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Review of Literature
15
Microorganisms are the very important source that have been
endowed the capacity to produce a vast array of enzymes. The produced
enzymes are exploited commercially over the years. Pectinases are one such
enzyme which shows great significance with tremendous potential to offer to
industry (Dayanand and Patil, 2003). They are one of the upcoming enzymes
of the commercial sector, especially the juice, vegetable and as such food
industry (Kashyap et al., 2001) and in the paper and pulp industry (Beg et al.,
2001; Viikari et al., 2001). Pectinases are also known as Pectinolytic enzymes
and are a heterogeneous group of related enzymes that hydrolyze the pectic
substances which are mostly present in plants. Pectinolytic enzymes are
widely distributed in higher plants and microorganisms such as moulds,
yeasts and bacteria (Whitaker, 1990). As of now, only limited information is
available on the pectinolytic enzyme systems. Studies on pectinolytic
actinomycetes have mainly revealed the presence of pectate lyases (Sato
and Kaji, 1980 and 1981; Spooner and Hammerschmidt, 1989). Recently, in
Streptomyces viridochromogenes, a pectin lyase has been detected (Agate et
al., 1962). The commercial preparations of pectinases are produced mainly
from fungi, especially Aspergillus niger (Torres et al., 2005).
It has been reported that microbial pectinases account for 25% of the
global food enzymes sales (Jayani et al., 2005; Elagovan Namasivayam et
al., 2011). This review includes different types of pectinolytic enzymes, their
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classification, mode of action and the substrates on which they act and finally
the possible applications of these enzymes in industrial sector.
Structure, classification and nomenclature of pectic substances:
Pectins are high molecular weight acid polysaccharides which are
primarily made up of α-(1-4) linked D-galacturonicacid residues with a small
number of rhamnose residues in the main chain and arabinose, galactose
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17
and xylose on its side chain (Deul and Stutz, 1958; Whitaker, 1984; Singh et
al., 1999; Kapoor et al., 2000; Lang and Dornenberg, 2000). Pectic
substances are widely distributed in fruits and vegetables like (10-30%) in
turnips, peels of orange and in pulps of tomato, pineapple and lemon; hence
they form important natural substrates for pectinases (Gummadi and Panda,
2003).
Structure of pectin molecule (Rexova and Markovic, 1976)
Pectic substance is the generic name used for the compounds that are
acted upon by the pectinolytic enzymes. They are also negatively charged,
acidic, complex glycosidic macromolecules. They are present as the major
components of middle lamella between the cells in the form of calcium
pectate and magnesium pectate (Rastogi, 1998). The middle lamella is
largely composed of pectic substances that have been confirmed by the
comparable uptake of ruthenium red by known pectic substances (Sterling,
1970) and from the estimation of pectin by the use of alkaline hydroxylamine
(McCready, 1955; Gee, 1959; Albersheim and Killias, 1963). Pectic
substances account for 0.5–4.0% of the fresh weight of plant material (Sakai
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et al., 1993). Contrary to the proteins, lipids and nucleic acids, being
polysaccharides, pectic substances do not have a defined molecular weight
as shown below. The relative molecular masses of pectic substances range
from 25 to 360 kDa.
Molecular weights of some pectic substances (Sakai et al., 1993) are,
Source Molecular weight (kDa)
Apple and Lemon 200–360
Pear and Prune 25–35
Orange 40–50
Sugar beet pulp 40-50
The term “pectins” encompasses a group of acidic
heteropolysaccharides with distinct structural domains. They are subjected to
both biosynthetic and cell wall-based modifications. The chemical structure of
pectins has been the subject of many scientific investigations for decades
(Perez et al., 2003). On the other hand, pectinases are a group of related
enzymes involved in the breakdown of pectin from a variety of plants. These
enzymes are classified based on their preferred substrate (pectin, pectic acid
or oligo-D-galacturonate), the degradation mechanism (transelimination or
hydrolysis) and the type of cleavage, random (endo) or terminal (exo)
(Kashyap et al., 2001).
