Introduction

1

Pectin

Pectin is a structural polysaccharide present in primary cell wall and

middle lamella of fruits and vegetables (Voragen et al., 2003; Prathyusha and

Suneetha, 2011). Pectin or pectic substances are heterogeneous group of high

molecular weight, complex, acidic structural polysaccharides with a backbone of

galacturonic acid residues linked by α-(1-4) linkages (Kapoor et al., 2000;

Kashyap et al., 2001; de Vries and Visser, 2001; Torres-Fanela et al., 2003;

Alphons et al., 2009; Biswapriya das et al., 2011; Elagovan Namasivayam et al.,

2011). They constitute major components of the middle lamella, a thin layer of

adhesive extracellular material found between the primary cell walls of adjacent

young plant cells (Naidu and Panda, 1998; Singh et al., 1999; Kapoor et al.,

2000; Hoondal et al., 2000).

Pectin is known to contain neutral sugars which are present in side chains.

The most common side chain sugars are xylose, galactose and arabinose

(Shembekar et al., 2009). Pectins show widespread commercial use, especially

in the textile industry (Henriksson et al., 1999) and in the food industry as

thickener, texturizer, emulsifier, stabilizer, filler in confections, dairy products, and

bakery products etc (Liu et al., 2006). It is also studied for its potential in drug

delivery, in the pharmaceutical industry (Chambin et al., 2006; Chapin et al.,

2009) and is interesting as a dietary supplementation to humans due to its

possible cholesterol-lowering effect (Pilnik and Voragen, 1970; Fernandez et al.,

1994; Thakur et al., 1997; Schols et al., 2009; Morris et al., 2010). Pectin also

Introduction

2

has a potential in making biodegradable films (Hoagland and Parris, 1996).

Despite these applications, pectins are similar to cellulose and hemicelluloses, in

converting common waste materials to soluble sugars, ethanol (Doran et al.,

2000), and biogas (Hutnan et al., 2000).

Pectins function as ’glue’ that holds the other cell wall polysaccharides,

like cellulose and hemicellulose (i.e. xyloglugan or glucuronarabinoxylan) and

proteins, such as hydroxyproline-rich glycoprotein extension, together. Two

antiparallel pectin chains can be condensed in the cell wall by cross-linking with

Ca2+ ions to form 'junction zones' or the so-called multiple 'eggbox'. In some

species, pectins may be cross-linked to other pectins or non-cellulosic

polysaccharides by ester linkages with dihydroxycinnamic acids such as diferulic

acid. In plants, pectins are present in all stages of development. The composition

depends not only on the species but also on tissue, stage of growth,

maturity and growth conditions. Pectins are heterogeneous with respect to both

chemical structure and molecular weight.

Pectic Substances

Pectic substances are basically classified into four main types based on

the type of modifications of the backbone chain. They are, Protopectin, Pectic

acid, Pectinic acid and Pectin (Kashyap et al., 2001). Protopectin is the water

insoluble parent pectin substance found in the middle lamella of plant tissues

Introduction

3

which yields soluble pectic substances like pectin or pectinic acid upon restricted

hydrolysis. Pectic acid is a group designation applied to pectic substances which

are mostly composed of galacturonans containing negligible amounts of

methoxyl groups. The salts of pectic acid are generally known as pectates.

Pectinic acids are the galacturonans containing various amounts of methoxyl

groups. The salts of pectinic acids are either normal or acid pectinates. Under

suitable conditions, pectinic acids are capable of forming gels with sugars and

acids or if suitably low in methoxyl content with certain metallic ions. Pectins are

the soluble polymeric materials containing pectinic acids as the major

component. They can form insoluble protopectins with other structural

polysaccharides and proteins located in the cell wall (Kashyap et al., 2001).

Pectin substance consists of pectin and pectic acid. Demethylated pectin

is known as pectic acid or polygalacturonic acid. Pectic substances are

commonly amorphous with a degree of polymerization. Compared with young

actively growing tissues, lignified tissues have a low content of pectic

substances. The content of the pectic substances is very low in higher plants.

They are mainly found in fruits and vegetables, constitute a large part of some

algal biomass (up to 30%) and occur in low concentration in forestry or

agricultural residues. Polysaccharides from cell walls of ripe pears were reported

to contain 11.5% pectic substances, 16.1% lignin, 21.4% glucosan, 3.5%

galactan, 1.1% mannan, 21% xylan and 10% arabinan (Horikoshi, 1990).

Introduction

4

There are basically three types of pectic enzymes; de–esterifying

enzymes (pectinesterases), depolymerizing enzymes (hydrolases and lyases),

and protopectinases. They can be further classified according to the following

criteria; whether they cause random cleavage (endo or liquefying or

depolymerizing enzymes) or the cleavage is endwise (exo or saccharifying

enzymes) (Alkorta et al., 1998; Kashyap et al., 2001).

