� Review of literature

8

Chapter 2 Review of literature

2.1 Historical perspectives of enzymes

Enzymes occur in every living cell, hence in all microorganisms. Enzymes are biocatalysts

produced by living cells to bring about specific biochemical reactions generally forming

parts of the metabolic processes of the cells. Enzymes are highly specific in their action on

substrates and often many different enzymes are required to bring concerted action, the

sequence of metabolic reactions performed by the living cell. Almost all enzymes which

have been purified are protein in nature, and may or may not possess a non protein

prosthetic group for their biological activity. The practical application and industrial use of

enzymes to accomplish certain reactions apart from the cell, dates back many centuries and

practiced long before the nature or function of enzymes was understood. Use of barley

malt for starch conversion in brewing and treatment of hides in leather making are

examples of ancient use of enzymes. It was not until nearly the turn of this century that the

causative agents or enzymes responsible for bringing about such biochemical reactions

became known. Then crude preparations of enzymes from certain animal tissues such as

pancreas and stomach mucosa, or from plant tissues such as papaya fruit, were prepared

which found technical applications in the textile, leather, brewing, and other industries.

Once the favorable results of employing such enzyme preparations were established, a

search began for better properties (stability and activity against alkaline, acidic, high

temperature, metal ions, surfactants, protease resistant, etc), less expensive and more

readily available sources of such enzymes. It was found that certain microorganisms

produce enzymes similar in action to the amylases of malt and pancreas, or to the proteases

of the pancreas and papaya fruit. This led to the development of processes for producing

9

such microbial enzymes on a commercial scale. Great diversity exists in microbial enzyme

production, each single strain of a organism produces a large number of enzymes such as

hydrolases, oxido/reducatases, transferases, isomerases, lyases and ligases, which are

metabolic in nature. But the absolute and relative amounts of the various individual

enzymes produced will vary depending on cellular need and it differs markedly between

species and even between strains of the same species. Hence, it is customary to select

microbial strains for commercial production of specific enzymes that have the capacity for

producing highest amounts of the particular enzymes with properties desired.

The roots of modem enzymology may be traced back to the last century when scientists

such as Payen and Pesoz, showed that an alcohol precipitate of malt extract contained a

thermolabile substance which converted starch into fermentable sugars. The enzyme

responsible was proved to be diastase because of its ability to yield soluble dextrins from

insoluble starch granules. The existence of several additional enzymes including pepsin,

polyphenol oxidase, peroxidase and invertase was recognized by the mid-nineteenth

century. The first enzyme preparation to be patented for industrial use was termed Taka-

Diastase, an amylolytic preparation produced by A. oryzae when grown on rice. The patent

was granted to Dr. Takamine, a Japanese immigrant to the U.S in 1884 (Miles Inc. 1988).

Microbial biotechnology of enzymes production has substantially made an impact on

healthcare, production and processing of food, basic molecular studies, genetic

engineering, agriculture and forestry, environmental protection, and biotransformation of

chemical compounds. Among the major new technologies that have appeared since 1970s,

microorganisms for biotechnological enzymes production perhaps attracted the most

attention. Microbes proved capable of generating enormous wealth and influencing every

10

significant sector of the economy. In a conservative estimate, microbial enzymes represent

almost 90% of the total market. Industrial enzymes are currently manufactured by three

major suppliers, Novozymes A/S (headquartered in Denmark), Genencor International Inc.

(headquartered in the US), recently acquired by Danisco A/S (headquartered in Denmark)

and DSM N.V. (headquartered in the Netherlands). Industrial uses of enzymes have

increased greatly during the past few years. Prospects are excellent for continuing

increased usage of presently available enzymes in different applications.

Enzymes have several distinct advantages for use in industrial processes:

1. They are of natural origin and are nontoxic.

2. They have great specificity of action; hence can bring about reactions not otherwise

easily carried out.

3. They work best under mild conditions of moderate temperature and near neutral pH,

thus not requiring drastic conditions of high temperature, high pressure, high

acidity/alkaline, which necessitate special expensive equipment.

4. They act rapidly at relatively low concentrations, and the rate of reaction can be

readily controlled by adjusting temperature, pH, and amount of enzyme employed.

5. They are easily inactivated when reaction completed as far as desired.

Because of these inherent advantages, many industries are keenly interested in

adapting enzymatic methods to the requirements of their processes. Examples of some

applications under intensive investigation of different sectors include biofuel, food

processing, textile, leather, pulp and paper, fruit juice and beverages etc,. Nowadays

advances in extremophilic microbiology, isolation, identification and industrial

exploitation of extremophiles i.e. microbes adapted to exist and grow at extreme

11

environments, are getting considerable attention. Eventhough such extremophiles are

difficult to grow under laboratory conditions, their genes can be cloned into suitable

mesophilic hosts as illustrated by the cloning of thermophilic enzyme genes from archaea

and bacteria, and psychrophilic enzyme genes into mesophilic expression host systems

(Feller et al. 1998; Bertoldo and Antranikian 2002; Viikari 2007). In fact, the advances in

molecular genetics and genetic engineering in the last few decades have made possible to

clone and express virtually any gene in a suitable microbial host, so that now enzymes

from other microorganisms and also from higher organisms can be produced in convenient

representative microbial hosts like bacteria (Escherichia coli), yeasts (Pichia pastorius)

and fungi (A.niger) (Glick and Pasternak 2003). This fact has contributed significantly to

limitless application of enzymes that can be produced by microbial fermentation and also

to increase the productivity of the fermentation and the quality of the enzyme product. It

was estimated that about 50% of the industrial enzymes (on a mass basis) were produced

from genetically engineered organisms (Hodgson 1994). This proportion might have

increased significantly in the last decade because of the advances in recombinant DNA

technology and protein engineering and also because of the increasing production of

speciality enzymes for the pharmaceutical and fine-chemicals industries (Dannert and

Arnold 1999; Schulein 2000; McCoy 2001; Rasor and Voss 2001; Thomas et al. 2002).

2.2 Cellulose source materials and their derivatives

Industrial sustainability aims to achieve sustainable production and processing within the

context of ecological and social sustainability (Miyamoto 1997). Compared to

conventional production, sustainable processes and production systems should be more

profitable because they require less energy, result in less emissions of greenhouse gases

12

and other pollutants, enable greater and more efficient use of renewable resources and to

lessen dependence on nonrenewable resources. Lignocellulosic biomass is the major

sustainable resource, comprising around half of the plant matter produced by

photosynthesis and representing the most abundant renewable organic matter.

