues. These linker regions vary considerably in length (6 to 59 amino acids). It is apparent that many plants cell wall hydrolases have a modular structure in which the various domains are not located in equivalent positions in the different enzymes. In addition, catalytic domains which are structurally unrelated are linked, in some cases to identical highly conserved cellulose-binding domains (CBD).

1.3.1.1 Catalytic Binding Domain (CD)

The adsorption of cellulase to cellulose is a prerequisite step for hydrolysis. The overall binding efficiency of the cellulases to the cellulose is enhanced by the presence of the catalytic binding domain, and this correlates clearly with higher activity towards insoluble cellulose (Reinikainen et al., 1995). At the same time, the strong binding via catalytic binding domain to the cellulose surface can lead to a population of non-productive bound enzymes (Stahlberg et al., 1991). In addition to anchoring the enzyme molecules to the cellulose surfaces, the disruption of cellulose microfibrils by family II  CBDs has  been reported (Din et  al., 1991). Simultaneous addition of separated CBD and catalytic domain resulted in synergy between these domains in the hydrolysis of cotton cellulose (Esteghlalian et al., 2001). It is probable that different catalytic binding domains bind to different regions on the cellulose surface. CBDs can promote the enzyme activity towards different regions on the cellulose surface, thereby determining the substrate specificity (Carrard et al., 2000).

1.3.1.2 Cellulose Binding Domains (CBD)

Most cellulases contain, in addition to the catalytic domain (CD), a carbohydrate binding module (CBM), more specifically called cellulose-binding domain (CBD). The CBDs are believed to play an important role in cellulose hydrolysis. Although these domains do not affect the activity of cellulases toward soluble and amorphous substrates, they significantly enhance the capacity of the enzymes to hydrolyse crystalline cellulose (Carrard and Linder, 1990; Gilkes et al., 1991). There are several families of cellulose-binding domains resulting in a large number of their possible combinations. Removal of the cellulose-binding domain drastically reduces the binding capacity of cellulases to insoluble cellulose while the catalytic efficiency on soluble substrates is usually maintained. Isolated cellulose-binding domains bear most of the binding properties of cellulases but do not hydrolyse cellulose. The multiple types of synergy that cellulases display when acting in combination on cellulose appear to result from their different activities and selectively from the substrate micro heterogeneity, and sometimes from both (Lynd et al., 2002). The efficient hydrolysis of cellulose needs interplay between the two domains.

1.3.2   Properties of Cellulases

Most cellulases studied have similar pH optimal, solubility and amino acid composition. Thermal stability and exact substrate specificity may vary. However, it should be noted that cellulose preparations generally contain other enzymatic activities besides cellulase and this may also affect the properties of the preparations. The optimum pH for cellulase preparations is effective between pH 3 and 7 and 5. Besides that, the optimum temperature for cellulase production is between 40-50oC (Henrissat, 1985). The activity of cellulase preparations has been found to be completely destroyed after 10-15 minutes at 800C. Solutions of Cellulase at pH 5-7 are stable for 24 hours at 40C. These products should be stored at 40C, in a dry place in tightly closed containers. If stored in this manner,

lyophilized preparation is stable for several months without significant loss of activity (Henrissat, 1985).

1.3.3   Microorganisms Producing Cellulases

Cellulolytic microbes are primarily carbohydrates degraders and are generally unable use protein  or  lipid  as  energy  sources  for  growth,  cellulolytic  microbes  notably  the  bacteria, Cellulomonas and Cytophage and most fungi can utilize a variety of other carbohydrates in addition to cellulose while the anaerobic cellulolytic species have a restricted carbohydrate range limited to cellulose and or its hydrolytic products (Ashok and Carlos 2010). The ability to secrete large amount of extracellular protein is characteristic of certain fungi and such strain are most suited for production of higher levels of extracellular cellulases. One of the most extensively studied fungi is Trichoderma reesei,  which converts native  as  well as  derived cellulose  to  glucose. Most  commonly studied cellulolytic organism include fungal species Trichoderma, Humicola, Pennicillium, Aspergillus, bacteria  including bacilli, Pseudomonas, Cellulomaonas and Actinomycetes. While several fungi metabolize cellulose as an energy source only few strains are capable of secreting a complex of cellulose enzyme which could have practical application in the enzymatic hydrolysis of cellulose. Besides Trichoderma reesei, other fungi like Humicola, Aspergillus and Penicillium have the ability to yield high levels of extracellular cellulases. Aerobic bacteria such as Cellulomonas, Cellovibrio, and Cytophaga are capable of cellulose degradation in pure cultures. However the microbes commercially exploited for cellulose preparations are most limited to Trichoderma reesei, Humicola insolens, Aspergillus niger, Thermomonospora fusca, Bacillus sp and a few other organisms (Ojumu et al., 2003).