Role of Microbes in Pectinase Production

Enzymes are the Bio-active compounds that regulate many chemical

changes in living tissues (Prathyusha and Suneetha, 2011). Pectinases are a

group of at least seven different enzymatic activities that contribute to the

breakdown of pectin which is a structural polysaccharide found in primary cell

wall and middle lamina of fruits and vegetables. Pectolysis is one of the most

important processes for plant as it plays a role in cell elongation and growth as

well as fruit ripening. Microbial pectolysis is important in plant pathogenesis,

symbiosis and decomposition of plant deposits (Lang and Dornenberg, 2000).

The main source of the microorganisms that produce pectinolytic enzymes are

yeast, bacteria and large varieties of fungi, insects, nematodes and protozoas

(Luh et al., 1951; Whitaker, 1991; Patil and Dayanand, 2006b; Yadav et al.,

2009; Jayani et al., 2010). Thus by breaking down pectin polymer for nutritional

purposes, microbial pectolytic enzymes play an important role in nature (Yadav

Introduction

5

et al., 2009). These enzymes are inducible, produced only when needed and

they contribute to the natural carbon cycle (Hoondal et al., 2000).

Pectic enzymes have two classes namely, pectinesterases and pectin

depolymerases. Pectin esterase has the ability to de-esterify pectin by the

removal of methoxy residues. Pectin depolymerases readily split the main chain

and it was further classified as polygalacturonase (PG) and pectinlyases (PL)

(Haidar and Fazaelipoor, 2010). Thus, pectinases are hydrolytic enzymes, which

hydrolyze the pectin molecules and are readily soluble in water (Ramanujam et

al., 2008).

Microbial pectinases account for 10-25% of the global food and industrial

enzyme sales (Stutzenberger, 1992; Singh et al., 1999; Jayani et al., 2005;

Murad and Azzaz, 2011) and their market is increasing day by day. These are

used extensively for fruit juice clarification, juice extraction, manufacture of pectin

free starch, refinement of vegetable fibers, degumming of natural fibers, waste-

water treatment, curing of coffee, cocoa and tobacco and as an analytical tool in

the assessment of plant products (Alkorta et al., 1998; Singh et al., 1999).

Many enzymes are involved in pectin degradation and they are referred by

several different names, which can be quite confusing. They may be acting either

by hydrolysis or by trans-elimination; the latter performed by lyases (Kashyap et

al., 2001). Polymethylgalacturonase, endopolygalacturonase (pectin

Introduction

6

depolymerase, pectinase, EC 3.2.1.15), exopolygalacturonase (EC 3.2.1.67) and

exopolygalacturanosidase (EC 3.2.1.82) hydrolysing the polygalacturonic acid

chain by addition of water, are all classified under GH28 and are the most

abundant among all the pectinolytic enzymes (Kashyap et al., 2001; Jayani et al.,

2005; da Silva et al., 2005). α-L-rhamnosidases (EC 3.2.1.40, in GH family 28, 78

and 106) hydrolyze rhamnogalacturonan in the pectic backbone. α-L-

arabinofuranosidases (EC 3.2.1.55) found in 5 different GH families hydrolyze

the L-arabinose side-chains and endo-arabinase (EC 3.2.1.99, GH43) acts on

arabinan side-chains in pectin (Takao et al., 2002). These two enzymes operate

synergistically in degrading branched arabinan to yield L-arabinose (Spagnuolo

et al., 1999).

Polysaccharide lyases (PL) which is like GH have been classified under

sequence-related families, cleave the galacturonic acid polymer by α-elimination

and comprises, e.g. polymethylgalacturonate lyase (pectin lyase, EC 4.2.2.10),

polygalacturonate lyase (pectate lyase, EC 4.2.2.2), and exopolygalacturonate

lyase (pectate disaccharide-lyase, EC 4.2.2.9) (http://www.cazy.org; Marin-

Rodriguez et al., 2002; Jayani et al., 2005). Pectinesterase (pectinmethyl

esterase, pectinmethoxylase, (EC 3.1.1.11) de-esterify the methyl ester linkages

of the pectin backbone (Jayani et al., 2005).

Many acidic and alkaline pectinases are found in microbes. Acidic

pectinases are mainly produced by fungi and are widely used in the production

Introduction

7

and clarification of fruit juices, in maceration and solubilization of fruit pulps

(Naidu and Panda, 1998). Bacillus sps. generally produce alkaline pectinases

and are used in several areas, like retting and degumming of fiber crops, textile

proccessing, coffee and tea fermentations, paper and pulp industry, and in oil

extraction (Hoondal et al., 2000).