Lignocellulosic residues from wood, grass, agricultural and forestry wastes and municipal

solid wastes are particularly abundant in nature and have a potential for bioconversion and

the production on global scale of these sources are given in table 2.1. They constitute a

renewable resource from which many useful biological and chemical products can be

derived (Zosel 1994; Berkel 2000; Gavrilescu 2004; Gavrilescu and Nicu 2004). Industry

is truly sustainable only when it is economically viable, environmentally compatible, and

socially responsible (OECD 1998; UNEP 1999). Lignocellulosic biomass consists of three

types of polymers, cellulose, hemicellulose and lignin that are strongly intermeshed and

chemically bonded by non-covalent forces and by covalent cross linkages. The major

component is cellulose, followed by hemicellulose and lignin. The composition and

proportions of these compounds vary between plants and chemical composition of some of

lignocellulosic residues are given in table 2.2 (McKendry 2002; Malherbe and Cloete

2002; John et al. 2006, Prassad et al. 2007; Carmen 2009). Only a small amount of the

cellulose, hemicellulose and lignin produced as by-products in agriculture or forestry is

used, the rest being considered waste.

Many microorganisms are capable of degrading and utilizing cellulose and

hemicelluloses as carbon and energy sources. However, a much smaller group of

filamentous fungi has evolved with the ability to break down lignin, the most recalcitrant

component of plant cell walls. These are known as white-rot fungi, which possess the

13

unique ability of efficiently degrading lignin to CO2. Accumulation of lignocellulose in

large quantities in places where agricultural residues present a disposal problem results not

only in deterioration of the environment but also in loss of potentially valuable material

that can be used in paper manufacture, biomass fuel production, composting, human and

animal feed among others (Pandey et al. 2000).

Table 2.1 Lignocellulosic residues generated from different agricultural sources

Source: Carmen 2009

14

Table 2.2 Composition of some lignocellulosic materials

Lignocellulosic

residues

Lignin (%) Hemicellulose

(%)

Cellulose (%) Ash (%)

Hardwood stems 18–25 24–40 40–55 NA

Softwood stems 25–35 25–35 45–50 NA

Nut shells 30–40 25–30 25–30 NA

Corn cobs 15 35 45 1.36

Paper 0–15 0 85–99 1.1–3.9

Rice straw 18 24 32.1 NA

Cotton seed hairs 0 5–20 80–95 NA

Newspaper 18–30 25–40 40–55 8.8–1.8

Waste paper from

chemical pulps 5–10 10–20 60–70 NA

Switch grass 12.0 31.4 45 NA

Grasses (average

values for grasses) 10–30 25–50 25–40 1.5

Sugarcane bagasse 19–24 27–32 32–44 4.5–9

Wheat straw 16–21 26–32 29–35 NA

Barley straw 14–15 24–29 31–34 5–7

Oat straw 16–19 27–38 31–37 6–8

Rye straw 16–19 27–30 33–35 2–5

Bamboo 21–31 15–26 26–43 1.7–5

Bast fiber Kenaf 15–19 22–23 31–39 2–5

Bast fiber Jute 21–26 18–21 45–53 0.5–2

Leaf Fiber Abaca

(Manila) 8.8 17.3 60.8 1.1

Leaf Fiber Sisal

(agave) 7–9 21–24 43–56 0.6–1.1

Leaf Fiber Henequen 13.1 4–8 77.6 0.6–1

Banana waste 14 14.8 13.2 11.4

NA = Not available. Source: Carmen 2009

15

2.3 Cellulose chemistry

Cellulose is the end product of photosynthesis in terrestrial environments, and the most

abundant renewable bioresource produced in the biosphere (~100 billion dry tonns/year)

(Jarvis 2003; Zhang and Lynd 2004). Cellulose is an organic compound with the formula

(C6H10O5)n, a polysaccharide consisting of a linear chain of several hundred to over ten

thousand β(1→4) linked D-glucose units. Payen (1795 - 1871) was a French chemist

known for discovering the carbohydrate cellulose. Although it took many decades after the

identification of cellulose, it was later shown to be a long chain polymer with repeating

units of D-glucose, a simple sugar. In the cellulose chain, the glucose units are in 6-

membered rings, called pyranoses. They are joined by single oxygen atoms (acetal

linkages) between the C-1 of one pyranose ring and the C-4 of the next ring. Since a

molecule of water is lost when an alcohol and a hemiacetal react to form an acetal, the

glucose units in the cellulose polymer are referred to as anhydroglucose units (Fig. 2.1 A &

B).

2.4 Enzymatic hydrolysis of cellulose

The physical heterogeneity of cellulosic substrates together with the complexity of

cellulase system produced by different microorganisms have led to the development of

several assay procedures for the measurement of cellulase activities. All existing

cellulolytic enzymes activity assays can be divided into three types: (1) the accumulation

of products after hydrolysis, (2) the reduction in substrate quantity, and (3) the change in

the physical properties of substrates. The considerable difference in the nature of substrates

used, variation in assay procedures adopted for measuring different cellulase components,

and the synergistic action of cellulase components have made the comparison of results

among laboratories difficult. Therefore, in 1984, the IUPAC

16

Fig. 2.1 A. Schematic structures of cellulose chains. B. The structure of cellulose

showing the chains held together by hydrogen bonds.

Sources of links A. http://www.doitpoms.ac.uk/tlplib/wood/figures/cellulose.png

B. http://chempolymerproject.wikispaces.com/Cellulose-D-TPNR

17

Commission on Biotechnology published standard assay procedures for measuring

cellulase activities (Ghosh 1984). Some of these recommendations have been readily

accepted, but many of these procedures are quite restricted and not satisfactory for

understanding the mechanism of action and substrate specificities of cellulases in detail.

Consequently, Wood and Bhat (1988) reviewed the cellulase assays used by laboratories

working on fungal cellulases. The majority of assays involve the accumulation of

hydrolysis products, i.e. quantification of reducing sugars, and chromophores. The most

common reducing sugar assays include the dinitrosalicyclic acid (DNS) method (Miller

1959; Ghosh 1984) and the Nelson-Somogyi method (Nelson 1944; Somogyi 1952). The

traditional protocols for quantification of cellulolytic enzymes, substrates and assay are

given in table 2.3. The synergistic action of cellulolytic enzymes on cellulose degradation

was shown in figure 2.2.

2.5 Sources of cellulases

Microbial cellulases are the most economic and available sources to meet the

industrial scale, because microorganisms can grow on an inexpensive media such as

agriculture and food industries by-products. Certain microbial sources that are studied for

cellulase production are listed in table 2.4.

18

Table 2.3 Substrates and assay procedures for mesuring cellulolytic enzymes activity.

Enzyme Substrate Assay

Endo-β-1,4-D-glucanase

(endoglucanase, CM-

cellulase, endocellulase)

Carboxymethyl cellulose

Hydroxyethylcellulose

Release of reducing sugars (Miller

1959).

Decrease in viscosity (Wood and Bhat

1988; Tolan and Foody 1999).

Exo- 1,4-13-D-glucanase

(exocellulase or Avicelase or

FPase)

Filter paper

Avicel

Hydrocellulose

Dyed Avicel

Release of reducing sugars (Miller

1959)

-do-

-do-

Release of dyed cellobiose (Nummi et

al. 1981; Wood and Bhat 1988 )

β-D-Glucosidase or

Cellobiase

Cellobiose

p-Nitrophenyl- β -D-

glucopyranoside

Release of glucose and Release of o- or

p-nitrophenol (Wood and Bhat1988).

