BULE HORA UNIVERSITY

COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCE

DEPARTMENT OF BIOTECHNOLOGY

NAME ID NO.

BY ERMIYAS BIHON .......................................RU 5612/13

TITLE: EXTRACTION OF PECTINASE FROM ASPERGILLUS NIGER

ADVISOR: Mr. NUGUSE HOTESSA

A PROPOSAL SUBMITED TO THE DEPARTMENT OF

BIOTECHNOLOGY FOR THE PARTIAL FULFILLMENT OF
BACHELOR OF DEGREE IN BIOTECHNOLOGY (B.SC.)

BULE HORA ; ETHIOPIA

JANUARY,2016E.C
 Catalog
CHAPTER ONE:.........................................................................................................................................1

1.INTRODUCTION....................................................................................................................................1

1.1. Background of the study................................................................................................................1

1.2. Statement of the problem..............................................................................................................2

1.3. Objectives.....................................................................................................................................2

1.3.1. General objective................................................................................................................2

1.3.2. Specific objectives..............................................................................................................2

1.4. Significance of the study...............................................................................................................2

CHAPTER TWO.........................................................................................................................................3

2.: LITERATURE REVIEW.......................................................................................................................3

2.1. Pectinase enzyme..........................................................................................................................3

2.2. The substrate.................................................................................................................................3

2.3. APPLICTION OFPECTINASE....................................................................................................4

2.3.1. Fruit juice extraction...........................................................................................................4

2.3.2.Wine processing...................................................................................................................4

2.3.3. Purification of plant virus...................................................................................................4

2.3.4. Textile processing and bio bleaching of cotton...................................................................4

2.3.5. Waste water treatment........................................................................................................4

2.3.6. Animal feed........................................................................................................................5

2.3.7. Paper and pulp industry......................................................................................................5

2.3.8. Degumming and bast fiber..................................................................................................5

2.4. Microbial Source...........................................................................................................................5

2.5. Culturing Method..........................................................................................................................6

CHAPTER THREE....................................................................................................................................7

3. METHODOLOGY (METHOD AND MATERIAL)...............................................................................7

I
 3.1. Description of Study area..............................................................................................................7

3.2. Research design.............................................................................................................................7

3.3. Sample collection..........................................................................................................................7

3.4. Material and equipment.................................................................................................................7

3.5. Chemicals and reagents.................................................................................................................7

3.6. Procedure......................................................................................................................................7

3.6.1. Media preparation...............................................................................................................7

3.6.2. Isolation and identification of Aspergillus niger.................................................................7

3.6.3. Observation of zone of clearance........................................................................................8

3.6.4. Pectinase enzyme production by solid state fermentation...................................................8

3.6.5. Observation of the effect of the crude pectinase.................................................................8

3.7. TIME SCHEDULE & BUDGET BREAKDOWN....................................................................................9

3.7.1.Time schedul........................................................................................................................9

Table-1........................................................................................................................................................9

3.7.2.Budget estimation..............................................................................................................10

Table-2......................................................................................................................................................10

REFERENCE............................................................................................................................................11

II
CHAPTER ONE:

1.INTRODUCTION
1.1. Background of the study
Enzymes are highly efficient biological catalysts which perform all synthetic and degradative reactions in
living organisms. The enzymes are preferred to chemicals in commercial endeavor mainly because of
their high catalytic power, specific mode of action, stereo specificity and eco-friendly nature and reduce
energy requirement. There is increasing demand to replace some traditional chemical processes with
biotechnological processes involving microorganisms and enzymes such as pectinases. Pectinase or
pectinolytic enzymes are today one of the upcoming enzymes of the commercial sector. It has been
reported that microbial pectinases account for 25% of the global food enzymes sales (Jayani et al.,
2005). Primarily, these enzymes are heterogeneous group of related enzymes that hydrolyzes the pectic
substances, present mostly in plants.

