EXTRACTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF CELLULASE FROM Aspergillus fumigatus AND Aspergillus flavus IN SUBMERGED FERMENTATION SYSTEM USING BREADFRUIT HULLS AS CARBON SOURCE

CHAPTER ONE

INTRODUCTION

 

Enzymes are the catalysts of biological processes. Like any other catalyst, an enzyme brings the reaction catalyzed to its equilibrium position more quickly than would occur otherwise. An enzyme cannot bring about a reaction with an unfavourable change in free energy unless that reaction can be coupled to one whose free energy change is more favourable (Nelson and Cox, 2000). The activities of enzymes have been recognized for thousands of years. However, only recently have the properties of enzymes been understood properly (Wolfgang, 2007). Indeed, research on enzymes has now entered a new phase with the fusion of ideas from protein chemistry, molecular biophysics, and molecular biology which have given rise to applications in fields ranging from agriculture to industry (Wolfgang, 2007).

 

The enzyme industry as we know it today is the result of a rapid development seen primarily over the past four decades and thanks to the evolution of modern biotechnology (Ole et al., 2002). Enzymes found in nature have been used since ancient times in the production of food products, such as cheese, sourdough, beer, wine and vinegar, and in the manufacture of commodities such as leather, indigo and linen (Ole et al., 2002). All of these processes relied on either enzymes produced by spontaneously growing microorganisms or enzymes present in added preparations such as calves’ rumen or papaya fruit. The development of fermentation processes during the later part of the last century, aimed specifically at the production of enzymes by use of selected production strains, made it possible to manufacture enzymes as purified, well-characterized preparations even on a large scale (Wolfgang, 2007)

 

Microbial cellulases have shown their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing and agriculture. Due to the complexity of the enzyme system and immense industrial potential, cellulases have been a potential candidate for research by both academic and industrial research groups (Shang, 2013). The growing concerns about depletion of crude oil and the emissions of greenhouse gases have motivated the production of bioethanol from lignocellulose, especially through enzymatic hydrolysis of lignocellulosic materials (Bayer et al., 2004; Himmel et al., 1999)

 

• Cellulose

 

Cellulose is a linear polymer of β-D-glucose units linked through 1,4-β-linkages with a degree of polymerization ranging from 2,000 to 25,000 (Kuhad et al., 1997). Cellulose chains form numerous intra- and intermolecular hydrogen bonds, which account for the formation of rigid, insoluble, crystalline microfibrils (Golan, 2011). Natural cellulose compounds are structurally heterogeneous and have both amorphous and highly ordered crystalline regions (Morana et al., 2011). The degree of crystallinity depends on the source of the cellulose and the highly crystalline regions are more resistant to enzymatic hydrolysis (Morana et al., 2011). Cellulosic materials are particularly attractive because of their relatively low cost and abundant supply. As the most abundant polysaccharide in nature, cellulose decomposition plays not only a key role in the carbon cycle of nature, but also provides a great potential for a number of applications, most notably biofuel and chemical production (Lynd et al., 2012). The central technological impediment to more widespread utilization of this important resource is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass.

 

1.1.1 Structure of Cellulose

 

1.1.1.1 Chemical Structure

 

Payen first used the term cellulose for this plant constituent which is the most widespread organic compound on Earth (Payen 1938; Guo et al., 2008]). The total amount of this polysaccharide on our planet has been estimated at 7 × 1011 tons (Coughlan, 1985) and constitutes the most abundant and renewable polymer resource available today. Cellulose is an insoluble crystalline substrate, flavourless, odourless, hydrophilic, insoluble in water and in most organic solvents, chiral, and with a wide chemical variability (Coughlan, 1985). It is a structural component of the cell wall of green plants accounting for almost 33% of the total biomass. It is also biosynthesized in other living systems such as Bacteria and Algae. Cellulose produced by plants usually exists within a matrix of other polymers primarily hemicellulose, lignin, pectin and other substances, forming the so-called lignocellulosic biomass, while microbial cellulose is quite pure, has a higher water content, and consists of long chains (Jagtap and Rao, 2005) . It is a carbohydrate polymer with formula (C₆H₁₀O₅)n , consisting of a linear chain of several hundred to over ten thousand 1,4-β-D-glucose units linked through acetal functions between the equatorial -OH group of C4 and the C1 carbon atom (Jagtap and Rao, 2005). The high stability of this conformation leads to a decreased flexibility of the polymer, so this is usually described as a real tape.

 

There are two different types of intra- and one interchain hydrogen bonds in the structure, and it has been considered that the intrachain hydrogen bonds determine the single-chainconformation and the stiffness of cellulose, while the interchain hydrogen bond is responsible for the sheetlike nature of cellulose (Watanabe and Tokuda, 2001; Klemm et al., 2002; Klemm et al., 2005). The chains are arranged parallel to each other and form elementary fibrils that have a diameter between 1.5 and 3.5 nm (microfibrils), the length of the microfibrils is about of several hundred nm (Watanabe and  Tokuda, 2001; Klemm et al., 2002; Klemm et al., 2005) .

 

 

 

Fig. 1: Structure of Cellulose (Matheus et al., 2013)

 

1.1.1.2 Crystalline structure

 

The high degree of hydrogen bonds within and between cellulose chains can form a 3-D lattice-like structure, while amorphous cellulose lacks this high degree of hydrogen bonds and the structure is less ordered (Morana et al., 2011). The physical and chemical properties of cellulose are defined by intermolecular interactions, cross-linking reactions, polymer lengths, and distribution of functional groups on the repeating units and along the polymer chains (Morana et al., 2011).

 

Initially, crystalline structure of native cellulose (cellulose I) has been studied by X-ray diffraction and has been defined as monoclinic unit cells with two cellulose chains with a twofold screw axis in a parallel orientation forming slight crystalline microfibrils (Gardner and Blackwell, 2004; Klemm et al., 2005). Moreover, there are other types of crystal structures: cellulose II, III, and IV (Gardner and Blackwell, 2004); the cellulose I, result the less stable thermodynamically, while the cellulose II is the most stable structure (Klemm et al., 2005). The cellulose I can turn into other forms using different treatments; for example, by mercerization, using aqueous sodium hydroxide or dissolution followed by precipitation and regeneration (formation of fiber and film) (O’Sullivan, 1997; Nishiyama et al., 2002). However, additional information on the structure of noncrystalline random cellulose chain segments are needed because it is very important for the accessibility and reactivity of the polymer and the characteristics of cellulose fibers (Paakkari et al., 1989).