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Pectic substances mainly consist of galacturonans and rhamno-
galacturonans in which the C-6 carbon of galactate is oxidized to a carboxyl
group, the arabinans and the arabinogalactans (Whitaker, 1990). These
substances are a group of complex colloidal polymeric materials, composed
largely of a backbone of anhydrogalacturonic acid units (Cho et al., 2001;
Codner, 2001). The carboxyl groups of galacturonic acid are partially
esterified by methyl groups and partially or completely neutralized by sodium,
potassium or ammonium ions (Kashyap et al., 2001). Some of the hydroxyl
groups on C2 and C3 may be acetylated (Alkorta et al., 1998). The primary
chain consists of α-D-galacturonate units linked α-(1-4), with 2–4% of L-
rhamnose units linked β-(1-2) and β-(1-4) to the galacturonate units
(Whitaker, 1990). The rhamno-galacturonans are negatively charged at pH 5.
The side chains of arabinan, galactan, arabinogalactan, xylose or fucose are
connected to the main chain through their C1 and C2 atoms (Pilnik and
Voragen, 1970; Rombouts and Pilnik, 1980; Blanco et al., 1999; Van der et
al., 2000; Sathyanarayana et al., 2003; Mohnen, 2008; Caffall and Mohnen,
2009). This description indicates that the pectic substances are present in
various forms in plant cells and this is the probable reason for the existence of
various forms of pectinolytic enzymes.
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The American Chemical Society classified pectic substances into four
main types as follows (Alkorta et al., 1998). Pectinases hydrolyse pectin by
different mechanisms and they are divided into, (I) Protopectin: It is the water
insoluble pectic substance present in intact tissue. Protopectin on hydrolysis
yields pectin or pectic acids. (II) Pectic acid: It is the soluble polymer of
galacturonans which contains negligible amount of methoxyl groups. Normal
or acid salts of pectic acid are called pectates. These act on de-esterified
pectin. (III) Pectinic acid: It is the polygalacturonan chain that contains >0
and <75% methylated galacturonate units. Normal or acid salts of pectinic
acids are referred to as pectinates. These act on methyl-esterified pectin. (IV)
Pectin (Polymethyl galacturonate): It is the polymeric material in which, 75%
of the carboxyl groups of the galacturonate units are esterified with methanol.
It confers rigidity on cell wall when it is bound to cellulose in the cell wall.
Pectinase is a generic name for a family of enzymes that catalyse
hydrolysis of the glycosidic bonds in the pectic polymers (Collee et al., 1996;
Castilho et al., 1999 and 2000; Reid and Ricer, 2000; Gummadi and Panda,
2002; Bai et al., 2004). Pectinases are classified into polygalacturonase (EC
3.2.1.15), pectin esterase (EC 3.1.1.11), pectin lyase (EC 4.2.2.10) and
pectate lyase (EC 4.2.2.2) on the basis of their mode of action (Alkorta et al.,
1998; Hoondal et al., 2000; Kapoor and Kuhad, 2002). The complete
degradation of pectin is due to the synergistic action of methylesterase
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(EC.3.1.11.1), endo-polygalacturonase (EC.3.2.1.15), exopolygalacturonase
(EC.3.2.1.67), endo-pectate lyase (EC.4.2.2.2), exo-pectate lyase
(EC.4.2.2.9) and pectinlyase (4.2.2.10) (Goodman et al., 1986; Agrios, 1988;
Kashyap et al., 2001; Koponen et al., 2008).
Most extracellularly induced enzymes are known to be synthesized in
higher quantities when inducers are present in the cultivation medium (Alkorta
et al., 1998; Lang and Dornenburg, 2000). The production of pectolytic
enzymes using different sources and the effect of physical parameters such
as temperature, aeration rate and type of fermentation were investigated and
reported in literature (Nair et al., 1995; Naidu and Panda, 1998). Pectolytic
enzymes have been reported to be induced by several substances. In many
cases pectin itself has been used. Many investigators had used complex
media such as beet sugar, wheat bran, ground nut meal, citrus fruit peels etc
(Kilara, 1982; Hoondal et al., 2000). In the industrial market pectolytic
enzymes contribute to at most 25% of the global enzyme sales, where its
contribution is imagined to increase further by the year 2009 (Tari et al.,
2007). Due to the potential and wide applications of pectinases, it is
necessary to study on several aspects related to pectinase production. The
idea of using cheaper raw materials for pectinase production is an important
parameter in technological development (Panda et al., 2004). Thus here we
used orange and banana peel powder for our study. Now, the main aim is to
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present an overview of the pectinase activity values obtained by Bacillus
subtilis from different substrates like how Friedrich et al., (1989) and Bailey,
(1990) have used.
