12_chapter 2.pdf - shodhganga

Review of Literature - 23 -

he combustion of petroleum based fossil fuels has become a concern

with respect to global climate change due to accelerated carbon

emissions. Burning of fossil fuels has also created a concern for

unstable and uncertain petroleum sources, as well as, the rising cost of fuels.

These apprehensions have shifted global efforts to utilize renewable resources

for the production of a ‘greener’ energy replacement which can also meet the

high energy demand of the world (Maki et al., 2009). As a result lignocellulosic

biomass has been identified as a renewable resource for the production of the

green energy alternative, ‘ethanol’ and has resulted in large investments in the

biofuel industry in the recent past (Schubert, 2006; Sheridan, 2008).

2.1 Lignocellulosic biomass and production of ethanol

Lignocellulosic biomass is a great potential resource for the production of

biofuels because it is abundant, inexpensive and use of such resources is

environmentally sound. Approximately 70% of plant biomass is locked up in 5

and 6-carbon sugars (Maki et al., 2009). These sugars are found in

lignocellulosic biomass, which is comprised of mainly cellulose, hemicelluloses

and lignin. The major component cellulose is a homopolysaccharide comprised

of glucose units, linked by β-(1→4) glycosidic bonds. A prerequisite to convert

the lignocellulosic biomass into ethanol is its hydrolysis into simple sugar

molecules.

Nature is abound with bacteria and fungi that can produce lignocellulose

degrading enzymes to solubilize these complex components to simple

molecules. Most of organisms isolated from soil, waste and composting waste

material are capable of producing a spectrum of cell wall degrading enzymes,

collectively known as cellulases. The hydrolysis of cellulose is accomplished by

components of cellulase including randomly acting endoglucanase (1,4-β-D

T

Chapter 2 - 24 -

glucan-4-glucanohydrolases; EC 3.2.1.4) that cleaves the internal β-1,4-

glycosidic bonds, cellobiohydrolase (1,4-β-D glucan glucanohydrolases or

exoglucanases; EC 3.2.1.91) which releases cellobiose from reducing and non-

reducing ends and β-glucosidase (β-glucoside glucohydrolases; EC 3.2.1.21)

that cleaves the cellobiose into glucose units.

In the biofuel industry lignocellulosic biomass is converted to simple

sugars and fermented to ethanol through chemical and biological processes. At

present the process consist of sequential steps of thermo-chemical

pretreatment, enzymatic saccharification, fermentation and product recovery.

However, this process stream is inefficient and expensive. The major bottleneck

in the conversion technology is the recalcitrance of plant cell walls to hydrolysis

to simple sugars. The cost of bioethanol production from lignocellulosic

materials is relatively high when based on current technologies, and the main

challenges are the low yield and high cost of the hydrolysis process. A cocktail

of lignocellulolytic enzymes are essential for the second step and their cost is a

major economic constrain in the current technology. It has been recognized by

experts that major improvements have to be made in the enzymatic hydrolysis

of cellulosic biomass for cellulosic ethanol to compete economically with corn

ethanol and the fossil fuels (Galbe and Zacchi, 2002; Sun and Cheng, 2002;

Lynd et al., 2002). In order to be cost competitive with the other fuels the

enzymes used for biomass hydrolysis must become more efficient and far less

expensive. Cost-competitive technology can be developed by improving the

lignocellulolyitc enzyme machinery, as well as by rendering the cellulosic

substrates more susceptible to hydrolysis (Himmel et al., 2007).

2.2 Enzymes involved in large scale production of bioethanol and their

limitations

In the commercial scale plants, involved in ethanol production

Trichoderma reesei cellulase is used. The hydrolytic efficiency of the


multienzyme mixture in the process of lignocellulose saccharification highly

depends on the ratio of enzyme components, endoglucanase, exoglucanases

and β -glucosidase, because of the synergetic effect among them. The cellulase

cocktail produced by T.reesei generally contains two exoglucanases (CBHI and

CBHII) and two endoglucanases (EG1 and EG2), in a rough proportion of

60:20:10:10, while the β-glucosidase component typically makes up less than

1% (Lynd et al., 2002). Low levels of β-glucosidase leads to the accumulation of

cellobiose, which inhibits the cellulolytic enzyme action. β-Glucosidases

hydrolyze cellobiose which is an inhibitor of cellulase activity. Therefore to

increase the efficiency of T.reesei cellulase additional supply of β-glucosidase is

essential. It has been proved that the addition of β-glucosidases into the T.

reesei cellulases system can achieve better saccharification than the system

without β-glucosidases (Excoffier et al., 1991; Xin et al., 1993).

At present Aspergillus spp. are considered as the most promising fungi

for β-glucosidase production (Solovyeva et al., 1997). Jäger et al., (2001)

compared the β-glucosidase production potential of three species of Aspergillus

and A. niger proved to be the best enzyme producer on solid-state medium. But

A. niger β-glucosidase is inhibited by glucose. This restricts its application in

commercial scale cellulose degradation ventures (Riou et al., 1998; Gunata and

Vallier, 1999). Glucose tolerant β-glucosidases would improve the process of

saccharification of lignocellulosic materials and are therefore currently in great

demand. Therefore the search for β-glucosidases insensitive to glucose

inhibition has increased recently and enzymes with this characteristic would

improve the process of saccharification of lignocellulosic materials (Bhatia et

al., 2002).

2.3 β-glucosidases and their natural roles

β-Glucosidases (β-D-glucoside glucohydrolase, EC 3.2.1.21) constitutes a

major group among glucoside hydrolases. They catalyze the selective hydrolysis

Chapter 2 - 26 -

of glycosidic bond in oligosaccharides, and their conjugates. β-glucosidases

occur ubiquitously in plants, animals, fungi and bacteria (Esen, 1993) and

play important roles in fundamental biological processes. In cellulolytic

organisms, such as fungi and bacteria, β-glucosidase is a component of the

cellulase complex, and is responsible for the hydrolysis of short chain

oligosaccharides and cellobiose (Beguin, 1990; Bhatia et al., 2002). In plant

physiology, β-glucosidases are implicated in growth regulation, stress

response, cellobiose degradation, lignification, and defense (Poulton, 1990;

Brzobohaty et al., 1993; Leah et al., 1995). In humans, membrane bound

lysosomal acid β-glucosidase is involved in the hydrolysis of glycosylceramides.

Individuals with β-glucosidase deficiency exhibits enlargement of organs such

as spleen, liver and lymph nodes, a condition known as Gaucher’s disease

(Esen, 1993). β-glucosidases have been the focus of much research recently

because of their important roles in commercial scale bioethanol production and

a variety of fundamental biological processes (Hansson and Ablercreutz, 2002).

2.4 Classification of β-glucosidases

β-glucosidases, the heterogeneous group of hydrolytic enzymes, have

been classified according to various criteria. There is no single well defined

method for the classification of this diverse group of enzymes. In general two

systems of classification have been followed in the literature. They are based on

substrate specificity and nucleotide sequence identity (Henrissat and Bairoch,

1996).