Fig. 2.2 Cellulose hydrolysis by a cellulase system (Winkelmann 1992).

19

Table 2.4 Certain microbial sources for cellulolytic enzymes production.

Organism Enzyme Reference

I. Bacteria

Acidothermus cellulolyticus Endoglucanase Ding (2006), Himmel et al. (1994),

Sakon et al. (1996)

Alkalophilic Streptomyces Cellulase,

Endoglucanase,

Nakai et al. (1988), Damude et al.

(1993)

Anaerocellum thermophilum Endoglucanase Zverlov et al. (1998)

Bacillus sp. KSM-S237 Endoglucanase Hakamada et al. (1997)

Caldocellulosiruptor

saccharolyticus

Endoglucanase

Exoglucanase

Bergquist et al. (1999), Te’o et al.

(1995)

Caldocellum saccharolyticum Endoglucanase Te’o et al. (1995)

Cellulomonas flavigena β-D-glucosidases Gaspar et al. (2007)

Clostridium stercorarium Endoglucanase

Exoglucanase

Bronnenmeier and Staudenbauer

(1990), Bronnenmeier et al. (1991),

Clostridium thermocellum Endoglucanase Fauth et al. (1991),

Romaniec et al. (1992)

Lactobacillus plantarum β-D-glucosidase Sestelo et al. (2004)

Pseudomonas fluorescens Cellulase Yamane et al. (1970)

Pyrococcus furiosus Cellulase Voorhorst et al. (1999)

Rhodothermus marinus Endoglucanase Hreggvidsson et al. (1996)

Sulfolobus solfataricus Cellulase Moracci et al. (2001)

II. Fungi

Aspergillus aculeatus Cellulase Murao et al. (1988)

Aspergillus glaucus XC9 Cellulase

(endoglucanase)

Chang et al. (2006)

Aspergillus nidulans β-D-glucosidases Kwon et al. (1992)

Aspergillus nidulans β-D-glucosidases Bagga et al. (1990)

Aspergillus niger Cellulase Okada (1988)

20

Aspergillus niger B-glucosidase Johanssona and Reczey (1998

Aspergillus oryzae β-D-glucosidases Zhang et al. (2007)

Aspergillus sojae β-D-glucosidases Kimura et al. (1999)

Chaetomium thermophilum Endoglucanase Li et al. (2003)

Cladosporium sp. Endoglucanase

Exoglucanase

Abrha and Gashe (1992)

Humicola insolen,

Humicola grisea

Cellulase

Cellulase

Hayashida et al. (1988)

Melanocarpus albomyces Endoglucanase Oinonen et al. (1996)

Metschnikowia pulcherrima β-D-glucosidase Pombo et al. (2008)

Mucor circinelloides Endoglucanase Nakamura et al. (2001)

Penicillium pinophilurn Cellulase Wood et al. (1989),

Wood and McCrae (1986)

Rhizopus oryzae Endoglucanase

β-D-glucosidase

Murashima et al. (2002)

Thermoascus aurantiacus Cellulase,

β-D-glucosidase

Hong et al. (2007)

Trichoderma reesei Cellulase Bhikhabhai et al. (1984)

Trichoderma viride Cellulase Voragen et al. (1988)

21

2.6 Application of cellulases

Cellulose biodegradation by cellulolytic microorganisms represents a major carbon flow

from fixed carbon sinks to atmospheric CO2 (Falkowski et al. 2000; Melillo et al. 2002;

Berner 2003) is very important in several agricultural and waste treatment processes

(Russell and Rychlik 2001; vanWyk 2001; Hamer 2003; Angenent et al. 2004; Haight

2005), and could be widely used to produce sustainable biobased products and bioenergy

to replace depleting fossil fuels (Lynd 1996; Mielenz 2001; Galbe and Zacchi 2002; Lynd

et al. 2002; Wyman 1994; 1999; 2003; Angenent et al. 2004; Kamm and Kamm 2004;

Demain et al. 2005; Reddy and Yang 2005). Commercial production of cellulase enzymes

by submerged culture fermentation began in the early 1970s, with cellulase made by

Trichoderma sold for use in research and pilot studies. The mid-1980s saw the first large

industrial uses of cellulase for stonewashing denim and as an additive for animal feeds.

This was accompanied by the introduction of commercial cellulases made by fungi of the

genera Aspergillus, Penicillium, and most importantly Humicola, introduced by Novo in

1986 (Nielsen 1995).

Cellulosic wastes have great potential as a feedstock for producing fuels and

chemicals. Cellulose is a renewable resource that is inexpensive, widely available and

present in ample quantities. Large amounts of waste cellulose products are generated by

commercial and agricultural processes. Concerns about diminishing resources, and the

excessive production of greenhouse gases continue to motivate the search for alternatives

to fossil fuel. The present challenge is to substantially increase the production and use of

biofuels for the transport sector. In order to reach the future goals of substituting fossil

based fuels, it will be necessary to promote the transition towards second generation

22

biofuels. The hydrolysis of lignocelluloses to fermentable sugars remains the greatest

challenge in the development of economical plant biomass feedstock for the biorefinery

industry (Brown 2003). These can be produced from a wider range of feedstock, including

lignocellulosic raw materials. Biomass resources can be broadly categorised as agricultural

or forestry-based, including secondary sources derived from agro- and wood industries,

waste sources and municipal solid wastes. Fuels from lignocellulosic biomass have a

higher potential to reduce greenhouse gas emissions, and hence are an important means to

fulfil the CO2 emission targets, as compared with first generation biofuels. Lignocellulosic

raw materials comprise an abundant source of carbohydrates (cellulose and hemicellulose)

for a variety of biofuels, including bioethanol. The conversion technologies of

lignocellulosic raw materials are, however, more complex and need novel enzyme systems

and advanced fermentation technologies. The rate-limiting step in the conversion of

cellulose to fuels is hydrolysis, especially the initial attack on the highly ordered cellulose

structure. In spite of recent achievements, further developments are still needed to improve

the overall economy of the lignocellulose-to-ethanol process. These novel conversion

techniques would also be applicable for the production of other sugar platform-based

chemicals, the schematic representation of biological conversion of lignocellulosic material

to value added products is shown in fig. 2.3.

2.6.1 Textile industry

The textile industry is one of the first to benefit from targeted use of biotechnology. The

introduction of cellulases in the 1980s truly revolutionized denim garment processing

(Stewart 1996; Kumar and Yoon 1997). Denim finishing with cellulases emerged in the

1980s, radically changed the conventional process by reducing the need for pumice stones,

23

and created the largest market segment for enzyme technology in textiles (Schafer et al.