In nature, pectin is degraded by enzymic systems produced by a wide
variety of saprophytic and phytopathogenic micro-organisms like bacteria and
fungi (Rombouts and Pilnik, 1980). Pectinases play an important role in the
invasion of plant tissues by phytopathogens, the spoilage of fruits,
vegetables, in food processing and also in plant biotechnology applications.
Bacillus subtilis is the best-studied bacterium in terms of biotechnology,
biochemistry and genetics, and is also a valuable model system for molecular
research.
Fermentation methods
Generally, pectinases are produced by submerged (SmF) and solid-
state fermentation systems (SSF) (Beg et al., 2000; Kavitha and Umesh-
Kumar, 2000; Kaur et al., 2004; Martin et al., 2004; Couto and Sanromàn,
2006) whereas slurry-state fermentation (SLSF) has rarely been reported (De
Gregorio et al., 2002). Studies have been conducted on comparative
production of pectinases in systems of SmF and SSF (Maldonada and Saad,
1998). SSF has several advantages over SmF system such as higher
concentration of products, less effluent generation, requirement for simple
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23
equipments etc. (Pandey et al., 1994). Reports are very few on the
comparison of SmF, SSF and SLSF for the production of pectinases.
The price of commercially available enzymes which are produced
mostly by submerged fermentation is usually too high for agro-
biotechnological applications (Philippidis, 1994; Ruth et al., 1999). An
alternative technique of enzyme production is solid state cultures (Fonseca et
al., 1991; Lonsane and Ohildyal, 1993).
Solid state cultures have more advantages than liquid cultures: they
show higher product yield; better product quality; cheaper product recovery
and cheaper technology (Doelle et al., 1992). Solid state cultures are
becoming a viable alternative technique for large-scale industrial processes
for enzyme production (Cen and Xin, 1999). It is the efficient and competitive
method for bioconversion of solid substrates (Gervais et al., 1988; Oriol et al.,
1988 a, b).
The degradation of pectic substances involves the combined action of
different pectinases namely, esterases and depolymerases (hydrolases and
lyases). Pectic transeliminases or pectic lyases are one among the
pectinases which degrade pectic substances by 3-elimination mechanism
yielding 4:5 unsaturated oligogalacturonates. Pectin lyase acting on pectin
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and polygalacturonate lyase or pectate lyase acting on polygalacturonic acid
is two important transeliminases acting on pectic substances.
Substrates used
Production of enzymes from agrowastes is very important because
they contain large amounts of cellulose, hemicellulose and pectin, which
could serve as inducers for the production of cellulase, xylanase, and
pectinases, respectively. Several agrowastes, mainly citrus peel (Jansen and
MacDonnell, 1945), apple pomace (Hours et al., 1988) and coffee pulp
(Boccas et al., 1994) have been studied for the production of pectinases.
Here, we have used orange and banana peel powder for our present study.
Microorganisms are widely accepted as the best sources for the
production of enzymes from agrowastes. Bacteria and fungi are known to
produce industrial enzymes. Among those, fungi are preferred (Sumantha et
al., 2005). Recently, the production of pectinases from agrowastes (Aguilar
and Huirton, 1990; Acuna Arguelles et al., 1995; Sebastian et al., 1996;
Gholifar et al., 2010) by fungi has been described as more attractive. In the
present study, bacteria are taken and studied. Therefore, an attempt was
made to examine the utility value of pectin rich regional agrowastes for the
production of pectinases by Bacilus subtilis. The effect of the addition of
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25
carbon and nitrogen sources on the production of pectinases was also
studied.
Occurrence
Pectinase production has been reported from bacteria including
actinomycetes (Cao et al., 1992; Brigitte et al., 1994; Beg et al., 2000), yeast
(Blanco et al., 1999; Reid and Ricard, 2004) and fungi (Elegado and Fujio,
1994; Huang and Mahoney, 1999). However, industrial production of
pectinases makes use almost exclusively of Aspergillus niger strains
(Gummadi and Panda, 2003).
