Helix Vol.

2: 673-677 (2015)

PRODUCTION, PURIFICATION AND ASSAY OF PECTINASE

ENZYME FROM ASPERGILLUS NIGER
*1Dasari Beulah, 2E. M. Sunitha, 3T.Srilakshmi
* Loyola Academy Degree and P.G College, 2Raja Bahadur Venkat Rama Reddy Womens College, 3Government
1

Degree College, Chevella, Ranga Reddy District

Email: bewlah91@gmail.com

Received: 6th Nov 2014, Accepted: 12th Dec 2014, Published: 1st March

Abstract fruits. Therefore, pectinase enzymes are commonly

Production of microbial enzymes at the industrial used in processes involving the degradation of plant
scale and their commercialization has gained a lot of materials, such as speeding up the extraction of fruit
focus and importance. Use of Microbes as bioreactors juice.
was also started due to these developments. Some of
the industrially important enzymes from microbial At present, almost all the pectinolytic enzymes used
origin include Lipases, Amylases, proteases, for industrial applications are produced by the fungi,
Xylynases and Pectinases. The current work involved namelyAspergillus sp., Rhizopusstolonifer, Alternaria
the use of mice\robes isolated from the decaying fruit mali, Fusariumoxysporum,
peel for the extraction of the Pectinase enzyme. The Neurosporacrassa, Penicillium italicum ACIM F-
organisms were isolated from the fruit peel and were 152, and many others. Orange peels are used as
cultured onto SDA agar Plate as the isolation is of substrate for the growth of Aspergillus niger. The
Fungi. The organisms were screened for the high cost used in the importation of industrial
production of the Pectinase enzyme using Pectin agar extracellular enzymes has led to the high cost of
media. Microscopic identification and Morphology finished industrial products. Besides, orange peels
study revealed that the Fungi isolated was cause waste disposal problems since they are being
Aspergillus niger Pectinase Production media was thrown around indiscriminately. This study is
later used for the Lab scale production of Pectinase therefore aimed at the production of pectinase from
enzyme by inoculating A. niger and incubating for 10 A. niger, purifying enzyme and assaying the enzyme
days. The enzyme thus produced was purified by activity.
several steps including Salt precipitation and
Dialysis. The enzyme thus produced was assayed and Materials and methods
compared with the normal fruit juice extraction Isolation of the Fungi from the Fruit Peels:
production. Quantitative estimation of protein was Few orange peels have been collected from the dump
done using Lowry’s method at660nm to know the of fruit wastes near a local fruit market in L.B Nagar,
concentration of protein. The results thus obtained Hyderabad; The peels were kept damp for few days
can conclude that the use of Aspergillus niger in the in the Zip Lock Bag with minute openings for aim
industrial production of Pectinase is highly passage. Once the decaying of the peels was started
beneficial. The amount of juice extracted in the the growth of fungi started. The peels were cut into
control samples was less compared to the samples small pieces and ground into fine liquid adding dist
extracted using Pectinase enzyme. The enzyme water. This was used as the inoculums for the
pectinase was then preserved using isolation of the organisms from the peel.
microencapsulation technique by Sodium Alginate
and stored at4°C. Pure Culture Preparation:
Sabouraud Dextrose Agar is used specifically for the
Keywords: growth of dermatophytes and other types of fungi.
Extracellular Enzyme, Pectinase, Aspergillus niger, Sabouraud agar typically contains 40 g/L dextrose;
Fruit wastes, purification, Enzyme assay, 10g/L peptone; 20 g/L agar; pH -5.6. The media was
Immobilization. prepared as per the composition and was autoclaved
to sterilize. After the sterilization the media was
Introduction poured into sterile petridishes and was solidified. The
The Pectinases are the enzymes that are capable of previously prepared inoculums was than used to
hydrolyzing the Pectin polysaccharide into smaller inoculate the plates and were incubated at room
fragments. All the peels of the fruits are made of temperature for 4 to 5 days for the mycelia to
pectin layer which can be easily digested by the develop.
microbial pectinases so as to extract the juices of the

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 Helix Vol. 2: 673-677 (2015)

