INTRODUCTION:
Pectinolytic enzymes
or pectinases are a heterogeneous group of enzymes
that hydrolyze the pectic substances present in
plants [1]. They include polygalacturonases, pectin lyase, and pectin methyl esterase that hydrolyze the glycosidic bonds of pectic
substances.
At present almost all the pectinolytic
enzymes used for industrial applications are produced by fungi namely, Aspergillus
sp., Aspergillus japonicus,
Rhizopusstolonifer, Alternariamali,
Fusariumoxysporum, Neurosporacrassa,
Penicillium italicum
ACIM F-152, and many others [2, 3]. Industrial production of this enzyme is carried out using mostly Aspergillus niger.There
are a few reports of pectinase production by
bacterial strains. Some of the bacterial species producing pectinases
are Agrobacterium tumefaciens, Bacteroides thetaiotamicron, Ralstonia solanacearum, and Bacillus sp [4, 5].
Fermentation
technology plays a very important role in the field of Biotechnology for the
production of various enzymes, antibiotics, single cell proteins, various food
products, etc.
In this
study we produce pectinase from biowaste
(orange peels) by solid state fermentation using Aspergillus
niger at 30°C and pH
4.5. Production of this enzyme was affected by nature of solid substrate, level
of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin
supplements to obtain the
maximum yield of Pectinase. Pectinase is extensively used in food processing
industry, souring of cotton, degumming of plant fibers, waste water treatment,
vegetables oil extractions, tea and coffee fermentation, bleaching of paper, in
the alcoholic beverage [7].
As we know the pectinases
used in food industry are mainly produced from the different fungal species
[6]. Most of the fungal pectinase have optimum pH
range between 3.0 and 6.0. This pH range is suitable for fruit juices, which
have almost the same pH, but these enzymes are not suitable for the vegetable
purees or the other preparations which need almost neutral pH range. The
objective of this work is to investigate the production of pectinolytic
enzymes by fungal strains isolated from soil and rotten vegetable samples,
selecting the best species for polygalacturonase
production and optimizing the culture conditions to maximize the enzyme
production as well as their improved pH tolerance. Solid-state fermentation
(SSF) is an attractive technology for enzyme production [8]. In this work, a
SSF process is described for the production of pectinase
by Aspergillus niger and orange peels use as substrate and
carbon source in a solid-state bioreactor. The process consists of three steps.
(1) Selection of wet orange peels for SSF; (2) Selection of dry orange peels
for SSF; (3) Orange peels cut in particle size of 0.7 –2mm and SSF process
was operated in a column-tray bioreactor at 30°C and 75% moisture content for
72 hrs. Maximum dry biomass and maximum specific growth rate of A. niger in SSF suggest as a very
promising process for pectinase production [16].
MATERIALS AND METHODS:
Different
parameters were studied during the work. Fungal species have an ability of
using any kind of nutrient source for its growth. Aspergillus niger strain
ATCC 16404TMwas
collected from Dabur India Ltd. Ghaziabad in lyophilized form use in this study. Pure
culture was grown on PDA
plate, repeatedly subcultured and maintained for one
month for enzyme production studies.
Strain subculture:
Screening and subculturing of the Aspergillus niger strain was done on agar plates. It was formulated
the pectinase screening agar medium (PSAM): 2 gm pectin;
0.6gm Diammonium orthophosphate; 0.4gm KH2PO4;
0.6 gm K2HPO4; 0.02 gm MgSO4 and 5.0 gm agar (for
200 ml). The initial pH of medium was adjusted to 4.5 [13]. This medium was sterilized
for 15 minutes and distributed aseptically in Petri dishes. The Petri dishes
containing PSAM were inoculated and incubated at 30oC for 24 hrs. At
the end of the incubation period, plates were stain with 50 mM
iodine for result.
Substrate
Sources:
A. niger required a lot amount of carbohydrate
source for enzyme productions. Two substrates sources were used for the
productions of Pectinase, (1) Pectin and (2) Wheat
bran. Pectin as substrate source was actually the chemical source available at
various laboratories but in our study we were used the orange peels biowaste as a pectin substrate source, which was used as
major substrate source for the pectinase production.
Wheat bran as substrate was agriculture waste which was used as major substrate
source of carbohydrate.
