EXTRACELLULAR PECTINASE PRODUCTION
BY Aspergillus niger HFM 8 THROUGH SOLID
SUBSTRATE FERMENTATION USING
POMELO PEELS AS A SUBSTRATE
NOR HAWANI BINTI SALIKIN
UNIVERSITI SAINS MALAYSIA
2014
EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus niger
HFM 8 THROUGH SOLID SUBSTRATE FERMENTATION USING
POMELO PEELS AS A SUBSTRATE
by
NOR HAWANI BINTI SALIKIN
Thesis submitted in fulfilment of the requirement for the degree of Master
of Science
August 2014
ii
ACKNOWLEDGEMENT
By The Name of Allah The Al-Mighty
First of all, Alhamdulillah, the most sincere gratitude to Allah The Al-
Mighty. Only with His blessing and endless help, this research could be successfully
completed even though life is sometime harsh and wretched. Think positively,
willing to face the saddle and only with His permission everything is going
smoothly.
A big appreciation goes to my inspiring supervisor, Professor Darah Binti
Ibrahim. The quality of her supervision is the best. Thank you for the precious
advice, guidance and motivation that brace my enthusiasm in accomplishing this
research. Thank you indeed.
The deepest gratitude also goes to Associate Professor Dr. Rosma Binti
Ahmad for her precious motivations and indulgence which instigate me to do the
best. Also for Puan Noor Hasmah Binti Idris and Puan Nazariah binti Noordin from
School of Industrial Technology who are very supportive and thoughtful. Really
appreciate all of your kindness.
My fully appreciation is also dedicated to Universiti Sains Malaysia and
Malaysia Ministry Of Higher Education for the splendid opportunity given to me in
pursuing this Master degree with fully financial support under the scheme of ASTS
USM or SLAB. I also would like to thank all of the lecturers and staffs in School Of
Biological Science especially in the Microscopy Unit for their support and help.
iii
Also, my full appreciation is devoted to my beloved parents, En. Salikin Bin
Haji Ihsan and Puan Marinah Binti Bibit who are inspiring me to be a positive-
minded individual and always standing beside me in every moment of my life. Also
to my beloved husband, En. Hasrul Hafizan Bin Rahmatullah, thank you very much
for your understanding and endless encouragement. Indeed, this victory is a reflect to
their blessing, love and endless prayer that I received from them. To my lovely kids;
Nur Iman Batrisya and Fawwaz Ziqri, both of you are heartening my life and
fortifying me to struggle.
My special thanks also goes to my understanding and happy family of IBRL
(Industrial Biotechnology Research Laboratory) especially Dr. Lim Sheh Hong (Post
Doctorate), Dr. Lee Chee Keong, Syarifah, Lee Kok Chang, Tong Woei Yen, Nisha,
Hareetharan, Anuradha, Taufiq, Norhaida, Tisha, Afifah, Chong Chai Lee, Nor
Azlinah, Husna, Yu, Angeline, Olivia, Mat and Gee who have been sharing the joys
and tears together. The moments we share are definitely unforgettable.
My apologies and request of forgiveness for those names never mentioned. I
would have never been able to complete this project without all of the blessings and
support from you. Again, thank you all for making this as a reality.
Wassalam
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iv
LIST OF TABLES xiv
LIST OF FIGURES xvi
LIST OF PLATES xix
LIST OF SYMBOLS AND ABBREVIATION xxi
ABSTRAK xxiii
ABSTRACT xxv
CHAPTER ONE : INTRODUCTION
1.1 General introduction on pectinase research 1
1.2 Rational of study 4
1.3 Objective of research 5
CHAPTER TWO : LITERATURE REVIEW
2.1 Growth of agricultural industry worldwide 6
2.2 Agro-industrial residues as a detrimental issue 7
2.3 Utilization of agro industrial residues in pectinase production 10
2.4 Pomelo peel as a potential substrate for pectinase production 13
2.5 Pectic substances 15
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2.5.1 Classification of pectic substances 17
2.5.1.1 Protopectin 17
2.5.1.2 Pectic acid 18
2.5.1.3 Pectinic acid 18
2.5.1.4 Pectin 19
2.5.2 Structure of pectin 21
2.5.2.1 Primary structure of pectin 21
2.5.2.2 Secondary structure of pectin 22
2.6 Pectinase 23
2.7 Classification of pectinase enzyme 26
2.7.1 Polygalacturonase (PG) 26
2.7.2 Polymethylgalacturonase (PMG) 27
2.7.3 Protopectinase 27
2.7.4 Pectate lyases (PGL) 28
2.7.5 Pectin lyases (PL) 28
2.7.6 Pectin methyl esterase (PME) 29
2.7.7 Rhamnogalacturonan rhamnohydrolases 30
2.7.8 Rhamnogalacturonan acetylesterases 30
2.8 Microorganisms as potential pectinase producer 33
2.8.1 Fungi as a potential pectinase producer 34
2.8.2 Bacteria as a potential pectinase producer 36
2.8.3 Yeasts as a potential pectinase producer 37
2.9 Biotechnological application of microbial pectinase 41
2.9.1 Acidic pectinase 41
2.9.1.1 Fruit juices extraction and clarification 41
2.9.1.2 Industrial wines making and manufacturing 44
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2.9.1.3 Peel oil extraction 45
2.9.1.4 Preparation of dried animal feed from
citrus fruits 45
2.9.2 Alkaline pectinase 46
2.9.2.1 Retting and degumming of plant fiber 46
2.9.2.2 Pectic wastewater treatment 47
2.9.2.3 Vegetable oil extraction 48
2.9.2.4 Coffee and tea fermentation 49
2.10 General introduction on solid substrate fermentation (SSF) 50
2.10.2 Fungal interaction in solid substrate fermentation (SSF) 55
2.10.3 General considerations on enzyme production through
solid substrate fermentation (SSF) 59
2.10.3.1 Selection of substrate 59
2.10.3.2 Screening of potential microorganism 60
2.10.3.3 Physical parameters governing the
enzyme production 62
2.10.3.4 Chemical parameters governing the enzyme
Production 68
2.11 Concluding remarks 72
CHAPTER 3 : MATERIALS AND METHODS
3.1 Microorganisms 73
3.2 Preparation of inoculums 73
3.3 Preparation of substrate 74
3.4 Primary screening for potential fungal pectinase producer 74
3.5 Secondary screening for potential fungal pectinase producer 75
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3.6 Extraction of crude pectinase 76
3.7 Identification of isolate HFM 8 as a potential fungal pectinase producer 76
3.7.1 Macroscopic observation of fungal isolate HFM 8
on different agar media 76
3.7.2 Microscopic observation on fungal isolate HFM 8
under light microscope 77
3.7.3 Microscopic observation on fungal isolate HFM 8
under scanning electrone microscope (SEM) 77
3.7.4 Identification of fungal isolate HFM 8
through molecular approach 78
3.7.4.1 Extraction of DNA 78