 

 

Fig. 2: Crystalline forms of Cellulose I (Matheus et al., 2013)

 

1.1.2  Biosynthesis of Cellulose

 

Cellulose is synthesized by a variety of living organisms, including plants, algae, bacteria, and animals. It is the major component of plant cell walls with secondary cell walls having a much higher content. The biosynthesis of cellulose essentially proceeds by the polymerization of glucose residues using an activated substrate UDP-glucose (Saxena et al., 200).

 

In plants, cellulose is synthesized on the plasma membrane by the enzyme cellulose synthase that is present in the membrane. In the bacterium Acetobacter xylinum, the enzyme cellulose synthase is present on the cytoplasmic membrane, and the cellulose is obtained extracellularly. However, in other organisms, cellulose is found to be synthesized in other regions of the cell. In the alga Pleurochrysis, cellulose scales are formed in the Golgi apparatus and then deposited on the cell surface (Saxena et al, 2000).

 

The biosynthesis of Cellulose proceeds in at least two stages – polymerization and crystallization. The first stage is catalyzed by the enzyme cellulose synthase, and the second stage is dependent on the organization of the cellulose synthases possibly with other proteins such that the glucan chains are assembled in a crystalline form (Saxena et al, 2000).

 

Fig. 3: Biosynthesis of Cellulose (Peter, 2008)

 

1.1.3 Sources of Cellulose

 

The plant cell wall is the major source of cellulose. Cellulose therefore abounds in agricultural wastes of plant origin.

 

 

 

 

Table 1: Lignocellulose composition of several agricultural wastes

Lignocellulosic materials    Cellulose (%)     Hemicellulose(%)  Lignin (%)

Hardwood                               40-55                24-40                 18-25

Softwood                                 45-50               25-35                  25-35

Nut shell                                  25-30               25-30                  30-40

Chestnut shell                          27.4                  10                       44.6

Grape stalk                              38                      15                       33

Corn stover                              36.7                   13.33                  33

Wheat straw                             30                      50                       15

Rice straw                                32.1                   24                       18

Brewer‘s spent grain                16.8                   28.4                    27.8

Paper                                        85-99                 0                          0-15

Leaves                                      15-20                80-85                    0

Cotton seeds hairs                    80-95                 5-20                     0________

(Jorgensen et al., 2007)

 

• Breadfruit (Treculia africana)

 

Treculia africana is a multipurpose tree species commonly known as African breadfruit. It belongs to the family Moraceae and it grows in the forest zone, particularly the coastal swamp zone (Agbogidi and Onomeregbor, 2008).  African breadfruit is a traditionally important edible fruit tree in Nigeria (Okafor, 1985) whose importance is due to the potential use of its seeds, leaves, timber, roots and bark. It is increasingly becoming commercially important in Southern Nigeria. Baiyeri and Mbah (2006) described African Breadfruit as an important natural resource which contributes significantly to the income and dietary intake of the poor. The seeds are used for cooking and are highly nutritious as pointed out by various authors including; Okafor and Okolo (1974), Okafor (1990) and Onyekwelu and Fayose (2007). However an important  by-product of processing breadfruit seed is the seed hull (seed coat or seed shell) and this may pose a risk to health as well as environment ( Atuanya et al., 2012). These hulls are particularly rich in cellulose that could be harnessed for cellulase production using microbes (Sonde and Odomelam, 2012)

 

Fig. 4: Breadfruit seed hulls (Atuanya et al., 2012)

 

• Hydrolysis of Cellulose

 

Cellulose and can be hydrolyzed to sugars and microbially fermented into various products such as ethanol or chemically converted into other products (Wyman, 1999). The primary challenge is that the glucose in cellulose is joined by beta bonds in a crystalline structure that is far more difficult to depolymerize than the alpha bonds in amorphous starch (Wyman, 1999). There are 2 broad based approaches to cellulose hydrolysis. These are acid based hydrolysis and enzymatic hydrolysis.

 

1.3.1 Acid hydrolysis

 

When heated to high temperatures with dilute sulfuric acid, long cellulose chains break down to shorter groups of molecules that release glucose that can degrade to hydroxymethyl furfural (McParland et al., 1982). Generally, most cellulose is crystalline, and harsh conditions (high temperatures, high acid concentrations) are needed to liberate glucose from these tightly associated chains. Furthermore, yields increase with temperature and acid concentration, reaching about 70% at 260 C (McParland et al., 1982). However, pyrolysis and other side reactions become very important above about 220⁰C, and the amount of tars and other difficult to handle by-products increases as the temperature is raised above these levels (Brennan et al., 1986). In addition, controlling reaction times for maximum glucose yields of only about 6 seconds at about 250ºC with 1% sulphuric acid presents severe commercial challenges (Brennan et al., 1986).

 

1.3.2 Enzyme hydrolysis

 

Enzymatic hydrolysis has a potential to overcome many of the drawbacks of acid hydrolysis. The conversion is carried out under mild conditions, thus greatly reducing the cost of hydrolysis equipment. Sugars decomposition is avoided, thus eliminating this cause for loss in yield (Hinz et al., 2009). Costly neutralization and purification equipment is unnecessary, and disposal of waste streams from acid neutralization are eliminated. Balancing these potential savings, extensive pre-treatment to breakdown to lignin and increase cellulase accessibility is required to achieve good yields. The cost of high activity cellulolytic enzymes is at very present very high (Wyman, 1999). Mutations and selection methods have been used to develop and isolate Fusarium and Trichoderma strains of high cellulolytic activity (Wyman, 1999).. In all, enzyme hydrolysis is a better alternative to industrial acid hydrolysis, given the relatively low cost of enzyme hydrolysis and also higher yields of products obtained via enzyme hydrolysis.

 

1.4 Cellulases

 

Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze the cellulolysis (or hydrolysis) of cellulose (Golan, 2011). Cellulases catalyze the hydrolysis of 1,4-β-D-glucosidic linkages in cellulose, and play a significant role in nature by recycling this polysaccharide, which is the main component of the plant cell wall. Cellulases work in synergy with other hydrolytic enzymes in order to obtain the full degradation of the polysaccharide to soluble sugars, namely cellobiose and glucose, which are then assimilated by the cell (Morana et al, 2011).

 

The enormous potential that cellulases have in biotechnology is the driving force for continuous basic and applied research on these biocatalysts from fungi and bacteria. Cellulases are found in many fields, such as animal feeding, brewing and wine, food, textile and laundry, pulp and paper products. The growing interest in the conversion of lignocellulosic biomass into fermentable sugars has generated an additional interest for cellulases and their related enzymes (Morana et al, 2011).