Pectic substances are polysaccharides of high molecular weight with negative charge, appearing mostly
in the middle lamella and the primary cell wall of higher plants, found in the form of calcium pectate and
magnesium pectate. During ripening process, plants generally use pectinase enzymes to hydrolyze some
of the pectin in and between cell wall making the cell weaker and therefore soft and edible .Pectinases
are heterogeneous group of enzymes that act specifically on pectic substances by decreasing
intracellular adhesivity and tissue rigidity. Pectinases have varied important uses in the industries
especially in the juice and food industry. Reports have shown that the treatment of fruit pulps with
pectinases increases juice volume from fruits with 50% reduction in filtration time (Kashyap et al., 2001).
Pectinases has been reported to be useful in the paper and pulp industry (Beg et al., 2001) as well as in
the decomposition and recycling of plant wastes (Jayani et al., 2005).

Pectinolytic enzymes are derived from different sources such as plants, animals and microorganisms.
Different types of microorganisms have been exploited for the production of pectinases because
microorganisms are not influenced by climatic seasonal factors and can be subjected to genetic and
environmental manipulations to increase yield (Vibha and Neelam, 2010). Among the microbial
pectinase the majority are from fungal sources in the global food enzyme sales (Jayani et al., 2005).
Pectinase from fungal sources produce best under acidic pH and low temperature exceeding 450C are
required. From bacteria, Bacillus species have been reported to produce as high as 20-25g/L of pectinase
as compared to other bacterial isolates (Namasivayam et al., 2011). Based on the economical and
ecological roles pectinase enzyme play, highly productive strains are required in the industries to reduce
production cost. As stated earlier almost all the commercial preparation of pectinases are produced
from fungal species. For industrial production of pectinase enzymes, Aspergillus nigeris the most
commonly used fungal species. The present study will be also aimed to extract pectinase from
Aspergillus niger.

1
1.2. Statement of the problem
The ever increasing of environmental pollution poses hazards to the life of humans. In order to make our
life good and healthy, the environment should be protected from pollution. Some industries in Ethiopia
release waste water containing pectinaceus substances to the environment which causes pollution and
bring or create a problem in human health and life . On the other hand other industries like textile
industries uses chemicals in the process which are polluting, toxic and non-degradable like waste
material found the enter gate of our campus . The other thing is, the juice and wine producing industries
faces a problem during the pressing and clarification process and as a result, their product will be low in
yield and quality. as we know debre berhan town is on the way of industrialization and fast growing so
future aspect will be come protected from pollution

1.3. Objectives
1.3.1. General objective
 The objective of this study is the isolation crude pectinase enzyme from Aspergillus niger.

1.3.2. Specific objectives

 To isolate Aspergillus niger from the ground nut (Arachis hypogeal).
 To observe zone of clearance.
 To apply the crude pectinase to the pectin

1.4. Significance of the study

This project shows the importance of pectinase in some industries like juice and textile industries. For
juice industry it contributes to increase the yield and production of various types of juices. It also has an
importance in textile industries by replacing chemicals by pectinase enzyme for scouring purpose which
is nontoxic and biodegradable. The other problem is waste water that is released in to the environment
which contains pectic substances. This waste water pollutes the environment and it is a risk to the
health. So this study is important to show the use of pectinase enzyme to degrade the pectinaceous
substances in the waste water in eco-friendly way.

CHAPTER TWO

2
 2.: LITERATURE REVIEW

2.1. Pectinase enzyme

The enzyme which hydrolyzes specific pectic substances is known as pectic enzymes, pectinase or
pectinolytic enzymes (Blanco et al., 1999). According to the cleavage site, pectinases are
divided into three groups: (1) hydrolases consisting of polygalacturonase, PG (EC 3.2.1.15); (2)
lyase/transeliminases comprising pectinlyase, PNL (EC 4.2.2.10), and pectatelyase, PL (EC
4.2.2.2); (3) pectin esterase, PE (EC 3.1.1.11) (Yadav et al., 2009; Osborne, 2004).

Pectinesterase (PE)

Pectin methyl esterase or pectinesterase (EC 3.1.1.11) catalyzes deesterification of the methoxyl group
of pectin forming pectic acid and methanol. The enzyme acts preferentially on a methyl ester group of
galacturonate unit next to a non-esterified galacturonate unit. It acts before polygalacturonases and
pectatelyases which need nonesterified substrates (Kashyap et al. 2001).