3.7.4.2 Polymerase chain reaction (PCR) amplification 80
3.7.4.3 Purification of PCR product 81
3.8 Analytical methods 82
3.8.1 Determination of pectinase activity 82
3.8.2 Determination of fungal growth 83
3.8.3 Determination of total protein concentration 84
3.9 Improvement of cultural conditions for pectinase production
by Aspergillus niger HFM 8 through a flask system 85
3.9.1 Improvement of physical conditions 85
3.9.1.1 Initial profiles of pectinase production and fungal
growth of Aspergillus niger HFM 8 before the
improvement of physical conditions 86
3.9.1.2 Effect of different particle size of substrate 87
3.9.1.3 Effect of different initial moisture content 87
3.9.1.4 Effect of different incubation temperature 88
3.9.1.5 Effect of different initial pH of moisturizing agent 88
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3.9.1.6 Effect of different size of inoculum 89
3.9.1.7 Effect of mixing frequency 90
3.9.1.8 Effect of different extracting solvent during
enzyme recovery 90
3.9.1.9 Profile of pectinase production and fungal
growth of Aspergillus niger HFM 8
after physical improvement 91
3.9.2 Improvement of chemical conditions 91
3.9.2.1 Effect of different carbon source supplementation 92
3.9.2.2 Effect of different nitrogen source supplementation 92
3.9.2.3 Effect of different concentrations of the selected
nitrogen source supplementation 93
3.9.2.4 Profile of pectinase production and fungal
growth of Aspergillus niger HFM 8
after physical and chemical improvement 93
3.10 Improvement of pectinase production and fungal growth
of Aspergillus niger HFM 8 in a tray system 94
3.10.1 Initial profiles of pectinase production and growth
of Aspergillus niger HFM 8 under different substrate thickness 94
3.10.2 Effect of different initial moisture content 95
3.10.3 Effect of different inoculum size 95
3.10.4 Effect of different mixing frequency 96
3.10.5 Profiles of pectinase production and growth of
Aspergillus niger HFM 8 in a tray system after the
improvement of physical conditions 96
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3.11 Statistical analysis 97
3.12 Purification of pectinase 97
3.12.1 Ultrafiltration 97
3.12.2 Anion exchange column chromatography 98
3.12.2.1 Determination of appropriate pH for chromatography 98
3.12.2.2 Determination of elution condition for chromatography 99
3.12.2.3 Fraction collection through anion exchange
column chromatography 99
3.12.3 Gel filtration 100
3.13 Pectinase purification analysis with Sodium Dodecyl
Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 101
3.14 Silver staining 102
3.15 Determination of molecular weight 103
3.16 Characterization of purified pectinase 104
3.16.1 Determination of optimum temperature 104
3.16.2 Temperature stability of purified pectinase 104
3.16.3 Optimal pH determination 105
3.16.4 pH stability of purified pectinase 105
3.16.5 Substrate specificity of purified pectinase 106
3.16.6 Effect of different reagents and metal ions on
purified pectinase activity 106
CHAPTER 4 : RESULTS
4.1 Primary screening of potential fungal pectinase producer 107
4.2 Secondary screening of potential fungal pectinase producer 113
4.3 Identification of isolate HFM 8 as a potential fungal pectinase producer 116
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4.3.1 Macroscopic observation of fungal isolate HFM 8
on different agar media 116
4.3.2 Microscopic observation of fungal isolate HFM 8
through Light Microscope (LM) 126
4.3.3 Microscopic observation on fungal isolate HFM 8 under
Scanning Electrone Microscope (SEM) 129
4.3.4 Identification of fungal isolate HFM 8 through molecular approach 132
4.4 Improvement of cultural conditions for extracellular pectinase
production by Aspergillus niger HFM 8 through a flask system 135
4.4.1 Initial profile before the improvement of physical conditions 135
4.4.2 Improvement of physical condition for extracellular
pectinase production by Aspergillus niger HFM 8 139
4.4.2.1 Effect of different size of substrate 139
4.4.2.2 Effect of different initial moisture content 141
4.4.2.3 Effect of different incubation temperature 143
4.4.2.4 Effect of different initial pH 145
4.4.2.5 Effect of different inoculum size 147
4.4.2.6 Effect of mixing frequency 149
4.4.2.7 Effect of different extracting solvent during
the enzyme recovery 151
4.4.3 Profile of pectinase activity and growth of Aspergillus niger
HFM 8 after the improvement of physical conditions 153
4.4.4 Improvement of chemical condition for extracellular
pectinase production by Aspergillus niger HFM 8 156
4.4.4.1 Effect of external carbon source supplementation 156
4.4.4.2 Effect of external nitrogen source supplementation 159
A
C
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4.4.4.3 Effect of different concentration of urea supplementation 161
4.4.5 Profile of pectinase activity and fungal growth of
Aspergillus niger HFM 8 after chemical improvement
of cultural condition 163
4.4.6 Comparison of pectinase activity and growth of
Aspergillus niger HFM 8 before and after the improvement
of physical and chemical conditions 166
4.5 Improvement of physical cultural conditions for extracellular
pectinase production by Aspergillus niger HFM 8 through
a tray system 169
4.5.1 Initial profile in different substrate thickness before the
improvement of physical conditions 169
4.5.2 Improvement of physical conditions for extracellular
pectinase production through a tray system 173
4.5.2.1 Effect of initial moisture content 173
4.5.2.2 Effect of inoculum size 175
4.5.2.3 Effect of mixing frequency 177
4.5.3 Final profile after the improvement of physical conditions
in a tray system 179
4.6 Purification of pectinase 183
4.7 Molecular weight determination of purified pectinase 188
4.8 Characterization of purified pectinase 191
4.8.1 Determination of optimal temperature 191
4.8.2 Stability of purified pectinase in different temperature 192
4.8.3 Determination of optimal pH 194
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4.8.4 Stability of purified pectinase in different pH 195
4.8.5 Determination of substrate specificity 197
4.8.6 Effects of reagents and metal ions 198
CHAPTER 5 : DISCUSSION
5.1 The feasibility of pomelo peel as a cheap substrate for fungal
pectinase production through solid substrate fermentation (SSF) 200
5.2 Screening of potential fungal pectinase producer 202
5.2.1 Primary screening of potential fungal pectinase producer 202