2007). Enzymes are applied during the preparation, dyeing, and finishing (“wet process”)

stages of textile production, during which the fibers are exposed to water for depilling,

softening and denim abrasion of cellulose (Fig. 2.4). Biopolishing is a treatment in which

cellulase is used to remove small fuzz and fibrils from the surface of the fabric. This

versatile treatment can be carried out as a separate step or in combination with other

enzymatic preparation processes on cellulose-containing (cotton, viscose rayon, lyocell)

yarns, fabrics and garments to reduce the pilling (or fibrillation) tendency of the fabric and

provide a softer feel. Frequently, improvements in dye uptake are also observed. Enzyme

inactivation is needed after treatment with cellulase to stop the hydrolysis before damage

occurs. Typical inactivation is carried out by raising the liquid pH to 10 with sodium

carbonate and heating at 80 ◦C for 10 min (Kumar and Harden 1999).

24

Fig. 2.4 Enzyme treatments in denim process: amylase desized (left),

cellulase abrasion (center), and laccase/mediator decolorization (right).

(Roland and Sell 2007)

Fig. 2.3 Schematic representation of biological conversion of

lignocellulosic materials to value added products. (Jorgensen et al. 2007)

25

2.6.2 Laundry detergents

The use of cellulase in household laundry detergent originated in 1993 with the

introduction of Humicola endoglucanase. Cellulase in laundry detergent removes the hairs,

known as pills that occur on cotton clothes after repeated wearing and machine washing.

The cellulase removes the existing pills, and conditions the surface of new or unpilled

clothes. The result is an appearance that more closely resembles a new garment in

sharpness of color and smoothness of appearance. The use of cellulase can eliminate the

need for cationic fabric softeners, which have disposal and cost problems (Eriksen 1996).

Several types of cellulases are used in laundry detergents. Cellulases can both boost

cleaning performance and provide fabric care benefits (Kirk et al. 2004; Gormsen et al.

1998).

2.6.3 Pulp and paper industry

Over the last two decades the application of enzymes in the pulp and paper industry has

increased dramatically. The cellulase mainly acts by strengthening, refining, deinking and

drainage improvement of pulp. To name a few, lipase for pitch control, esterase for stickies

removal and pectinases for charge control (Schafer et al. 2007). Historically, cellulases

were perceived as having negative impact on paper strength and yield. However,

remarkable improvement in dry tensile and tear strength of bleached kraft pulps in paper

preparation were observed after the treatment by a few selected cellulases (Lonsky and

Negri 2003).

26

2.6.4 Fruit juice and beverage processing

Cellulase enzymes break down cellulose and beta-glucan associated with the cell walls,

thereby decreasing the viscosity of the mash and increasing the ease of the juice recovery.

The conversion of cellulose and beta-glucans into soluble sugar provides another increase

in the overall juice solids yield. The enzyme treatment can also increase the clarity of the

juice by solubilizing small particles. The enzyme treatment can enhance the flavor of the

juice by increasing the extractability of flavor compounds in the mash. Where disposal of

the solid residue is costly, cellulase helps to decrease waste disposal costs. Another

concern in using cellulase in beverages, particularly beer and wine, is the possibility of

changes in flavor. Although increasing flavor extractability is often desirable, many beer

and wine brand names maintain a constant flavor that is undesirable to change. Most

cellulase used in the juice industry is Trichoderma cellulase,because of the typically low

pH present in the mash. Cellulase from Aspergillus niger is also used (Tolan and Foody

1999).

2.6.5 Alcohol production

Ethanol from cellulose represents an enormous opportunity to make a transportation fuel

that is an alternative to gasoline (Lynd 1996; Mielenz 2001; Lynd et al. 2002; Galbe and

Zacchi 2002; Wyman 2003; Angenent et al. 2004; Kamm and Kamm 2004; Demain et al.

2005; Reddy and Yang 2005). Development of such a fuel is motivated by 1) an increased

cleanliness of automobile exhaust with decreased levels of carbon monoxide and nitrous

oxides 2) a need for a fuel that does not contribute to an increase in the greenhouse effect,

3) the desire to decrease the dependence on imported petroleum, and 4) the possibility of

creating wealth in regions where cellulose is a prevalent natural resource. Cellulose is

27

converted to ethanol by making glucose and then fermenting the glucose to ethanol using

yeast (Tolan and Foody 1999).

2.7 Aspergillus sp. as source of enzymes

The species of the genus Aspergillus most abundant and widely distributed microfungi in

decomposing organic material of soil, water, air, stored seeds, food, etc. The Aspergillus

was first identified by Micheli (1729). The genus Aspergillus consists of more than 180

officially recognized species, and comprises a particularly important group of filamentous

ascomycete species. Although it includes the major pathogen of humans. ex. Aspergillus

fumigatus (Brookman and Denning 2000; Latge 1999) and industrially and

environmentally useful members for degradation of plant polysaccharides (deVries et al.

2000; deVries 2003). Aspergillus species are also important for the large-scale production

of both homologous and heterologous enzymes (Fawole and Odunfa 2003; Wang et al.

2003). The biochemical and physiological methods are important in the systematics of

Aspergillus species, besides morphological properties that are commonly used for initial

identification (Christensen et al. 2000; Klich 2002; Asan 2004). The typical outline

microscopic morphology of Aspergillus sp. is shown in fig. 2.5.

2.7.1 Aspergillus flavus – a source for enzymes of biotechnological applications

Aspergillus, having inherent property of high capacity for secreting extracellular enzymes

and organic acids, played an important role in commercial production of industrially

valuable enzymes and other products (vanKuyk et al. 2000; Nutan et al. 2002; Kumar et

al. 2004). Its genes and genomes are being extensively investigated in an effort to

understand the associated cellular mechanisms and to expand these applications

28

(Aleksenko et al. 2001; Mabey et al. 2004; Hombergh et al. 1997). Ojumu et al. (2003)

attempted cellulase (FPase) production in submerged fermentation by A. flavus L. NSPR

101 fermented using sawdust, bagasse and corncob. Long et al. (1997) isolated A. flavus

and used for mycelium bound lipase to modify the triglyceride structure of vegetables oils.

Awe and Akinyanu (1997) reported amylase production by A. flavus and A. niger using

cassava peels. Rosfarizan et al. (1998) exploited A. flavus for direct fermentation of

gelatinised sago starch to kojic acid. Rosfarizan, and Ariff, (2006) A. flavus used for kojic

acid fermentation using sucrose as a carbon source.

29

Fig. 2.5 Out line microscopic morphology of Aspergillus sp.