 

1.4.1 Classification of Cellulase

 

Enzymes are designated according to their substrate specificity, based on the guidelines of the international union of biochemistry and molecular biology (IUBMB). The cellulases are grouped with many of the hemicellulases and other polysaccharidases as o-glycoside hydrolases (EC 3.2.1.x) (Morana et al., 2011). Since the substrate specificity classification is sometime little informative, because the complete range of substrates is only rarely determined for individual enzymes, an alternative classification of glycoside hydrolases (GH) into families based on  amino acid sequence similarity has been suggested (Henrissat, 1991; Henrissat and Bairoch, 1993; Henrissat and Bairoch, 1996). In addition, Henrissat et al. (1998) have proposed a new type of nomenclature for glycoside hydrolases in which the first three letters designate the preferred substrate, the number indicates the glycoside hydrolase family, and the following capital letter indicates the order in which the enzymes were first reported. For example, the enzymes CBHI, CBHII, and EGI of trichoderma reesei are designated cel7a (CBHI), cel6a (CBHII), and cel6b (EGI).

 

Due to the great increase of identified glycoside hydrolases, Coutinho and Henrissat have

created an integrated database which is continuously updated (http://www.cazy.org/) (Coutinho and Henrissat, 1999). At the 13 july 2010 update, glycoside hydrolases were grouped into 118 families. In addition, 876 glycoside hydrolases have not yet assigned to a family (glycoside hydrolase family ―non-classified‖) because some of them display weak similarity to established GH families, but they are too distant to allow a reliable assignment. Cellulases are found in several different GH families (5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61, and 74), suggesting convergent evolution of different folds resulting in the same substrate specificity (Morana et al., 2011). Gh family 9 contains cellulases from bacteria (aerobic and anaerobic), fungi, plants and animals (protozoa and termites). Other families only group hydrolases from a specific origin, as GH family 7 which contains only fungal hydrolases and gh family 8 which contains only bacterial hydrolases. Cellulases from the same microorganism can also be found in different families (e.g. The Clostridium thermocellum cellulosome contains endoglucanases and exoglucanases from families 5, 8, 9, 44, and 48) (Morana et al., 2011).

 

1.4.2 Types of cellulases

 

Cellulases, responsible for the hydrolytic cleavage of cellulose, are composed of a complex mixture of enzymes with different specificities to hydrolyse glycosidic bonds. Cellulases can be grouped into three major classes viz. Endoglucanase, exoglucanase and β-glucosidase.

 

1.4.2.1 Endoglucanase (EC 3.2.1.4)

 

Endoglucanases, often called carboxy methyl cellulases (CMCase), are proposed to initiate random attack at multiple internal sites in the amorphous regions of the cellulose fiber to open up sites for subsequent attack of cellobiohydrolases (Sunil et al., 2011). This action results in a rapid decrease of the polymer length and in a gradual increase of reducing sugars concentration (Morana et al., 2011)

 

1.4.2.2 Exoglucanase (EC 3.2.1.91)

 

Exoglucanase, better known as cellobiohydrolase, is the major component of the microbial cellulase system accounting for 40-70% of the total cellulase proteins and can hydrolyse highly crystalline cellulose. It removes mono-and dimers from the end of the glucose chain (Sunil et al., 2011).

 

1.4.2.3 β-glucosidase (EC 3.2.1.21)

 

Β-glucosidase also known as cellobiase hydrolyses glucose dimers (cellobiose) and in some cases cello-oligosaccharides to release glucose units (Sunil et al., 2011). These enzymatic components act sequentially in a synergistic system to facilitate the breakdown of cellulose and the subsequent biological conversion to β-glucose.

1.5 Mechanism of action of cellulases

 

Cellulolytic enzymes hydrolyze the 1,4-β-glycosidic bonds in cellulose, but they differ in their specificities based on the macroscopic features of the substrate. They are progressive  enzymes when they interact with a single polysaccharide strand continuously, and non-progressive types when they interact once and then, the polypeptidic chain disengages to attack another polysaccharide strand (Morana et al., 2011) the enzymatic hydrolysis of cellulose requires a carbohydrate binding module (CBM) that binds and arranges the catalytic components on the surface of the substrate. Cellulases from fungi have a two-domain structure with one catalytic domain, and one cellulose binding domain, that are connected by a flexible linker. However, there are also cellulases that lack cellulose binding domain (Morana et al., 2011)

 

The following are three types of reaction catalyzed by cellulases:

1) breakage of the non-covalent interactions present in the crystalline structure of

cellulose (endo-cellulase)

2) hydrolysis of the individual cellulose fibers to break it down into smaller sugars

(exo-cellulase)

3) hydrolysis of disaccharides and tetrasaccharides into glucose (beta-glucosidase).

 

Fig. 5: Mechanism of cellulolysis (Zhang et al., 2006).

 

1.6 Molecular biology of cellulase

 

Here, we shall briefly consider cellulase e4 from Thermomonospora fusca. T. Fusca is a filamentous thermophilic soil bacterium and an important species degrading cellulose and hemicelluloses in plant residues (Lykidis et al., 2007). Cellulase produced by T. Fusca is unusual in that it has characteristics of both exo- and endo- cellulases (Sakon et al., 1997).

 

 

Fig. 6: The 3D structure of cellulase E4 from T. fusca ( Sakon et al., 1997)

 

The complete genome sequence shows that T. fusca has a single circular chromosome of 3,642,249 bp predicted to encode 3,117 proteins and 65 rna species with a coding density of 85% (Lykidis et al., 2007). Genome analysis reveals the existence of 29 putative glycoside hydrolases in addition to the previously identified cellulases and xylanases. T. fusca has been the source organism for isolating and studying multiple secreted cellulases and other carbohydrate degrading enzymes (Hu and Wilson, 1988). Using classical biochemical methods, six different cellulases have been identified. Four endocellulase genes (Hu and Wilson, 1988) and two exocellulase (Irwin et al., 2000). In addition an intracellular β-glucosidase and extracellular xyloglucanase have been cloned and characterized (Irwin et al., 2000).

 

1.7 Production of Cellulase

 

Cellulases are well established in different industrial areas, and are currently the third largest industrial enzyme worldwide, by dollar volume, mainly because of their use in cotton processing and paper recycling, as detergent industry enzymes, and in juice extraction and animal feeding additives as well (Nascimento and Coehlo, 2011). Here, we discuss cellulase producing organisms, methods of fermentation and factors affecting enzyme production.

 

1.7.1 Cellulase Producing Microganisms

 

Microganisms involved in cellulase production will be grouped into two broad groups- mesophilic microorganisms and thermophilic microorganisms.