5.2.2 Secondary screening of potential fungal pectinase producer 204
5.3 Identification of fungal isolate HFM 8 207
5.4 Improvement of physicochemical conditions for pectinase
production through solid substrate fermentation employing
a shake flask system 209
5.4.1 Improvement of physical cultural conditions 209
5.4.2 Improvement of chemical cultural conditions 222
5.5 Improvement of pectinase activity by Aspergillus niger
HFM 8 under physicochemical optimized conditions 228
5.6 Large scale pectinase production employing a tray system 230
5.7 Improvement of pectinase activity by Aspergillus niger
HFM 8 under physicochemical optimized conditions employing
a tray system 240
5.8 Purification of pectinase and molecular weight determination 241
5.9 Characterization of the purified pectinase 248
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CHAPTER 6 : CONCLUSION AND RECOMMENDATION
6.1 Conclusion 255
6.2 Future recommendation 257
REFFERENCES 258
APPENDICES
Appendix A : Standard curve for pectinase activity determination
at 520 nm based on Galacturonic acid as a reducing sugar
Appendix B : Standard curve of glucosamine content for fungal growth
determination at 530 nm
Appendix C : Standard curve for protein determination based on Bovine
Serum Albumin (BSA) at 750 nm
Appendix D : Preparation of SDS-PAGE reagents
Appendix E : The consensus sequence of Aspergillus niger HFM 8
LIST OF PUBLICATIONS
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LIST OF TABLES
Page
Table 2.1 Agricultural waste generation in Asia 8
Table 2.2 Consumption of assorted agro industrial residues in pectinase
production through solid substrate fermentation (SSF) 11-12
Table 2.3 Percentage of pectin in different fruits and vegetables 20
Table 2.4 Classification of pectinase enzyme 31
Table 2.5 Acidic and alkaline microbial pectinase producer 39-40
Table 2.6 Contribution of solid substrate fermentation towards
economical sector 51
Table 2.7 Application of microorganisms in solid substrate
fermentation process 56
Table 4.1 Diameter of hydrolysis zone and fungal colony on pectinase
screening agar medium after 24 hours incubation period 108-109
Table 4.2 Diameter of hydrolysis zone and fungal colony on pectinase
screening agar medium after 48 hours of incubation period 110-111
Table 4.3 Pectinase activity and growth of 43 fungal isolates on
pomelo peel substrate through solid substrate fermentation 114-115
Table 4.4 Macroscopic features of fungal isolate HFM 8 cultivated
on different agar medium 117-119
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Table 4.5 Review on comparison of pectinase activity and growth of
Aspergillus niger HFM 8 in non-improved and improved
cultural conditions 168
Table 4.6 Comparison of the highest pectinase activity and growth of
Aspergillus niger HFM 8 depicted under the cultivation in
different substrate thickness 172
Table 4.7 Summary of non-improved and improved physical condition
in cultivation system employing a tray system 182
Table 4.8 Summary of pectinase enzyme purification 187
Table 4.9 The effects of reagents and metal ions towards purified pectinase 199
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LIST OF FIGURES
Page
Figure 2.1 Primary cell wall structure in most flowering plants 16
Figure 2.2 Primary structure of pectin 21
Figure 2.3 Secondary structure of pectin 22
Figure 2.4 Pectinase reaction against pectic substances 32
Figure 2.5 Pectinase application in the production of apple juice 43
Figure 2.6 Model or schematic drawing of fungal growth in solid
substrate fermentation system 58
Figure 4.1 Initial profile of pectinase activity and growth of Aspergillus
niger HFM 8 before physical condition improvement 136
Figure 4.2 Effect of different size of substrate on pectinase activity and
growth of Aspergillus niger HFM 8 140
Figure 4.3 Effect of different initial moisture content on pectinase
activity and growth of Aspergillus niger HFM 8 142
Figure 4.4 Effect of different incubation temperature on pectinase
activity and growth of Aspergillus niger HFM 8 144
Figure 4.5 Effect of different initial pH on pectinase activity and
growth of Aspergillus niger HFM 8 146
Figure 4.6 Effect of different inoculum size on pectinase activity
and growth of Aspergillus niger HFM 8 148
xvii
Figure 4.7 Effect of different mixing frequency on pectinase activity
and growth of Aspergillus niger HFM 8 150
Figure 4.8 Effect of different extracting solvent consumption on
pectinase activity and growth of Aspergillus niger HFM 8 152
Figure 4.9 Profile of pectinase activity and growth of Aspergillus niger
HFM 8 after the improvement of physical condition 154
Figure 4.10 Effect of supplementation of different carbon source on
pectinase activity and growth of Aspergillus niger HFM 8 157
Figure 4.11 Effect of supplementation of different nitrogen source on
pectinase activity and growth of Aspergillus niger HFM 8 160
Figure 4.12 Effect of urea supplementation in different concentration on
pectinase activity and growth of Aspergillus niger HFM 8 162
Figure 4.13 Profile of pectinase activity and growth of Aspergillus niger
HFM 8 after the improvement of chemical condition 164
Figure 4.14 Comparison of pectinase activity and growth of Aspergillus
niger HFM 8 before and after the improvement of
physical and chemical conditions in solid substrate
fermentation through a flask system 167
Figure 4.15 Initial profile of pectinase activity and growth of Aspergillus
niger HFM 8 in tray system with different substrate thickness 170
Figure 4.16 Effect of different initial moisture content on pectinase
activity and growth of Aspergillus niger HFM 8 in a tray system 174
xviii
Figure 4.17 Effect of different inoculum size on pectinase activity and
growth of Aspergillus niger HFM 8 in a tray system 176
Figure 4.18 Effect of different mixing frequency on pectinase activity and
growth of Aspergillus niger HFM 8 in a tray system 178
Figure 4.19 Final profile of pectinase activity and growth of Aspergillus
niger HFM 8 in a tray system after the improvement
of physical conditions 180
Figure 4.20 Elution profile of pectinase produced by Aspergillus niger
HFM 8 in initial stage of purification employing anion
exchange column chromatography (DEAE Sephadex) 184
Figure 4.21 Elution profile of pectinase produced by Aspergillus niger
HFM 8 in second stage of purification employing
