INTRODUCTON

Introduction
Pectinase is an enzyme that breaks down pectin, a polysaccharide found in plant cell walls.
Commonly referred to as pectic enzymes, they include pectolyase, pectozyme,
and polygalacturonase, one of the most studied and widely used commercial pectinases. It is
useful because pectin is the jelly-like matrix which helps cement plant cells together and in
which other cell wall components, such as cellulose fibrils, are embedded. Therefore, pectinase
enzymes are commonly used in processes involving the degradation of plant materials, such as
speeding up the extraction of fruit juice from fruit, including apples and sapota. Pectinases have
also been used in wine production since the 1960s.[1] The function of pectinase in brewing is
twofold, first it helps break down the plant (typically fruit) material and so helps the extraction of
flavors from the mash. Secondly the presence of pectin in finished wine causes a haze or slight
cloudiness. Pectinase is used to break this down and so clear the wine.

They can be extracted from fungi such as Aspergillus Niger. The fungus produces these enzymes
to break down the middle lamella in plants so that it can extract nutrients from the plant tissues
and insert fungal hyphae. If pectinase is boiled it is denatured (unfolded) making it harder to
connect with the pectin at the active site, and produce as much juice.

Pectinases are also used for retting. Addition of chelating agents or pretreatment of the plant
material with acid enhance the effect of the enzyme.

The enzyme which hydrolyze pectic substances are known as pectic enzymes, pectinases or
pectinolyitc enzymes. Pectolytic enzymes are a group of hydrolyses found in higher plants and
several microorganisms that hydrolyses the glycosidic bonds of pectic substances. There is
following substrate involve in pectolytic enzymes: 1) Pectic substances are carbohydrate
polymers usually foundd in the middle lamella of the cell wall of higher plants which confer
structural texture to the plant organ. 2) In young fruits, the pectic substance is known as
protopactin which is insoluble and has no organized structure. 3) Maturation leads to its
conversion to pectin. Ripening converts pectin into soluble products which causes fruits to
soften.

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Types of reaction and sources: -

There are 4 major pectolytic enzymes. These are

1. Pectin methyl esterase(PME) [ methoxygalacturonide] lyase

2. Polygalacturonase (PG) (poly [ 1, 4 –α- D galacturonide glucano hydrolase]
3. Pectin lyase (pectin depolymerase, PL)
4. Pectate (pectic acid) lyase

Pectolytic enzymes play an important role in food processing industries and alcoholic
beverage industries. These enzymes degrade pectin and reduce the viscosity of the solution
so that it can be handled easily. These enzymes are mainly synthesized by plants and
microorganism. It also deals with the effect of operating parameters such as temperature,
aeration rate, agitation and type of fermentation on the production of these enzymes

Fig.1 Structure of pectin

Fig. 2: Reaction of Pectolytic enzyme.

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Pectolytic enzymes:

The pectolytic enzyme consist of a main backbone containing a variable but normally large
proportion of partially methyl-esterified galacturonic acidsubunits linked by

α-1, 4 glycosidic linkages demethylated compound

Pectin pectic

The enzymes which hydrolyze pectic substances are known as pectic enzymes, pectinases or
pectinolytic enzymes [Hankin L., 1971]. Based on its mode of action and substrate preference
these enzymes are classified into three types:

I. Protopectinases, which solubilize protopectin forming soluble pectin.

II. Esterase’s (pectin methyl esterase’s and pectin acetyl esterase’s), which eliminate
methoxy and acetyl residues from pectin giving rise to polygalacturonic acid.
III. III. Depolymerases, which break the glycosidic α- (1- 4) bonds between galacturonic
residues Via:

1. Hydrolysis (polygalacturonase)

2. Trans elimination (pectin lyases and pectate lyases)

Also, the latter enzymes are subdivided into endo- if its pattern of action is random or exo- if
its pattern of action is at the terminal end [Fogarty, 1983].