Pectinases are constitutive or inducible enzymes that can be produced by

both submerged (Aguilar and Huirton, 1990) and solid state fermentation (Acuna-

arguelles et al., 1995). Submerged fermentation means cultivation of

microorganisms on liquid broth. It requires high volumes of water, continuous

agitation and generates lot of effluents. SSF incorporates microbial growth and

product formation on or within particles of a solid substrate (Mudgett, 1986)

under aerobic conditions, in the absence or near absence of free water, and does

not generally require aseptic conditions for enzyme production. Many bacteria

are used in both submerged as well as solid state fermentation for production of

various industrially important products such as citric acid and ethanol which are

generally regarded as safe (GRAS) by United States Food and Drugs

Administration (USFDA) are employed in food industry (Pariza and Foster, 1983).

But comparatively, SSF is serious of advantageous as large quantities of enzyme

being produced (Ramanujam et al., 2008).

Introduction

8

Substrates and various parameters used for the production of Pectinases

Substrates that are employed in the production of enzyme should be solid,

as solid substrate can give good encourage to the growing cells. Substrates

should provide all needed nutrients to the microorganisms for their growth. Other

factors like particle size, moisture levels (Leda et al., 2000; Martin et al., 2004),

concentration of nutrients, pH, temperature, are also to be taken for

consideration. Generally agro-industrial wastes are employed for the pectinase

production. Various substrates that are being used are sugarcane bagasse,

wheat bran, rice bran, wheat straw, rice straw, sorghum stems, saw dust, corn

cobs, sun flower heads, coconut coir pith, banana waste, tea waste, sugar beet

pulp, apple pomade, orange peel, soya bean pulp powder, lemon peel etc (Smith

and Aidoo, 1988; Pilar et al., 1999). The effect of carbon and nitrogen sources

on the productivity of pectinases also can be studied (Friedrich et al., 1992; Maria

et al., 2000; Catarina and Almeida et al., 2003; Patil and Dayanand, 2006a).

Actual and potential industrial applications of pectinases were reviewed

(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 also reviewed (Gummadi and Panda, 2003; Jayani et al., 2005;

Favela-Torres et al., 2005).

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9

Because of the potential and wide applications of pectinases, there is a

need to highlight recent developments on several aspects related to their

production. Microbial production of pectic transeliminases was reviewed

(Gummadi and Kumar, 2005). However, aspects regarding the most common

microorganisms and processes for hydrolytic depolymerising pectinase (PGase)

production have not been considered until now. 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. In this study, we report the

nutritional and environmental conditions required for the production of Pectinase

by Bacillus subtilis using Orange peel powder and Banana peel powder.

The genus Bacillus includes aerobic or facultatively anaerobic, rod

shaped, Gram +ve (Gram variables), endospore forming bacteria that are widely

distributed in the environment (Slepecky and Hemphill, 1991; Goto et al., 2000;

Nazina, et al., 2001). There are many kinds of species which have thermophilic,

psychrophilic, acidophilic, alkalophilic and halophilic properties in the genus

(Nazina et al., 2001). The reclassification of genus Bacillus began in 1991 and

yielded eight genera: Alicyclobacillus, Aneurinibacillus, Bacillus, Brevibacillus,

Gracilibacillus, Paenibacillus, Salibacillus and Virgibacillus (Goto et al., 2000).

These eight genera include more than 100 species that have similar phenotypic

characteristics. Thus identification of them is not easy. In the past Bacillus

species have been identified mainly by morphological and physiological criteria.

However, the discrimination power of phenotypic methods is limited. Randomly

Introduction

10

amplified polymorphic DNA (RAPD) method and the hybridization methods were

effective for detection of a small number of Bacillus species. Over the years, a

data base of 16S rRNA gene has been constructed and it was successfully used

in the differentiation of bacteria (Goto et al., 2000).

The pectinolytic enzymes from microorganisms have generally focused on

induction enzyme production under various conditions, fermentation process,

various substrate purification and characterization and use of this enzyme for

different industrial processes. The enzyme system used by microbes for

metabolizing and for complete breakdown of pectin are most important tools for

elaborating the economical, ecofriendly and green chemical technology for using

pectin polysaccharide in nature.

As there are several biotechnological applications of pectinases, the study

was presently undertaken which is aimed at screening and isolating pectinolytic

bacteria from the soil samples, collected from different areas of Guntur district

and identification of the isolates based on staining, biochemical and molecular

characterization. Further, efforts were also made to optimize the cultural and

environmental conditions for maximizing the yield of an enzyme. Computational

studies of the pectinase enzymes were also carried out using bioinformatic tools.

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11

Objectives of the present study are:

• To isolate bacteria from soil samples that produce pectinase enzyme

using a selective medium after enrichment.

• To identify the bacterial isolates based on biochemical and molecular

characterization by sequencing the 16S rRNA coding gene.

• To optimize various physico-chemical factors such as temperature and pH

that influence growth and production of the pectinase.

• To study the influence of different concentrations of natural substrates and

organic and inorganic nitrogen sources on the rate of enzyme production.

• To undertake the homology modeling of the Polygalacturonase of Bacillus

subtilis by computational studies.

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