gel filtration column chromatography (Sephadex G-100) 185
Figure 4.22 Molecular weight determination of purified pectinase using
SDS-PAGE 190
Figure 4.23 The effect of different temperatures on the purified
pectinase activity 191
Figure 4.24 Stability of the purified pectinase in different temperatures 193
Figure 4.25 The effect of different pH on the purified pectinase activity 194
Figure 4.26 Stability of the purified pectinase in different pH conditions 196
Figure 4.27 Substrate specificity of the purified pectinase 197
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LIST OF PLATES
Page
Plate 4.1 Hydrolysis zone formation 112
Plate 4.2 Morphology of isolate HFM 8 on Potato Dextrose Agar (PDA)
plates after seven days of incubation period 120
Plate 4.3 Morphology of isolate HFM 8 on Oatmeal Agar (OA) plates
after seven days of incubation period 121
Plate 4.4 Morphology of isolate HFM 8 on Czapex Yeast Agar (CYA)
plates after seven days of incubation period 122
Plate 4.5 Morphology of isolate HFM 8 on Creatine Sucrose Agar (CREA)
plates after seven days of incubation period 123
Plate 4.6 Morphology of isolate HFM 8 on Malt Extract Agar (MEA)
plates after seven days of incubation period 124
Plate 4.7 Morphology of isolate HFM 8 on Yeast Extract Agar (YES)
plates after seven days of incubation period 125
Plate 4.8 Microscopic features of the Fungal Isolate HFM 8 under
a light microscope observation 127 - 128
Plate 4.9 SEM micrographs of the fungal isolate HFM 8
microscopic features 130 - 131
Plate 4.10 Purified PCR product of Aspergillus niger HFM 8 133
Plate 4.11 Top 16 blast hit sequence of sample HFM 8 through
BLAST 2.2.26+ Search Tool database 134
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Plate 4.12 Growth of Aspergillus niger HFM 8 on pomelo peel
substrate 138
Plate 4.13 SDS-PAGE of purified pectinase produced by
Aspergillus niger HFM 8 with 12.5% polyacrylamide 189
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LIST OF SYMBOLS AND ABBREVIATION
% Percent
± Plus minus
oC Degree Celcius
α Alpha
β Beta
® Registered Trade Mark
µ Micro
∆ Delta
A Absorbance
g Gram
L Litre
mg Milligram
ml Millilitre
mm Milimeter
cm Centimeter
µg Microgram
µmol Micromol
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M Molar
nm Nanometer
v/v Volume over volume
w/v Weight over volume
U Unit of activity
U/ml Unit of activity per millilitre enzyme solution
U/g Unit of activity per gram substrate
V Volt
g/L Gram per litre
g/mol Gram per mol
bp Base pair
Rf Relative mobility
kDa Kilo Dalton
SDS Sodium dodecyl sulphate
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SEM Scanning Electron Microscope
CMC Carboxymethylcellulose
rpm Revolution per minute
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PENGHASILAN PEKTINASE EKSTRASEL OLEH Aspergillus niger HFM 8
MELALUI FERMENTASI SUBSTRAT PEPEJAL MENGGUNAKAN KULIT
LIMAU BALI SEBAGAI SUBSTRAT
ABSTRAK
Penimbunan kulit limau bali (Citrus grandis) sebagai sisa industri tani setelah
pemprosesan buah-buahan sewajarnya dieksploitasi sepenuhnya dengan bantuan
mikroorganisma untuk menghasilkan produk lain yang mempunyai nilai komersil
seperti enzim. Kulit limau bali telah dibuktikan mengandungi pektin pada kadar yang
tinggi dan berperanan sebagai penggalak untuk penghasilan pektinase oleh
mikroorganisma melalui kaedah fermentasi substrat pepejal (SSF). Pencilan HFM 8
telah dipilih sebagai kulat penghasil pektinase yang terbaik dengan aktiviti
maksimum sebanyak 112.79 ± 17.2 U/g substrat setelah dua kali proses penyaringan
melibatkan penyaringan primer dan penyaringan sekunder. Pencilan ini dikenalpasti
sebagai Aspergillus niger HFM 8 setelah melakukan proses identifikasi berdasarkan
sifat-sifat koloni dan juga secara molekul. Penambahbaikan keadaan fizikal dan
kimia adalah ditekankan dalam kajian ini dengan objektif untuk meningkatkan
produktiviti pektinase oleh mikroorganisma tersebut. Hasilnya, peningkatan
pektinase sebanyak 48.82% telah berjaya dihasilkan oleh Aspergillus niger HFM 8
apabila ditumbuhkan di dalam sistem fermentasi keadaan pepejal menggunakan
kelalang di dalam keadaan fizikal dan kimia yang telah dioptimumkan iaitu dengan
menggunakan substrat bersaiz 0.75 mm, kadar kelembapan sebanyak 60% (i/b)
(menggunakan air suling steril pada pH 5.0), saiz inokulum sebanyak 1 x 104
bilangan spora /ml, pengeraman pada suhu bilik (30 ± 2oC), penambahan sumber
nitrogen iaitu 0.1% (b/b) urea, pengeraman tanpa kesan pengadukan dan penggunaan
xxiv
larutan penimbal asetat (pH 4.5, 0.1 M) untuk pengekstrakan pektinase. Penghasilan
pektinase oleh Aspergillus niger HFM 8 telah dilakukan pada skala besar dengan
menggunakan sistem dulang. Dalam keadaan fizikal yang telah dioptimumkan iaitu
dengan ketebalan substrat sebanyak 1.0 cm bersamaan 60 g serbuk kulit limau bali
(saiz 0.75 mm), 40% (i/b) kelembapan (air suling steril pada pH 5.0), saiz inokulum
sebanyak 1 x 106 bilangan spora/ml, pengeraman pada suhu bilik (30 ± 2
oC),
penambahan 0.1% (b/b) urea sebagai sumber nitrogen tambahan, kesan pengadukan
sekali pada setiap 24 jam dan penggunaan larutan penimbal asetat (pH 4.5, 0.1M)
untuk pengekstrakan pektinase, didapati produktiviti pektinase telah meningkat
sebanyak 23.00%. Secara perbandingan, penghasilan enzim pektinase telah
meningkat sebanyak 191.41% daripada pemfermentasian di dalam sistem kelalang ke
sistem dulang yang membuktikan potensi Aspergillus niger HFM 8 untuk
menghasilkan lebih banyak pektinase pada skala industri. Penulenan enzim
seterusnya dijalankan dengan melaksanakan kaedah kromatografi pada dua peringkat
melibatkan penukaran ion (DEAE Sephadex) dikuti penapisan gel (Sephadex G-
100). Prosedur SDS-PAGE mengesahkan bahawa berat molekul pektinase tulen ini
adalah 55.37 kDa. Enzim ini bertindak-balas secara optimum pada 40oC dan pH 4.0
di samping stabil pada suhu 35oC ke 45
oC dalam julat pH 3.5 ke pH 4.0. Pektinase
tulen ini didapati bertindak-balas sangat spesifik terhadap pektin tanpa melibatkan
peranan agen pelarut atau ion logam untuk aktiviti yang optimum. Sebaliknya, FeCl3
dan AgNO3 didapati telah merencatkan tindak-balas pektinase. Secara jelasnya, kulit
limau bali terbukti dapat diaplikasikan untuk penghasilan pektinase secara komersil
melalui kaedah fermentasi substrat pepejal di mana enzim pektinase yang dihasilkan
berpotensi untuk digunakan dalam industri pengekstrakan dan penjernihan jus buah-
buahan.
