**4.1 Source of cellulase**

Cellulases are the hydrolytic enzymes which are produced by a diversity of microbes like actinomycetes, bacteria and fungi when grown on cellulosic substrates [61]. Among these organisms fungi are studied most extensively [62]. Filamentous fungi are the chief sources known for producing cellulases and hemicellulases [63]. Crude cellulases from *Trichoderma* and *Aspergillus* genera production are commercially available for agricultural and industrial use [64]. Representatives of *Trichoderma* genus secretes comparatively large quantities of endo-β-glucanase and exo-β-glucanase but low level of β-glucosidase, while those of Aspergillus genus secretes moderately high level of endo-β-glucanase and β-glucosidase with low level of exo-β-glucanase [65]. Cellulases isolated from thermophilic fungi are of great interest because of their industrial application on account of thermo stability. Thermophilic fungi producing cellulases include *Chaetomium thermopile*, *Humicola insolens*, *Humicola agrisea*, *Myceliopthora thermophila*, *Talaromyces emersonii* and *Thermoascus aurantiacus* [66]. Unlike thermophiles, cellulase producing alkaliphilic fungi are very rare [67]. The alkaline tolerated cellulases producing marine fungi Chaetomium sp. (NIOCC36) from mangrove leaves. Surprisingly, no any thermophilic archaea showing cellulolytic behavior have been described [68]. Bacterial cellulase generally forms complex systems (cellulosomes). Historically fungal cellulases have been easier to study than bacterial system, as the bacterial enzyme tend to form aggregates. *Cellulomonas*, *Bacillus* and *Micrococcus* spp. isolated from coir retting effluents of estuarine environment were also employed to study endoglucanase activity [69]. Gaor and Tiwari reported organic solvent thermostable cellulases from *Bacillus vallismortis* RG-07 [70]. *Bacillus thuringiensis* strains [71], *Bacillus pumilus* EB3 [72] are also reported as good cellulase producers. Wild-type and mutants stains of *Pseudomonas fluorescens* were used by Bakare and co-workers to produce cellulases [73]. Interestingly, research findings are reported even for the production of cellulases from several species of insects in the orders of dictyoptera, orthoptera, and coleoptera by their own in the mid gut or salivary glands. These findings challenged the traditional view of cellulose digestion that it is mediated by microbial cellulases in the gut of insect [74]. The first endogenous cellulase

**67**

*Overview of the Process of Enzymatic Transformation of Biomass*

from insect was discovered in 1998 in the termite (*Reticulitermes speratus*), which was found to be capable for feeding wood even after the removal of its gut fauna [75]. Acquisition of digestive enzymes has also been explored in other xylophagous arthropods, molluscs, including snails, a sea slug, a periwinkle and some bivalves. Various possible sources are reported for these endogenous enzymes such as the hepatopancreas, gastric teeth, and crystalline styles (needlelike structures made of

crystalline proteins forming a motor organ in the stomach of bivalves [76].

Two fundamental approaches used for measuring cellulase activity are:

1.Measuring individual cellulase (endoglucanases, exoglucanases and

The first one is ideal for measuring individual cellulase activity within a short time however the third one is a chosen for measuring total enzyme activity within a

Total cellulase activity assay is always performed using insoluble substrates having pure cellulosic substrates such as Whatman No. 1 filter paper. The filter paper activity (FPase activity) is the key method for analysis of total cellulase activity which was developed by Mandels, cotton linter, microcrystalline cellulose, bacterial cellulose, algal cellulose and cellulose-containing substrates such as pretreated lignocellulose [78]. This standard filter paper method has been revised by Ghose which was established and published by the International Union of Pure and Applied Chemistry (IUPAC) [79]. He used Whatman No. 1 filter paper (1 × 6 cm strip) as the substrate. It is used as the standard substrate because of its readily availability and

Commercial avicel is also used for measuring exoglucanase activity because it has a low degree of polymerization (DP) and it is moderately hard to be attacked by endoglucanases [81]. Endoglucanase activity can be measured using a soluble cellulose derivative with a high degree of polymerization (DP) such as carboxymethyl cellulose (CMC). It can be measured by both methods, i.e., reduction in substrate viscosity/increase in reducing sugar. CMCase activity using CMC is measured by determining reducing sugars released after 5 min of enzyme reaction with 0.5% CMC at pH 4.8 and 50°C [78]. Exoglucanases are known to cleave the easily accessible ends of cellulose molecules liberating glucose and cellobiose. β-glucosidases cleaves soluble cellobiose and other cellodextrins having DP up to 6 and liberates glucose as end product [82]. Various chromogenic and nonchromogenic substrates could be evaluated. In chromogenic method, p-nitrophenol-β-glucoside (P-NPG) can be used as the substrate. However, in the case of nonchromogenic substrates different methods used are based on nature of substrates. For example, when oligo or disaccharides (such as cellobiose) are used, released glucose can be evaluated by the GOD (glucose oxidase)

2.Measuring the total cellulase (FPase) activity [77].

1.Accumulation of products after hydrolysis.

3.Change in the physical properties of substrates.

2.The reduction in substrate quantity.

Quantitatively cellulase activity can be assayed in three ways:

*DOI: http://dx.doi.org/10.5772/intechopen.85036*

**4.2 Cellulase activity assay**

given time [77].

inexpensiveness [80].

β-glucosidases) activities.