Pectic substances:
Pectin substance consists of pectin and pectic acid. The main chain of pectin is partially
methyl-esterified-1, 4, D-galacturonan. Demetylated pectin is known as pectic acid or
polygalacturonic acid [Arunachalam and Asha, 2010]. Sixty-seven cultures of plant
pathogenic bacteria, of the genera Agrobacterium, Corynebacterium, Erwinia, Pseudomonas
and Xanthomonas, and 56 cultures of non-pathogenic bacteria were tested for production of
pectic enzymes in culture. γ-Pectin glycosidase wasproduced by 22 strains of pathogenic

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bacteria and by 15 non-pathogens. Pectin methyl esterase was produced by a few pathogens:
the soft-rot erwinias, X.campestris and one of three strains of X. vasculorum [W.K. Smith,
1958]. The synthesis of pectic substances occurs in the Golgi apparatus from UDP-D-
galcturonic acid during early stages of growth in young enlarging cell walls [Sakai, T., 1993].
The preferred substrate is polygalacturonic acid rather than pectin; the enzyme, provisionally
named polygalacturonic-trans-eliminase, is activated by calcium. [Pilar Blanco, 1999].

Fig. 3: structure of primary cell wall which degrade by Pectolytic enzyme

Role of Microbes in Pectinase Production:

Bacillus:
Bacillus is known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive
bacterium. A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form
tough, protective endospore, allowing the organism to tolerate extreme environmental condition

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Fig. 4: Microscopic image of Bacillus

Pectolytic enzymes are wide spread in nature and are produced by Bacteria, Fungi, Yeast,
Insects, Nematodes and Protozoa. For example, Bacteria like Bacillus species, Clostridium
species, Fungi like Aspergillus species, Penicillin species, Yeast like Saccharomyces, Candida
etc., microbial Pectolysis is important in plant pathogenesis, symbiosis and decomposition of
plant deposits [Lang, C. 2000].
 Pectin is also known to contain other neutral sugars which are present in side chains. The
most common side chain sugars are xylose, galactose andarabinose [Shembekaret al., 2009]
Pectinase 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 . The main source of the microorganisms that
produce pectinolytic enzymes are yeast, bacteria and large varieties of fungi sendo
polygalacturonase production was first reported in 1951 using Saccharomyces fragilis [Luh,
B.S.,1951]
 Pectic enzymes have two classes namely pectinesterases and pectin depolymerases. Pectin
esterase has the ability to de-esterifypectin by theremoval of methoxy residues. Pectin
depolymerases readily split the main chain and it wasfurtherclassified as polygalacturonase
(PG) and pectinlyase (PL). Thus on the whole pectinases are hydrolytic enzymes, which
hydrolyze the pectin molecules and are readily soluble in water [Ramanunjam, N., 2008].

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Pectin substance consists of pectin and pectic acid. The main chain of pectin is partially
methyl esterified-1, 4, D-galacturonan. Demetylated pectin is known as pectic acid or
polygalacturonic acid. Pectic substances are commonly amorphous; with a degree of
polymerization of about 200-400 substituent’s and be found at the C-2 OR C-3 position of
the main chain. The synthesis of pectic substances occurs in the Golgi apparatus from UDP-
D-galcturonic acid during early.

DENTAL CARIES:

Dental caries is one of the most common chronic infectious diseases in the world. Despite
advancements in oral disease science, dental caries continues to be a worldwide health concern,
affecting humans of all ages, especially children where caries disease is on the rise.1 According
to a recent national study, dental caries continues to affect a large number of Americans in all
age groups, with tooth decay in primary teeth increasing among children aged 2-5 years.2 This
National Health and Nutrition Examination Survey (1999-2004) reported that 42% of children,
aged 2-11, have had carious lesions in their primary teeth and 21% of children, aged 6-11, have
had carious lesions in their permanent dentition. Approximately 59% of adolescents, aged 12-19,
have experienced dental caries and by adulthood, aged 20-75+, well over 92% of those surveyed
have experienced dental caries in their permanent dentition. These statistics demonstrate that
although it is not always obvious in an exam (a snapshot in time), the war on tooth decay is being
lost as we age. Current science demands that dental caries be treated as a disease, rather than
with tooth repair alone.