Screening for the Pectinase Enzyme Production: cold condition. Incubate the solution in ice-cold
Pectinase Screening Agar Medium (PSAM) is used conditions for overnight. This technique is useful to
for the selective growth of those microbes which remove large amounts of contaminant proteins and
release pectin. It was prepared as per the following obtain the partially pure protein.
composition
0.3gms/100ml, (NH4)2HPO4,0.2gms/100ml- Dialysis:
KH2PO4,0.3gms/100ml,K2HPO4,0.01gms/100ml- This technique involved activation of the Dialysis
MgSO4, 2.5gms/100ml–Agar ,1gm/100ml –Pectin membrane using sodium bi carbonate and
and PH-4.5 alternatively boiling it in the hot water. Once the
membrane was activated the partially pure extract
After the preparation and sterilization of the media, was added into the dialysis tubing and the tube was
loopful of microbial culture from the master plate sealed to be leak proof. The sealed tubing was
was taken and transferred to petridish with PSAM suspended in water and was incubated overnight to
medium by simple streak plate technique. The plates remove the salt impurities present in the Protein
were Stored at room temperature in inverted position extract.
for microbial growth. After incubation the plates
were screened for the identification of Zones of Pectin Enzyme Assay:
hydrolysis indicating the positive result for Pectinase Pectinesterase activity is measured by increase in free
production. Carboxyl group by titrating against NaOH in the
present of a ph indicator like Phenolphthalein.
Colony Morphology of the Culture and
Microscopy: For assaying pectinesterase activity 20ml of 1%
Different zones of microbial growth are found on the pectin was dissolved in 0.15M NaCl (pH -7) and 4ml
screening plate. Loopful of culture from each zone of crude enzyme extract is taken in a beaker and
was taken and teased onto a glass slide, This was incubated for 1 hour. After incubation, the solution is
stained using Lacto phenol Cotton Blue dye and titrated against 0.02 N NaOH to reach PH 7 using
observed by covering with cover slip under an Phenolphthalein as indicator (colour change from
electron microscope. Morphology of each Mycelia colourless to pink) the heated crude enzyme extract is
was observed under the 40x magnification. The green used as control.
color sprangiophore with the Sproraniospores on the
sporangium indicates the organism to be Aspergillus Pectin esterase activity = Vs – V b (Normality of
niger. NaOH) ×100/Vt

Pectinase enzyme production by liquid state Where, Vs- Volume of NaOH used to titer sample
fermentation: (ml), V b- Volume of NaOH used to titer blank (ml),
Aspergillus niger is placed in a basal medium used V - Volume of incubation mixture (ml), t - Reaction
for pectinase production, the medium consist of: time (min). Pectin esterase activity is expressed as
0.3% (NH4)2HPO4, 0.2%KH2PO4, 0.01%K2HPO4, milli equivalents of NaOH consumed min -1ml -1of
0.01%-MgSO4, 2.5%- Pectin. The type of crude enzyme extract under the assay conditions.
fermentation used was submerged liquid state
Estimation of the Protein Concentration by
fermentation. The culture was incubated for 10-12
Lowry's Method:
days at 25°C.
0.2 ml and 0.4 ml of BSA working standard was
taken in 2 test tubes and made up the volume to 1ml
Crude Enzyme Extraction by Filtration:
using distilled water. Ccrude enzyme, filtered sample
To separate the liquid enzyme mixture and the
and dialysis sample were taken in different
mycelia mat along with the spore bodies’ filtration
concentrations and made up to 1ml using distilled
technique was employed. Filter broth through
water. The test tube with 1 ml distilled water serves
Whatman No. 1 filter paper and then centrifuge at
as blank. 5 ml of Reagent I was added and incubated
10,000 rpm for 15 minutes. This crude enzyme
for 10 minutes at room temperature, after incubation
extract is used for measuring pectinase activity.
0.5 ml of reagent II was added and incubated for 30
minutes at Dark conditions.
Ammonium sulfate precipitation:
The absorbance was measure at 660 nm and the
44 grams of Ammonium sulfate was weighed and
standard graph was plotted. The amount of protein
used for 100ml of suspension. Pinch-by-pinch
present in the given sample was estimated from the
Ammonium sulphate was added in to the enzyme
standard graph.
suspension for complete saturation maintaining ice

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 Helix Vol. 2: 673-677 (2015)

Application of Pectinases in Fruit Juice Fig 1: The colony Morphology of Aspergillus niger
Extraction: on SDA Media:
The aim here is to monitor enzyme activity by
measuring the amount of apple juice released by
pectinase use and compare it with the normal
protocol in the absence of the enzyme.
Apples were chopped into cubes that are roughly
5 mm on a side. It is important to chop the apple into
very small pieces—added surface area helps the
enzyme break down the pectin in the plant cell walls,
releasing more juice. The chopped apples were
divided equally between the two beakers. Sensitive
balance was used to weigh equal amounts of chopped
apple (20gms.) into each beaker. 3 ml of the enzyme
Pectinase was added to one beaker, and 3 ml of water
to the other. The beakers were labeled as "pectinase"
and "water" respectively. Chopped apple pieces were
stirred in each beaker with a glass-stirring rod. Be
sure to wet all of the pieces. The beakers were Growth of Pectin hydrolyzing fungi on SDA media
covered with aluminum foil and were incubated at a with fruit peels inoculums.
40°C water bath for 25–30 minutes. After removing
jars from water bath, a wooden spoon was used to Screening for the Pectin Hydrolysis by the Fungi:
gently stir/squeeze the apple pieces in each. To filter The PSAM medium allowed selective growth of
the juice from the filter paper was used in the funnels. Pectinolytic microbes, different coloured zones were
observed on the plate, microscopy on cells from each
A graph was made using the total volume of juice zone revealed the presence of Aspergillus niger.
produced by each treatment and the weight of apples
used. Fig 2: Growth of Fungi in the Screening Media
PSAM:
Enzyme Immobilization
0.3g of sodium alginate is dissolved in 10ml of
distilled water and vortexed until it dissolves
completely, 3ml of purified pectinase enzyme is
added to the sodium alginate prepared; 0.5g of
calcium chloride is dissolved in 25ml of distilled
water. The pectinase-alginate mixture is dropped into
2%calcium chloride solution with a syringe. In the
calcium chloride solution, alginate polymerizes to
give bead like structure and contains enzyme trapped
in it.