Source:http://www.mould.ph/aspergillus

mould.htm

30

Naganagouda et al., (2009) reported the isolation and identification of mannanase-

producing A. flavus and A.niger. They studied the mannanase production using cheaper

sources and partially characterized the enzyme. Mariana et al. (2008) screened nine

thermophilic fungi for production of protease in solid and submerged cultivation media

and the tested fungi were Thermoascus aurantiacus, Thermomyces lanuginosus,

T.lanuginosus, A. flavus, Aspergillus sp Aspergillus sp Aspergillus sp Rhizomucor

pusillus and Rhizomucor sp. Banga and Tripathi (2009) reported the isolation and

identification of a novel heparinase producing fungal culture A. flavus (MTCC-8654), and

its production and purification. Khoo et al. (1994) studied the purification and

characterization of the α-amylase produced by A. flavus grown on raw low-grade tapioca

starch as a fermentation substrate.

2.8 Exploitation of microbial sources for production of cellulolytic

enzymes

The cellulolytic enzymes have attracted considerable attention in recent years due to their

great biotechnological and industrial potential. Conversions of food industries and

agriculture wastes to valuable sugars are the great uses of cellulase enzymes (Bothast and

Saha 1997). Enzymatic treatment of cellulosic materials to produce carbohydrates, that can

be used as source for alcohol fermentation, or for production of industrial chemicals and

beverages.

31

2.8.1 Submerged fermentation

Submerged fermentation involves the growth of a microorganism as a suspension in liquid

medium, in which various nutrients are either dissolved or suspended as particulate solids

in many cases of commercial media (Frost and Moss 1987). Filamentous fungi are

preferred for commercially important enzyme productions, because the level of enzymes

produced by these cultures is higher than those obtained from yeast and bacteria (Bakri et

al. 2003). A. niger and T. viride are important and safe organisms for industrial use and

they are good producers of cellulases (Berka et al..1992; Oxenboll 1994). Commercial

production of cellulase enzymes by submerged culture fermentation began in the early

1970s with cellulase made by Trichoderma sold for use in research and pilot studies.

Almost all commercial cellulases produced by submerged fermentation are made by the

fungi Trichoderma, Aspergillus, and Penicillium (Tolan and Foody 1999).

The cellulase producing microorganisms are abundantly present but the main

objective remains selection of promising strains that can produce the acceptable yield to

meet the industrial requirements. There is a very huge demand to improve the stability of

the enzymes to meet the requirements set by specific applications, especially with respect

to temperature and pH. Synthesis of enzymes depends on the type of nutrients available to

the organism and besides an adequate carbon source, other nutrients may be equally

important to the composition of the medium. Besides the carbon source, it has been

suggested that the nitrogen source can also control cellulolytic enzymes production. The

induction of cellulolytic enzymes requires substrates having β-1,4 glycoside bond, include

cellulose, CMC, cellobiose, filter paper etc. The past fifty years have witnessed remarkable

progress in (a) isolation of microorganisms producing cellulases; (b) improving the

32

enzyme productivity by medium engineering by submerged and solid state fermentation (c)

purifying and characterizing the cellulase components; (d) understanding the mechanism

of cellulose degradation; (e) cloning and expression of cellulase genes; and (h)

demonstrating the industrial potential of cellulases. Optimization of the medium for

cellulase production by selecting the best nutritional and environmental conditions is

important to increase the produced cellulase yield (Gomes et al. 2000). Optimized culture

conditions for cellulolytic enzymes production by certain microorganisms are given in the

table 2.5. The typical unit operations involved in screening, selection, optimization of

fermentation cultural conditions for submerged fermentation and solid state fermentation

are shown in fig. 2.6.

Table 2.5 Optimized cultural conditions for certain cellulolytic enzymes production by submerged fermentation

Si.No Microorganism Cellulolytic enzyme

Optimum conditions Medium

Referance Temperature

(oC)

pH Carbon source

(s)

Nitrogen

source (s)

1 Mucor

circinelloides

(NRRL 26519),

Endoglucanase,

Cellobiohydrolase,

and β-D-glucosidase)

30 5

Lactose,

Cellobiose, or

Sigmacell 50

Corn steep

liquor Badal 2004

2. Aspergillus niger

β-D-glucosidase 30 5.5-6

Glucose

Malt extract

Johansson and

Reczey 1998

3. Aspergillus niger

VKMF-2092

β-D-glucosidase 29 4.5-5.5 NA

NaNO2,

(NH4)2SO4

NH4 NO2

Urea

Kerns et al. 1987

4. Trichoderma

reesei Rut C 30

Filter paperase

β-D-glucosidase 30 6.0

Cellulose

powder

Dry yeast

Szengyel 2000

5. Aspergillus niger

NCIM 1207

Endoglucanase

β-D-glucosidase 28 3-5.5

Cellulose

powder

(NH4)2SO4N

H4H2PO4 and

corn-steep

liquor

Gokhale 1991

6 Aspergillus niger

(CBS 55464),

Aspergillus

niger (420)

Aspergillus oryzae

(CBS 12559)

β-D-glucosidase 30 NA

Quercetin, rutin,

Cellobiose

Glucose

(NH4)2HPO4

(NH4)2SO4

Gunata and Vallier

1999

7 Aspergillus

nidulans β-D-glucosidase 37 NA

Glucose

NA Lee et al. 1996

8 Lactobacillus

plantarum USC1

β-D-glucosidase 30 NA Glucose

NA Sestelo et al. 2004

34

9 Aspergillus

nidulans

Endoglucanase,

Cellobiohydrolase,

and β-D-glucosidase)

37 NA

Sugarcane

bagasse

NA Bagga 1990

10 Aspergillus

terreus

Endoglucanase,

Cellobiohydrolase,

and β-D-glucosidase)

40 5.2

Cellulose

powder,

wheat bran

Urea,

(NH4)2SO4 Bastawde 1992

11 Trichoderma

reesei Rut C-30

(Mutant strains)

Endoglucanase,

Cellobiohydrolase,

and β-D-glucosidase)

28 5.5 Lactose Yeast cream Jun et al. 2009

NA – Not available

Fig. 2.6 Schematic representation of traditional fermentation unit process for microbial enzyme

production and characterization

Selection of sites rich

with cellulose getting

microbial degradation

36

2.8.2. Soild state fermentation

Solid state fermentation (SSF) is generally defined as the growth of the microorganism on solid

material in the absence or near absence of free flowing water. Microbial enzymes are produced

mainly by submerged fermentation under tightly controlled environmental conditions (Rose

1980; Chahal 1985; Lonsane et al.. 1992; Singh et al. 2008). However, SSF also has good

potential for the production of enzymes, especially those from filamentous organisms that are

particularly suited for surface growth (Raimbault 1998; Pandey et al. 1999). The high cost of

microbial enzyme production and low yields are the major problems for industrial application.

Therefore, investigations on the ability of the cellulose and hemicelluloses hydrolyzing microbial

strains to utilize inexpensive substrates have been carried. Much work has been directed to

develop hyper producing microbial strains while also focusing on improvement of the

fermentation processes (Esterbauer et al. 1991; Haltrich et al. 1996).