Solid State
Fermentation:
Orange Peels
as Substrate in Wet Form:
Wet orange peels cut in particle size
of 0.7–2mm.The medium was
composed of 2%NaNO3, 1% K2HPO4, 5% MgSO4,
5%KCl, 0.1% CaCl2, 0.001% FeSO4, 10% wet orange peels,
20% wheat bran and 2% sucrose(for 1000 ml). The initial pH of medium was
adjusted to 4.5.This medium was sterilized at 121oC for 20 minutes
and distributed aseptically in trays. In three trays 300 ml of the medium was
added and inoculated with spore suspensions. The strain culture was incubated
at 30oC for 3 days (72 hrs) in close trays [14].After every 24 hrs
(till 72 hrs) 10 ml medium was separated, filter and culture filtrate used as
source of crude enzyme [9]. After 3 days incubation, culture mediums
(in cake form) were transfer from the tray to hot water containing sodium azide for inhibition the growth of Aspergillus niger and
filter it. After that we obtained pectinase enzyme
with the mixture of other enzymes like amylase, cellulase.
Then separate it.
Orange Peels
as Substrate in Dry Form:
First dry the orange peels in oven, cut the peels in
particle size of 0.7–2mm and finally grind to made in powder form. The medium was composed of 2% NaNO3,
1% K2HPO4, 5% MgSO4, 5% KCl,
0.1% CaCl2, 0.001% FeSO4, 10% wet orange peels, 20% wheat
bran and 2% sucrose (for 1000 ml). The initial pH of medium was adjusted to
4.5. This medium was sterilized at 121oC for 20 minutes and
distributed aseptically in trays. In three trays 300 ml of the medium was added
and inoculated with spore suspensions. The strain culture was incubated at 30oC
for 3 days (72 hrs) in close trays [14]. After every 24 hrs (till 72 hrs) 10 ml
medium was separated, filter and culture filtrate used as source of crude
enzyme [9]. After 3 days incubation, culture mediums (in cake form) were
transfer from the tray to hot water containing sodium azide
for inhibition the growth of Aspergillus niger and filter it. After
that we obtained pectinase enzyme with the mixture of
other enzymes like amylase, cellulase. Then separate
it.
Downstream Processing:
I) Partial
Purification: Culture filtrate was cooled to 40oC for 30 min.
Treated with three volumes of chilled ethanol and allowed to stand for 15 min.
The precipitation obtain by centrifugation (5000 rpm for 10 min) was dissolved
in distilled water and used for further investigation.
II) Extraction
of crude enzyme: culture filtrate centrifuged at 10000 rpm for 10 min at 27oC.
Supernatant was taken in test tube. Supernatant is used for further
investigation.
Enzyme Assay:
The Standard
protocol of Sigma Quality Control Department was used for the enzyme Assay. 0.5
% pectin solution, 50 mM iodine with 200 mM potassium iodide, 1 M sodium carbonate, 2 M sulfuric
acid, 100 mM sodium thiosulfate,
1% starch indicator, Pectinase solution.
Table 1-
Enzyme Assay
|
Regent |
Test |
Blank |
|
Pectin
solution |
4.90 |
5 .00 |
|
Pectinase |
0.10 |
|
|
I2/KI |
5.0 |
5.0 |
|
Na2CO3 |
1.0 |
1.0 |
|
H2SO4 |
2.0 |
2.0 |
Mix by swirling
the mixture. Titrated the Test and Blank with reagent Na2S2O3
until it is light yellow. Then added 1 drop of Starch indicator and
continuously titrate it with reagent Na2S2O3
until solution becomes colourless [10].
Ammonium Sulfate
Fractionation:
Various
percentage of Ammonium Sulfate was being used for the precipitation of the
enzyme sample.10 ml crude enzyme solution (Cell free filtrate) was taken in
centrifuged tube. Then added (40%, 50 %, 65 %, and 70%)
ammonium sulphate to enzyme solution. Keep at
4oC for overnight. Then centrifuged at 12000 g for 15mins Pellet was
dissolved using sodium acetate buffer. Precipitated enzyme was purified and
separated by using filtration, SDS PAGE and chromatography procedures.
By Ultra filteration:
After filtering, 1600 mL
crude solution of pectinases was concentrated to 300 mL using the hollow fibre cross-flow ultra-filtration
module fit with a polysulfone membrane.