xxv
EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus niger
HFM 8 THROUGH SOLID SUBSTRATE FERMENTATION USING
POMELO PEELS AS A SUBSTRATE
ABSTRACT
The accumulation of pomelo peels (Citrus grandis) as the agro-industrial
residues after the fruit processing and manufacturing should be maximally exploited
for the microbial bioconversion of the detrimental wastes into a value-added by
product such as enzymes. Pomelo peels are authenticated as rich in pectin, acting as
the main inducer for pectinase enzyme production by microorganism through solid
substrate fermentation system (SSF). Isolate HFM 8 revealed as the best fungal
pectinase producer after the implementation of double steps screening processes
(primary and secondary screenings) with maximal activity of 112.79 ± 17.2 U/g
substrate. Based on the phenotypic features and molecular identification, this novel
isolate was denoted as Aspergillus niger HFM 8. The improvement of
physicochemical cultural conditions was emphasized in this current research with an
attempt to enhance the pectinase productivity by the microorganism. An
augmentation of 48.82% on pectinase yield by Aspergillus niger HFM 8 in a shake
flask system was recorded after being cultivated in an enhanced physicochemical
condition constituted of 0.75 mm particle size of substrate, 60% (v/w) of initial
moisture (sterilized distilled water, pH 5.0), inoculum size of 1 x 104 spores/ml,
incubation in room temperature (30 ± 2oC), inclusion of 0.1% (w/w) urea as the
external nitrogen source, no mixing effect (static condition during fermentation) and
utilization of acetate buffer (pH 4.5, 0.1 M) as the extracting solvent during the
xxvi
enzyme recovery. A large scale pectinase production by Aspergillus niger HFM 8
was then conducted employing a tray system. Under the optimized physical
conditions comprised of 1.0 cm substrate depth equalized to 60 g pomelo peels
substrate (0.75 mm of particle size), 40% (v/w) of initial moisture (sterilized distilled
water, pH 5.0), inoculum size of 1 x 106 spores/ml, incubation in room temperature
(30 ± 2oC), inclusion of 0.1% (w/w) urea as the external nitrogen source, inclusion of
mixing effect once for every 24 hours interval and utilization of acetate buffer (pH
4.5, 0.1 M) as the extracting solvent during the enzyme recovery, an amount of
23.00% increment on pectinase productivity was detected. Comparatively, the fungal
pectinase production boosted up to 191.41% of increment from a shake flask system
to a tray system indicating the potentiality of Aspergillus niger HFM 8 to be
industrially employed particularly in pectinase production. Further purification of the
crude pectinase was conducted by practising double steps chromatography
constituted of anion exchange (DEAE Sephadex) followed by gel filtration
(Sephadex G-100). SDS-PAGE procedure determined the molecular weight of the
purified pectinase as 55.37 kDa. This enzyme was found optimal at 40oC and pH 4.0
whereby the catalytic reaction was stable between 35oC to 45
oC and in condition of
pH 3.5 to pH 4.0. The purified pectinase was highly specific on pectin as the
substrate whilst no solvents or metal ions found enhancing the catalytic reaction.
Inversely, FeCl3 and AgNO3 prohibit the pectinase reaction. Apparently, pomelo
peels are markedly applicable for commercial pectinase production employing a
solid substrate fermentation system whereby the produced enzyme is highly
applicable to be incorporated into the fruit juice extraction and clarification process.
1
CHAPTER 1
INTRODUCTION
1.1 General introduction on pectinase research
Pectinase is one of the forthcoming enzymes at the moment which constitutes
25% of the global food enzyme market (Jayani et al., 2005). This group of
heterogenous enzymes catalyze the degradation of pectic substances in plant
structures such as pectin, pectic acid or oligo-D-galacturonate in different mode of
actions including transelimination and hydrolysis (Favela-Torres et al., 2006; Jayani
et al., 2010 ; Heidar et al., 2011). In nature, pectinase is mainly found in plant tissue
to facilitate the cell wall extension and softening some tissues during the maturation
of part of the plant such as fruits. In addition, pectinase also catalyze the
decomposition of plant residues and contribute to the carbon cycle and ecosystem
stabilization. During the phytopathogenic process, pectinase is secreted copiously by
microorganisms to infect the host plant (Siddiqui et al., 2012). For industrial sector,
pectinase is vastly applied in fruit juice extraction and clarification, textile and fiber
processing and manufacturing, coffee and tea fermentations, industrial pectic
wastewater treatment, paper making, purification of plant virus, poultry feed
production and oil extraction (Saito et al., 2004; Sharma and Satyanarayana, 2006;
Siddiqui et al., 2012). Furthermore, this novel enzyme is also utilized in the
extraction of specific plant bio-compounds from raspberry residues, tomatoes, grape
skin and also apple skin (Neagu et al., 2012). Undeniably, pectinase is a profitable
enzyme which has enormous significant contributions to the mankind.
2
In order to accomplish the increasing worldwide demand, industrial scale
production of pectinase enzyme has been generated either through solid substrate
fermentation (SSF) or submerged fermentation (SmF) (Neagu et al., 2012). It has
been proven that pectinase is an inducible enzyme specifically with the visibility of
pectic substances as the substrate (Reddy and Sreeramulu, 2012). In solid substrate
fermentation, the microorganisms grow together with the metabolites production
occur on the surface of the solid substrate in near absence of free water (low water
activity) (Heidar et al., 2011) whilst in submerged fermentation, the microorganisms
grow in free flowing liquid substrate such as broth and the metabolites are secreted
into the liquid medium (Subramaniyam and Vimala, 2012). Comparatively, in
producing bioactive compound such as pectinase enzyme, fungi is more applicable in
solid substrate fermentation rather than submerged fermentation which is more
suitable for bacteria and yeast due to the high water activity (aw). Low water activity
in solid substrate fermentation generally mimicing the nature environment favoured
by fungi to grow and produce various metabolites. Furthermore, the fungal
metabolism is enhanced since the bacterial contamination is prohibited due to the
low water activity in solid substrate fermentation (Favela-Torres et al., 2006).
Several studies conducted have significantly proven that the volume of
pectinase yielded through solid substrate fermentation is higher compared to
submerged fermentation (Favela-Torres et al., 2006). Moreover, the cost of
production through solid substrate fermentation is cheaper due to the exploitation of
agro-industrial residues as substrates and the pectinase generated has better
physicochemical properties, contradictory to submerged fermentation. In fact, the
application of agro wastes as the main carbon source in solid substrate fermentation
3
is an effective solution in solving detrimental problem arises due to the waste
disposal management. For example, every year in Egypt, large amount of orange
peels are generated as residues after the oranges canning process by the
manufacturing factories (Abdel-Mohsen, 1996). According to Mamma et al. (2008),
about 35 000 tons (dry weight) of citrus peels are generated every year in Greece. In
Spain, around 250 millions kilograms of grape pomace which consists of seed, stem
and skin are produced by the wine cellars (Chatterjee et al., 2013). While in
Karnataka, India, approximately, 3.1 tons/hectare husk of pulses and 14.3
tons/hectare deseeded sunflower head are being generated every year respectively
after the processing of sunflower oilseed. All of these agro-industrial residues are
rich in pectin and applicable as the pectin-containing substrate to be utilized in the
microbial pectinase production through solid substrate fermentation. In Malaysia,
approximately 1.2 million tonnes of agro-wastes are being accumulated each year,
especially from the palm oil and oil palm industries.
In order to produce pectinase enzyme, different types of microorganism
including fungi, bacteria and yeasts are incorporated into the fermentation system.
According to Favela-Torres et al. (2006), approximately 30 genera of filamentous
fungi, bacteria and yeast which are frequently employed in producing pectinase such
as Erwinia, Rhizopus, Penicillium, Fusarium, Aspergillus, Bacillus, Saccharomyces
and Kluyveromyces. These microorganisms must be non-pathogenic, non-toxic and
generally do not produce antibiotics which are the important characteristics of GRAS
(Generally regarded as safe) microorganism. One of the most frequently utilized
microorganisms in fungal pectinase production is Aspergillus niger which is
proclaimed possessing the GRAS status (Reddy and Sreeramulu, 2012).
4
Technically, commercial enzyme with enviable biochemical and physico-
chemical properties with low cost production has been the focus of researchers
currently (Silva et al., 2002). It is undeniable that the utilization of agro industrial
residues as pectin-rich substrate in pectinase production through solid substrate
fermentation is promising a low cost and eco-friendly enzyme industry (Suresh and
Viruthagiri, 2010). However, further research is crucial in screening the best
substrate to be incorporated into the fermentation system and its suitability for the
microbial growth and enzyme production.