1.3.4   Components of Cellulase

1.3.4.1 Endoglucanses (EC 3.2.1.4)

Endoglucanase is mostly found in the form of a complex that is made of three separate domains. Endoglucanase are one group of isoenzymes of the cellulase complex (Figure 4). They hydrolyze internal β-1, 4-glycosidic bonds of the cellulose polymer and show more affinity for the amorphous zones of the cellulose where weaker hydrogen bonds between cellulose  fibres exist (Murashima et al., 2002; Zhang and Lynd, 2004a). Endoglucanase is mostly found in the form of a complex that is made up of three separate domains. This main contains the large, globular catalytic domain which expresses the active site. A loop of the protein chain forms a tunnel that encloses the active site, which is attached to the O-glycosylated B block hinge region of the catalytic domain of the smaller globular CBM at its C terminal A block by a linker peptide made up of proline, serine, and threonine (Boraston et al., 2004).

Figure 4: A 3D illustration of the quaternary structure of the endoglucanase complex with cellulose in its active site (Wikipedia, 2011a).

1.3.4.2 Exoglucanase (EC 3.2.1.91)

Exoglucanase are key enzymes required for the efficient hydrolysis of crystalline cellulose. It has been proposed that exoglucanase hydrolyze cellulose chains in a manner to reduce primarily cellobiose (Yu-san et al., 2009).

Figure 5: Exoglucanse (Wikipedia, 2011b)

1.3.4.3 β-Glucosidase (EC 3.2.1.21)

β-Glucosidase (Figure 6) catalyze the hydrolysis of β-Glucosidic linkages, such as those in alkyl-  or  aryl-  β-Glucosides  as  well  as  diglucosides  and  oligosaccharides.  They  represent  an important group of enzymes because of their potential use in various biotechnological processes, including biomass degradation (Carl and Stephen, 1995), production of bio ethanol from cellulosic agricultural residues (Bothast and Saha, 1997), release of aromatic compounds in the flavour industry and synthesis of useful β-Glucosides (Makropoulous et al., 1998). The role of the β-Glucosidase in the saccharification of cellulose is to degrade cellobiose, an inhibitor of the depolymerising enzyme, and cellulo-oligosaccharides to glucose. However, β-Glucosidase is frequently a rate-limiting factor during enzymatic hydrolysis of cellulose and is very sensitive to glucose inhibition, which limits its activity.

Figure 6: Structure of β-glucosidases from bacterium Clostridium cellulovorans (Jeng et al., 2010).

1.3.5   Enzymatic Degradation of Cellulose

Enzymatic hydrolysis of cellulose is generally a slow and incomplete process. However, in relatively short time (up to 48 hour), the microbial consortium in the bovine rumen hydrolyzes cellulose to 60-65%, and the lower termites were even reported to assimilate wood cellulose to an extent greater than 90% (Begum and Lemaire, 1996). In complex biological systems like a rotting tree or plant debris in soil, cellulose is decomposed in a time scale of months, though at lower temperature. Enzymatic degradation of cellulase is of great biologic as well as economic importance. It constitutes one of the necessary steps in the balance of opposing synthetic and degradative forces in the carbon cycle, and is a major limitation to the usefulness of wood, paper, pulp, cotton, rayon, cellophane,  and  a  host  of other  cellulosic  materials of great  and  diverse utility.  Cellulases are enzymes that catalyze the hydrolysis of cellulose (Lee et al., 2003). Hydrolysis of cellulose can be achieved by enzymatic methods with lower temperature and weakly acidic conditions, ideal for coupled fermentation processes (Wang et al., 2003). All cellulases hydrolyze the β-1, 4 glucosidic bonds between glycosyl moieties by a general acid catalysis requiring a proton donor and a nucleophile base. They release the products either by overall retention or inversion of the anomeric configuration at carbon one (C1) (Henrissat, 1994). Cellulose degradation is brought about mainly by bacteria, fungi and protozoa, but the production of cellulases are documented also in plants and in a number of invertebrate that includes insects, crustaceans, annelids, molluscs, mussels and nematodes (Watanabe and Tokuda, 2001; Davison and Blaxter, 2005). Cellulose degradation typically involves the concerted activities of at least three enzymes; endo-β-glucanase, exo-β-glucanase (cellobiohydrolase) and β-glucosidase, which interact synergistically in producing glucose (Boisset et al., 2000). Endoglucanase randomly hydrolyze internal β-1, 4-glycosidic bonds to decrease the chain, exo-β-glucanase split off cellobiose from the shortened cellulose chain (Zhang and Lynd, 2004a; Riaan et al., 2007) and β-glucosidases hydrolyze cellobiose to render glucose. Synergism has been explained by the proposal that endoglucanase attack amorphous regions of cellulose fibres, forming sites for exoglucanase which can then hydrolyze cellobiose units from more crystalline regions of the fibres. Finally, β-glucosidases, by hydrolyzing cellobiose, prevent the accumulation of this disaccharide, which is an inhibitor of exoglucanase activity. Generally, enzymatic degradation of cellulose is characterised by a rapid initial phase followed by a slow secondary phase that may last until all substrate is utilized. This is explained most often by the rapid hydrolysis of the readily accessible fraction of cellulose, strong product inhibition and slow inactivation of absorbed enzyme molecules (Converse et al., 1988).