Most of the works on pectinase production have been focused on
either submerged fermentation where the pectin is used as the inducer to a
pre-formulated synthetic medium or through solid state fermentation using
pectin rich substrates like citrus peel, fruit wastes etc (Patil and Agasar
Dayanand, 2006). To our best knowledge much work has not been reported
on pectinase enzyme production from the dry orange peel and banana peel
powder. Hence, taken in the present study. The main sources for the
pectinolytic complex enzymes are yeast, bacteria and a large variety of
filamentous fungi, for which the most relevant ones are Aspergillus (Nair et
al., 1995; Ahmad et al., 1997; Solis et al., 1997; Perenirova et al., 1998).
Through studies with microorganisms, experimental assays demonstrated
that the enzyme synthesis is correlated with the quality and concentration of
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26
the carbon and nitrogen sources. In this area, research is more advanced
with bacteria than with fungi. The studies of pectinases synthesis from
Aspergillus, Fusarium and Verticillium and among others, show that it is
induced mainly by pectin or pectin associated to other substances (Call et al.,
1985; Aguilar and Huirton, 1987).
Although pectolyase from A. japonicus is commercially available from
SIGMA (USA), very few studies with the other pectinase complex have been
recently published (Aguilar and Huirton, 1987; Griffin, 1994). Pectin lyase
from A. japonicus was previously published (Mathieu et al., 1994; Rout-Mayer
et al., 1997). In light of importance, the regulation study of the enzyme
synthesis of pectinolytic complex, the influence of a variety of carbon and
nitrogen sources on the pectinases (endo and exo-polygalacturonase)
production of Bacillus subtilis was studied in order to obtain high enzymatic
levels.
Endo-PGases are widely distributed among fungi, bacteria and many
types of yeast (Luh and Pha, 1951). They are also found in higher plants and
some plant parasitic nematodes (Sakai, 1992). They have been reported in
many microorganisms, including Aureobasidium pullulans (Sakai and
Takaoka, 1984), Rhizoctonia solani Kuhn (Marcus et al., 1986), Fusarium
moniliforme (De Lorenzo et al., 1987), Neurospora crassa (Polizeli et al.,
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1991), Rhizopus stolonifer (Manachini et al., 1987), Aspergillus sps. (Nagai et
al., 2000), Thermomyces lanuginosus (Kumar and Palanivelu, 1999) and
Peacilomyces clavisporus (Souza et al., 2003). Endo- PGases have also
been cloned and genetically studied in a large number of microbial species
(Laing and Pretorius, 1993; Reymond et al., 1994; Centis et al., 1996 and
1997; Gao et al., 1996; Gainvors et al., 2000; Naumov et al., 2001).
In contrast, exo-PGases occur less frequently. They have been
reported in Erwinia carotovora (Paloma¨ki and Saarilahti, 1997),
Agrobacterium tumefaciens (Rodrigues et al., 1991), Bacteroides
thetaiotamicron (Tierny et al., 1994), E. chrysanthemi (Koboyashi et al.,
2001), Alternaria mali (Nozaki et al., 1997), Fusarium oxysporum (Maceira et
al., 1997), Ralstonia solanacearum (Huang and Allen, 1997), Bacillus sps.
(Koboyashi et al., 2001). Very few strains of Bacillus sps. (Soriano et al.,
2005) produce both pectate lyase (PL) and polygalacturonase (PGL) in
appreciable amounts. Pectate lyase (PL) activity has been detected
previously in supernatants of phytopathogenic bacteria and has been
described as the most important cause of soft rot disease (Chesson, 1980;
Tsuyumu and Chatterjee, 1984; Collmer and Keen, 1986; Liao, 1989). Pectin
lyase (PNL) and pectin methyl esterase (PME) are also cited as pectinolytic
enzymes produced by microorganisms (Chesson, 1980). P. marginalis has
been described as bacteria producing PL (Ceponis and Friedman, 1959;
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Nasuno and Starr, 1966), PNL (Nikaidou et al., 1992; Sone et al., 1988), and
small amounts of PME (Ceponis and Friedman, 1959; Nasuno and Starr,
1966). To quantify growth and enzyme production of P. marginalis during
storage at low temperature and to determine the influence of pH and NaCl
concentration on shelf life of a food product, we have chosen a predictive
microbiology approach.