Microbial enzymes, particularly those related to lignocellulose degradation, are produced

by SSF (Duenas et al. 1995; Pandey et al.. 2000; Kanga et al. 2004). Other hydrolases like

amylases (Bogar et al. 2002), proteases (George et al.. 1997) and phytase (Bogar et al. 2003;

Vohra and Satyanarayana 2003; Roopesh et al. 2006) are also produced by SSF. Thus SSF holds

tremendous potential for the production of enzymes, as this process has low capital investment,

superior productivity, less energy requirements and low effluent generation (Chahal 1985;

Nigam and Singh 1994). SSF compares favorably with submerged fermentation in terms of

energy requirements, volumetric productivity and product recovery; it represents a good option

when production costs should be reduced as is the case of the microbial enrichment of

agricultural residues or the production of bulk inexpensive enzymes (Illanes et al. 1992; Pandey

et al. 2000).

37

The cellulolytic enzymes are inducible enzymes, and that cellulose is the best inducer. In

SSF for cellulase production, cellulosic materials act as either the carbon source or the inducer.

The recent studies SSF process for cellulolytic enzyme production have explored a variety of

substrates varying from agro- residues to wastes of industries such as sugarcane bagasse, wheat

bran, rice bran, maize bran, gram bran, wheat straw, rice straw, rice husk, soyhull, sago hampas,

grapevine trimmings dust, saw dust, corncobs, coconut coir pith, banana waste, tea waste,

cassava waste, palm oil mill waste, sugar beet pulp, sweet sorghum pulp, apple pomace, peanut

meal, rapeseed cake, coconut oil cake, mustard oil cake, cassava flour, wheat flour, corn flour,

steamed rice, steam pre-treated willow, starch, etc (Pandey et al. 2000). The natural resources

such as wheat bran, rice hull, corn straw, corncob, fruit peels and seeds and effluents from paper

industry have increased as a result of industrialization, becoming a problem regarding space for

disposal and environmental pollution. However, the above residues represent alternative source

for the microbial growth aiming the production of biomass or enzymes (daSilva et al. 2005). The

wastes such as saw dust and wood chippings have a huge potential for cellulases and

hemicellulases production. In order to ensure viable commercialization of enzyme production via

SSF system, a cheap system with hyper enzyme producers has been established (Mitchell et al.

2006). Certain microbial sources of cellulolytic enzymes production using various

agricultural/industrial residues by SSF are given in table 2.6. If SSF used to produce cellulase,

the following advantages and disadvantages may be noted (Cen and Xia 1999):

1. The raw materials required to produce cellulase are cheap and abundant. Natural cellulosic

materials such as plant stems and corn cobs can be used as carbon source. The composition of

the medium is simple and of low cost. Because of the high capability of cellulosic materials to

buffer the pH value, it is not necessary to add additional expensive buffer solution.

38

3 There is no stirrer in most types of solid-state fermentor and the requirements for water and

aeration are less than that in submerged fermentation. Therefore, the energy consumption is

low and there is no waste water produced in the process.

4 In solid-state fermentation, the productivity per unit reactor volume is high and the solid

cellulase koji can be directly applied to hydrolyze cellulosic materials.

5 The equipment in the solid-state fermentation process is relatively simple and the capital

investment is low. However, new types of fermentors for large scale cellulase production

need to be developed.

6 The process is generally labor intensive and hard to control. The reproducibility is relatively

poor with batch-to-batch difference. In addition, more care is needed in order to prevent

contamination.

The composition of a fermentation medium influences the supply of nutrients and metabolism

of cells, therefore, the productivity of a fermentation process also depends on the culture medium

used. Of the major culture medium nutrients, carbon and nitrogen sources generally play a

dominant role in fermentation productivity. Traditional method for determining optimal

conditions in fermentation processes is ‘one variable at a time’ i.e. varying one parameter while

keeping others at a constant level (Adinaryana et al. 2003). This is a time consuming, laborious

and cost ineffective method, in addition, does not include interaction effects among variables.

Because of the large number of quantitative and qualitative variables involved in a bioprocess,

methods of statistical experimental design are used in many studies for optimizing fermentation

media. To reduce the experimental workload, simple batch cultures in a parallel approach are

carried out in shake flasks. Optimization using factorial design and response surface method can

overcome such drawbacks. Factorial design technique has been successfully used to optimize

and to evaluate the effects of process parameters in the production of enzymes and other

metabolites (Cordova 1998; Muralidhar 2001). To obtain a suitable medium for enzyme

39

production in SSF, a series of statistically designed studies were conducted to investigate the

effect of various cultural conditions and media components. The optimization process firstly

identify the significant factor preferably nutrients (carbon sources, nitrogen sources and essential

elements) for enzyme production. The basic research goal when studying industrial applications

is reduction of the enzyme production cost by optimization of the cultivation medium and

conditions. The statistical analysis offers many tools for optimizing medium components. The

response surface methodology (RSM) is probably the most extensively used statistical

optimization method. RSM can be used to determine the optimal production conditions and

ranges of controllable variables as well as to generate a polynomial equation. RSM can also be

used to estimate the relationships between controllable variables and observed results. Recently,

statistical designs for the optimization have been successfully employed, demonstrating that

these statistical methods are powerful and useful tools (Dey et al. 2001; Park et al. 2002; Wejse

et al. 2003; Chandrika and Fereidoon 2005; Chen et al. 2005). The significant factor optimized

by varying one factor at a time while keeping the others constant, then focus on the most

important factors and then focus on the critical subset of cultural conditions and media

components, finally a RSM was derived to optimize the critical components and maximize the

enzyme production. RSM was proved to be a powerful tool in optimizing the fermentation

process. In general, the procedure applied can be subdivided into four steps: identification of the

most important medium components, identification of the variable range, search for the optimum

value of the variables and experimental verification of the optimum (Botz 2000). Certain

previous reports of statistical optimization of microbial fermentations such as production of

glucoamylase by a thermophilic mold Thermomucor indicae-seudaticae (Kumar and

Satyanarayana 2004), γ- polyglutamic acid production by Bacillus subtilis ZJU-7 (Shi et al.

2006), increase in xylanase production by Aspergillus niger XY-1 (Xing et al. 2008) and ethanol

from sugarcane bagasse (Sasikumar and Viruthagiri 2008).

