review of literature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/8282/8/08_chapter...

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


16

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


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


18

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).


19

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.


20

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


21

(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


22

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


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


24

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


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


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.,


27

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;


28

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).


29

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).


30

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


31

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.


32

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


33

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


34

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;


35

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


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).


37

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.


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


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


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


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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