Affinity Chromatography:
The crude enzyme was purified by affinity chromatography
on a column (1.6 cm×20 cm) packed with pectic biomass
powder cross-linked by epichlorhydrin [17].
Determination of Protein:
The protein was determined by the method of Lowry et
al.[12] with bovine serum albumin (Pharmacia) as a
standard. Protein in the column effluents was monitored by an online UV
detector at 280 nm.
SDS−Polyacrylamide Gel
Electrophoresis (PAGE):
SDS−PAGE was performed on a 10% gel according to
Laemmli[11]
to check the homogeneity of the enzyme and determine the molecular weight.
Protein bands were visualized by staining with coomassie
brilliant blue R250. Low range (14.4∼97.4 kDa) molecular weight markers (Pharmacia) were used for
molecular weight of purified endo-polygalacturonase.
RESULTS AND DISCUSSION:
Screening of Strains:
Aspergillus niger (ATCC 16404TM) demonstrated a large zone of hydrolysis
around the large colony on pectin agar medium. The zone of clearance was seen,
degradation was evidenced by a clear zone around fugal growth (Diameter of zone
of clearance was 4.4 mm).
Maximum specific growth rate of A. niger was obtained when
culture medium pH adjust to 4.5. A. niger culture medium pH 4.5 in
SSF was suggest as a very promising process for pectinase
production.
Figure1-Aspergillus niger culture (ATCC 16404TM).
Enzymatic
Assay:
Crude enzyme:
After 3 days
incubation, culture mediums we obtained pectinase
enzyme with the mixture of other enzymes like amylase, cellulase.
Figure 2-
Graphical Comparison of enzymes obtained from dry (orange peels).
The strain obtained
from ATCC shows similar activity when pectin was used as substrate. The enzyme
used for enzymatic assay was crude enzyme.The enzyme Pectinase showed maximum activity after 48 hours ofincubation and later on started depleting when the
incubation time was increased. Partially Purified enzyme.
Figure 3-
Graphical Comparison of pectinase obtained from wet
and dry (orange peels).
The enzyme shows
maximum activity when dry substrate used in comparison of wet substrate used.
The enzyme used for enzymatic assay was partially purified. The enzyme Pectinase showed maximum activity after 48 hours of
incubation and later on started depleting when the incubation time was
increased. Results showed that high levels of pectinase
activities were obtained from dry substrate after 48hrs incubation period of A. niger.
The maximum pectinase activity obtained was
70 U/ml.
Ammonium
Sulfate Fractionation Result:
It is often used
to remove proteins other than the one being purified. The technique involves
adding increasing concentrations of ammonium sulfate to the protein solution
and centrifuging out the precipitated material in certain concentration ranges.
Highest degree of precipitations was achieved by 65 % concentration of ammonium
sulphate. Ammonium sulfate fractionation; proteins
can be further purified by the sequential application of chromatography.
SDS PAGE:
The Samples with
the highest O.D. readings were used for inoculating on the SDS –PAGE. The
Molecular weight of the Protein was found to be 40KDa .The samples used for the
inoculation on the PAGE are mentioned below:
Lane 1:
Molecular Marker
Lane 2: 0.05 M TrisHCl + 0.1 M NaCl (Fraction
No.2)
Lane 3: 0.05 M TrisHCl + 0.2 M NaCl (Fraction
No.2)
Lane 4: 0.05 M TrisHCl + 0.4 M NaCl (Fraction
No.2)
Lane 5: 0.05 M TrisHCl + 0.5 M NaCl (Fraction
No.5)
Lane 6: 0.05 M TrisHCl + 0.2 M NaCl (Fraction
No.3)
Lane 7: 0.05 M TrisHCl + 0.4 M NaCl (Fraction
No.3)
1 2 3
4 5 6
7
Figure 4-
Lane 3, 4 and 5 did not show any bands. Lane 6 and 7 were showing light bands
that could be other proteins. Lane 2 showed only one band which had molecular
weight between 36 – 50Kda.
According to
studies [15] and result which was obtained from SDS-PAGE that shows the molecular
weight of Pectinase is 40 KDA.