Based on substrate specificity β-Glucosidases have been subdivided into

three classes. Class 1 includes enzymes with glycosyl β-glycosidase and aryl β-

glycosidase activity; these enzymes have the ability to hydrolyze cellobiose,

lactose, β-p-nitrophenylglucoside (β-pNPG), β-p-nitrophenylgalactoside (β-

pNPGal), β-p-nitrophenylfructoside (β-pNPFru) and other similar substrates.

Class 2 includes those with only glycosyl β-glucosidase activity; therefore, they


can only hydrolyze substrates such as cellobiose and lactose. Class 3 includes

enzymes with only aryl (or alkyl) β-glucosidase activity; these enzymes would

have significant activity towards β-pNPG and similar substrates (Terra and

Ferreira, 1994).

On the basis of amino acid sequence identity, β-glucosidases are grouped

along with all other glycosylhydrolases, which have been classified into 108

families and the β-glucosidases are placed in family 1 and 3 (GH1 and GH3).

Both GH families hydrolyze their respective target substrates with a net

retention of configuration of the anomeric carbon (Coutinho and Henrissat,

1999).

Family 1 glycoside hydrolases (GH-1) include enzymes with different

activities abundantly distributed among all sort of living organisms, which have

in common the ability to hydrolyse β-glycosidic linkages of disaccharides,

oligosaccharides or conjugated saccharides (Coutinho and Henrissat, 1999).

Among the members of this family are bacterial and fungal cellobiases that

play an important role in cellulolysis; phospho-β-galactosidases from lactic

acid bacteria; plant enzymes involved in defense mechanisms against grazing

herbivores; and intestinal lactase-phlorizin hydrolase from mammals (Sanz-

Aparicio et al., 1998a). The number of GH-1 enzymes whose three-dimensional

structure has been solved has increased sharply in the last decade. Among the

enzymes of known structure there are several from archaea, [Sulfolobus

solfataricus (Aguilar et al., 1997), Thermosphaera aggregans (Chi et al., 1999),

Pyrococcus horikoshii (Akiba et al., 2004)] bacteria, [Lactococcus lactis

(Wiesmann et al., 1995), Paenibacillus polymyxa (Sanz-Aparicio et al., 1998b),

Bacillus circulans (Hakulinen et al., 2000), Thermus nonproteolyticus (Wang et

al., 2003), Thermotoga maritime (Zechel et al., 2003)] plants [Trifolium repens

(Barrett et al., 1995), Sinapsis alba (Burmeister et al.,1997), Zea mays (Czjzek

et al.,2001; Zouhar et al., 2001), Sorghum bicolor (Verdoucq et al., 2004),

Chapter 2 - 28 -

Triticum aestivum (Sue et al., 2000a)] and from the insect Brevicoryne brassicae

(Husebye et al., 2005). Although the structures of GH1 enzymes from archaea,

bacteria, plants, and an insect have been described there has been very little

information on the 3D structure of fungal GH1 enzymes except a recent report

on the crystal structure of family 1 β-glucosidase from Phanerochaete

chrysosporium (Hrmova and Fincher, 2007).

Although different in quaternary structure, the monomeric form of all

GH-1 enzymes have a common eight fold β/ barrel motif with a molecular

mass of approximately 50 kDa. The hydrolysis of the β-glycosidic bond is

carried out by a catalytic mechanism that retains the conformation of the

anomeric carbon and two conserved glutamate residues acting as nucleophile

and proton donor (Isorna et al., 2007).

Family GH3 consists of β-glucosidases of bacterial, mold and yeast

origin. The family 3 enzymes may be subdivided further into two classes. AB

and AB’. All the family 3 enzymes consist of two domains A and B. In AB class

both the domains are equally prominent but in AB’ class B domain is

compressed, but conserved sequences have still been retained (Bhatia et al.,

2002). At the molecular level, the genes of the family 3 β-glucosidase enzymes

consist of five distinct regions, the N-terminal residues, an N-terminal catalytic

domain, a nonhomologous region, a C-terminal domain of unknown function

and the C-terminal residues.

2.5 Sources of β-glucosidases

β-Glucosidases exist widely in nature. Besides microorganisms, such as

fungi and bacteria, they are found in plants and animals.


2.5.1 Bacterial Sources

β-Glucosidases from several bacterial species have been purified and

characterized. The list is growing fast and table 2.1 presents a sample of

bacterial species whose β-Glucosidases have been investigated in detail.

Table 2.1. Examples of bacterial species whose β-glucosidases have been purified and characterized

2.5.2 Fungal Sources

The enzyme β-glucosidase has been identified, isolated and characterized

from several fungal species also (table 2.2).

Organism Reference

Agrobacterium faecalis Wang et al., 1995

Bacillus circulans Paavilainen et al., 1993

Bacillus polymyxa Painbeni et al., 1992

Bacillus sp. Hashimoto et al., 1998

Caldicellulosiruptor saccharolyticus Love et al.,1988

Clostridium thermocellum Romaniec et al.,1993

Leuconostoc mesenteroides Gueguen et al.,1997

Microbispora bispora Wright et al., 1992

Oenococcus oeni Grimaldi et al., 2000

Pyrococcus horikoshii Matsui et al., 2000

Pyrococcus furiosus Kengen et al.,1993

Ruminococcus albus Ohmiya et al., 1985

Spingomonas paucimobilis Marques et al., 2003

Sulfolobus solfataricus D'Auria et al.,1996

Thermoanaerobacter brockii Breves et al., 1997

Thermobifida fusca Spiridonov and Wilson, 2001

Thermotoga maritima Goyal et al., 2001

Chapter 2 - 30 -

Organism Reference

Aspergillus aculeatus Sakamoto et al., 1985; Murai et al., 1998

Aspergillus nidulans Hang and Woodams, 1994

Aspergillus niger Yan and Lin , 1997; Dan et al.,2000

Aspergillus ornatus Yeoh et al., 1986

Aspergillus oryzae Riou et al.,1998

Aspergillus phoenicis Wen et al., 2005

Aspergillus tubingensis Decker et al., 2001

Candida molischiana Gonde et al., 1985; Vasserot et al., 1991

Candida peltata Saha and Bothast ,1996

Debaryomyces hansenii Yanai and Sato, 1999

Fusarium oxysporum Christakopoulos et al.,1994

Lentinula edodes Makkar et al., 2001

Lentinus edodes Morais et al., 2001; Zheng and Shetty, 2000

Monascus purpureus Liu et al.,2004; Daroit et al., 2007 Phanerochaete chrysosporium

Kawai et al., 2003; Igarashi et al., 2003

Saccharomyces cerevisiae Spagna et al., 2002a; Rosi et al., 1994

Thermoascus aurantiacus Gomes et al., 2000

Trichoderma atroviride Kovács et al., 2008

Trichoderma koningii Wood and MeCrae , 1982

Trichoderma reesei Kubicek ,1981; Fowler et al., 2002

Table 2.2. Examples of fungal species whose β-glucosidases have been purified and characterized

In most of the fungi they form a component of the cellulase enzyme

machinery. In addition to this β-glucosidase have additional roles in many

phytopathogenic fungi. For example, saponin hydrolyzing enzymes, such as

avenacinases, which are a subset of β-glucosidases, have been identified as

essential molecular tools for the pathogenicity of phytopathogenic fungi like

Gaeumannomyces graminis var. avenae (Osbourn et al., 1995; Bouarab et al.,

2002). Saponins are antifungal molecules often found in plants and constitute

an important part of the plant defense artillery. This enzyme removes both β-

1,2 and β-1,4-linked terminal D-glucose residues from avenacin A-1 yielding

products that are less toxic to fungal growth and allows invasion of host plant

tissues (Bowyer et al., 1995). The saponin-hydrolysing enzymes, avenacinases


belong to GH3 (Osbourn et al., 1995; Sandrock et al., 1995; Bouarab et al.,

2002) and β-glucosidases from fungal sources such as Humicola grisea and

Hypocrea jecorina (Takashima et al., 1999) belong to GH1.