1.2 Rational of study
Pomelo (Citrus grandis) is massively cultivated in Asia especially in
Malaysia, China, Southern Japan, Vietnam, Indonesia and Thailand (Hameed et al.,
2007). Commonly in Malaysia, during the trimming stage, some of the immature
pomelo fruits are removed to improve the quality of the others and disposed to the
environment subsequently. This scenario will only lead to the detrimental pollution
problem to the community (Saikaew et al., 2009). Evidenced by research conducted
by Norziah et al. (2000) and Burana-Osot et al. (2010), pomelo peel is rich in pectin
and applicable in pectinase production through solid substrate fermentation
employing fungal strains. Since the biotechnological research on pectinase
production using pomelo peel is still scanty (Foo and Hameed, 2011), this vigorous
study is implemented to maximize its potential as the main substrate in fermentation
system to generate commercialized pectinase enzyme employing local fungal isolate.
5
1.3 Objective of research
With an aim to use pomelo peels as a substrate for pectinase production by a
local fungal isolate via solid substrate fermentation system, these objectives are
employed;
i. To identify and screen the best fungal pectinase producer based on its
capability to produce extracellular pectinase qualitatively and
quantitatively.
ii. To optimize the physical and chemical conditions for a maximal
pectinase production by the selected fungal isolate in a shake flask system
and a tray system.
iii. To purify and characterize the purified pectinase
6
CHAPTER 2
LITERATURE REVIEW
2.1 Growth of agricultural industry worldwide
With an aim to stimulate the agricultural growth, combating poverty and
hunger for human worldwide and at the same time to encourage environmental
sustainability, agricultural sector is believed as the efficient tool.
In United States Of America, agriculture is the major industry with 2.2
millions farms which dominate an area of 922 million acres in 2007. Corn, tomatoes,
potatoes, peanuts and sunflower seeds are among the major crops being cultivated.
Meanwhile, in China, over 300 millions farmers are employed in agricultural
industry which endowed this country as the first place in worldwide farm output.
Primarily, China is the main producer of rice, wheat, sorghum, potatoes, peanuts, tea,
millet, barley, oilseed and cotton. Besides, India ranks as the second largest
contributor of farm output after China. According to FAO (Food And Agriculture
Organization) 2010 Worldwide Statistics, India is the largest supplier of many fresh
fruits and vegetables, spices, milk, jute, millet and castor oil seeds. India is also the
second largest producer of wheat and rice and also ranked as one of the five largest
contributors of 80% of agricultural products including coffee and cotton in 2010
(FAO, 2012).
7
The same scenario is also taking place in Malaysia where agricultural sector
plays an imperative role to the economy, providing 16% of employment to the local
citizen. Rubber, palm oil and cocoa are among the foremost commercial crops being
cultivated locally. In 1999, about 10.55 million matric tons of palm oil was generated
which ranked Malaysia as the world‟s largest producer. Besides, paddy, banana,
coconut, durian, pineapple, rambutan and other local fruits and vegetables are also
cultivated for domestic market (Encyclopedia Of The Nation, 2013).
In future, it is assumed that the expansion of agricultural sector will be
boosting up throughout the year in order to accomplish the worldwide population‟s
demand. Nonetheless, the disposal management of agro-industrial residues remains
as problematic issue to be solved by the developing countries.
2.2 Agro-industrial residues as a detrimental issue
Despite of soaring development depicted by the global agricultural sector
yearly, the accumulation of assorted disposed agro-industrial residues remain a major
concern to the public and the ecosystem as well. According to Agamuthu (2009),
around 998 million tonnes of agricultural wastes were produced around the world.
While in Malaysia, approximately 1.2 million tonnes of agro wastes were being
disposed every year. Table 2.1 shows the agricultural wastes generated in Asian
countries as reported by Agamuthu (2009). The table also demonstrates the predicted
increasing numbers of agricultural wastes in the year of 2025.
8
Table 2.1 : Agricultural waste generation in Asia
Country In Asia
Agricultural Waste
Generation
(kg/capita/day)
Projected Agricultural
Waste Generation In
2025 (kg/capita/day)
Brunei
0.099
0.143
Cambodia 0.078 0.165
Indonesia 0.114 0.150
Laos 0.083 0.135
Malaysia 0.122 0.210
Myanmar 0.068 0.128
Philipines 0.078 0.120
Singapore 0.165 0.165
Thailand 0.096 0.225
Vietnam 0.092 0.150
Nepal 0.060 0.090
Bangladesh 0.040 0.090
Adapted from : Agamuthu (2009)
9
Commonly, for agro-industrial waste management in developing countries,
crop residues, animal garbages, forest litter and grasses are being burnt as fuel which
leads to problematic air pollution. In general, the main source for these horrendous
agro industrial wastes comes from the processed animal products and crops.
Brewer‟s waste, maize milling by-product, molasses, oilseed cakes and bagasse are
among the common wastes produced by the agro-industrial processing sector. While
for the crop residues such as straw, stem, peel, shell, leaves, husk, seed, pulp, lint and
also stubble which are derived from cereals (which include rice, maize or corn,
millet, barley, wheat and sorghum), legumes (soy bean and tomatoes), jute,
groundnut, cotton, coffee, cocoa, tea, olive, fruits (banana, mango, cashew) and palm
oil are also defined as agricultural waste (Pandey et al., 2009).
Undeniably, managing agro-industrial waste as one of the solid waste is a big
challenge for a developing country around the world. Without an effective step, the
accumulation of these residues will only lead to the public health hazard and
detrimental pollution to the environment (Hwa, 2007). Apparently, converting the
„waste‟ into „wealth‟ through the utilization of agro-industrial residues in profitable
enzyme production is one of the practical steps in encountering such problem.
10
2.3 Utilization of agro industrial residues in pectinase production
Abundance of agro-industrial waste as by-product in agricultural sector
(around 3.5 billion tonnes/year) is promising a low cost enzyme yield especially in
microbial pectinase production employing solid substrate fermentation. These
renewable residues can be utilized as source of energy for microbial growth during
fermentation and also as carbon supply during the synthesis of biomass and other
products (Jacob, 2009). In addition, these agro industrial residues also serve as an
anchorage for the cells and provide sufficient nutrients for the microbial growth.
However, for certain nutrients that are available in sub-optimal concentration or even
absent, the substrate should be supplemented externally (Pandey et al., 1999). In
consideration of agro-industrial residues‟ suitability and availability to be exploited
as the substrate in solid substrate fermentation, the problem regarding the pollution
as a result to its accumulation and disposal method should be overcome effectively
(Hoondal et al., 2002).
In nature, variety of substrates come from agro-industrial wastes which are
applicable in producing profitable value added products especially pectinase enzyme
through solid substrate fermentation. However, the number and type of potential
residues varies between the crops and districts in the world (Cohen and Hadar, 2001).