 

1.7.1.1 Mesophilic microorganisms

 

Microorganisms growing best at moderate temperatures (between 10ºC and 45°C) are named mesophiles. They represent the majority of microbial species on Earth, and their habitats include the soil, the human body, the animals, etc. There are many mesophilic Bacteria and Fungi that play a significant role in the carbon cycle on Earth, and there is increasing interest in the enzymes from these microorganisms, since they have a key function in the conversion of plant biomass into useful products (Morana et al., 2011).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2. Some mesophilic cellulolytic Bacteria

 

___________________________________________________________________________

Microorganism         Gram reaction               Growth (°C)                         Growth conditions

                                                                       Temperature

___________________________________________________________________________

Acetivibrio cellulolyticus        –                                     37                                         Anaerobic

Bacillus megaterium               +                                     30                                        Aerobic

Bacillus pumilus                     +                                     30                                        Aerobic

Bacteroides cellulosolvens      –                            35                                  Anaerobic Butyrivibrio fibrisolvens         +                                    37                                        Anaerobic

Cellulomonas fimi                   +                                    30                                         Aerobic

Cellulomonas fermentans        +                                    30                                          Aerobic

Cellulomonas flavigena          +                                     30                                         Aerobic

Cellulomonas gelida                +                                    30                                         Aerobic

Cellulomonas iranes             +                            28                                     Aerobic Cellulomonas persica             +                                     28                                          Aerobic

Cellulomonas uda                   +                                     30                                          Aerobic

Cellvibrio mixtus                    –                                      20                                          Aerobic

Clostridium acetobutylicum   +                                     37                                          Anaerobic

Clostridium cellulolyticum     +                                   35-37                                      Anaerobic

Clostridium cellulofermentans –                                    40                                          Anaerobic

Clostridium cellulovorans        –                                    37                                          Anaerobic

Clostridium herbivorans          +                                    37                                          Anaerobic

Clostridium hungatei               –                                     30                                          Anaerobic

Clostridium josui                     –                                     45                                          Anaerobic

Clostridium papyrosolvens     –                                      25                                          Anaerobic

___________________________________________________________________________

(Morana et al., 2011)

 

Identification, purification and characterization of cellulases are continuously increasing and always in progress, with incessant research and isolation of new microorganisms able to produce novel cellulolytic activities. As an example, a bacterial strain, TR7-06(T), showing high sequence similarity (98.5 %) to Cellulomonas uda DSM 20107(T), was isolated from compost at a cattle farm near Daejeon, Republic of Korea. The isolated type strain of a novel Cellulomonas species, named Cellulomonas composti sp. nov., possesses endoglucanase and

β-glucosidase activities (Kang et al., 2007). A microorganism capable of hydrolyzing rice hull, one of the major cellulosic waste materials in Korea, was isolated from soil and identified as Bacillus amyloliquefaciens DL-3 (Lee et al., 2008). Based on the characteristics of this novel strain of Bacillus, Lee et al., (2008) aimed to develop an economical process for production of cellulases by using cellulosic waste as inexpensive and widely distributed carbon source. The new isolate produced an extracellular cellulase with an estimated molecular mass of about 54.0 kDa. The deduced amino acid sequence of the cellulase from B. amyloliquefaciens DL-3 showed high identity to cellulases from other Bacillus species, a modular structure containing a catalytic domain of the GH family 5, and a cellulose-binding module type 3 (CBM3). The purified enzyme was optimally active at 50°C and pH 8.0, and showed broad thermal and pH stability ranging from 40 to 80°C and from 4.0 to 9.0, respectively (Lee et al., 2008)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3. Some mesophylic cellulolytic fungi

 

___________________________________________________________________________

Microorganism Growth                    Growth (°C)                       Growth conditions

                 Temperature

__________________________________________________________________________

 

Acremonium cellulolyticus                 24                                      Aerobic

Anaeromyces mucronatus                   37                                      Anaerobic

Aspergillus glaucus                             30                                      Aerobic

Aspergillus niger                                 30                                      Aerobic

Aspergillus terreus                              35                                      Aerobic

Caecomyces communis                      37                                       Anaerobic

Ceratocystis paradoxa                         20                                     Aerobic

Chrysosporium lucknowense            25-43                                   Aerobic

Cyllamyces aberensis                          37                                      Anaerobic

Fusarium solani                                   25                                      Aerobic

Neocallimastix frontalis                      37                                       Anaerobic

Neocallimastix patriciarum                 37                                      Anaerobic

Orpinomyces sp.                                  37                                      Anaerobic

Penicillium funiculosum                     24                                       Aerobic

Penicillium pinophilum                       24                                       Aerobic

Phanerochaete chrysosporium             35                                      Aerobic

Piptoporus betulinus                            25                                      Aerobic

Piromyces sp.                                      39                                       Anaerobic

Piromyces equi                                    39                                       Anaerobic

Pycnoporus cinnabarinus                     24                                     Aerobic

Rhizopus oryzae                                  30                                       Aerobic

___________________________________________________________________________

(Morana et al., 2011)

 

Fungal cellulases are well-studied enzymes used in various industrial processes (Bhat, 2000). A variety of aerobic and anaerobic Fungi are producers of cellulose-degrading enzymes. The aerobic Fungi play a major role in the degradation of plant materials and are found on the decomposing wood and plants, in the soil, and on the agricultural residues. The cellulase systems of the aerobic Fungi Trichoderma reesei, T. koningii, Penicillium pinophilum, Phanerochaete chrysosporium, Fusarium solani, Talaromyces emersonii, and Rhizopus oryzae are well characterized (Bhat and Bhat, 1997). Much of the knowledge on enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase system. In particular, the cellulase system of T. reesei (initially called T. viride) has been the focus of research for 50 years (Reese and Mandels, 1971). A lot of work on cellulases has been directed toward this fungus since it produces readily, and in large quantities, a complete set of extracellular cellulases, and consequently, it has a high commercial value (Claeyssens et al., 1998; Miettinen-Oinonen and Suominen 2002). In fact, T. reesei is capable of secreting more than 30 g/L of protein into the extracellular medium (Conesa et al., 2001). It has been reported that T. reesei possesses two CBH (cellobiohydrolase) genes, cbh1-2, and eight EG (endoglucanase) genes, egl1-8, and that CBH I–II and EG I–VI are secreted proteins (Foreman et al., 2003).