1.3.6   Adsorption Characteristics

The active site of a cellulase consists of multiple binding sites for glucose units, which enhances the probability for the enzyme to remain bound to the substrate after a catalytic and thereby work progressively. The adsorption of cellulase onto cellulose is necessarily the first step of the hydrolytic process and corresponds to  a phase transfer of the  free enzyme (in  solution) to the insoluble substrate (solid phase). A long time before the multi-domain structure of cellulases was discovered, one of the first observed effects of the binding of these enzymes to microcrystalline cellulose was the swelling effect or fragmentation to release short fibres. Another early recognized feature of the adsorption of cellulases onto cellulose is that some cellulases adsorb more tightly than others and that the tight-binding enzymes are the most efficient for the hydrolysis of cellulose. There are several surfaces and intermolecular forces which determines the power of adsorption of protein onto solid/liquid interface. These interactions are usually non covalent and is mediated by hydrogen bond, electrostatic or hydrophobic interactions. Proteins are polyampholytes containing both positive and negative charges which makes them intrinsically surface active molecules. The most hydrophobic residues  in  protein  are  those  containing  large  aromatic  groups  such  as  tryptophan  (Trp), phenylalanine (Phe) and tyrosine (Tyr). As a rule, when the protein is folded, the non-polar hydrophobic amino acid residues are preferentially located on the surface of the protein molecules, therefore forming binding sites for hydrophobic substances through hydrophobic interaction and hydrogen bonding. The affinity of protein increases as the hydrophobicity of the surface increases (Davies and Henrissat, 1995). Generally, adsorption of hydrophilic surfaces is more irreversible than adsorption of hydrophobic surface. Cellulose surface is often considered highly hydrophilic because of the hydrogen bonding of the cellulose inter-chains.

1.3.7   Synergism of Cellulases

Cellulose  is  hydrolysed  by  essentially  two  types  of  enzyme  system  defined  as  non- aggregating and aggregating enzymes. The three types of non- aggregating enzyme, endoglucanase (EC 3.2.1.4) which cleaves  internal β-1,  4-glycosidic  bonds, exoglucanase (EC 3.2.1.91) which release  cellobiose  from the  non-reducing end  of cellulose.  β-glucosidases (EC 3.2.1.21), which hydrolyses cellobiose to glucose. These three enzymes act in synergy to degrade cellulose (Han et al.,

1995; Fujita et al., 2004; Haq et al., 2006). This synergy is necessary due to the alternating crystalline and amorphous regions which occur in cellulose. The crystalline regions in particular are resistant to degradation by any single enzyme due to their rigid strongly bonded structure. Effective degradation of cellulose requires cooperation between these cellulases. This cooperation, resulting in higher total

