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
Review of Literature - 25 -
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
Review of Literature - 27 -
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.
Review of Literature - 29 -
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
Review of Literature - 31 -
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
Review of Literature - 33 -
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
Review of Literature - 35 -
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
Review of Literature - 37 -
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
Review of Literature - 39 -
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
Review of Literature - 41 -
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
Review of Literature - 43 -
(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
Review of Literature - 45 -
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.
Review of Literature - 47 -
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
Review of Literature - 49 -
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
Review of Literature - 51 -
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
Review of Literature - 53 -
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.