 

1.7.1.2 Thermophilic Microorganisms

 

The thermophilic microorganisms represent a unique group growing at temperatures that may exceed 100°C. More precisely, thermophilic microorganisms thrive at temperatures from 65 to 85°C, and hyperthermophiles grow at temperatures of above 85°C (Morana et al., 2011). Hyperthermophiles are microorganisms within the Archaea domain although some bacteria are able to tolerate temperatures around 100°C. An extraordinary heat-tolerant hyperthermophile is Methanopyrus kandleri, discovered on the wall of a black smoker from the Gulf of California at a depth of 2000 m, at temperatures of 84-110°C. It can survive and reproduce at 122°C (Takai et al., 2008). Thermophilic and hyperthermophilic microorganisms have received considerable attention as sources of thermostable cellulolytic enzymes, as the properties of these biocatalysts make them interesting candidates for industrial applications. Running biotechnological processes at elevated temperatures has many advantages. High temperature has a significant influence on the solubility of the substrates (especially if viscous or polymers) and on the reaction rate. Moreover, problems of microbial contamination can be avoided when a reaction is performed at elevated temperature (Takai et al., 2008).

 

 

 

Table 4: Some (hyper) thermophilic cellulolytic Bacteria and Archaea

 

___________________________________________________________________________

Microorganism                           Gram reaction           Growth                        Growth condition

Temperature (°C)

___________________________________________________________________________

 

Acidothermus cellulolyticus                         +                         55                       Aerobic

Alicyclobacillus acidocaldarius                    +                         60                      Aerobic

Anaerocellum thermophilum                         +                        75                       Anaerobic

Aquifex aeolicus                                             –                       85-95                   Aerobic

Caldibacillus cellulovorans                            +                        68                      Aerobic

Caldicellulosiruptor saccharolyticus              –                         70                     Anaerobic

Clostridium stercorarium                                +                        65                     Anaerobic

Clostridium thermocellum                              +                        60                      Anaerobic

Dictyoglomus thermophilus                            –                        73                       Anaerobic

Dictyoglomus turgidus                                    –                        72                       Anaerobic

Pyrococcus abyssi                                           –                         96                      Anaerobic

Pyrococcus furiosus                                        –                          98                     Anaerobic

Pyrococcus horikoshii                                    –                           98                     Anaerobic

(Morana et al., 2011)

 

Thermostable cellulases are of great biotechnological interest (Hongpattarakere, 2002). A number of cellulolytic thermophilic Bacteria have been isolated, and many cellulose degrading enzymes have been identified, characterized, cloned and expressed (Bergquist et al., 1999). Conversely, screening of hyperthermophilic Bacteria for cellulose-degrading enzymes has revealed that the presence of such enzymes is rather rare in this group. In addition, among the Archaea, only the genus Pyrococcus and Sulfolobus have been found to process thermoactive cellulases. Few aerobic thermophilic microorganisms have been described to produce cellulases in comparison with the anaerobic ones. Acidothermus cellulolyticus, isolated from 55-60°C acidic water and mud samples collected in Yellowstone National Park, produces at least three thermostable endoglucanases (Mohagheghi, 1986). One of them, E1 belonging to GH family 5, was crystallized, while properties and application of the other enzymes are protected by patents (Sakon et al., 1996). The aerobic thermophilic bacterium Rhodothermus marinus, isolated from a submarine hot spring at Reykjanes, NW  Iceland (Alfredsson et al., 1988), produces one higly thermostable cellulase (Cel12A) which retains 50% activity after 3.5 h at 100°C (Hreggvidsson et al., 1996).

 

 

 

Table 5: Some (hyper) thermophilic cellulolytic Fungi

 

___________________________________________________________________________

Microorganism               Growth Temperature (°C)                  Growth conditions

___________________________________________________________________________

Chaetomium thermophilum        45-55                                    Aerobic

Humicola grisea                             45                                      Aerobic

Humicola insolens                      40-50                                     Aerobic

Melanocarpus albomyces           45-55                                     Aerobic

(Morana et al., 2011)

 

Among the thermophilic Fungi, only a few number is described to be cellulase-producer The thermophilic filamentous fungus Humicola sp. has been known to produce several cellulases, and some of the genes have been cloned, sequenced and expressed (Takashima et al., 1996). The cellulase system of the thermophilic fungus Humicola insolens possesses a battery of enzymes that allows the efficient utilization of cellulose. This system, homologous to that of T. reesei, contains five endoglucanases: EGI (Cel7B), EGII (Cel5), EGIII (Cel12), EGV (Cel45A), and EGVI (Cel6B) in addition to two cellobiohydrolases: CBHI (Cel7A), and CBHII (Cel6A) (Schulein, 1997).

 

The thermophilic fungus Chaetomium thermophile var. dissitum, was able to produce in the culture medium all the enzymes involved in cellulose breakdown, namely endoglucanase (41.0 kDa), exoglucanase (67.0 kDa) and β-glucosidase (Eriksen and Goksoyr, 1977). Lu et al. (2002) reported that C. thermophile secreted in the culture medium a glycosylated endocellulase with an apparent molecular weight of 67.8 kDa, as determined by SDS-PAGE. The enzyme was optimally active at pH 4.0-4.5 and 60°C, and it retained 30% activity after 60 min at 70°C.

 

Melanocarpus albomyces, a rare true thermophilic Ascomycete capable of growing copiously at 50°C, has been documented to produce high levels of endoglucanases under optimized culture conditions (Jatinder et al., 2006). The endoglucanases from this fungus have been recognized as potentially important in denim washing. In fact, the supernatant from M. albomyces worked well in biostoning, with low backstaining. Three cellulases were identified and purified to homogeneity, and two of them were endoglucanases with apparent molecular masses of 20.0 kDa (Cel45A) and 50.0 kDa (Cel7A) (Miettinen-Oinonen et al., 2004).

 

The thermophilic fungus Thermoascus aurantiacus produces high levels of cellulase components when grown on lignocellulosic carbon sources such as corncob and cereal straw (Khandke et al., 1989). As these enzyme components are remarkably stable over a wide range of pH and temperatures, they appear to have great commercial potential. A major extracellular endoglucanase, with a molecular mass of 34.0 kDa, was purified and characterized (Parry et al., 2002). It was optimally active at 70-80°C and pHs 4.0-4.4, and it was stable at pH 5.2 and up to 60°C for 48 h. At 70°C and pH 5.2 the enzyme retained 40% of the original activity after 48 h (Parry et al., 2002). The cellulase exhibited the highest activity toward CMC; barley β-glucan and lichenan were also hydrolyzed, but the enzyme was inactive on laminarin, confirming that it was an endoglucanase and was specific toward β-1,4 linked polysaccharides (Parry et al., 2002).

 

1.7.2 Aspergillus spp

 

A large number of fungi have been reported in municipal solid waste. The Aspergillus sp. was predominantly high among the other fungal species (Gautam et al., 2010). Most of the work on fungal Cellulase is centered on the saccharification of cellulose by Aspergillus (Milala et al., 2005) although Cellulase production on different carbon sources by Aspergillus and other fungi have been reported (Ruijter and Visser, 1997).