Applications of Pectinolytic Enzymes
In Fruit Juices, Jams, Jellies and Other Food Industries
Pectinases have been applied for more than 60 years in the food and
beverage industry (Pilnik and Rombouts, 1985). While in the case of the
production of clear juices the pectinolytic enzymes are added to increase
yield during pressing and for clarification (Whitaker, 1990a; Prade et al., 1999;
Jayani et al., 2005; Niture, 2008; Jose et al., 2008; Mohnen, 2008; Tochi et
al., 2009; Ribeiro et al., 2010). The stabilisation of clouds in orange juices is
achieved by the use of pectic enzymes with high levels of polygalacturonase
activity (Whitaker, 1984). By applying these enzymes on fruit pulp, it
degrades pectin thereby reducing the viscosity and the fruit juice can be
handled easily (Blanco et al., 1999; Kashyap et al., 2000; Blunt, 2000;
Sarioglu et al., 2001; Maria et al., 2000; de Gregorio et al., 2002; Souza et al.,
2003; Fernandez-Gonzalez et al., 2004; Ribeiro et al., 2010).
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These enzymes play an important role in maceration and solubilization
of fruit pulps and in clarification. The traditional method of clarification of
pectin containing juice involves a number of steps, including centrifugation to
remove suspended solid, enzymatic treatment for depectinization, finding
agents such as bentonite and gelatin to remove haze and finally filtration by
the diatomaceous earth to remove the finding agents. With membrane
technology, juice can be clarified using depectinization followed by ultra
filtration or micro filtration. Pectins are used in the manufacturing of jams and
jellies too. Treatment of fruit pulps with pectinases also showed an increase in
fruit juice volume from banana, grapes and apples (Will et al., 2000; Kaur et
al., 2004). Pectinases in combination with other enzymes namely, cellulases,
arabinases and xylanases have been used to increase the pressing efficiency
of the fruits for juice extraction (Gailing et al., 2000; Ribereau-Gayon et al.,
2006). Vacuum infusion of pectinases has a commercial application to soften
the peel of citrus fruits during removal. This technique is used in future to
replace hand cutting for the production of canned segments (Baker and
Wicker, 1996). Infusion of free stone peaches with pectin methylesterase and
calcium results in four times firmer fruits. This may be applied to pickle
processing where excessive softening may occur during fermentation and
storage (Baker and Wicker, 1996). Pectinases are also responsible for the
texture of fruits and vegetables during growth, maturation and their storage
(Alkorta et al., 1998; Caffall and Mohnen, 2009).
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Low methylesterified pectin is used in combination with carrageenan to
produce sugar-free jams for diabetics and in fruit preparations for yoghurt. It is
also used as a thickening agent for sauces, ketchups, flavoured syrups and
as a texturing agent in fruit flavoured milk desserts (Girdharilal et al., 1998).
In Dairy Industries
High methylesterified pectin is known to prevent the formation of
casein clumps in acidified dairy products at pH below the isoelectric pH (4.6).
In Wine Industries
In wine industry pectinases are mainly used for decreasing astringency
by solubilizing anthocyanins without leaching out procyadin polyphenols and
pectinases also increase pigmentation by extracting more anthocyanins
(Tucker and Woods, 1991; Carmen Sieiro et al., 2012).
In Textile Industries
Textile processing has benefited greatly in both environmental and
product quality aspects through the use of enzymes. Pectinases are used in
combination with amylases, lipases, cellulases and hemicellulases to remove
sizing agents from cotton in a safe and eco-friendly manner, replacing toxic
caustic soda which was used earlier (Henriksson et al., 1999; Hoondal et al.,
2000; Jayani et al., 2005; Niture, 2008). Bioscouring is a novel process for
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removal of noncellulosic impurities from the fiber with specific enzymes.
Pectinases are used for this purpose without any negative side effect on
cellulose degradation (Hoondal et al., 2000).
Prior to weaving of yarn in to fabric, the warp yarns are coated with a
sizing agent to lubricate and protect the yarn from abrasion during weaving.