40

Table 2.6 Cellulolytic enzymes production by certain microbial sources grown on substrates

by SSF

Si.No Microorganism Substrate Referance

1 Penicillium echinulatum

Sugarcane bagasse,

Wheat bran Camassola and Dillon. 2007

2. Trichoderma harzianum Wheat straw and bran Deschamps et al. 1985

3. Penicillium decumbens Wheat straw and

Wheat bran Moet et al. 2004

4. T. reesei LM-UC 4 and

Aspergillus phoenicis

Sugarcane bagasse Correa and

Tengerdy 1997

5 Thermoascus aurantiacus Dry wheat straw Kalogeris et al. 2003

6. Myceliophthora sp.

Rice straw, wheat straw

Wheat bran, bagasse and

Corn cob

Badhan et al. 2007

7. A. niger

Wheat straw and wheat

bran

Jecu 2000

8. A. ustus

Rice straw and wheat

bran

Shamala and Sreekantiah

1986

9 Aspergillus niger

Sugarcane bagasse

Correa et al. 1999

10 Aspergillus niger KK2, Rice straw and wheat

bran Kang et al. 2004

11 Aspergillus ellipticus and

Aspergillus fumigatus

Sugarcane bagasse, dry

wheat straw, wheat bran,

rice bran and groundnut

shell

Gupta and Madamwar 1997

12

Trichoderma reesei LM-

UC4

Aspergillus phoenicis

QM329

Sugarcane bagasse Correa and Tengerdey 1997

41

2.9 Purification of cellulolytic enzymes

To study the biochemical and physical properties of enzymes, it is necessary to purify and

concentrate the preparation. The level of downstream processing to which any enzyme or other

protein to be subjected is largely dependent on the intended application of the finished product

(Bruton 1983; Wheelwright 1987). Generally the optimized conditions of physical and medium

compostion are used for enzyme production. Purification to desired level of enzymes normally

involves several steps and they are chosen according to its intended use. Enzymes application in

pharmaceutical, clinical diagnostic and basic molecular biological sectors requires high-purity

grade enzymes, where as concentrated and partially purified enzymes used for industrial

biotechnological conversions of lignocellulosic materials to biofuels, text tile, paper and pulp,

fruit juice clarification, and detergents need concentrated highly active enzyme. Thus, it is

significant to develop economic processes for their purification to obtain concentrated enzymes

with maximum activity (Headon 1994).

Microbial enzymes to be exploited as reagents in any field, be it analytical or industrial, it

must first be concentrated and purified to a degree that removes any other enzyme capable of

catalysing undesirable side-reactions. This may or may not mean purification to homogeneity.

Traditionally the purification of cellulase from fermentation media has been done in several

steps, which include centrifugation of the culture, and selective concentration of the supernatant,

usually by selective precipitation of the enzyme by ammonium sulphate or organic solvents such

as ethanol in the cold. Then the crude enzyme is subjected to various preparative

chromatographic techniques to meet desired level of purity. The traditional purification steps

reported for microbial cellulolytic enzymes are given in the table 2.7.

42

Table 2.7 Schemes used for purification of cellulolytic enzymes

Si.no Organism

(Enzyme)

Purification

scheme

Final

purification

fold

Reference

1 Cellulomonas flavigena

(β-D-glucosidase)

• wild-type

• Mutant PN-120

Crude extract

Q Sepharose anion

exchange column

Bio Gel P60 column

Bio Gel P100 column

28.85

15.63

Gaspar et al., 2007

2

Mucor circinelloide

(endoglucanase)

Culture supernatant

Ethanol precipitation

CM Bio-Gel A

Bio-Gel A-0.5m

408

Badal 2004

3

Candida sake

(β-D-Glucosidase)

Culture supernatant

Sephacryl S-300

Chromatography

Q-Sepharose

Chromatography

35.5

Gueguen et

al.2001

4

Aspergillus niger

(β-D-Glucosidase)

Culture supernatant

Ammonium sulphate

precipitation

Sephadex G-75 fraction

6.0

Peshin et al. 1999

5.

Aspergillus nidulans

(Endoglucanase/

Exo gluconase/

β-D-Glucosidase)

Culture supernatant

Ammonium sulphate

precipitation

NA

Bagga et al. (1990)

43

Sephadex G-200

DEAE Sephadex A-50

6.

A. nidulans

(β-D-Glucosidase)

Ultrafiltration

Non denaturing

polyacrylamide slab gel

Ion exchange

chromatography

NA

Kwon et al. 1992

7.

Aspergillus niger No. 5.1

(β-D-Glucosidase)

Culture supernatant

Ammonium sulphate

precipitation

Chitopearl-DEAE

Sephadex G-100

NA

Xie et al. 2004

8

Aspergillus sojae

(β-D-Glucosidase)

Culture supernatant

Sephacryl S-200 HR

Q-Sepharose HP

G-l

G-2

G-2 purification

Sephacryl S-300 HR

Methyl HIC

Bio gel HPT

NA

Kimura, et al.

1999

NA – Not available

44

2.10. Properties of cellulases

Looking into the depth of microbial diversity, there is always a chance of finding microorganism

producing novel enzyme with improved properties for commercialization. Microbial enzymes

show great diversity in relation to their properties and biological activities. As there is growing

demand of cellulase application on different industrial sectors, it is necessary to identify and

standardize the novel features of cellulolytic enzymes. The cellulase system from the fungal

strain contains all three enzyme activities needed to produce glucose from cellulose and has great

potential to be used in enzymic saccharification of various lignocellulosic substrates. The use of

these cellulolytic enzymes as agents in bioconversion of plant residues or other wastes to

valuable products (such as fuel alcohols) has been the subject of intense study in recent years. At

present there is considerable interest for cellulolytic enzymes with highest activity and stability

against temperature, pH, cationic metals and resistance against feedback inhibition.

The concentrated and purified enzyme is necessary to evaluate the biochemical properties

of enzymes such as SDS-PAGE analysis to know the homogeneity and molecular mass of the

selected enzyme, optimum physiological conditions for activity, temperature and pH stability,

and kinetic parameters to know the Km and Vmax . The biological activity of cellulolytic enzyme

complex is of great importance, as revealed by studies carried out on hydrolysis of cellulosic

substrates. Such processes, if they are to be economically feasible, will require stable enzymes

able to retain their activities during prolonged reactions and recycling and in the presence of high

concentrations of end-products. The reported cellulolytic enzymes of bacterial and fungal

sources and their biochemical properties are summarized in table 2.8.

Table 2.8 Optimum bio-chemical properties of certain extracellular cellulase components from microbial sources

S.

No Organism

Cellulolytic

enzyme

Molecular mass

(kDa)

Optimum assay

conditions

Kinetic

parameters Referance

Tempera

ture (oC)

pH Km

(mM)

Vmax

(U)

Bacterial source

1. Cellulomonas flavigena β-D-glucosidase 210 37-40 6 114 90.1 Gaspar et al. 2007

2. Lactobacillus

plantarum β-D-glucosidase 40 45 5.0 1.82 4.89 Sestelo et al. 2004

3. Streptomyces

albaduncus

Endoglucanase

Exoglucanase

β-D-glucosidase

-

-

-

50

6

6

6.5

40

92

1.7

0.606

33.33

0.109

Harchand and Singh

1997

Fungal source

4. A.niger USDB 0827

A. niger USDB 0828

β-D-glucosidase

β-D-glucosidase -

65

65

4.6

4.6

0.75

0.89

3067

3629 Hoh et al. 1992

5. Aspergillus aculeatus Endoglucanase

25

38

66

68

50

65

70

60

4.5

4.0

5.0

2.5

- - Murao et al. 1988

6. Aspergillus niger Endoglucanase 31 - 4 - - Okada 1988

7. Aspergillus niger No.

5.1 β-D-glucosidase 67.5 60 6 5.34 2.57 Xie et al. 2004

8. Aspergillus ornatus β-D-glucosidase - 60 4.6 0.76 - Yeoh et al. 1986

9. Coriolus versicolor Endoglucanase 29.5 55 5 - - Idogaki and

Kitamoto 1992

10. Dichomitus squalens

Endoglucanase I

Endoglucanase II

Endoglucanase III

42

56

47

55-60

55-60

55-60

4.8-5.0

4.8

4.6-4.8

Rouau and Foglietti

1985

46

11. Humicola insolen

Humicola grisea

Endoglucanase

Endoglucanase I

Endoglucanase II

57

63

58

50

50

50

5

5

5

- -

Hayashida et al.