Fungal β-glucosidases are also known for their transglycosylation activity

(Vaheri et al., 1979), which is thought to be important for the formation of

soluble cellulose inducer compounds. For instance, a gene (bgl2) from

Trichoderma reesei, encoding a β-glucosidase, has been expressed in

Escherichia coli and has been shown to produce sophorose from glucose via

transglycosylation (Saloheimo et al., 2002). Sophorose is a β-1,2-linked glucose

disaccharide and a potent cellulase inducer in Trichoderma sp.

2.5.3 Plant Sources

Plant β-glucosidases have been the subject of much work (table 2.3)

because of their importance in (i) numerous biological processes such as

growth and development through the release of phytohormones (auxins,

gibberellins, cytokinins) from their inactive glucoconjugated forms (Duroux et

al., 1998), host-parasite interactions (Osbourn,1996; Sue et al., 2000b),

lignifications (Hosel et al., 1978; Dharmawhardana, et al., 1999), cell wall

degradation in the endosperm during germination (Leah et al., 1995), circadian

rhythm of leaf movements (Ueda and Yamamura, 2000) and (ii) in

biotechnological applications: food detoxification (Birk et al.,1996), biomass

conversion (Pemberton et al., 1980; Woodward and Wiseman, 1982) and, over

the past decade, flavor enhancement in beverages (Gunata et al., 1993). β-

Glucosidase from olive fruit tissues is reported as a key enzyme in fruit

ripening and defense response (Goupy et al., 1991; Balbuena et al., 1992;

Konno et al., 1999).

Chapter 2 - 32 -

Organism Reference

Apple Podstolski and Lewak, 1970

Barley Leah et al.,1995

Black cherry Kuroki and Poulton, 1987

Catharanthus roseous Geerlings et al., 2000

Chick pea Hosel et al., 1978 Corn Han and Chen, 2008

Dioscorea caucasica Gurielidze et al., 2004

Flax Fan and Conn, 1985 Grapes Lecas et al., 1991

Hevea sp. Selmar et al., 1987

Lima beans Frehner and Conn, 1987 Maize Czjzek et al., 2001

Oat Kim et al., 1996 Olive Mazzuca, 2006

Pine Dharmawardhana et al.,1995

Rapeseed Höglund et al., 1991

Rice Opassiri et al., 2003 Rye Sue et al., 2000b

Sicilian blood oranges Barbagallo et al., 2007

Sinapis alba Eriksson et al., 2001

Sorghum Cicek and Esen,1998 Soybean Hsieh and Graham, 2001

Sweet Almond Grover et al.,1977; Li et al.,1997

Sweet cherry Gerardi et al., 2001

Tapioca Keresztessy et al., 1994

Tea Mizutani et al., 2002

Thai rosewood Toonkool et al., 2006

Tomato Pressey, 1983

Trifolium repens Kakes, 1985; Barrett et al.,1995

Vicia angustifolia Ahn et al., 2007

Wheat Sue et al., 2000a

Table 2.3. Examples of plant species whose β-glucosidases have been purified and characterized

Indeed the intensive research carried out over the past two decades has

demonstrated that, in a great number of fruits and other plant tissues,

important flavor compounds accumulate as non-volatile and flavorless

glycoconjugates. These compounds make up a reserve of aroma and have


immense potential to be used as natural flavoring compounds. To make use of

the plant glycoconjugates there exists a need for the exploration of plant

derived β-glucosidases (Stahl-Biskup et al., 1993; Winterhalter and

Skouroumounis, 1997). The identification of β-glucosidases, their substrates,

and the nature of their interactions will not only shed light on the structure

and function of the enzymes, but also help define their biological significance in

vivo.

2.5.4 Animal Sources

There are several reports available on the β-glucosidase activity in

animals (table 2.4). Most of the works concentrates on the digestive β-

glucosidases in insects. β-glucosidase has been reported from various orders

and families of insects, with the majority having been isolated from the

intestinal tract (Terra and Ferreira, 1994). Most of the animal β-glucosidases

have been studied in their crude form because of the difficulties in purification.

Gilliam et al., (1988) detected the presence of β-glucosidase activity in

the midgut and hindgut of honey bee (Apis mellifera). Ferreira et al., (1998)

isolated β-glucosidase from the midgut of Scaptotrigona bipunctata, the same

family (Apidae) as the honey bee. β-Glucosidase activity has been detected in

honey also (Low et al., 1986), and this activity was correlated with the

formation of β-O-glycosidic linked oligosaccharides in this food (Low et al.,

1988). The origin of β-glucosidase activity in honey has not been identified, but

it could be from the honey bee. If the origin of β-glucosidase activity in honey is

from the bee, then β-glucosidase should be present in the honey sac and in the

organs secreting digestive enzymes in the mouth. Several glands discharging

secretions in the mouthparts of the honey bee have been identified including

thoracic, head and hypopharyngeal glands (Snodgrass and Erickson, 1992).

The presence of α-glucosidase in the hypopharyngeal glands of the honey bee

Chapter 2 - 34 -

also has been confirmed and has been related to α-glucosidase activity in

honey (Simpson et al., 1968).

Table 2.4. Examples of animal species whose β-Glucosidases have been purified and characterized

In humans, several β- glucosidases have been described and for most of

them, the role and physiological substrates are known. For example, the

lysosomal β-glucosidase (also named ‘acid β-glucosidase’) hydrolyses

glucocerebrosides (glycosphingolipids) present in the lysosomal membranes,

and a lack of this enzyme is the cause of the various forms of Gaucher’s

Organism Reference

Abracris flavolineata Marana et al., 1995

Apis mellifera Gilliam et al., 1988

Diatraea saccharalis Ferreira et al., 1997

Dysdercus peruvianus Silva et al., 1996

Erinnyis ello Santos and Terra, 1985

Guinea pig Gopalan et al., 1992

Human Fleury et al., 2007;Daniels et al., 1981

Locusta migratoria Morgan,1975

Pheropsophus aequinoctialis Ferreira andTerra,1989

Phoracantha semipunctata Pig

Chararas and Chipoulet, 1982 Lambert et al., 1999

Pyrearinus termitilluminans Colepicolo et al., 1986

Rhagium inquisitor Chipoulet and Chararas,1985

Rhodnius prolixus Terra et al., 1988

Rhynchosciara americana Ferreira and Terra, 1983

Scaptotrigona bipunctata Schumaker et al., 1993

Sitophiulus oryzae Baker and Woo, 1992 Snail Hu et al., 2007

Sophrorhinus insperatus Adedire and Balogun, 1995

Spodoptera frugiperda Marana et al., 2000

Tenebrio molitor larvae Terra et al., 1985

Thaumetopoea pityocampa Pratviel et al., 1987


disease, one of the hereditary lysosomal storage disorders (Neufeld, 1991). The

cytosolic β-glucosidase is one of the best-characterized enzymes of its category

and belongs to family GH1 (Daniels et al., 1981; Lambert et al., 1999; de Graaf

et al., 2001). It has been shown to hydrolyze β-D-galactoside and β-D-glucoside

substrates with comparable efficiencies (Glew et al., 1993). Its physiological

role is still unclear but its ability to hydrolyze several non-physiological and

dietary xenobiotics glycosides of plant origin has led to the hypothesis of a role