Polygalacturonase (EC 3.2.1.15)

Polygalacturonases (PGases) are the pectinolytic enzymes that catalyse the hydrolytic cleavage of the
polygalacturonic acid chain with the introduction of water across the oxygen bridge (Kashyap et al.
2001).

Pectatelyase (EC 4.2.2.2)1).

Pectatelyase (PGL) cleaves glycosidic linkages preferentially on polygalacturonic acid forming

unsaturated product through trans-elimination reaction. PGL has an absolute requirement of Ca2+ ions.
Hence it is strongly inhibited by chelating agents as EDTA (Jayani et al., 2005).

2.2. The substrate

Pectin a major component of plant cell wall occurs primarily in the non woody parts of which have parts
that are most likely to consume. It also contributes to the firmness and structure of
plant tissues (Sathyanarayana and Panda, 2003).
The generic name of pectic substances is used for referring to four types of molecules: protopectin
(pectic substance in intact tissues), pectinic acid (polygalacturonan containing >0 – 75% methylated
galacturonate units), pectic acid (polygalacturonan that contains negligible amounts of methoxyl groups)
and pectins (pectinic acid with at least 75% methylated galacturonate units). Protopectins are insoluble
in water, while the rest are wholly or partially soluble in water (Alkorta et al., 1998). Pectic substances
represent between 0.5 – 4% of fresh weight of plant material (Jayani et al., 2005; Sakai et al., 1993). In
addition to their role as cementing and lubricating agents in the cell wall of higher plants, they are
responsible for the texture of fruits and vegetables during growth, maturation and their storage (Caffol
and Mohnen, 2009).Furthermore, specific substances are involved in the interaction between plant
hosts and their pathogens (Prade et al., 1999).

3
Pectins have numerous and important application in the feed and pharmaceutical industries. In the feed
sector, it is primarily used as gelling agent, replacing sugar and fats in low calorie food and as nutritional
fiber (Sakai et al., 1993).

2.3. APPLICTION OFPECTINASE

2.3.1. Fruit juice extraction
Blanco et al. (1999) reported that a mixture of pectinase and amylase is used to clarify fruit juice and it
decreases filtration time up to 50%. Similarly Kaur and Kumar (2004) reported that treatment of fruit
pulp with pectinase also showed an increase in fruit juice volume from banana, grapes and apples.
Gailing et al. (2000) demonstrated that pectinase in combination with other enzyme such as cellulase
the pressing efficiency of the fruits for juice extraction. Alkorta et al. (1998) explained that pectinases
that can depolymerize highly esterified found in apples are the major types of enzymes used in apple
juice processing and when it combines with cellulase has been reported to give a juice yield up to 100%.

2.3.2.Wine processing
Wine processing industry also recognizes the importance of acidic pectinases (Roldán et al. 2010), where
the enzyme can be applied at different stages. The addition of pectinases during crushing of the fruits
increases the juice yield and also accelerates the release of anthocyanin into the juice. Pectinase
treatment at the pre-fermentation or fermentation stage, settles out suspended particles. After
fermentation, enzyme is added to the wine to increase its clarity and filtration rate (Kashyap et al. 2001).

2.3.3. Purification of plant virus

Pectinases have also been reported to work on purification of viruses. 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 (Reid
and Ricard, 2000).

2.3.4. Textile processing and bio bleaching of cotton

Hoondal et al.(2000) reported that pectinase have been used in conjugation with amylase, lipase,
cellulase and hemicellulase to remove sizing agents from cotton in a safe and eco-friendly manner,
replacing toxic caustic soda used for the purpose earlier. Bio scouring is a novel process for removal of
non-cellulosic impurities from the fiber with specific enzymes. They concluded that pectinase have been
used for this purpose without any negative side effect on cellulose degradation.