1988

12. Metschnikowia

pulcherrima β-D-glucosidase 49 50 4.2 1.5 0.8 Pombo et al. 2008

13. Mucor circinelloides Endoglucanase 27 55 4-6 - - Saha 2004

14. Rhizopus oryzae Endoglucanase I

Endoglucanase II

41

61

55

55

5-6

5-6 - -

Murashima et al.

2002

15. Thermoascus

aurantiacus

Endoglucanase 34 7.-80 4.0-4.4 - -

Parry et al., 2002

16. Thermoascus

aurantiacus β-D-glucosidase - 70 5 - - Hong et al. 2007

17. Trichoderma koningii

Endoglucanase I

Endoglucanase II

Endoglucanase III

Endoglucanase IV

13

31

48

48

- - - - Wood and McCrac

1978

18. Trichoderma reesei

Endoglucanase I

Endoglucanase II

Endoglucanase III

Endoglucanase IV

Endoglucanase V

20

43

48

55

56

- - - - Bhikhabhai et al.

1984

19. Trichoderma viridae QM

9414

Endoglucanase I

Endoglucanase II

Endoglucanase III

Endoglucanase IV

Endoglucanase V

23.5

45

50

52

57

5.5

4.0

5.1

4.5

4.5

- - Voragen et al. 1988

‘-’ Not available

47

2.11 Immobilization of cellulase

The use of microbial enzymes in industrial applications has been limited by several factors,

mainly the high cost of the enzymes production, their instability, and availability in small

amounts. Also the enzymes are soluble in aqueous media and it is difficult and expensive to

recover them at the end of catalytic process. This restricts the use of soluble enzymes to batch

operations. Over the last few decades, intense research in the area of enzyme technology has

provided many approaches to overcome these limitations to facilitate their practical applications.

Among them, the newer technological developments in the field of immobilized biocatalysts can

offer the possibility of a wider and more economical exploitation of biocatalysts in industry,

waste treatment, medicine, and in the development of biosensor (Souze 1980). Immobilization of

biocatalysts helps in their economic reuse and in the development of continuous bioprocesses.

The variety of chemical transformations catalyzed by enzymes has made these catalysts a prime

target of exploitation by the emerging biotech industries.

In general, the term immobilization refers to the act of the limiting movement or

restricting movement to a confined space (Qung et al. 2004). Immobilization of biocatalysts

helps in their economic reuse and in the development of continuous bioprocesses. Biocatalysts

can be immobilized either using the isolated enzymes or the whole cells. Several methods that

have been used to immobilize the cells/enzymes, include cross linking, physical adsorption, ionic

binding, metal binding, covalent binding and entrapment methods. All of these experimental

biocatalyst immobilization systems normally fall into one of three categories: 1. Biocatlyst

entrapment in polymer gels or porous supports. 2. Adhesion on micro carrier surface and 3.

Capture behind membrane (encapsulation). Sometimes the distinction between these different

categories may not be very clear, depending on the particular immobilization system employed.

However, every method has its advantages and drawbacks. The major components of an

48

immobilized biocatalyst include enzyme, matrix and mode of interaction of the enzyme with the

carrier. A large number of enzymes have been immobilized on inorganic carriers like porous

glass (Adamich et al. 1978), ceramics (Dale and White 1979), carbon (Cho and Bailey 1978) and

sand (Puls et al. 1977) by different techniques. The most extensively studied immobilization

method is the entrapment of microbial cells/enzymes in polymer matrices. The matrices used are

agar, alginate, carrageenan, cellulose and its derivatives, collagen, gelatin, epoxy resin, photo

cross-linkable resins, polyacrylamide, polyester, polystyrene and polyurethane. Among the

above matrices, entrapment of cells/enzymes in alginate gel is popular because of the

requirement for mild conditions and the simplicity of the used procedure. Several reports on

employing alginate gel are available (Kierstan and Bucke 1977).

Enzymes such as cellulases and amylases are currently used by several industries to

hydrolyze cellulose or starch to products such as dextrins, syrups, and sugars. Such reactions

represent the key first step toward the production of a variety of useful chemicals and sweeteners

and are also useful in the pulp and paper industry for fiber modification. Enzymatic hydrolysis of

cellulosics, the most abundant renewable resource on earth, offers an attractive alternative, if the

process can be made economically competitive. Several previous studies have considered the

advantages of immobilized enzymes with soluble substrates, and a few studies have also

investigated the properties of immobilized enzymes with insoluble substrates. The performance

of cellulase and amylase immobilized on siliceous supports was evaluated with respect to

immobilization conditions and thermal stability (Bradley et al. 2004). Enzyme immobilization

has been reported to improve the thermal stability of enzymes and may also affect binding of

substrates and inhibitors to the enzyme, thereby affecting the Michaelis constant and enzyme

inhibition (Wiseman 1994, Lim and Macdonald 2003). Sadhukhan et al. (1993) observed that

immobilization of amylase onto Sepharose, alginate beads, and polyacrylamide gels could

49

increase the optimum reaction temperature to about 70°C, compared with about 60°C for the

soluble form. Natural cellulases from most fungal sources are limited in their overall activity by

low amounts of β-glucosidase (Klyosov 1986) and by cellobiose inhibition of cellobiohydrolase

(Sundstrom et al.1981). Although advantageous to the growth of the fungus, these characteristics

are inconvenient for the industrial use of cellulase in cellulose processing. Improved cellulose

hydrolysis and increased glucose yield can be achieved under laboratory or industrial conditions

by combining additional β-glucosidase with fungal cellulases (Sundstrom et al. 1981). However,

β-glucosidase is an expensive enzyme to produce and, if used in a soluble form that cannot be

recycled, is not cost effective for industrial use. The coimmobilization of cellulase and β-

glucosidase and resulted in the improved kinetic properties of the enzyme system and the

improved glucose yield as compared to cellulase function alone (Chakrabarti and Storey 1989). It

is therefore conceivable that the use of immobilized enzyme or cells may offer a solution

towards a reduction in the cost of cellulase production.