activity, is called synergism. The synergy factor (SFp) is defined as the ratio of the activity of the combined enzymatic action to the sum of the activities of individual components. Two classes of synergism  between  cellulase  have  been  described:  the  cooperation  between  endoglucanase and exoglucanase (endo-exo synergism) and that between two cellobiohydrolases (exo-exo synergism). Generally, synergism between endo- and exo-enzymes is highest on semi crystalline cellulose of high degree  of polymerization,  lower  on  amorphous  cellulose  and  non-existent  on  soluble  cellulose derivatives  (Nidetzky  et  al.,  1993).  The  molecular  basis  of  synergism  is  not  yet  completely understood, largely  because the  modes of action of the  individual enzymes are  not  clear.  The synergism has been found to be dependent on the relative proportions of the enzyme components (Henrissat et al., 1985) and also on the degree of saturation of the substrate with the enzymes (Woodward and Wiseman, 1988). It is generally assumed that the mechanism of endo-exo synergism can be discussed in terms of sequential actions where by the random endoglucanase initiates attack and the new chain ends generated are then hydrolysed by the cellobiohydrolase. Furthermore, β- glucosidase can work in synergy with cellulase by removing the cellobiose produced. Exoglucanase then cleaves cellobiose units from these exposed non-reducing ends in a hydrolysis reaction which continues  into  the  crystalline  regions.  β-glucosidases cleaves  the  cellobiose  into  its  component glucose residue ensuring that the cellobiohydrolase activity is not inhibited by disaccharide build-up.

1.3.7.1 Synergy between Exoglucanase and β-glucosidase

This results in the removal of glucose, cellobiose and cellodextrin as product of the action of the two enzymes. The catalysis by exo-glucanase produce glucose and cellobiose unit (Perez et al.,

2002) while β-glucosidase cleaves the β-1,4-glucosidic bonds in cellobiose and other cello- oligosaccharide producing glucose (Haq et  al., 2006; Murashima et  al.,  2002). The  synergistic interactions between these enzymes lead to the production of reducing sugar.

1.3.7.2 Endo-exo Synergism

The synergism involves endoglucanase and exoglucanase. The mechanism of reaction is such that endoglucanase initially cut the crystalline cellulose surface, followed by exoglucanase liberating cellodextrin, cellobiose or glucose (Murashima et al., 2002). The synergism follows a sequential manner, i.e. endoglucanase initiate the reaction followed by exoglucanase reaction (Wood and McCrae, 1986).

1.3.7.3 Exo-exo Synergism

This type  of synergism involves  exoglucanase.  Wood and  McCrae  (1986)  used  DEAE- sepharose to separate cellobiohydrolase I and cellobiohydrolase II of Trichoderma reesei. They found out that they hydrolyze cellulase from the opposite ends, i.e. the reducing end and the non reducing end of cellulose microfibrils respectively. Wood and McCrae (1986) suggested a hydrolytic action method in which one of the cellobiohydrolase involves in the removal of cellobiose units successively from one type of non reducing chain and exposed another neighbouring chain, a non reducing chain end exposed another neighbouring chain, anon reducing end group, with correct configuration for attack by another stereo-specific cellobiohydrolase.

1.3.8   Cellulase Assays

The  two  basic  approaches  to  measuring  cellulase  activity  are  measuring  the  individual cellulase (endoglucanase, exoglucanase and β-glucosidase) activities and then measuring the total cellulase activity. In general, hydrolases activities are expressed in the form of the initial hydrolysis rate for the individual enzyme component within a short time, or the end-point hydrolysis for the total enzyme mixture to achieve a fixed degree of hydrolysis within a given time (Sheehan and Himmel,

1999). All existing cellulase activity assays can be divided into three types: the accumulation of products after hydrolysis, the reduction in the amount of substrate, and the change in the physical properties of substrate. Most of the assays involve the accumulation of hydrolysis products, which include reducing sugars total sugars, and chromophores. According to Alexander et al. (2011), the most common reducing sugar assays include the dinitrosalicyclic acid (DNS) method (Miller, 1972), an alternative to Nelson-Somogyi method (Nelson, 1944; Somogyi, 1952). Major reducing sugar assays depend on the reduction of inorganic oxidants such as cupric ions (Cu2+) or ferricyanide, which accepts electrons from the donating aldehyde groups of reducing cellulose chain ends. The DNS and Nelson-Somogyi methods are two of the most common assays for measuring reducing sugars for cellulase activity assays because of their relatively high sugar detection range (i.e. no sample dilution required) and low interference from cellulase (i.e. no protein removal required). However, the primary drawback for this method is the poor stoichiometric relationship between cellodextrins and the glucose standard (Coward-Kelly et al., 2003; Zhang and Lynd, 2005).

1.3.8.1 Substrate for Cellulase Activity Assays

Substrate for cellulase activity assays can be divided into two categories, based on their solubility in water.