Historically, the main sizing agent used for cotton fabrics was starch because
of its excellent film-forming capacity, availability and low cost. Before the
fabric can be dyed, the applied sizing agent and the natural non-cellulosic
materials present in the cotton must be removed. Before the discovery of
amylase enzymes, the only way to remove the starch-based sizing was
extended treatment with casting soda at high temperature. The chemical
treatment was not totally effective in removing the starch and also result in a
degradation of the cotton fiber resulting in distraction of the natural soft feel or
'hand' of the cotton. The use of enzyme such as pectinase, in conjugation
with amylases, lipases, cellulases and other hemicellulolytic enzymes to
remove sizing agents has decreased the use of harsh chemicals in textile
industry, resulting in a lower discharge of waste chemicals to the
environment, improving both the safety of working conditions for textile
workers and the quality of the fabric.
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Degumming Of Plant Fibers
The most upcoming application of pectinolytic enzymes is their use in
the degumming of plant fibers such as ramie, sun hemp, jute and flax (Cao et
al., 1992; Bruhlmann et al., 1994; Henriksson et al., 1999; Kapoor et al.,
2001). Bast fibers are the soft fibers present in groups outside the xylem,
phloem or pericycle. These fibers contain gum which should be removed
before its use for textile making (Hoondal et al., 2000). Degumming treatment
by chemicals generally cause pollution, which is toxic and non-biodegradable.
Biotechnological degumming using pectinases in combination with xylanases
results in an eco-friendly and economic alternative to solve the above
problem (Kashyap et al., 2000; Kapoor et al., 2001). Pectinolytic enzymes
play a key role in the maceration of plant tissues by degrading the pectin
located in the middle lamella and in the primary cell wall of higher plants
(Collmer and Keen, 1986; Kotoujanky, 1987). They are involved in the retting
and degumming of jute, flax, hemp and ramie bast fibers (Bhattacharyya and
Paul, 1976; Sharma, 1987; Baracat et al., 1989; Gillespie, 1990). Cellulose
fibers obtained from ramie are considered as the longest, strongest and
silkiest plant fibers. The fibers are obtained by mechanical removal of bast
from stem of the plant (decortication). Decorticated ramie fibers contain about
20 to 30% incrusting material (gum) consisting mainly of pectin and
hemicellulose. This material is removed in a chemical degumming process by
treatment of the decorticated fibers with hot alkaline solutions with or without
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the application of pressure (Bhattacharyya and Paul, 1976; Das Gupta, 1976).
This process produces polluting effluents and can cause damage of the
fibers. The incrusting material could also be removed by polysaccharide-
degrading microorganisms or their enzymes in a biotechnical degumming
process.
The enzymatic processing result in no damage to the fibers and most
importantly in addition to being energy conservative is environmentally
friendly (Gurucharanam and Deshpande, 1986). A high pH optimum of
pectinase from microorganisms is reported to be desirable for degumming of
plant fibers since a high pH not only prevents contamination but also allows
an open fermentation system to be adopted (Zheng et al., 2001).
Retting Of Plant Fibers
In recent years, a few fundamental studies have been initiated on the
enzymatic retting process. During retting of flax, to separate the fibers and
eliminate pectins generally, Pectinases are used (Hoondal et al., 2000;
Kashyap et al., 2000; Zhang et al., 2000; Soriano et al., 2005).
These employ purified enzymes on defined substrates and
characterization of the resulting products. A pectinase from Rhizomucor
pumilis was used for flax retting (Henriksson et al., 1999). To ensure
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maximum strength of the thread manufactured from retted flax, only a small
fraction of the pectinases belonging to the fiber bundles needs to be
hydrolyzed. In developing nation and particularly in countries where forest
lands are endangered from over exploitation, better use might be made of
herbaceous fibers for paper production. Such feedbacks should be amenable
to enzymatic pulping and the resulting processes should give together yields
with fewer environmental problems.