in their metabolism (LaMarco and Glew, 1986; Gopalan et al.,1992; Berrin et

al., 2002). This cytosolic β-glucosidase, present in liver, kidney, intestine and

spleen of humans, has been purified and characterized to some extent (Daniels

et al., 1981; Lambert et al., 1999) and the respective genes are cloned and

expressed in COS7 cells and in the methylotrophic yeast Pichia pastoris (de

Graaf et al., 2001; Berrin et al., 2002). All those proteins presented similar

physical and enzymatic properties. It is a 53 kDa, monomeric protein with a

broad and near neutral pH optimum and it is not glycosylated (Daniels et al.,

1981; Lambert et al., 1999; de Graaf et al., 2001; Berrin et al., 2002). Berrin et

al., (2003) modeled the human cytosolic β-glucosidase (hCBG) on the Zea mays

β-glucosidase X-ray structure. They showed in this model that the active site of

hCBG is surrounded by a hydrophobic cluster of amino acids, confirming the

high affinity of the enzyme for hydrophobic matrices (Lambert et al., 1999).

2.6 Applications of β-glucosidases

The ability of β-glucosidase to cleave and synthesize glycosidic bonds

makes them suitable candidates in a number of biotechnological applications.

These applications can be broadly classified into two classes; applications

based on hydrolytic activity of the enzyme and applications based on synthetic

activity of the enzyme.

Chapter 2 - 36 -

2.6.1 Applications based on the hydrolytic activity

2.6.1.1 Application of β-glucosidase in the degradation of lignocellulosic

materials and the production of ethanol

Ethanol derived from lignocellulosic biomass is considered as a potential

alternative to fossil fuels. Conversion of lignocellulosic material to fermentable

sugars is the first step in the production of ethanol form lignocellulosic

biomass. A complex array of enzymes is needed for the conversion of

lignocellulosics to simple sugars and cellulase is the major one among them.

Cellulase is a complex mixture of enzymes with different specificities to

hydrolyze glycosidic bonds. Exoglucanase, endoglucanse and β-glucosidase are

the major components of the cellulase system. Action of both exo and

endoglucanse on cellulose results in the formation of cellobiose and

cellooligosaccharides. It is the hydrolytic activity of β-glucosidase that converts

cellobiose and cellooligosaccharides produced by the endo and exoglucanases

to glucose. Since cellobiose and cello-oligosaccharides are inhibitors of the

cellulose degrading enzymes their removal is crucial and essential for the

effective and continuous degradation of cellulose by the above-mentioned

enzymes.

2.6.1.2 Application of β-glucosidase as a dietary supplement

Woodward and Wiseman (1982) reviewed the beneficial effects of β-

glucosidase supplementation in the feed for pigs and chickens. Barlican, an

enzyme preparation from T.reesei has been reported to be safe for use as feed

additive for such animals. It has also been demonstrated that supplementing

cattle diets with fiber degrading enzymes such as cellulases including β-

glucosidase and xylanases has significant potential to improve feed utilization

and animal performance (Beauchemin et al., 1999). Improvements in animal

performance due to the use of enzyme additives can be attributed mainly to

improvements in ruminal fiber digestion and the resulting increased digestible


energy intake (Arambel et al., 1987). This approach offers exciting possibilities

for using enzymes to improve nutrient digestion, utilization, and animal

productivity and at the same time reduce animal fecal material and pollution.

Fiber degrading enzymes may also help to improve the digestion of cereal

grains with fibrous seed coats. Cellulase/xylanase enzymes sprayed onto a

barley and barley silage diet improved weight gain and feed efficiency in steers

(Beauchemin et al., 1999).

2.6.1.3 Application of β-glucosidase in the bioconversion of plant

glycoconjugates

Flavonoids are large class of polyphenolic-plant secondary metabolites

providing much of the color and flavor in plant foods. Consequently they occur

in many foods and beverages resulting in high human consumption. Recently

flavonoids have attracted considerable interest due to their antioxidative

activities and their capacity to inhibit enzymes such as cyclooxygenase and

protein kinases involved in cell proliferation and apoptosis. The flavonoids

exist in nature almost exclusively as β-glycosides. Most industrial and

domestic food processing procedures do not lead to cleavage of the glycosidic

linkage and hence flavonoids in foods are generally present as glycosides.

There is considerable interest in altering the form of dietary polyphenols

in order to positively affect their bioavailability and/or their biological activities

in humans. It is reported that the aglycone moiety, released as a result of

hydrolytic activity of β-glucosidase, has potent biological activity, with several

uses in the field of medicine as antitumer agents, in general biomedical

research and in the food industry. A simple route for altering the form of

polyphenols is through deglycosylation with the action of β-glucosidases. There

are several reports regarding the use of β-glucosidases for the hydrolysis of

flavanoid and isoflavanoid glucosides.

Chapter 2 - 38 -

Isoflavones are phytoestrogens, they have a structural/ functional

similarity to human estrogen (Brouns, 2002; Beck, et al., 2003) and therefore

are considered to play an important role in the prevention of cancers (Coward

et al., 1993; Adlercreutz, 2002), heart disease (De Kleijin et al., 2002),

menopausal symptoms (Messina, 2000) and osteoporosis. Of all plant

estrogens, soy isoflavones have been studied most due to the extensive

consumption of soy foods. Isoflavones are present in soy foods mainly as

glucosides, with the carbohydrate conjugated at the 7 position of isoflavone

and the sugar often being esterified with acetyl or malonyl groups at 6’ position

(Kudou et al., 1991). The most abundant isoflavones are glucosides of genistein

and daidzein, known as genistin and daidzin. The glucoside isoflavones are

very poorly absorbed in the small intestine as compared with their aglycones,

because of their greater molecular weight and higher hydrophilicity of the

glucosides (Chang and Nair, 1995). Furthermore, the isoflavone glucosides,

daidzin and genistin, are known to be less bioactive than their respective

aglycones, daidzein and genistein (Piskula et al., 1999; Xu et al., 1994). Human

isoflavone bioavailability depends upon the relative ability of gut microflora to

degrade these compounds. β-Glucosidases of intestinal microflora in lower

bowel can hydrolyze the glucoside isoflavones to aglycones and promote their

absorption (Hendrich, 2002). Therefore, bacteria with β-glucosidase activity are

potentially important in the production of compounds with higher estrogenicity

and better absorption, facilitating the bioavailability of isoflavones. Pyo et al.,