Some potentially utilized residues in pectinase production through solid substrate
fermentation using various microorganisms are recorded in Table 2.2
11
Table 2.2 Consumption of assorted agro industrial residues in pectinase production through solid substrate fermentation (SSF)
Agro Industrial Residue Microorganism Reference
Sugar cane bagasse Aspergillus niger Jacob (2009)
Aspergillus awamori Suresh and Viruthagiri (2011)
Penicillium sp. Jacob (2009)
Aspergillus niger CH4 Solis-Pereyra et al. (1996)
Moniliella sp. SB9 Martin et al. (2004)
Penicillium sp EGC5 Martin et al. (2004)
Monosodium glutamate waste water Aspergillus niger Bai et al. (2004)
Sugar beet pulp Aspergillus niger Bai et al. (2004)
Bacillus gibsonii S2 Li et al. (2005)
Penicillium oxalicum Neagu et al. (2012)
Wheat bran Aspergillus carbonarius Jacob (2009)
Aspergillus niger Castilho et al. (2000) ; Heidar et al. (2011)
Aspergillus sojae Heerd et al. (2012)
Thermoascus aurantiacus Jacob (2009)
Bacillus sp. DT7 Kashyap et al. (2003)
Penicillium sp. Jacob (2009) ; Heidar et al. (2011)
Streptomyces sp. RCK-SC Jacob (2009)
Penicillium viridicatum RFC3 Silva et al. (2005)
Streptomyces lydicus Jacob (2009)
Bacillus pumilus dcsr1 Sharma and Satyanarayana (2012)
Alternaria alternata Faten and Abeer (2013)
12
Table 2.2 Continue
Agro Industrial Residue Microorganism Reference
Rice bran Aspergillus niger Suresh and Viruthagiri (2011)
Aspergillus awamori Suresh et al. (2009)
Orange bagasse Thermoascus aurantiacus Jacob (2009)
Penicillium viridicatum RFC3 Silva et al. (2005)
Moniliella sp. SB9 Martin et al. (2004)
Penicillium sp EGC5 Martin et al. (2004)
Apple bagasse Penicillium sp Heidar et al. (2011)
Aspergillus niger Heidar et al. (2011)
Orange pulp Fusarium moniliforme Jacob (2009)
Orange peel Aspergillus niger Vasanthi and Meenakshisundaram (2012)
Coffee pulp Aspergillus niger Loera et al. (1999)
Aspergillus niger C28B25 Antier et al. (1993)
Aspergillus niger V22 B35 Boccas et al. (1994)
Coffe mucilage Erwinia herbicola Avallone et al. (2002)
Lactobacillus brevis Avallone et al. (2002)
Grape pomace Aspergillus awamori Botella et al. (2005)
Grape peel Saccharomyces cerevisiae CECT 11783 Arévalo-Villena et al. (2011)
Soy bran Aspergillus niger Castilho et al. (2000)
Citrus peel Aspergillus niger Dhillon et al. (2004)
Citrus waste Aspergillus foetidus Garzỏn (1992)
Aspergillus niger F3 Rodríguez-Fernàndez et al. (2012)
Corn cobs Alternaria alternata Faten and Abeer (2013)
13
2.4 Pomelo peel as a potential substrate for pectinase production
Pomelo or its scientific name Citrus grandis (better known in Malaysia as
limau bali or limau kedang) is the largest citrus fruit with average diameter of 30
centimetres and sometime weighs until 10 kilograms. Generally, it possesses very
thick rind with soft texture but yet still easy to peel off (Hameed et al., 2007). This
citrus fruit is also known as “shaddock” and believed as an ancient species to the
genus of Citrus. It is classified in family of Rutaceae, sub-family Aurantioidae, tribe
Citreae and sub-tribe Citrinae (Paudyal and Haq, 2008).
Pomelo is cultivated immensely in Malaysia, Indonesia, Thailand, China, Fiji,
India and Japan. Currently, this large citrus fruit is also grown widely in Caribbean
and United States Of America especially in California and Florida. It is believed that
pomelo or “shaddock” was introduced to the West Indies (Barbados) from the Dutch
East Indies by Captain Shaddock and latterly being spread to the other tropical
countries (Hameed et al., 2007).
Commonly, the juicy flesh of pomelo which is rich in Vitamin C, B1, B2,
B12 and protein is eaten raw or being extracted for commercial purpose. Meanwhile,
its thick rind is utilized widely in sweet manufacturing industry (covered with sugar)
and also in marmalade production (Hameed et al., 2007). The flowers, seeds and
fruits are also employed in medication and the flower itself is applied in perfumery
extraction. Meanwhile, the thick peel is extracted as the source of pectin and dietary
14
fiber (Saikaew et al., 2009 ; Burana-Osot et al., 2010). According to the statistics
provided by FAO (Food And Agriculture Organization), the total production of
pomelo and grapefruit amounted about 5.05 million tonnes which constituted 6.2%
of citrus fruit production worldwide in 1994 while in Thailand itself, 11% of its
exported fruit comes from pomelo (Paudyal and Haq, 2008).
In Malaysia, pomelo is principally cultivated in Johor, Kedah, Melaka and
Perak with approximately 1895 hectares of pomelo are cultivated commercially (Foo
and Hameed, 2011). Generally, pomelo is produced throughout the year with its peak
seasons between January to February and also from August to September
respectively. In order to ensure the well growth and quality assertion of certain
amount of pomelos, fruits that are unsuitable to be commercialized in fresh market
and also the immature fruits are eradicated from the trees (Norziah et al., 2000). Due
to the high consumption of pomelo in food manufacturing industry together with the
disposal of immature fruits during the trimming stage, this circumstance leads to a
severe pollution to the community and the environment as well (Hameed et al.,
2007).
As an efficient solution in managing the waste, pomelo peel as part of the
fruit could be employed as a low cost substrate in producing pectinolytic enzymes as
a value added by-product through solid substrate fermentation. According to Norziah
et al. (2000), pomelo peel is a good source of pectin which 20.8% of its total
extraction is constituted by pectin after being extracted through acetone precipitation
utilizing sodium hexametaphosphate. Yuxiang et al. (2012) also reported that
15
20.98% of pomelo peel was constituted of pectin after being extracted under the
optimum condition determined by Response Surface Methodology (RSM). In
addition, the raw material for the production of commercialized pectin nowadays is
mainly contributed by residues from fruit juice manufacturing such as citrus fruits
and apple pomace (Hoondal et al., 2002). This chemical component plays a vital role
in inducing the pectinase yielded by microorganisms during the fermentation period
(Jacob, 2009; Reddy and Sreeramulu, 2012). However, the exact potential of pomelo
peel as the raw material in pectinase production through microbial fermentation is
still yet undiscovered since less research was reported by the previous studies.
2.5 Pectic substances
Pectic substances are a complex, high molecular weight, acidic and
negatively charged biomacromolecule (Jayani et al., 2005; Bhardwaj, 2010) which
constituted of 17 different monosaccharides and at least seven different
polysaccharides (Jacob, 2009). In plant cell wall, pectic substances and
hemicelluloses act as the cementing agent for celluloses microfibrils in order to
provide strength and stability to the structure of cell wall (Geetha et al., 2012). The
structure of primary cell wall in most flowering plant is displayed in Figure 2.1.