 

Aspergillus is a genus consisting of several hundred mold species found in various climates worldwide (Bennet, 2010).  Aspergillus is a  potential producer of cellulases (Mohammed et al., 2005). The organisms are widespread in nature and are typically found in soil and decaying organic matter, such as compost heaps, where they play an essential role in carbon and nitrogen recycling (Bennet, 2010). The two species of aspergillus used for this work are A. fumigatus and A. flavus.

Fig. 7:  A. flavus (Winiati, 2013)

 

Fig. 8: A. fumigatus (Mirhendi, 2000)

 

 

1.8 Fermentation Methods

 

Fermentation is the technique of biological conversion of complex substrates into simple compounds by various microorganisms such as bacteria and fungi. In the course of this metabolic breakdown, they also release several additional compounds apart from the usual products of fermentation, such as carbon dioxide and alcohol (Subramaniyam and Vimala, 2012). These additional compounds are called secondary metabolites. Secondary metabolites range from several antibiotics to peptides, enzymes and growth factors (Machado et al., 2004). Two broad methods will be considered for the production  of cellulase enzyme. These are solid state fermentation (SSF) and submerged fermentation (SmF) processes.

 

1.8.1 Solid-State Fermentation (SSF)

 

SSF utilizes solid substrates, like bran, bagasse, and paper pulp. The main advantage of using these substrates is that nutrient-rich waste materials can be easily recycled as substrates (Subramaniyam and Vimala, 2012). In this fermentation technique, the substrates are utilized very slowly and steadily, so the same substrate can be used for long fermentation periods (Subramaniyam and Vimala, 2012). Hence, this technique supports controlled release of nutrients. SSF is best suited for fermentation techniques involving fungi and microorganisms that require less moisture content. However, it cannot be used in fermentation processes involving organisms that require high water activity (aw), such as bacteria. (Babu and Satyanarayana, 1996).

 

1.8.2 Submerged Fermentation (SmF)/Liquid Fermentation (LF)

 

SmF utilizes free flowing liquid substrates, such as molasses and broths. The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly;

Hence, they need to be constantly replaced/supplemented with nutrients (Subramaniyam and Vimala, 2012). This fermentation technique is best suited for microorganisms such as bacteria that require high moisture content. An additional advantage of this technique is that purification of products is easier (Subramaniyam and Vimala, 2012). SmF is primarily used in the production of secondary metabolites that need to be used in liquid form. Submerged fermentation method for enzyme production is usually preferred since the enzyme will be obtained in liquid form thus making for easy purification and characterization.

 

 

1.9 Factors Affecting Cellulase Enzyme Production

 

1.9.1 Chemical Factors

 

1.9.1.1 Effect of Carbon Sources

 

Since any cellulose biotechnological process is likely to base on crude enzymes, it is important to increase their activities in the culture supernatants by selecting the best carbon and nitrogen sources and optimizing their concentrations (Gomes et al., 2000). Cellulase production is dependent on the nature of the carbon source used in the culture medium. Various lignocellulose carbon sources have been tested for their ability to induce cellulase production. Besides, the efficiency of enzyme production also depends on the bare chemical composition of the raw material, accessibility of various components and their chemical and physical associations. Wheat straw, rice straw and corn stover have been known as ideal substrate for cellulose production (Panagiotou et al., 2003; Mishra and Nain, 2010). Several investigations have indicated that cellulases are inducible enzymes, and different carbon sources have been used to find their role in effecting the enzymatic levels. Cellobiose (2.95 mM) may act as an effective inducer of cellulases synthesis in Nectria catalinensis (Pardo and Forchiassin, 1999). An increased rate of endoglucanase biosynthesis in Bacillus sp. was reported in the presence of cellobiose or glucose (0.2%) in the culture medium (Paul and Verma, 1990). Yeoh et al. (1986) had reported the inhibition of β-glucosidase activity at higher concentrations of cellobiose to an extant of 80%; similarly, laminaribiose and glucose also led to a 55–60% reduction in the enzymatic activity. Later, Shiang et al. (1991) described a possible regulation mechanism of cellulose biosynthesis and proposed that sugar alcohols, sugar analogues, xylose, glucose, sucrose, sorbose, cellobiose, methylglucoside etc. at a particular concentration may induce a cellulose regulatory protein called cellulase activator molecule (CAM). The level and yield of CAM get affected possibly due to substrate concentration and some unknown factors imparted by moderators. Many different agro-industrial wastes, synthetic or natural, have been examined as the carbon source for the process. Among the cellulosic materials, sulfate pulp, printed papers, mixed waste paper, wheat straw, paddy straw, sugarcane bagasse, jute stick, carboxymethylcellulose, corncobs, groundnut shells, cotton, ball milled barley straw, delignified ball milled oat spelt xylan, larch wood xylan, etc. have been used as the substrates

for cellulase production (Singh et al., 1991; Mishra and Nain, 2010). The observations indicated that the production of cellulases increased with increase in substrate concentration up to 12% during solid-state-fermentation using Aspergillus niger. Further increase in substrate concentration decreased the production levels. This might have been due to limitation of oxygen in the central biomass of the pellets, and exhaustion of nutrients other than energy sources. Martins et al. (2008) and Steiner et al. (1993) also demonstrated that carboxymethycellulose or cereal straw (1%, w/w) would be the best carbon source compared to sawdust for CMCase and β-glucosidase production using Chaetomium globosum as the producer organism. In contrast, 3% malt extract or water hyacinth was found optimum for CMCase, FPase and β-glucosidase as observed with lactose as an additional carbon sources (Mukhopadhyey and Nandi, 1999). However, the saccharification of alkali-treated bagasse at higher substrate levels of 4% w/v was also reported (Singh et al., 1991). Interestingly, higher concentrations (2.5–6.2% w/v) of carbon source were observed to be suitable for maximum saccharification when cellobiose was supplemented in the medium containing delignified rice straw, news print or other paper wastes as substrates ( Ju and Afolabi, 1999).