(2005) demonstrated the application of β-glucosidase-producing lactic acid

bacteria as a functional starter cultures to obtain the bioactive isoflavones,

genistein and daidzein, in fermented soymilk. They have identified four β-

glucosidase-producing lactic acid bacteria that have great potential for the

enrichment of bioactive isoflavones in soymilk fermentation. Latter three

strains of Lactobacillus acidophilus, two of Lactobacillus casei and one of

Bifidobacterium sp. were identified as potent β-glucosidase producers by Otieno

et al., (2006) and their ability to breakdown isoflavone glucosides to the


biologically active aglycones in soymilk was also reported. In another

experiment Mamma et al., (2004) demonstrated the use of β-glucosidase from

Penicillium decumbens to cleave flavones (apigetrin), flavanones (naringenin-7-

glucoside) and isoflavones (daidzin) and quercetin-3-glucoside. Similarly

phloridizin was hydrolyzed to liberate the aglycon moiety, which is a precursor

of melanin. The latter is known to reduce the risk of skin cancer and promote

dark color of hair.

Saponins, glycosides with steroids or triterpenes as aglycons, are an

important class of physiologically active compounds occurring in many herbs.

Ginsenosides, the major active components of ginseng (the root of Panax

ginseng) is a good example and have been reported to show various biological

activities including anti-inflammatory activity and anti-tumor effects. However,

the absorption of naturally occurring ginsenosides including Rb1, Rb2, and Rc

by the gastrointestinal tract is very poor. Hu et al., 2007 demonstrated the use

of β-glucosidase from China white jade snail (Achatina fulica) to hydrolyse

ginsenosides Rb1, Rb2, Rb3 and Rc into their active metabolites, compound K,

compound Y, Mx, and Mc, respectively. The products of the biotransformation

were more readily absorbed into the bloodstream and exhibited excellent anti-

tumor activities.

Resveratrol is a polyphenol compound existing in a variety of plant

species, including grapes, peanuts, mulberries and other plants, especially

Polygonum cuspidatum. Resveratrol has the activity of cardiovascular

protection, owing to its oxidative modification of low-density lipoproteins, its

ability to act as an antioxidant and as an inhibitor of platelet aggregation, and

its action as phytoestrogen. It has been shown that resveratrol inhibits the

growth of several cancerous cell lines or has the ability to cause apoptosis in

these lines (Surh et al., 1999; Ahmad et al., 2001). Resveratrol also has anti-

Chapter 2 - 40 -

inflammatory, anti-microbial, anti-HIV effects (Heredia et al., 2000; Gao et al.,

2003).

In general, resveratrol is obtained by extraction from natural sources

such as Polygonum cuspidatum. But the main compounds in this plant are

piceid (3,5,4’-trihydroxystilbene-3-O-β-D-glucopyranoside, and resveratroloside

(3,5,4’-trihydroxystilbene-4-O-β-D-glucopyranoside) rather than resveratrol.

Recently Zhang et al., (2007) showed that the resveratrol content in Polygonum

cuspidatum crude extract can be increased by adding β-glucosidase from the

fungus, Aspergillus oryzae, which hydrolyzes the β-(1-3)-D-glucopyranoside

bond and transforms piceid to resveratrol.

Mazzeia et al., (2006) purified the β-glucosidase from Olea europaea fruit

and the enriched enzyme fraction was immobilized on polymeric membranes to

develop biocatalytic membrane reactors for the hydrlysis of oleuropein

(glycosylated heterosidic ester of elenolic acid and 3,4dihydroxy phenyl-

ethylethanol) that cause bitterness in unripe olives. The aglycone moiety

released due to cleavage is a pharmacologically active compound useful in the

prevention of coronary heart disease and cancer (Briante et al., 2000).

2.6.1.4 Application of β-glucosidase in improving the quality of food

products

In recent years a substantial increase in the use of glycosidases to

improve the aroma of food products has been witnessed. Aroma increase of

wine is typical example. Wine aroma is the outcome of a complex interaction

among the substances from the gapes, those produced during fermentation

and those produced during ageing. Terpenes are one of the major grape

components that contribute to wine aroma. They are present in two forms: a

free volatile form and a nonvolatile conjugated glycosidic form. Chemically, the


aglycone moiety of the precursor glucoside is linked to the disaccharides 6-o--

rhamnopyranosyl β-D-glucoside and 6-o--arabinopyranosyl β-D-glucoside

(Williams et al., 1992). The aglycone in the glycosidic form could be a volatile

phenole such as vanillin, aliphatic or cyclic alcohols like hexanol, 2-

phenylethanol, benzylalcohol, or terpenols like nerol, linalool, geraniol and

citronellol (Gunata et al., 1993). The nonvolatile compounds may be hydrolyzed

by the action of β-glucosidases which release volatile terpines from the

nonvolatile conjugated form (Sanchez-Torres et al., 1996). However, in nature

this process is generally slow and unable to liberate the entire flavor reservoir.

Supplementation with β-glucosidase from external source may enhance aroma

release, thus benefiting wine making process (Bhatia et al., 2002). At present,

aroma release is often enhanced using commercial enzyme preparations of

fungal origin, mainly Aspergillus spp. (Spagna et al., 2002b). Recently Villena et

al., (2007) showed that it is possible to use wine yeasts itself for this purpose,

by improving their ability to produce β-glycosidase, in place of the commercial

fungal enzyme preparations currently used in winemaking.

In food industry the application of gellan, an exopolysaccharide produced

by Sphingomonas paucimobilis is very limited owing to its high viscosity and

low solubility. Hydrolytic activity of β-glucosidase may be useful in the

production of low-viscosity gellan foods. For example, the intracellular β-

glucosidase produced by Bacillus sp. were shown to catalyse cleavage of the

trisaccharide glycosyl-rhamnosyl-glucose (produced by the action of gellan

lyase and extracellular glycosidases) to release glucose and rhamnosyl-glucose,

thereby reducing viscocity (Bhatia et al., 2002). β-glucosidases were also

associated with removal of bitterness from citrus fruit juices by catalyzing the

hydrolysis of naringin (4,5,7-trihydroxyflavanone-7-rhamnoglucoside) to

prunin (Romero et al., 1985)

Chapter 2 - 42 -

β-glucosidase from bacterial species such as Thermoanaerobacter brockii,

Thermotoga neopolitana and Cellovibrio mixtus can also act as lamnaribiase

and therefore can be used in the multi enzyme conversion of laminaridextrins

and laminaribiose to glucose (Breves et al., 1997). This property is important in

the production of algal biomass to fermentable sugars.

The possibility of using β-glucosidase in pigment metabolism is also

reported by several workers. Dried flowers of saffron (Crocus sativus) were

treated with β-glucosidase to isolate precarthamine pigment (Sarry and

Gunata, 2004). Similarly the deglycosylation of betacyanin by β-glucosidase in

Beta vulgaris is the first step towards the degradation of these compounds to

release the bioactive cellular metabolite, which has anti tumor activity. They

are also used as natural food dyes in confectionary products.