CONCLUSION:
In the present study, Aspergillus niger, a fungus isolated from soil,
produced good amount of pectinase activity after 48
hrs of
incubation in production medium at 30°C and pH 4.5. Maximum enzyme production
was obtained from dry substrate of orange peels as pectine
source and wheat bran as carbohydrate source used. So the methods used above
for commercial production of Pectinase can therefore
be used for industrial level production. Thus, enzyme prepared does not require
any specific environmental condition due to that this technology can be easily
adopted for commercialization. Its manufacturing is very cheaper. It can be a
milestone in enzyme industry to fulfil demand supply. Due to this excellent
quality this enzyme can be manufactured in any region of our country.
ACKNOWLEDGEMENT:
We are thankful to Dabur
India Ltd. Company, Ghaziabad, for providing Laboratory facilities to carry out
this work.
REFERENCES:
1.
Fogarty WM, Kelly CT. Pectic
enzymes. In: Fogarty WM, editor. Microbial Enzymes and Biotechnology. London,
UK: Applied Science Publishers; 1983. pp. 131–182.
2.
Souza JVB, Silva ÉS, Maia MLS, Teixeira MFS. Screening of fungal strains for pectinolytic activity: endopolygalacturonase
production by Peacilomycesclavisporus 2A.UMIDA.1. Process
Biochemistry.2003;39(4):455–458.
3.
Jayani RS, Saxena S, Gupta R. Microbial pectinolytic
enzymes: a review. Process Biochemistry.2005;40(9):2931–2944.
4.
Soares MMCN, Da Silva R, Gomes E. Screening of bacterial strains for pectinolytic activity: characterization of the polygalacturonase produced by Bacillus sp. Revista de Microbiologia.1999;30(4):299–303.
5.
Kashyap DR, Soni SK, Tewari R. Enhanced
production of pectinase by Bacillus sp. DT7 using
solid state fermentation. Bioresource
Technology. 2003;88(3):251–254.
6.
Loera O,
Aguirre J, Viniegra-González G. Pectinase
production by a diploid construct from two Aspergillus niger overproducing
mutants. Enzyme and Microbial Technology. 1999;25(1-2):103–108.
7.
Yugandhar
N.M., Ravi Kumar D.V.R., Prasanthi V., Kiran Kumar N. and Sri Rami Reddy
D. (2008) ResearchJournal of Microbiology, 3 (1),
9-16.
8.
Héctor A. Ruiz, Rosa M. Rodríguez-Jasso,Raúl Rodríguez, Juan C. Contreras-Esquivel, Cristóbal N. Aguilar, Pectinase production from lemon peel pomace as support
and carbon source in solid-state fermentation column-tray bioreactor; Biochemical
Engineering Journal, Volume 65,
15 June 2012, Pages 90–95.
9.
Gretty K. Villena and Marcel Gutiérrez-Correa
(2007) Electronic Journal of Biotechnology, 10, 1.
10.
Sigma
Quality control test procedure: Enzymatic assay of Pectinase
(1997) EC 3.2.1.15, 1-4.
11. Laemmli-SDS-PAGE Biochemistry,
Protein,
Protein electrophoresis; Bio-Protocols, 2011, 5340
12. Lowry
OH, Rosebrough NJ, Farr AL, Randall RJ (November
1951). "Protein measurement with the Folin
phenol reagent". J. Biol. Chem.193
(1): 265–75.
13. Aguilar
G, Huitron C. Stimulation of the production of
extracellular pectinolytic activities of Aspergillus sp. by galacturonic
acid and glucose addition. Enzyme and Microbial Technology.1987;9(11):690–696.
14. Maldonado
MC, Callieri DAS. Influence of environmental
conditions on the production of pectinesterase and polygalacturonase by Aspergillus niger. MIRCEN
Journal of Applied Microbiology and Biotechnology. 1989;5(3):327–333.
15.
KalyaniMondal, Payal Mehta and Munishwar
N. Gupta (2004) Protein Expression and Purification, 33, 104–109.
16. Aguilar
G, Huitron C. Application of fed-batch cultures in
the production of extracellular pectinases by Aspergillus sp. Enzyme and Microbial Technology. 1986;8(9):541–545.
17.
Chiba Y, Kobayashim;
Substrate binding ability of chemically inactivated Pectinase
for the substrate pectin acid. Biosci. Biotechnol. Biochem.; 1995 Jul; 59 (7); 1242-5.
Received on 05.09.2014 Modified on 16.09.2014
Accepted on
19.09.2014 ©A&V Publications All right reserved
Research J.
Science and Tech. 6(4):
Oct.
- Dec.2014; Page 194-198