Pretreatment of Pectic Waste Water
Environmentally, the treatment of waste water from citrus processing
industries containing pectic substances is carried out in multiple steps,
including physical dewatering, chemical coagulation, direct activated sludge
treatment (Hoondal et al., 2000; Jayani et al., 2005; Niture, 2008) and
chemical hydrolysis which lead to formation of methane. These have several
disadvantages, such as the high cost of treatment and longer treatment times
in addition to environmental pollution from the use of chemicals. Thus, an
alternative, cost effective, and environmentally friendly method is the use of
pectinases from bacteria which selectively remove pectic substances from the
waste water. The pretreatment of pectic wastewater from vegetable food
processing industries with alkaline pectinase and alkalophilic pectinolytic
microbes facilitates removal of pertinacious material and renders it suitable
for decomposition by activated sludge treatment (Tanabe et al., 1988;
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Horikoshi, 1990; Bruhlmann et al., 1994; Naidu and Panda et al., 1998;
Kashyap et al., 2000; Hoondal et al., 2000; Gummadi and Panda, 2003 and
2005; Lara-Márquez et al., 2011). An extracellular endopectate lyases from
an alkalophilic soil isolate Bacillus sps. GIR 621 was used effectively to
remove pectic substances from industrial waste water (Tanabe et al., 1988).
Coffee and Tea Fermentation
Pectinase treatment accelerates tea fermentation and also destroys
the foam forming property of instant tea powders by destroying the pectins
(Carr, 1985; Begelis, 1993; Kashyap et al., 2000; Jayani et al., 2005; Niture,
2008). Pectinolytic microorganisms are used in the fermentation of coffee to
remove the mucilaginous coat from the coffee beans (Amorim and Amorim,
1977; Alkorta et al., 1998; Singh et al., 1999a; Kashyap et al., 2001; Serrat et
al., 2002; Silva et al., 2005). Pectinases are some time added to remove the
pulpy bean layer consisting of pectic substances (Schwan and Wheals, 2004;
Ouattara et al., 2010).
Paper and Pulp Industry
With the advancement of biotechnology and increased reliance of
paper and pulp industries on the use of microorganisms and their enzyme for
biobleaching and paper making, the use of enzyme other than xylanases and
ligninases such as mannanase, pectinases is increasing in the paper and
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36
pulp industries in many countries (Kirk and Jefferies, 1996; Bajpai, 1999;
Saadoun et al., 2007). During paper making pectinase can depolymerize
polymers of galacturonic acids and subsequently lower the cationic demand
of pectin solutions and the filtrate from peroxide bleaching (Reid and Ricard,
2000; Viikari et al., 2001; Jayani et al., 2005; Niture, 2008).
An overall bleach-boosting of Eucalyptus kraft pulp was obtained when
alkaline pectinase from Streptomyces sps. QG-11-3 was used in combination
with xylanase from the same organism for biobleaching (Beg et al., 2000).
Thus, the ability of polygalacturonic acid to complex cationic polymers
depends strongly on the degree of polymerization.
Poultry Feed
Intensive research in to the use of various enzymes in animal and
poultry feeds started in the early 1980s. The first commercial success was
addition of α-glucanase in to barley-based feed diets. Usually a feed enzyme
preparation is a multi enzyme cocktail containing glutanases, xylanases,
proteinases, pectinases and amylases. Enzyme addition reduces viscosity
which increases absorption of nutrients, liberates nutrients either by
hydrolysis of non degradable fibers or by liberating nutrients blocking by these
fibers and thus reduces the amount of faeces (Hoondal et al., 2000; Murad et
al., 2011).
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Oil Extraction
Citrus oil such as lemon oil can be extracted with pectinases as this
enzyme destroys the emulsifying properties of pectin which interferes with the
collection of oils from citrus peel extracts (Scott, 1978; Jayani et al., 2005;
Niture, 2008). Plant cell wall degrading enzyme preparation has been used in
olive oil preparation. The enzyme is added during the process of grainding of
olives by which easy removal of oil is accomplished in subsequent separation
procedures. The enzyme treatment not only improves oil yield and stability
but also increases polyphenols and vitamin E content enhancing its
organoleptic quality (West, 1996; Kashyap et al., 2001; Hoondal et al., 2000;
Iconomou et al., 2010).
In Medical field
Pectin use is allowed all around the world and was recommended as a
safe additive by the joint FAO/WHO committee. It has a broad range of
applications as a gelling agent, thickener, texturizer, emulsifier, stabiliser, fat
or sugar replacer in low-calories foods and as a component of many
medicines. In medicine, pectin is used mainly because of its anti-diarrhoea
effect, for lowering of blood cholesterol levels and as a natural prophylactic
substance against poisoning with toxic cations. Because of its biodegradable
and recyclable character, pectin films nowadays find more applications apart
from those within the food and pharmaceutical industries.