2.3.5. Waste water treatment

The wastewater from the citrus-processing industry contains pectinaceous materials that are barely
decomposed by microbes during the activated-sludge treatment have tried to develop a new
wastewater treatment process by using an alkalophillic microorganism. Hoondal et al.(2000) concluded
that pretreatment of these waste water with pectinolytic enzymes facilitates removal of pectinaceous
materials and renders it soluble decomposition by activated sludge treatment.

4
2.3.6. Animal feed
Pectinases are used in the enzyme cocktail, used for the production of animal feeds. This reduces the
feed viscosity, which increases absorption of nutrients, liberates nutrients, either by hydrolysis of non
biodegradable fibers or by liberating nutrients blocked by these fibers, and
reduces the amount of faeces (Hoondal et al., 2000).

2.3.7. Paper and pulp industry

During paper manufacturing Pectinase is used in depolymerizing polymers of galacturonic acids and also
it lowers the cationic demand of pectic solutions and the filtrate from peroxide bleaching in paper
manufacturing process. Alkaline pectinase from Streptomyces sp. Qg-11-3 was utilized in bleach-
boosting of eucalyptus Kraft pulp. Pectinase was used in combination with xylanase for bio-bleaching
from same organism (Vikari et al., 2000), (Reid and Richard, 2004).

2.3.8. Degumming and bast fiber

Bast fibers are soft fibers were formed in groups outside the xylem, phloem like Ramie and sun hemp.
These fibers hold gum, so it has to be removed for further treatment for textile processing. But,
chemical treatment of degumming is toxic, polluting and non-biodegradable. It can be overcome with
the utilization of pectinase in combination with xylanase can be used for degumming in an eco-friendly
and bio-degradable manner (Jayani et al., 2005).

2.4. Microbial Source

Pectinolytic enzymes can be derived from different sources such as plants, animals and microorganisms
(Whitaker, 1990; Banu et al., 2010; Namasivayam et al., 2011). According to Vibha and Neelam (2010)
different types of microorganisms have been exploited for the production of pectinase because
microorganisms are not influenced by climatic and seasonal factors and can be subjected to genetic and
environmental manipulations to increase yield. As reported in Jayani et al. (2005) microbial pectinases
has been account for 25% of the global food enzymes and majority of these are from fungal sources.

Urmila et al.(2005) reported that the thermophilic Asprgillus fumigates expressed maximum pectinolytic
activities after 2 – 3 days of incubation in a medium containing wheat bran, sucrose, yeast extract and
(NH4)2 SO4 at 50 0C. Whereas Nazneen et al., (2011) concluded that the wild strain Aspergillus niger 1M
-6 has outstanding pectinase producing capability at 40 0C in 60% initial moisture content for 7 days of
incubation in solid state fermentation.

According to Schalleymey et al., (2001); Namasivayam et al. (2011) Bacillus species can produce as high
as 20 – 25 g/L of pectinase as compared to other bacterial isolates. Torimori et al. (2013) reported that
optimum temperature between 50 and 60 0C was obtained for the three Bacillus species with Bacillus
stearothermophilus showing highest activity at 60 0C while Bacillus cereus and Bacillus subtilis both
showed highest activities of pectinase at 50 0C. Elangova et al. (2013) reported that the production and
optimization study with Bacillus cereus requires 37 0C, PH 6.5, pectin (carbon source), yeast extract
(nitrogen source) and 36 hours of incubation time for higher pectinase enzyme production.

5
2.5. Culturing Method
Trejo-Hernandez et al. (1991) and Solis-Pariera et al. (1993) have compared the pectinase yields and
productivity of both solid state fermentation (SSF) and submerged state fermentation (SmF) techniques,
suggesting that solid stat fermentation (SSF) is more productive than submerged state fermentation
(SSF). Similarly, Acuna et al. (1995) reported that production and productivity of pectinase in solid state
fermentation (SSF) were higher than those obtained by submerged state fermentation (SmF).