1.3.8.1.1         Soluble Substrate

Soluble substrates include low degree of polymerization (DP) cellodextrins from 2 to 6 sugar units and their derivatives, as well as long DP cellulose derivatives (several hundreds of sugar units). They are often used for measuring individual cellulase component activity. Cellodextrins are very slightly soluble. Their solubility decreases drastically with increasing DP because of strong intermolecular hydrogen bonds and system entropic effects (Zhang and Lynd, 2005).

1.3.8.1.2         Insoluble Substrates

Insoluble cellulose-containing substrates for  cellulase  activity assays  include  nearly pure cellulose (cotton linter, Whatman No. 1 filter paper and amorphous cellulose) and impure cellulose- containing substrate (α-cellulose and pre-treated lignocelluloses).

1.3.9   Cellulase Activities

1.3.9.1 Total Cellulase Activity Assay

The total cellulase system consists of endoglucanase, exoglucanase, and β-glucosidase, all of which hydrolyze cellulose synergistically. Total cellulase activity assays are always measured using insoluble substrates, including pure cellulosic substrates such as Whatman No 1. filter paper, cotton linter,  microcrystalline  cellulose,  bacterial  cellulose,  algal  cellulose;  and  cellulose-containing substrate such as dyed cellulose, α-cellulose, and pre-treated lignocelluloses. The heterogeneity of insoluble cellulose and the complexity of the cellulase system cause problems in measuring total cellulase activity (Ghose, 1987).

1.3.9.2 Exoglucanase Activity Assay

Exoglucanases cleave the  accessible  ends of cellulose  molecules to  liberate glucose and cellobiose (Zhang and Lynd, 2004b). Avicel has been used for measuring exoglucanase activity because it has the highest ratio of FRE/Fa (fraction of the reducing end of all anhydroglucose units of cellulose/furfuryl alcohol) among insoluble cellulosic substrates.

1.3.9.3 Endoglucanase Activity Assay

The endoglucanase are commonly assayed by viscosity reduction in carboxymethyl cellulose (CMC) solutions. The modes of actions of endoglucanase and exoglucanase differ in that endoglucanase decrease the specific viscosity of CMC significantly with little hydrolysis due to

Intramolecular cleavage, whereas exoglucanases hydrolyze long chains from the ends in a progressive process (Teeri, 1997; Zhang and Lynd, 2004a).

1.3.9.4 β-glucosidase Activity Assay

β-glucosidases hydrolyze soluble cellobiose and other cellodextrins with a DP up to 6 to produce glucose in the aqueous phase. The rate of hydrolysis decreases as the substrate degree of polymerization increases (Zhang and Lynd, 2004b). β-glucosidase activities can be measured using cellobiose, which is not hydrolyzed by endoglucanase and exoglucanase (Ghose, 1987).

1.3.10 Factors affecting Cellulase Activities

Within the normal range, temperature, pH, concentration of substrate and enzyme concentration affect the rate of reaction in accordance with predictable interaction between enzymes and substrate molecule.

1.3.10.1 Effect of temperature on Cellulase Activity

Every enzyme exhibits its catalytic function maximally at a particular optimum temperature depending on its source. Above the temperature optimum, the tertiary structure of the enzymes will be altered thereby, affecting the enzyme’s binding ability, active sites, the binding of substrate (S) and subsequent catalysis to yield product.

1.3.10.2 Effect of pH on Cellulase Activity

The  hydrogen ion concentration is  another  major factor which affects  both the  activity, stability and the three dimensional structure of enzyme as well as proteins. Every enzyme has its optimum pH at which it exhibit maximum activity. The pH of a solution can have several effects on the structure and activity of enzymes (Anosike, 2001). For example, pH can have an effect on the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. In general enzymes have a pH optimum. However, the optimum pH is not the same for all enzymes. Cellulases have different pH optima depending mostly on the sources (Bajaj et al., 2009).

1.3.11 Application of Cellulases in Industries

Cellulase is of great potential in the industrial sector. One of the greatest potential is in ethanol production from biomass which lies in enzymatic hydrolysis of cellulose using cellulase enzyme. The applications of cellulases in the industry are numerous (Klemm et al., 2005; Rajeev et al., 2005). For example, cellulase is used for commercial food processing in coffee, hydrolysis of cellulose during drying of beans, used in textile industry (making cotton cloths softer by limited hydrolysis) and in laundry detergents, pulp and paper industry for various purposes, and for pharmaceutical applications.