Results and Discussion:

Isolation of Aspergillus niger from contaminated
Peel:
Light green and black colonies are formed on SDA
agar with fruit peel inoculums after 10th day of
inoculation. The cells were densely populated and Pectinase enzyme production by submerged liquid
filamentous growth was seen after 15 days of state fermentation:
inoculation on SDA media. The mycelia thus The Pectinase Production broth containing 2% Pectin
obtained was used for the staining with Lacto phenol was prepared and the fungal pure culture was
Cotton Blue and observed under 40X magnification. inoculated in the production broth. The broth was
Based on the Colony Morphology the Mycelia were incubated for 20 days for the complete production of
identified as Aspergillus niger. the enzyme into the extracellular media.

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 Helix Vol. 2: 673-677 (2015)

Fig 3: The Production Broth with the Fungal Mat: Fig 4: The graph showing the Pectinse Activity

0.05

0.045

0.04

meq.of NaOH consumed

0.035
meq.
0.03 of
NaO
0.025 H
0.02 cons
um…
0.015

0.01

0.005
A light green colored mat is observed on the liquid 0
broth (production medium) after 12 days of
pectin esterase
inoculation. This is the state of consistent growth and
release of secondary metabolites and extracellular enzyme
enzymes.
Fig 5: Quantitative estimation of protein by
Assay of pectinase activity: Lowry’s method:
In order to estimate qualitatively the activity if the
enzyme, Enzyme Assay was performed based on
titrimetric analysis. NaOH was used for titration and
the titer mixture contained Enzyme extract, BSA,
water and Phenolphthalein indicator.
Based upon the titer result and Calculations Pectin
esterase activity was calculated to be
0.043µmol/min/mg.

Pectin esterase activity = Vs – V b

(Normality of NaOH) ×100/Vt
Table 1: Showing the Assay of Pectinase Activity
by Titrimetry
Volume of NaOH used to titrate 45ml
the sample
Volume of NaOH used to titrate 7ml
the blank Concentration in µg/ml
Normality of NaOH 0.02N
The concentration of test sample was calculated to be
Volume of incubation mixture 29ml
24µg/ml
Time 60min
Application in fruit juice extraction:
This step was conducted to find the efficacy of
Based on the above titre values the Pectinase activity
pectinase in juice extraction. 6.3 ml of juice was
was calculated to be 0.043µmol/min/mg
extracted without using pectinase whereas 11 ml of
juice has been extracted using pectinase i.e there is a
twofold increase in extraction of juice as shown in
the Fig6.

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 Helix Vol. 2: 673-677 (2015)

Fig 6: The comparative juice extraction between several techniques like Filtration, Centrifugation, Salt
control and the test pectinase enzyme. precipitation and Dialysis. The purified product was
used for the Enzyme assay which showed that the
activity of the enzyme produced was
Juice extracted 0.043µmol/min/mg. Quantitative estimation of the
without Pectinase enzyme was performed by Lowry’s Method which
showed the concentration of the enzyme to be
24µg/ml. To confirm the applicability of the enzyme,
it was used for the extraction of the apple juice in
Juice extracted comparison with the normal juice extraction in the
with Pectinase
absence of the enzyme. The experiment revealed that
the juice produced in the presence of enzyme was
double the volume of the juice produced using
regular protocol.

The work concludes that the enzyme Pectinase can be

produced from Aspergillus niger and can be used for
There is an extensive increase in the volume of the the industrial extraction of fruit juices to increase the
juice extracted with the use of Pectinase enzyme yield.
when compared to the control sample.
Acknowledgements
Fig 7: Graphical representation of the yield of The Authors would like to express their deep sense of
apple juice: gratitude to Mrs D. Jyothsna of Bioaxis DNA
Research Centre (P) Ltd. for her overwhelming
15
support and guidance throught the project. They
Quantity of juice in ml.

extend their sincere thanks to Mrs. Zehra Samana,

10
Mrs. Suchitra Naidu and their parents for their
water constant and unfailing support.
5
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