*Overview of the Process of Enzymatic Transformation of Biomass DOI: http://dx.doi.org/10.5772/intechopen.85036*

from insect was discovered in 1998 in the termite (*Reticulitermes speratus*), which was found to be capable for feeding wood even after the removal of its gut fauna [75]. Acquisition of digestive enzymes has also been explored in other xylophagous arthropods, molluscs, including snails, a sea slug, a periwinkle and some bivalves. Various possible sources are reported for these endogenous enzymes such as the hepatopancreas, gastric teeth, and crystalline styles (needlelike structures made of crystalline proteins forming a motor organ in the stomach of bivalves [76].

## **4.2 Cellulase activity assay**

*Elements of Bioeconomy*

**4.1 Source of cellulase**

**Figure 10.** *Cellulases [59].*

(CD). Both the domains are found to be connected by a linker region (**Figure 10**) [57]. Till date, about 300 different CBMs have already been identified. CBMs are categorized into 45 families on the basis of their amino acid similarity [58]. This variation in affinity may be due to variation in spatial structure created by the presence of CBMs [60].

Cellulases are the hydrolytic enzymes which are produced by a diversity of microbes like actinomycetes, bacteria and fungi when grown on cellulosic substrates [61]. Among these organisms fungi are studied most extensively [62]. Filamentous fungi are the chief sources known for producing cellulases and hemicellulases [63]. Crude cellulases from *Trichoderma* and *Aspergillus* genera production are commercially available for agricultural and industrial use [64]. Representatives of *Trichoderma* genus secretes comparatively large quantities of endo-β-glucanase and exo-β-glucanase but low level of β-glucosidase, while those of Aspergillus genus secretes moderately high level of endo-β-glucanase and β-glucosidase with low level of exo-β-glucanase [65]. Cellulases isolated from thermophilic fungi are of great interest because of their industrial application on account of thermo stability. Thermophilic fungi producing cellulases include *Chaetomium thermopile*, *Humicola insolens*, *Humicola agrisea*, *Myceliopthora thermophila*, *Talaromyces emersonii* and *Thermoascus aurantiacus* [66]. Unlike thermophiles, cellulase producing alkaliphilic fungi are very rare [67]. The alkaline tolerated cellulases producing marine fungi Chaetomium sp. (NIOCC36) from mangrove leaves. Surprisingly, no any thermophilic archaea showing cellulolytic behavior have been described [68]. Bacterial cellulase generally forms complex systems (cellulosomes). Historically fungal cellulases have been easier to study than bacterial system, as the bacterial enzyme tend to form aggregates. *Cellulomonas*, *Bacillus* and *Micrococcus* spp. isolated from coir retting effluents of estuarine environment were also employed to study endoglucanase activity [69]. Gaor and Tiwari reported organic solvent thermostable cellulases from *Bacillus vallismortis* RG-07 [70]. *Bacillus thuringiensis* strains [71], *Bacillus pumilus* EB3 [72] are also reported as good cellulase producers. Wild-type and mutants stains of *Pseudomonas fluorescens* were used by Bakare and co-workers to produce cellulases [73]. Interestingly, research findings are reported even for the production of cellulases from several species of insects in the orders of dictyoptera, orthoptera, and coleoptera by their own in the mid gut or salivary glands. These findings challenged the traditional view of cellulose digestion that it is mediated by microbial cellulases in the gut of insect [74]. The first endogenous cellulase

**66**

Two fundamental approaches used for measuring cellulase activity are:


Quantitatively cellulase activity can be assayed in three ways:


The first one is ideal for measuring individual cellulase activity within a short time however the third one is a chosen for measuring total enzyme activity within a given time [77].

Total cellulase activity assay is always performed using insoluble substrates having pure cellulosic substrates such as Whatman No. 1 filter paper. The filter paper activity (FPase activity) is the key method for analysis of total cellulase activity which was developed by Mandels, cotton linter, microcrystalline cellulose, bacterial cellulose, algal cellulose and cellulose-containing substrates such as pretreated lignocellulose [78]. This standard filter paper method has been revised by Ghose which was established and published by the International Union of Pure and Applied Chemistry (IUPAC) [79]. He used Whatman No. 1 filter paper (1 × 6 cm strip) as the substrate. It is used as the standard substrate because of its readily availability and inexpensiveness [80].

Commercial avicel is also used for measuring exoglucanase activity because it has a low degree of polymerization (DP) and it is moderately hard to be attacked by endoglucanases [81]. Endoglucanase activity can be measured using a soluble cellulose derivative with a high degree of polymerization (DP) such as carboxymethyl cellulose (CMC). It can be measured by both methods, i.e., reduction in substrate viscosity/increase in reducing sugar. CMCase activity using CMC is measured by determining reducing sugars released after 5 min of enzyme reaction with 0.5% CMC at pH 4.8 and 50°C [78]. Exoglucanases are known to cleave the easily accessible ends of cellulose molecules liberating glucose and cellobiose. β-glucosidases cleaves soluble cellobiose and other cellodextrins having DP up to 6 and liberates glucose as end product [82]. Various chromogenic and nonchromogenic substrates could be evaluated. In chromogenic method, p-nitrophenol-β-glucoside (P-NPG) can be used as the substrate. However, in the case of nonchromogenic substrates different methods used are based on nature of substrates. For example, when oligo or disaccharides (such as cellobiose) are used, released glucose can be evaluated by the GOD (glucose oxidase)

method with a commercial kit but when polysaccharide are used a substrate, reducing sugars released is measured by the DNS (dinitrosalicylic acid) method [81].