 

1.9.1.2 Effect of Nitrogen Sources

 

The effects of nitrogen sources on cellulase production are variable with respect to the fungi used (Kachlishvili et al., 2006). Enzyme production is affected significantly under different concentrations of nitrogen sources (Panagiotou et al., 2003). With different nitrogen sources, enzyme activities are higher with organic nitrogen (Gao et al., 2008). Maximum cellulase activity has been obtained with yeast extract (Gao et al., 2008), though other researchers found that inorganic nitrogen sources produce an optimal result (Kalogeris et al., 2003). The effect of different inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium ferrous sulfate, ammonium chloride and sodium nitrate have been studied. Among these, ammonium sulfate (0.5 g /L) led to maximum production of cellulases (Singh et al., 1991). In contrast, Menon et al. (1994) observed a significant decrease in enzymatic levels in the presence of ammonium salts as the nitrogen source. However, an increase in the level of β-glucosidase was reported when corn steep liquor (0.8% v/v) was added into the production medium. Corn steep liquor also resulted in 3-5 fold induction of endo- and exoglucanase levels with synthetic cellulose, wheat straw and wheat bran as the substrates. Enzyme production was sensitive to corn steep liquor (0.88 g/L), and production increased significantly when mixed nitrogen sources (corn steep liquor and ammonium nitrate) were supplied (Steiner et al., 1993). However, additional incorporation of nitrogen sources into medium scale up the cost of the process (Sunil et al., 2011).

 

1.9.1.3 Phosphorus Sources

 

Phosphorus is an essential requirement for fungal growth and metabolism. It is an important constituent of phospholipids involved in the formation of cell membranes. Besides its role in linkage between the nucleotides forming the nucleic acid strands, it is involved in the formation of numerous intermediates, enzymes and coenzymes essential in carbohydrate metabolism, other oxidative reactions and intracellular processes (Singh et al., 1991). Different phosphate sources such as potassium dihydrogen phosphate, tetra-sodium pyrophosphate, sodium β-glycerophosphate and dipotassium hydrogen phosphate have been evaluated for their effect on cellulases production (Garg and Neelkantan, 1982). It has been widely known that potassium dihydrogen phosphate is the most favorable phosphorus source for cellulase production (Sunil et al., 2011)

 

1.9.2 Physical Factors

 

1.9.2.1 pH

 

Different physical parameters influence the cellulose bioconversion, and pH is an important factor affecting cellulase production (Pardo and Forchiassin, 1999). The effect of pH on cellulase production has been analysed using Aspergillus niger, and found that pH 5.5 was optimal for maximum cellulase production. On other side, the pH range of 5.5–6.5 was optimal for β-glucosidase production from Penicillium rubrum (Menon et al., 1994). Eberhart et al. (1977) has reported that production and release of cellulase from Neurospora crassa depends on pH of the medium and maximum release occurs at pH 7.0, whereas the enzyme remained accumulated in the cell at pH 7.5. Similarly, pH 7.0 is suitable for extracellular production of cellulase from the Humicola fuscoatra (Rajendran et al., 1994).  The adsorption behavior of cellulases has been found to be affected by pH of the medium. Kim et al. (1988) had reported maximum adsorption of cellulase from Aspergillus phoenicus at pH 4.8–5.5. The pH range 4.6–5.0 has been found suitable for CMCase, filterpaperase (FPase) and β- glucosidase production with Aspergillus ornatus and Trichoderma reesei (Mukhopadhyey and Nandi, 1999).

 

1.9.2.2 Temperature

 

Temperature has a profound effect on lignocellulosic bioconversion. The temperature for assaying cellulase activities is generally within 50–65 °C for a variety of microbial strains (Menon et al., 1994; Steiner et al., 1993), whereas growth temperature of these microbial strains was found to be in the 25–30 °C (Macris et al., 1989). Similarly Penicillium purpurogenum, Pleurotus florida and Pleurotus cornucopiae show higher growth at 28 °C but maximum cellulase activities at 50 °C (Steiner et al., 1993) and about 98, 59 and 76% of the CMCase, FPase and β-glucosidase activities, respectively, retained after 48 h at 40 °C. Researchers have shown that temperature influences the cellulose-cellulase adsorption behaviour. A positive relationship between adsorption and saccharification of cellulosic substrate was observed at temperature below 60 °C. The adsorption activities beyond 60 °C decreased possibly because of the loss of enzyme configuration leading to denaturation of the enzyme activity (Van-Wyk, 1997). Bronnenmeier and Staudenbauer (1988) reported that extracellular as well as cell bound β-glulcosidase from Clostridium stercorarium required an identical temperature of 65 °C for their activity. Further increase in temperature led to a sharp decrease in the enzyme activity. Some of the thermophilic fungi having maximum growth at or above 45–50 °C produce cellulase with wide temperature optima (50–78 °C) (Wojtczak et al., 1987).

 

 

 

 

 

 

1.10 Applications of Cellulases

 

1.10.1 Cellulases in Brewing and Wine Biotechnology

 

The macerating enzymes, comprising cellulases, hemicellulases and pectinases, hydrolyze the plant cell wall and, consequently, can be used in brewing and wine biotechnology to improve the quality of finished products and avoid the use of chemicals. Enzyme preparations are used in the brewing and distilling industries to decrease the viscosity of the mash and to improve the overall efficiency of the process. In fact, cellulolytic and hemicellulolytic enzymes allow the conversion of undigestible lignocellulosic biomass into fermentable sugars, with consequent increase of alcohol yield.

 

1.10.1.1 Beer Brewing Process

 

Barley is the most common cereal used for the production of beer although wheat, corn, and rice are also widely used. The main processes involved in beer production include milling to reduce the size of the dry malt in order to increase the availability of the carbohydrates; mashing where water is added to the malt; lautering where spent grains are removed from the

wort, boiling of the wort with flavouring hops, fermentation of the wort liquor, maturation, conditioning, filtration and packaging of the final product. The high concentration of β- glucan in the brewing process, resulting from unsuitable brewing process or low quality barley, produces high viscosity of beer, formation of gelatinous precipitate, decrease of the extract yield, and lower run-off of wort (Bamforth, 1994; Bhat, 2000; Guo et al., 2010). In brewing process, cellulases are used during the mashing stage in order to hydrolyze excess β-glucans and reduce the viscosity, thus improving the separation of the wort from the spent grains. Oksanen et al. (1985) observed that the endoglucanase and the cellobiohydrolase from the Trichoderma cellulase system produced a large reduction of the degree of polymerization of the β-glucans, and wort viscosity. Moreover, the increased addition of enzymes used resulted in improved filtering. A. niger, T. reesei, and P. funiculosum, which are generally recognized as food grade microorganisms, are the major source of cellulases currently used in the mashing step, as these enzymes provide technological benefit to beer manufacture  (Karboune et al., 2008).

 

1.10.1.2 Wine Production    

 

Wine manufacture is a biotechnological process in which yeast cells and enzymes are indispensable for ensuring a high quality product. The use of cellulases, hemicellulases and pectinases during wine making, allows a better skin maceration, and superior color extraction, particularly important in the production of red wine; in addition, it improves clarification, filtration, and the overall quality and stability of the wine (Galante et al., 1998). Pectinase preparations, used in wine making, were lately modified by addition of cellulases and hemicellulases in small quantities to realize a more complete breakdown of the cells with consequent fruit liquefaction in a moderately short time period (Plank and Zent, 1993). It has also been demonstrated that the mixture of macerating enzymes worked better than pectinases alone in grape processing (Haight and Gump, 1994).