2.6.2 Applications based on synthetic activity

The role of oligosaccharides and glycocongugates is being explicit in the

biological and pharmaceutical sciences, necessitating their availability on a

large scale. Unfortunately, chemical synthesis of these compounds remains as

a substantial challenge. This difficulty is because of the fact that glycosidic

bond formation requires fine control of both regio- and stereochemistry, the

former being made more challenging by the similar reactivities of the hydroxyl

groups of sugar molecules. In order to evade these difficulties, it is generally

necessary to employ extensive protecting group chemistry with all its inherent

difficulties. Increasing attention is therefore now being paid to the use of

enzymes for such syntheses, particularly for large scale operations (Akita et al.,

1999) and one of the important candidate enzymes is β-glucosidase. Although

β-glucosidase normally hydrolyzes glycosidic linkages, under certain

conditions, they are able to catalyze the stereospecific formation of glycosidic

linkages. Thus, by exploiting either their reverse hydrolysis activity


(thermodynamically controlled approach) or their transglycosylation potential

(kinetically controlled approach), the synthesis of a variety of oligosaccharides

and glycocongugates has been achieved (Prade et al., 1998). Enzymatic

synthesis of oligosaccharides and glycocongugates through β-glucosidase

catalyzed transfer or condensation reactions is preferred over chemical

synthesis because of the selectivity of the enzymes and the use of mild reaction

conditions. Use of enzymes also eliminates the protection and deprotection

steps essential for chemical synthesis (Kobayashi et al., 2000).

2.6.2.1 Applications of β-glucosidase in the synthesis of alkyl-glycosides

The synthesis of alkyl-glycosides from natural polysaccharides or their

derivatives, and alcohols by reverse hydrolysis or trans-glycosylation by β-

glucosidase is an emerging trend. Alkyl-glycosides offer potential industrial

applications as non-ionic surfactants (Kobayashi et al., 2000) and are the topic

of active research. The surge of renewed interest in alkyl glycosides stems from

the following: they are prepared from naturally occurring, renewable resources

(sugars and fatty alcohols); they are easily biodegradable; and they are more

stable under alkaline conditions than the corresponding sugar fatty acid esters.

Apart from their value as bulk detergents, pure alkyl glycosides have proved

useful in biomedical and pharmaceutical applications. These have been used

as drug carriers and as solubilizing agents for biological membranes,

particularly hexyl-, heptyl-, and octyl-glycosides (Basso et al., 2002).

The stereo specific preparation of alkylglycosides involves either a

multistep synthesis, through brominated monosaccharide per-acetates, or a

chromatographic separation of anomers, obtained after direct acid-catalysed

coupling (Balogh et al., 2004; Turner et al., 2007). However, the latter reaction

can be carried out enzymatically using inexpensive and readily available β-

glucosidases as catalysts (von Rybinski and Hill, 1998). This allows absolute

Chapter 2 - 44 -

control of the configuration of the anomeric bond. The ability of β-glucosidase

for synthesis of alkyl glucosides from glucose and corresponding alcohols in

one step has made this enzyme attractive for synthetic application (Lu et al.,

2007). Normally, these enzymes catalyze the hydrolysis of glycosides, but in an

environment containing high amounts of alcohols and relatively low amounts of

water, many of those enzymes can use the alcohols as acceptors (nucleophiles),

resulting in the formation of alkyl glycosides.

Table 2.5. β-glucosidases used in the synthesis of various commercially important compounds

Several family 1 (GH 1) were reported as useful in the synthesis of alkyl

glucosides (table 2.5). The benefit of using family 1 enzyme is that they are well

characterized and that a number of three-dimensional structures have been

determined by X-ray crystallography. The most widely used and characterized

representative is the commercially available almond β-glucosidase (Ljunger et

al., 1994; Vic and Crout, 1995; Kobayashi et al., 2000 ; Andersson and

Adlercreutz, 2001; Kouptsova et al., 2001; Basso et al., 2002; Thanukrishnan

et al., 2004; Ducret et al., 2006). There are also several reports about

Enzyme Source Product Reaction Type Reference

Almond o-alkyl or aryl β-D-glucoside Reverse hydrolysis Balogh et al., 2004; Lu et al., 2007

Almond p-nitrobenzyl β-D-glucopyranoside Reverse hydrolysis Tong et al., 2005

Almond Octyl glucopyranoside Transglycosylation Basso et al., 2002

Almond

Allyl β-D-glucopyranoside Reverse hydrolysis

Vic and Crout, 1995 Benzyl β-D-glucopyranoside Reverse hydrolysis

Allyl β-D-galactopyranoside Reverse hydrolysis

Apple seed Alkyl o-glucoside Reverse hydrolysis Yu et al., 2007

Cassava Alkyl glucoside Transglycosylation Svasti et al., 2003

Fusarium oxysporum Alkyl-β-D-glucopyranoside Transglycosylation Makropoulou et al., 1998

Pyrococcus furiosus Oligosaccharides Transglycosylation Bruins et al., 2003

Sclerotinia sclerotiorum

Alkyl-glycosides Transglycosylation Gargouri et al., 2004

Streptomyces sp Alkyl β-D-glucopyranosides Transglycosylation Faijes et al., 2006

Sulfolobus solfataricus β-glycosides Transglycosylation Petzelbauer et al., 2000

Thermotoga neapolitana

Alkyl glycosides Transglycosylation Turner et al., 20007


thermostable β-glucosidases from the family used in synthesis reactions for

example, β-glucosidase from Thermotoga maritima (Goyal et al., 2001) and

Pyrococcus furiosus (Hansson and Ablercreutz, 2001). The family 3 enzymes

(GH3) have not frequently been used in synthesis applications, although they,

like GH1, have a retaining mechanism and several members with substantial

transglycosylation activity (Goyal et al., 2001; Saloheimo et al., 2002; Seidle et

al., 2005). GH3 glucosidases are reported to have a broad substrate specificity

and are frequently active towards different kinds of glycosides, such as

xylosides and aryl glucosides, but otherwise this enzyme family is not as well

characterized as family 1 enzymes and this limits their application in large

scale synthesis.

2.6.2.2 Applications of β-glucosidase in the synthesis of butyl and allyl

glycosides

Butyl-glycoside is a valuable compound because it serves as a precursor

in the synthesis of Gemini surfactants and other pharmaceutical compounds.

The former are useful as liquid crystal generators (Turner et al., 2007).

Esterification of butyl-glycoside in presence of phenyl butyric acid in a coupled

β-glucosidase/ lipase reaction resulted in the synthesis of an aromatic n-alkyl

glucoside ester that was effective in the treatment of fever, rheumatism,

headache and other ailments. The synthesis of some natural compounds, like

aryl-glucosides with repellant and antifeedant properties was achieved with

thermostable β-glucosidase from Sulfolobus solfataricus (Vic and Crout, 1995).