6
 CHAPTER THREE

3. METHODOLOGY (METHOD AND MATERIAL)

3.1. Description of Study area
The study will be conducted at Bule hora University Biotechnology laboratory Oromo Ethiopia . Bule
hora town is located in southern guji, Oromo Regional State, at 5.6327692° south latitude and from
38.2188455° to longitude and found at 469km south of Addis Ababa(capital city of ethiopia)with an
elevation ranging between 1000 and 2000meters above sea level.Based on the 2007 national census
conducted by the Central Statistical Agency of Ethiopia (CSA).

3.2. Research design

Experimental research will be used because it helps to investigate the potential of Aspergillus niger to
produce pectinase.

3.3. Sample collection

In this study the ground nut (Arachis hypogeal) seeds will be collected from super market in debre
berhan town in order to culture the seed in the nutrient medium for isolation of Aspergillus niger.

3.4. Material and equipment

Petridish will be used for culturing purpose, loop for transferring inoculums to the media, incubator for
maintaining the growth condition, autoclave for sterilization of the media and equipment, centrifuge for
separation of the crude enzyme from the culture broth, Laminar air flow hood for aseptic transfer of
inoculums, Conical flask for media preparation and fermentation, Hot oven for heating the media,
measuring cylinder for measuring the water, Balance for measuring the PDA and salts.

3.5. Chemicals and reagents

PDA , water , pectin (fruit of juice) , NH4(SO4)2 , CaCl2, NaCl2 , MgCl2 ,Antibiotic (chloramphenicol),
Alcohol (70%).

3.6. Procedure

3.6.1. Media preparation

Media will be prepared using Potato Dextrose Agar (PDA) by the standard of 39 g of Potato Dextrose
Agar (PDA) for 1000 ml of water. The media will be boiled in a boiler and it will be autoclaved at 121 0C
for 15 minutes. When the temperature of the media reaches 45 – 50 0C, 0.011 gram of antibiotic
chloramphenicol will be added and then will be poured in to 9 different petridishes and will be solidified.

3.6.2. Isolation and identification of Aspergillus niger

The ground nut seed will be surface sterilized with 70% alcohol for 1 minute,will be followed by
immersion in three successive sterile distilled water for one minute, and then it will placed on potato
dextrose agar (PDA) plate and will be incubated at 25 0C for three days. The fungal colonies will be
transferred to a fresh potato dextrose agar (PDA) plate by using loops and will be incubated for 10 days

7
at 25 0C to obtain pure cultures of Aspergillus niger. The major destination currently separating
Aspergillus niger from other species of Aspergillus is the production of carbon black color.

3.6.3. Observation of zone of clearance

2.023 gram of Potato Dextrose Agar (PDA) and the same amount of fruits of mango will be mixed with
52 ml of distilled water. The mixture will be boiled in a boiler and will be sterilized at 121 0C for 15
minutes in autoclaved . After the media Will be cooled down to about 40-500C 0.0026 gram of
chloramphenicol will be added as antibiotics to inhibit bacterial growth. The media that contain the
substrate will be poured in to the petridishes and will be solidified. After that the pure culture of
Aspergillus niger will be inoculated in to three different regions of the plate and will be incubated at 25
0
C for three days.

3.6.4. Pectinase enzyme production by solid state fermentation

Aspergillus niger will be placed on a solid medium for pectinase production in a canonical flask. The
medium will consists 1g of NaCl2, CaCl2, MgCl2, NH4 (SO4)2 and 4g of pectin (fruits of mango). The type
of fermentation will be solid state fermentation. The mixture will be incubated for 9 days at 25 0C to
observe the production of the pectinase enzyme. After fermentation it will be centrifuged at 10,000 rpm
for 5 minutes.

3.6.5. Observation of the effect of the crude pectinase

The extracted crude enzyme will be applied to fruits of mango (as alternative pectin) to observe the
effect of the enzyme.