 

1.10.2 Cellulases in Pulp and Paper Biotechnology

 

1.10.2.1 Biomechanical Pulping

 

Mechanical pulping process is electrical energy intensive and results in low paper strength. Biomechanical pulping, defined as the enzymatic treatment of lignocellulosic materials before the mechanical pulping step, has shown at least 30% savings in electrical energy consumption, and significant improvements in paper strength properties. The potential of enzymatic treatments has been assessed and the processes have proved successful (Gubitz et al., 1998). Utilization of cellulases from fungal sources (T. reesei, Aspergillus sp.) (Buchert et al., 1998; Suurnakki et al., 2000) saves 33% electrical energy and significantly improves paper strength properties. A cellulase preparation produced by the ascomycete fungus Chrysosporium lucknowense for using in the pulp and paper industry represents, at present, an attractive alternative to the well-known cellulases from Fungi like Aspergillus sp. and T. reesei for protein production on a commercial scale (Bukhtojarov et al., 2004; Hinz et al., 2009).

 

 

 

 

1.10.2.2 Biodeinking

 

All over the world people give more attention to the environment and so, the recycle of waste paper has to be considered also as a necessity for the protection of forest and economy. Paper mill will gain profit from the utilization of recycled fiber, since it is profitable to decrease pollution, cost, and investment. Conventional deinking technology with alkali is characterized by a low efficiency on laser printed paper and is not considered environmentally friendly. Consequently, researchers have concentrated their attention on new deinking technologies (Moon and Nagarajan, 1998). The principle of enzymatic deinking is based on the weakening of the connections between toner and fibers due to the enzyme attack with separation of toner particles from fibers (Yingjuan et al., 2005; Shufang et al., 2005). The enzymatic deinking allows us to avoid the use of alkali; moreover, using enzymes at acidic pH it is possible to prevent the yellowing, modify the distribution of the ink particle size, improve fiber brightness strength, pulp freeness and cleanliness, reduce fine particles and reduce environmental pollution. Until 2000, the use of enzymes to perform biodeinking was only investigated at the laboratory scale (Buchert et al., 1998; Bhat, 2000). Subsequently, a mixture of cellulase, lipase, and amylase was employed in biodeinking process at industrial level (Morbak and Zimmermann, 1998). The effect of combined deinking technology with ultrasounds, UV irradiation and enzyme on laser printed paper was investigated. The results confirmed that the dose of alkali can be reduced using biodeinking technology. Cellulases from different microorganisms such as A. niger, T. reesei, Humicola insolens, Myceliophtora fergusii, Chrysosporium lucknowense, Fusarium sp. were used for this purpose (Marques et al., 2003).

 

 

1.10.3 Cellulases in Textile and Laundry Biotechnology

 

Since the early part of the last century, enzymes such as the cellulases have been used for a wide range of applications in textile processing in replacement of the traditional methods.

 

 

 

 

1.10.3.1 Biostoning and Biopolishing

 

Jeans manufactured from denim are one of the world’s most popular clothing items. In the late 1970s and early 1980s, industrial laundries developed methods for producing faded jeans by washing the garments with pumice stones, which partially removed the indigo dye revealing the white interior of the yarn, which leads to the faded, worn and aged appearance. This process was designated as ―stone-washing (Cavaco- Paulo, 1998). The use of 1-2 kg stones per kg of jeans for 1 h during stone-washing met the market requirements, but caused several problems including rapid consumption of washing machines, and unsafe working conditions. As an alternative to the stone-washing, biostoning is by far the most economical and environmental friendly way to treat denim. The cotton fabrics treated with the enzymes loose the indigo, which later is easily removed by mechanical abrasion in the wash cycle (Cavaco- Paulo, 1998; Yamada et al., 2005). The substitution of pumice stones by an

enzymatic treatment has many advantages: washing machines lower consumption and elevated productivity, short treatment times and less intensive working conditions. Moreover, it is possible to operate in a more safe environment because pumice powder is not produced, and the process can be mechanized controlling, with the use of computer, the dosing devices of liquid cellulase preparations (Bhat, 2000).

 

In the textile wet processing, the biopolishing is usually carried out with desizing, scouring, bleaching, dyeing and finishing by utilization of cellulases. However, there are no clear indications about the best cellulase mixture to use. (Miettinen-Oinonen and Suominen, 2002). The use of these enzymes allow many improvements such as the removal of short fibers, surface fuzziness smooth, polished appearance, more color uniformity and brigthness, improved finishing, and fashionable effects. At last, due to increasing environmental concerns and constraints being imposed on textile industry, cellulase treatment of cotton fabrics is an environmentally friendly way of improving the property of the fabrics. In 2007, Anish et al. (2006) isolated an endoglucanase from the alkalothermophilic bacterium Thermomonospora sp. The enzyme which is used for denim biofinishing under alkaline conditions, was effective in removing hairiness with negligible weight loss and imparting softness to the fabric. Higher abrasive activity with lower back-staining was a preferred property for denim biofinishing exhibited by the Thermomonospora endoglucanase.

 

1.10.3.2 Laundry

 

The most important reason to use enzymes in detergents is that they are biodegradable and a very small quantity of these inexhaustible biocatalysts can replace very large quantity of chemicals. Since detergents hold ionic and anionic surfactants, and bleaching agents (oxidizing agents) that can partially or completely denature proteins, the enzymes for laundry

must be resistant to anionic surfactants and oxidizing agents. The accumulation of microfibrils on the surface of the fabrics makes the fabrics look hairy and scatters incident light, thereby lessening the brightness of the original colors. In detergent industry, cellulases are used to remove microfibrils from the surface of cellulosic fabrics, enhancing color brightness, hand feel and dirt removal from cotton garments that during repeated washings can become fluffy and dull.

 

Other notable applications of cellulase are found in the treatment of wastes, production of biofuels and also in the animal feed industry.

 

1.11 Aim and Objectives of study

 

1.11.1 Aim of study

 

This study is aimed at using microorganisms cultivated on agricultural waste to produce cellulase enzyme with industrial potential.

       

          1.11.2 Specific Objectives of the Study

 

          This work is therefore designed to achieve the following specific objectives:

• Isolation of crude cellulase secreted by Aspergillus
• Determination of the protein content of the enzyme.
• To Assay for activity of the cellulase enzymes
• Partial purification of cellulase.
• Characterization of purified cellulase.

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