Allyl β-D-glucopyranosides are the important starting intermediates in

carbohydrate chemistry as temporary anomeric protected derivatives. They are

also used in the synthesis of glycopolymers. The chemical synthesis of these

compounds is a multi-step procedure where at least three steps are necessary

starting from n-glucose or n-galactose. β-glucosidase catalyzed synthesis can

Chapter 2 - 46 -

be used instead of the multi-step chemical synthesis. Vic and Crout (1995)

described the enzymatic syntheses of three ally1 β-D-glucopyranoside using

reverse hydrolysis process starting directly from D-glucose or D-galactose and

the corresponding alcohol in presence of almond β-glucosidase. Recently, the

enzyme has also been used in reactions involving biosynthesis of short-chain

cellodextrins (Painbeni et al.,1992; Kuriyama et al.,1995) and β-mercaptoethyl-

glycoside (Dintinger et al., 1994).

2.6.2.3 Applications of β-glucosidase in the synthesis of oligosaccharides

Similarly the important role of oligosaccharides and their conjugates in

biology has been increasingly recognized in recent years, leading to an upsurge

of interest in this field. Oligosaccharides can be synthesized from

monosaccharides or disaccharides, using β-glucosidase as a catalyst.

Crittenden (1999) described the use of β-glycosidase from Pyrococcus furiosus

for the synthesis of oligosaccharides from cellobiose, lactose, glucose and

galactose. The oligosaccharides that are produced can be used as prebiotic food

ingredients. Non-digestible oligosaccharides have a positive influence on the

growth of essential microorganisms in the human gut flora.

2.6.2.4 Applications of β-glucosidase in the derivatisation of thiamin

In a recent experiment Ponrasu et al., (2009) demonstrated the use of β-

glucosidase for the derivetisation of thiamin. Thiamin (3-(4-amino-2-methyl-5-

pyrimidinylmethyl)–5-(2-hydroxyethyl)-4-methyl-1,3-thiazol-3-ium, vitamin B1)

is a water soluble vitamin belonging to the B complex group and is an

important cofactor of decarboxylase, transketolase and carboxylase. The

characteristic odor and a strong tongue-pricking taste of thiamin could be

reduced by preparing derivatives of this vitamin (Suzuki and Uchida, 1994).

The work by Ponrasu et al., (2009) describes the preparation of thiamin

glucoside using immobilized β-glucosidase.


2.6.3 Other applications

Levels of serum glycosylhydrolases, β-glucosidase and β-galactosidase

have been used as sensitive markers in post-diagnosis of hepatic ischemia-

reperfusion injury and recovery, because there is a marked increase in the

concentration of these enzymes following liver injury. The alterations in the

levels of lysosomal β-glucosidases, β-galactosidases and β-glucuronidass have

been used as diagnostic tool to detect premalignant and malignant lesions of

oral mucosa in hamsters, as activities of these enzymes were elevated markedly

only in the carcinoma stage. The H-antigen of Histoplasma capsulatum (a

fungus causing respiratory disease) was found to exhibit β-glucosidase activity.

It could elicit cell mediated immunity and humoral immunity and thus was

used for serodiagnosis of histoplasmosis (Bhatia et al., 2002).

2.7 Mechanism of Action of β-glucosidase

β-glucosidases hydrolyze cellobiose and other cello-oligosaccharides to

glucose. β-Glucosidases belonging to the family 1 glycoside hydrolases catalyze

the hydrolysis of the glucosidic bond between the anomeric carbon (C1 of the

glucose) and the glucosidic oxygen by a mechanism in which the anomeric

configuration of the glucose is retained (Davies and Henrissat, 1995). Two

conserved glutamic acid residues serve as a catalytic nucleophile and a general

acid/base catalyst, respectively. In retaining β-glucosidases, the catalytic

Fig 2.1. The reaction mechanism in β-glucosidase catalyzed reactions

(Source:Davies and Henrissat, 1995)

Chapter 2 - 48 -

glutamic acid residues are situated on opposite sides of the β-glucosidic bond

of the docked substrate at a distance of ~5.5Ǻ (Davies and Henrissat, 1995). As

the initial step in catalysis, the nucleophile performs a nucleophilic attack at

the anomeric carbon, which results in formation of a glucose–enzyme

intermediate. In this process, aglycone departure is facilitated by protonation of

the glucosidic oxygen by the acid catalyst.

During the second catalytic step (deglucosylation), a water molecule is

activated by the catalytic base to serve as a nucleophile for hydrolysis of the

glucosidic bond and release of the glucose under suitable conditions, β-

glucosidases can perform a transglucosylation in which the covalently bound

glucose in the enzyme–glucose intermediate is transferred to an alcohol or a

second sugar group (Davies and Henrissat, 1995). Under suitable conditions,

β-glucosidases can perform a transglucosylation in which the covalently bound

glucose in the enzyme–glucose intermediate is transferred to an alcohol or a

second sugar group.

Most β-glycosidases have one or more tryptophan residues at their active

sites that are important for sugar binding. Sugars rest on the indole ring but

other bonds are also involved in holding the sugars in place. The sugar-indole

interactions probably involve: (1) van der Waals interactions that occur with

the hydrophobic indole, since most sugars have one face that is somewhat non-

polar (2) weak electrostatic interactions that occur between the π-electron

clouds of the indole group and protons of the sugar which have small net

positive charges because the oxygens of the hydroxyls attached to the same

carbons are electron withdrawing and (3) hydrogen bonding that can occur

between the indole nitrogen and a hydroxyl group of the sugar. In addition, a

tryptophan at the active site can be important for conformation since it can

interact with other residues in the vicinity. Because of the large size tryptophan

can also serve an important function by filling space and preventing a cavity

that would otherwise change the conformation (Seidle et al., 2005). Through


site directed mutagenesis of Aspergillus niger, GH3 β-glucosidase gene Seidle et

al., (2005) showed that a Trp-262 is the key residue for determining the ratio of

the enzyme’s hydrolytic and transglucosidic activities.

Most plant β-glucosidases are glycosyl hydrolase family 1 (GH1)

members that catalyze the hydrolysis of their substrates via a double-

displacement mechanism (Henrissat and Davies, 2000). Although the active

site residues have not been precisely known for all β-glucosidases, the two

glutamate residues present in the highly conserved TL/FNEP and I/VTENG

motifs in all GH1 β-glucosidases are likely to act as the catalytic acid/base and

nucleophile residues (Ly and Withers, 1999). While the mechanism of catalysis

has been studied extensively for the plant β-glucosidases, the molecular basis

of substrate specificity is not as well understood. Glucosidic substrates

naturally occurring in plants contain a broad range of aglycone groups,

including cyanogenic glucosides (Eksittikul and Chulavatanatol, 1988; Barrett

et al, 1995), cellobiose (Ferreira and Terra, 1983), phenolic glucosides

(Podstolski and Lewak, 1970), thioglucosides (Durham and Poulton, 1989), and

isoflavonoid glucosides (Svasti et al., 1999). Differences in the aglycone

specificity-determining sites have been studied in maize and sorghum β-

glucosidases, whose sequences show 70% identity. Enzymatic studies of

chimeric β-glucosidases and X-ray crystallographic structures suggested that

the determinants of substrate specificity in the maize ZmGlu1 and sorghum

SbDhr1 enzymes include both homologous and nonhomologous residues

(Gebler et al., 1995).