3.7. TIME SCHEDULE & BUDGET BREAKDOWN

3.7.1.Time schedul

Table-1
Activity December January February March April May June

8
Material and
chemical

purchase

Literature
review 
Sample 
collection

Media 
preparation

Inoculation 
and transfer


Identification
of aspergillus
niger

Observation 
of zone of
clearance

data analysis

Proposal
comment by

advisor


PROPOSAL
APPROVAL

Preparetion
for defense

Proposal
defense


9
3.7.2.Budget estimation

Table-2
Items Quantity Price Total cost

Agar 4 Gram 500 2000

NH4(SO4)2 2 Gram 550 1100

CaCl,NaCl2 3 Gram 600 1800

MgCl2 4 Gram 700 2100

Antibiotic 0.5 Gram 400 200

Alcohol 3 Litter 250 750

Autoclave ,boiler
centrifugal ,balance
weight ,laminar air
flow(equipment)

Printing,binding, 1 Package 500 500

copy,stationary
materials

Print 50 page 3 150

Transport 10 100 1000

Total 9600

REFERENCE
Alkorta I, C. Garbisu, M.J. Liama and J.L. Serra (1998). Industrial application of pectinase enzymes: A
review. Proc. Biochem. 33:21 – 28.
Banu R.A, Devikm, Gnanaprabhal GR, Pradep Bv, Paliniswami (2010). Production and characterization of
pectinase enzyme from penicillin chrysogenum. Ind.s.sci. Technol.3:377–381.

10
Beg Q.K, Kapporm M, Tiwari R.P., Hoondal G.S. (2001). Bleach boosting of eucalyptus Kraft pulpusing
combination of xylanase and pectinase from Streptomyces Spp. QG-11-3 RasBull. Biochemistry.40:2931-
2944.
Blanco P., SeiroC, and Villa TG (1999).Production of pectic enzymes in yeast. FEMs Microbiology
Letters.11:1-9.
Central statistical agency report. 2007. WWW.csa.gov.com
Chesson A. and Codner RC (1978).The maceration of vegetable tissue by a strain of Bacillus
subtilis.J.Appl.Bacteriol.44:347-364.
Codner R.C (2001). Pectinolytic and cellulolytic enzymes in the microbial modification of plant
tissues.J.Applied.Bacteriol.84:147-160.
Dasari Beulah, E.M. Sunitha, T. Srilaashmi (2015). Production, purification and assay of pectinase enzyme
from Aspergillus niger. Helix.2:673-677.
Elangovan Namasivayam, John Ravindar D, Marioppan K, AkillJiji, Mukash Kumar and RicardJayara
(2013). Production of extracellular pectinase by Bacillus cereus isolated from market solid waste. J.
Bioanal Biomed.3:3.
Fontana, R.C.; Salvador, S.; Silviera, M.M. (2005). Influence of pectin and glucoses on growth and
polygalactronase production by Aspergillus niger in solid state cultivation. J. Ind. Microbial. Biotechnol.
32(8): 371-377.
Gailing M.F, A Guibert and D. Combes (2000). Fractional design applied to enzymatic sugar beet pulp
pressing improvement. Bioprocess Eng. 22:69-74.
Holom B, R.Ekman, R. Sijoholm, C. Eckerman and J. Thoraton (19991). Chemical change in peroxide
bleaching of mechanical pulp. Das paplar A. 45:116-22.
Hoondal GS, RP. Thiwari, N, Dashiyan and Q.K Beg (2000). Microbial alkaline pectinase and their
applications: A review. Appl. Microbiol. Biotechnol. 59:409-418.
Jayani RS, Saxana S, and Gupta R. (2005).Microbial pectinolytic enzyme: A review. Process
Biochemistry.40: 2931-2944.
Kashyap DR, PK. Volhra, S. Chopra and R.Tewari (2001). Application of pectinase in the commercial
sector: A review. Bioresource. Technol. 77:215-227.
Kaur G, S. Kumar and T. Sathyanarayana (2004). Production, characterization and application of
thermostable polygacturonase of a thermophilic mould sporotrichum thermophile Apinis. Bioresource.
Technol. 94: 239-243.
UrmilaPhutela, VikramDhuna, Shohana Sandhu, B.S. Chandha (2005).Pectinase and
polygalacturonaseProduction by a thermophilic Aspergillus fumigatus isolated from de composting
orange Peels. Brazilian Journal of Microbiology.36:63-69

11