Dental caries is an infectious bacterial biofilm disease which is expressed in a predominantly

pathologic oral environment. Although acid generating bacteria are the etiologic agents, dental
caries has been thought of as multifactorial since it is influenced by dietary and host factors as
well.5 In addition, the role of saliva as a defense system against dental caries is well
documented. These defense systems include clearance, buffering, antimicrobial agents, and
calcium and phosphate delivery for remineralization to name a few.

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Fig. 5: Destruction of a tooth by dental caries.

Dental caries is also known as tooth decay, cavities, or caries, is a breakdown ofteeth due to
activities of bacteria. The cavities may be a number of different colors from yellow to black. [2]
Symptoms may include pain and difficulty with eating. Complications may include inflammation
of the tissue around the tooth, tooth loss, and infection or abscess formation.

The cause of caries is bacterial breakdown of the hard tissues of the teeth (enamel, dentin and
cementum). This occurs due to acid made from food debris or sugar on the tooth surface. Simple
sugars in food are these bacteria's primary energy source and thus a diet high in simple sugar is a
risk factor. If mineral breakdown is greater than build up from sources such as saliva, caries
results. Risk factors include conditions that result in less saliva such as: diabetes mellitus,
Sjogren's syndrome and some medications. Medications that decrease saliva production include
antihistamines and antidepressants among others.Caries is also associated with poverty, poor
cleaning of the mouth, and receding gums resulting in exposure of the roots of the teeth.

Prevention includes: regular cleaning of the teeth, a diet low in sugar, and small amounts of
fluoride. Brushing the teeth two times per day and flossing between the teeth once a day is
recommended by many. Fluoride may be from water, salt or toothpaste among other sources.
Treating a mother's dental caries may decrease the risk in her children by decreasing the numbers
of certain bacteria. Screening can result in earlier detection. Depending on the extent of
destruction, various treatments can be used to restore the tooth to proper function or the tooth
may be removed. There is no known method to grow back large amounts of tooth.[7] The
availability of treatment is often poor in the developing world.[3] Paracetamol (acetaminophen) or
ibuprofen may be taken for pain.

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Disc diffusion method:

The Kirby-Bauer test, known as the disk-diffusion method, is the most widely used antibiotic
susceptibility test in determine what treatment of antibiotics should be used when treating an
infection. This method relies on the inhibition of bacterial growth measured under standard
conditions. For this test, a culture medium, specifically the Mueller-Hinton agar, is uniformly
and aseptically inoculated with the test organism and then filter paper discs, which are
impregnated with a specific concentration of a particular antibiotic, is placed on the
medium. The organism will grow on the agar plate while the antibiotic “works” to inhibit the
growth. If the organism is susceptible to a specific antibiotic, there will be no growth around the
disc containing the antibiotic. Thus, a “zone of inhibition” can be observed and measured to
determine the susceptibility to an antibiotic for that particular organism.

Antimicrobial:

An antimicrobial is an agent that kills microorganisms or inhibits their growth. Antimicrobial

medicines can be grouped according tothe microorganisms they act primarily against. For
example, antibacterial (commonly known as antibiotics) are used against bacteria and antifungal
are used against fungi. They can also be classed according to their function. Antimicrobials that
kill microbes are called microbicidal; those that merely inhibit their growth are called micro
biostatic. Disinfectants suchas bleach are non-selective antimicrobials.

Antagonistic activity:

One who opposes and contends against another; an adversary. Antagonistic activity was detected
of pectolytic enzyme which against biofilm forming bacteria isolated from dental carries by disc
diffusion method and observed the susceptibility of microbes against oral pathogen.

With the above literature theirs keeping in my mind my work plan and I designed my work is
Antagonistic activity of pectolytic enzyme can also observed and I worked the activity of
pectolytic enzyme against E. coli and pseudomonas.

Optimum environment:

As with all enzymes, pectinases have an optimum temperature and pH at which they are most
active. For example, a commercial pectinase might typically be activated at 45 to 55 °C and work

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well at a pH of 3.0 to 6.5.Pectinase is commonly used in fruit industries to speed up fruit juice
extraction.

Uses:

Pectinase enzymes are used for extracting juice from purée. This is done when the enzyme
pectinase breaks down the substrate pectin and the juice is extracted. The enzyme pectinase
lowers the activation energy needed for the juice to be produced and catalyzes the reaction.

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