Crystal structures of a number of GH1 enzymes, including β-

glucosidases have been determined, revealing details of their reaction

mechanism (retaining mechanism), glycone binding site (subsite _1), and

aglycone binding site (subsite +1) (Verdoucq et al., 2004). Many structural

features are common to the catalytic site of all the GH-1 enzymes, even though

Chapter 2 - 50 -

they are active with a large variety of substrates. On the other hand, even

minor changes in the substrate structure may bring about large alterations of

enzymatic behavior. The specificity of family GH-1 for the monosaccharide at

the −1 subsite is known through the crystal structure of several complexes

containing a ligand at this subsite. These studies showed a highly conserved −1

subsite, with a common pattern in sugar recognition. Conversely, structural

data for the +1, aglycone-binding site is limited to two plant β-glucosidases

from maize (ZmGlu1) and Sorghum bicolour (SbDhr1) with ligands occupying

this subsite. These studies gave some clues about the molecular basis of the

aglycone specificity which, however, were not confirmed by mutagenesis

experiments. Therefore, the available information is still insufficient to have a

complete picture of the molecular basis of substrate specificity, as the

aglycone-binding site is essential for defining substrate preference, and in the

correct positioning of the substrate into the active site for the reaction to

proceed. The characterization of the aglycone-binding site also demands

localization of the additional subsites (+2, +3…) found in some enzymes

(Verdoucq et al., 2004).

2.8 Production of β-glucosidase through fermentation

Submerged (SMF) and solid state fermentation (SSF) are being used

extensively for the production of β-glucosidase. Kovacs et al., (2008) reported

the production of the cellulase enzymes including β-glucosidase on pretreated

willow wood chips using a mutant strain of Trichoderma atroviride. They

observed that T. atroviride mutants produced high levels of extracellular

cellulases as well as β-glucosidase, rendering the need for β-glucosidase

supplementation in hydrolysis of cellulose or pretreated willow unnecessary.

Wang et al., (2009) produced β-glucosidase using mutant strain Trichoderma

viride T 100-14 in shake flasks. They used combined biochemical and

immunocytochemical techniques to monitor the intracellular and extracellular


distribution of β-glucosidase in different culture conditions in T. viride by using

activity assay and transmission electron microscopy method. Under constant

pH 4.8, highest intracellular enzyme activity, total enzyme activity and specific

activity were observed at 24 hours of fermentation. After 72 hours, the

extracellular and total activities fluctuated little and the maximal activity in

extracellular fraction was 2.7 times higher than control.

Dogaris et al., (2009) used Neurospora crassa for the production of β-

glucosidase through SSF using wheat straw-wheat bran mixture. Urea,

ammonium sulfate and potassium nitrate were found to be the nitrogen

sources preferred by the fungus. A pH range of 4-5 and 70.5% initial medium

moisture were found to be optimum for the production of the enzyme.

Production of cellulase enzymes by Trichoderma reesei RUT C30 on

steam pretreated spruce, willow, corn stover and delignified lignocellulose was

compared (Juhasz et al., 2005).Their experiments demonstrated that pretreated

corn stover is the good substrate for the production of cellulases including β-

glucosidase. Daroit et al., (2007) screened various agro-industrial residues in

combination with peptone, NH4Cl and soy bran as substrates for extracellular

β-glucosidase production by Monascus purpureus NRRL1992 on submerged

fermentations. Higher BGL production was achieved when the agro-industrial

residues were combined with peptone. The combination between grape waste

and peptone was found to be the best for enzyme production.

Extracellular β-glucosidase and other cellulolytic enzymes were produced

under solid state fermentation by the thermophilic fungus Thermoascus

aurantiacus using various agricultural waste materials such as wheat straw,

rice straw, corn cobs, wheat bran and oat bran (Kalogeris et al., 2003). Of the

various carbon sources wheat straw was found to be the one causing highest

enzyme titer.

Chapter 2 - 52 -

A radically different approach in the offing is to produce the enzyme in

genetically engineered plants rather than in fungi [Sticklen, 2006; Gray et al.,

2009]. Genetically modified plants can be used to express β-glucosidase. If the

enzyme could be produced at a high level without hurting the yield of a

productive crop, its cost could be reduced. A major advantage of producing

enzymes in plants over fungal production is that it is much easier to adjust the

amount of enzyme that is produced to meet the demand, as the amount of

land planted can be adjusted to the demand, whereas once a fermenter is built,

its capacity is always there. A problem with plant production of cellulases is

the multiplicity of proteins that are needed to efficiently degrade plant cell

walls. Fungi produce large numbers of proteins and we do not always know

which proteins are required to degrade a given substrate so that it is difficult to

engineer a plant to produce a mixture with equal activity to that produced by a

cellulolytic fungus (Wilson, 2009).

2.9 Statistical Design of Experiments (DOE) to improve fermentation yield

The success of any fermentation process depends on a harmonious blend

of various process parameters contributing to product formation. Optimization

of process parameters is therefore of pivotal importance. The aim of

optimization is to determine suitable fermentation conditions (pH, temperature,

medium composition etc.) for the respective biological system in order to

maximize or minimize economically or technologically important process

variables such as product concentration, yield, raw material cost etc (Weuster-

Botz, 2000).

The classical approach to optimize the process parameters is a ‘one

dimensional search’ by successively varying one variable at a time while fixing

all others at a certain level. The most important drawback of this approach is

that it seldom considers the interaction effects of variables. Hence the classical


approach is not adequate enough to achieve the optimum medium in a limited

number of experiments. To obtain an optimum fermentation medium through a

manageable number of experiments statistical design of experiments (DOE) is

being used. There are a few reports regarding the use of DOE for the

optimization of process variables connected with cellulase and β-glucosidase

production.

Hao et al., (2006) reported the use of response surface methodology to

optimize the fermentation conditions for the production of cellulase enzymes

using a mutant strain of Trichoderma reesei WX-112. By using a fractional

factorial design they could identify concentration of Avicel and soybean cake

flour in the medium as the factors influencing cellulase production

significantly. By using the fermentation medium optimized with response

surface methodology they could increase the production of cellulase from 7.2 to

10.6 IU/mL.

Production of cellulolytic and xylanolytic enzymes by a thermophilic

fungal isolate Myceliophthora sp. using a medium containing rice straw and

chemically defined basal medium under solid-state culture was reported by

Badhan et al., (2007). A combination of one factor at a time approach followed

by response surface methodology using Box–Behnken design of experiments

resulted in 1.28 fold increase in β-glucosidase activity.

Scytalidium thermophilum isolated from composting soil was optimized

for cellulase enzyme production by solid state fermentation (Jatinder et al.,

2006). Initial experiments showed that culture medium containing rice straw

and wheat bran (1:3) as carbon source prepared in a synthetic basal medium

supported maximal enzyme production at 450C. Further optimization of

enzyme production was carried out using Box-Behnken design of experiments

to study the influence of process variables (inoculum level, (NH4)2SO4 and pH)

on enzyme production. Under optimized conditions the fungus produced 151

8.194 U/gm substrate of β-glucosidase.

12_chapter 2.pdf - shodhganga

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