1.3.11.1 Textile and Laundry Biotechnology

Cellulases have achieved their worldwide success in textile and laundry because of their ability to modify cellulosic fibres in a controlled and desired manner, so as to improve the quality of fabrics. Although, cellulases were introduced in textile and laundry only a decade ago, they have now become the third largest group of enzymes used in these applications. Bio-stoning and bio-polishing are the best-known current textile applications of cellulases. Cellulases are also increasingly used in household washing powders, since they enhance the detergent performance and allow the removal of small, fuzzy fibrils from fabric surfaces and improve the appearance and colour brightness.

1.3.11.2 Pulp and Paper Biotechnology

Pulp and paper industries have increased considerably during the last decade through the application of cellulase. The mechanical pulping processes such as refining and grinding of the woody raw material lead to pulps with high content of fines, and bulk. While biomechanical pulping using cellulases resulted in substantial energy savings (20-40%) during refining and improvement in hand-sheet strength properties (Bhat, 2000). Mixtures of cellulases and hemicellulases have also been used for biomodification of fibre properties with the aim of improving drainage and beatability in the paper mills before or after beating of pulp. Endoglucanase have the ability to decrease the pulp viscosity with a  lower  degree  of hydrolysis.  Cellulase  have  also  enhances the  bleachability of softwood kraft pulp producing a final brightness scores comparable to that of xylanase treatment.

1.3.11.3 Bioethanol Industry

Enzymatic saccharification of lignocellulosic materials such as switch grass, saw dust, corn cob, sugarcane bagasse, rice straw and forest residues by cellulases for biofuel production is perhaps the most popular application currently being investigated (Lynd et al., 2002). The utility cost of enzymatic hydrolysis may be low compared with acid or alkaline hydrolysis because enzyme hydrolysis is usually conducted at mild conditions and does not have corrosion issues (Riaan et al., 2007).

1.3.11.4 Food Processing Industry

Cellulases have a wide range of potential applications in food biotechnology as well. The production of fruit and vegetable juices requires improved methods for extraction, clarification, and stabilization. Cellulase also have an important application as a part of macerating enzymes complex (cellulases, xylanase, and pectinases) used for extraction and clarification of fruits and vegetable juices to increase the yield of juices (Whitaker, 1990). Macerating enzymes increases both yield and process performance without additional capital investment. The macerating enzymes improve cloud stability and texture and decrease viscosity of the nectars and purees from tropical fruits such as mango, apricot, peach and papaya and pear. Aroma, texture and flavour properties of fruits and vegetable can be improved by reducing excessive bitterness of citrus fruits by infusion of enzymes such as pectinase and β-glucosidases.

1.3.11.5 Animals Feed Industry

Cellulase has the ability to eliminate anti nutritional factors present in the feed grains, degrade certain  feed constituents to  improve the  nutritional value,  and  provide  supplementary digestive enzymes such as proteases, amylases, and glucanases.

1.3.12 Economic feasibility of Cellulase

In general, the adoption and implementation of bioconversion technology will depend largely upon the economic  feasibility of integrating these practices  into  existing agricultural and  agro- industrial residue  management  schemes.  In  most  cases  of rural  agricultural and  agro-industrial operations, collection of residues in a large enough quantity to be economical to process is a major problem. Transportation adds to raw material costs and leads to problem such as need for adequate storage facilities and deterioration in quality. In many instances, transportation facilities are not adequate. Where recovery or treatment of residues is necessary largely for environmental reasons, potential economic benefit is rarely realized. In such cases, it is anticipated that at least operating costs can be recovered.

A  survey was  conducted in  Thailand to  identify  socio-economic issues surrounding the practical feasibility of wide-scale promotion of biogas generation. It was found that most farmers were generally aware of the technology, but economics where the primary concern. They were found to be understandably conservative regarding additional capital expenditure. This attitude is also true among industrial entrepreneurs in the country, with the exception of those operating joint ventures with foreign counterparts (Zhuang et al., 2007).

1.4 Aspergillus niger

Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments, where its black colonies can be confused with those of Stachybotrys (species of which have also been called “black mould”). It is a cosmopolitan fungus which is primarily isolated from compost, plant material and from soil. Aspergillus niger is more common in tropical or sub- tropical areas.

₦5,000.00 – DOWNLOAD FULL PROJECT DOWNLOAD FULL PROJECT Added to cart

---

---

---

---

Related Project Topics :