Review of Literature
38
Purification of plant viruses
Pectinases have also been reported to work on purification of
viruses (Salazar and Jayasinhe, 1999; Viikari et al., 2001; Reid and Richard,
2004). But they are yet to be commercialized. When virus particle is restricted
to phloem, to release the virus from the tissues, alkaline pectinases and
cellulases are used. This gives very pure preparations of the virus (Salazar
and Jayasinghe, 1999).
Improvement of chromaticity and stability of red wines
Pectinolytic enzymes are added to macerated fruits before the addition
of wine yeast in the process of producing red wine, though they show low
activity (Ducasse et al., 2011). This results in the improvement of colour and
turbidity of wine when compared to the untreated wines. Enzymatically
treated red wines showed chromatic characteristics, which are considered
better than the control wines. These wines also showed greater stability as
compared to the control (Revilla and Ganzalez-san jose, 2003; Jayani et al.,
2005; Niture, 2008). The cell walls degradation in the skin of grapes through
pectolytic enzyme treatment results in an increased release of phenolic
compounds that are responsible for colour (Pinelo et al., 2006; Busse-
Valverde et al., 2011). During fermentation process, it was observed that
there was an improvement in the clarification of the product (van Rensburg
and Pretorius, 2000) and also enhancement in the aroma of the wine (Piñeiro
Review of Literature
39
et al., 2006; Vilanova and Sieiro, 2006). Thus, pectic enzymes help the
breakdown of cell walls in grapes to extract the aromatic precursors, thereby
enhancing the aroma of wines (Gómez-Plaza et al., 2000; Pinelo et al., 2006;
Comitini et al., 2011; du Toit et al., 2011).
Other Uses
Since the 1940s, pectinases have been exploited for many industrial
applications. Pectinases are of prime importance for plants as they help in
cell wall extension (Ward and Moo, 1989; Jacob et al., 2008; Gayathri et al.,
2011) and softening of some plant tissues during maturation and storage
(Aguilar and Huirton, 1990; Sakai, 1992). They also help in maintaining
ecological balance by causing decomposition and recycling of waste plant
materials (Jayani et al., 2005).
In addition, the understanding of the regulation process of the
production of polygalacturonases contributes not only to improve enzyme
production, but also to get insights in the molecular dialogue between the host
and the pathogen during the microbial invasion of plant cell wall (Prade et al.,
1999; Esquerré-Tugayé et al., 2000; Lang and Dörnenburg, 2000).
Pectinases are also used in wood preservation and in maceration,
liquefaction and extraction of vegetable tissues (Bohdziewiez and Bodzek,
1994; Bai et al., 2004). Various literature reports and reviews are available on
Review of Literature
40
the production and applications of pectinases (Sakai et al., 1993; Kashyap et
al., 2001). Few reviews have highlighted the biological and technological
importance of pectinases (Reid and Ricard, 2000; Kashyap et al., 2001;
Favela-Torres et al., 2006; Ahlawat et al., 2007; El-Sheekh et al., 2008).
Actual and potential industrial applications of pectinases were
reviewed earlier (Alkorta et al., 1998; Lang and Dörnenburg, 2000; Kashyap
et al., 2001; Hoondal et al., 2000). The classification of pectinolytic enzymes
as well as some aspects related to their assay methods, purification, and
physico-chemical and biological properties were reviewed (Gummadi and
Panda, 2003; Jayani et al., 2005; Favela-Torres et al., 2005).
So, in recent years there has been also a growing interest in studying
pectic enzymes with very interesting properties from their application point of
view. These may include thermostable pectinases (Swain and Ray, 2010; Kar
and Ray, 2011) or pectinases with optimal activity at low temperatures
(Nakawawa et al., 2004; Cabeza et al., 2011; Merin et al., 2011; Padma et al.,
2011).
Microbial production of pectic transeliminases was reviewed earlier
(Gummadi and Kumar, 2005). However, aspects regarding the most common
microorganisms and processes for hydrolytic depolymerising pectinase
Review of Literature
41
(PGase) production have not been considered until now. Because of the
potential and wide applications of pectinases, there is a need to highlight
recent developments on several aspects related to their production. The aim
of this review is to present an overview of the pectinase activity obtained by
Bacillus subtilis as well as the strategies used to obtain higher activities.
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