Generally, pectic substances are ubiquitous to Plant Kingdom and copiously found in
the lamella between the primary cell walls of adjacent young plant cells in form of
calcium pectate and magnesium pectate which are responsible for the cell cohesion
(Jayani et al., 2005). In young and developing cell wall, pectic substances are
16
synthesized in the Golgi apparatus from UDP-D-galacturonic acid during the early
stage of plant growth (Hoondal et al., 2002).
Figure 2.1 Primary cell wall structure in most flowering plants
(Adapted from Parenicova, 2000)
The cellulose microfibrils are interweaved with xyloglucan polymers, and this
structure is entrenched in a matrix of pectic polysaccharides, polygalacturonic acid
(PGA) and rhamnogalacturonan (RG).
Cellulose microfibrils
17
2.5.1 Classification of pectic substances
According to Kashyap et al. (2001), pectic substances are classified based on
the modification type of its backbone chain. Hence, different type of pectinolytic
enzymes will react accordingly to this variety of pectic substances depending on its
specificity and mode of action (Yadav et al., 2009). The details of pectic substances
are further elaborated in the following description.
2.5.1.1 Protopectin
Protopectin is the “parents” of the pectic substances. The hydrolysis of
protopectin yield pectin or pectinic acid (Kashyap et al., 2001; Jayani et al., 2005).
This kind of pectic substances is a water–insoluble component and mainly found in
the plant tissue predominantly in the middle lamella between the plant cells. The
insolubility of this protopectin is due to its large molecular weight, salt bonding
between the carboxyl group and also the ester bond structure between the carboxylic
group of protopectin or the hydroxyl group of other constituents in the cell wall
(Hoondal et al., 2002).
18
2.5.1.2 Pectic acid
According to Jayani et al. (2005), the soluble polymer of galacturonans and
basically free from the methoxyl groups is called as pectic acid. The salt of pectic
acid which is either neutral or acidic is known as pectates (Kashyap et al., 2001;
Hoondal et al., 2002).
2.5.1.3 Pectinic acid
Pectinic acid is composed of polygalacturonan chain that consists of more
than 75% of methylated galacturonate units. The salt of pectinic acid which is either
neutral or acidic is known as pectinates (Jayani et al., 2005). Pectinic acid alone has
a typical property in forming gel with acid or sugar under suitable condition. In
addition, gel can also be formed with certain metallic ion or other compounds such as
calcium salts if the pectinic acid is low in methoxyl content (Hoondal et al., 2002).
19
2.5.1.4 Pectin
Pectin is also known as polymethyl galacturonate. It is a soluble polymeric
material which confers rigidity to the cell wall structure after binding together with
cellulose (Jayani et al., 2005). Generally, 75% of the carboxyl groups of the
galacturonate monomer are esterified with methanol (Hoondal et al., 2002).
Previous research had reported that pectin is copiously found in plants
including fruits and vegetables (Bhardwaj, 2010; Chaudhri and Suneetha, 2012).
Approximately, 0.5% to 4% of total plant fresh weight was constituted of pectin
(Jayani et al., 2005). According to Jacob (2009), pectic substances play an
imperative role in the plant cell growth and cell differentiation during the
development of the plant tissue. New materials will be laid down and the old
materials will be removed or degraded by the enzymes. While in an immature fruit,
the pectin binds to the cellulose microfibrils which contribute to the stability and
rigidity of the cell wall. However, during the ripening stage, the main backbone
chain or the side chain of pectin is changing due to the naturally occurring
pectinolytic enzymes reaction (Martin et al., 2004). Consequently, the rigid cell wall
is softened since the pectin becomes more soluble resulting from the natural chain
modification when the fruit turns to ripe (Kashyap et al., 2001). The estimation of
pectin content in assorted fruits and vegetables are different to each other as
described in Table 2.3.
20
Table 2.3 : Percentage of pectin in different fruits and vegetables
Fruit or vegetable Tissue Pectic substances (%)
Apple Fresh 0.6 – 1.6
Banana Fresh 0.7 – 1.2
Peach Fresh 0.1 – 0.9
Strawberry Fresh 0.6 – 0.7
Cherry Fresh 0.2 – 0.5
Pea Fresh 0.9 – 1.4
Carrot Dry matter 6.9 -8.6
Orange pulp Dry matter 12.4 – 28.0
Potato Dry matter 1.8 – 3.3
Tomato Dry matter 2.4 – 4.6
Sugar beet pulp Dry matter 10.0 – 30.0
(Adapted from Jayani et al., 2005)
21
2.5.2 Structure of pectin
2.5.2.1 Primary structure of pectin
According to Chaudhry and Suneetha (2012), chemically, pectic substances
are a complex colloidal acid polysaccharides with a long unbranched galacturonic
acid backbone chain and linked together by α, 1-4 linkages (Figure 2.2). This is the
basic unit of pectic substances which is also known as homogalacturonan (HG).
Galacturonic acid which is the monomer of this polysaccharide (Reddy and
Sreeramulu, 2012) can be methyl esterified at C-6 and the hydroxyl group at C-2 or
C-3 can be acetylated.
Figure 2.2 : Primary structure of pectin (adapted from Jacob, 2009)
22
2.5.2.2 Secondary structure of pectin
The secondary structure of pectin (Figure 2.3) is composed of two main
regions which are “smooth region” (homogalacturonan) and “hairy regions”
(rhamnogalacturonan I and II) (Pedrolli et al., 2009). Homogalacturonan constitutes
the backbone chain of pectin, composed of α, 1-4 linked D-galacturonic acid
residues. Rhamnogalacturonan I is a highly branched region which comprised of a
huge number of side chain α, 1-2 linked L-rhamnopyranose residues. Meanwhile,
the more complex Rhamnogalacturonan II is predominantly found in primary cell
wall of plants and plays a significant role as a signal molecule in the development of
plant cell wall. This biomacromolecule form a very flexible, extended and curve
(worm like) structure in solution (Yadav et al., 2009)
Figure 2.3 : Secondary structure of pectin (adapted from Yadav et al. (2009)
23
2.6 Pectinase
Pectinase is a kind of heterogeneous enzyme and occasionally known as
pectic enzyme or pectinolytic enzyme. This enzymatic constituent exhibits pectin-
containing substances degradation which is mainly found in plant structures. This
inducible enzyme can be produced extensively either through solid substrate
fermentation (SSF) or submerged fermentation (SmF) (Solis-Pereyra et al., 1993;
Maldonado and Saad, 1998; Geetha et al., 2012) employing various species of
saprophytic microorganism including fungi, bacteria and yeast (Jacob, 2009; Janani
et al., 2011).
Historically, in 1930, the profitable potential of pectinase enzyme was
principally applied in the home made preparation of fruit juices and wines. Thirty
years later, in 1960s, the understanding of chemical nature and enzymatic reaction
catalysed by pectinase in plant tissue was basically improved. With this awareness,
research on the application of pectinase enzyme for commercial purpose was
implemented extensively which in turn, endowed pectinase as one of the impending
enzymes in commercial sector nowadays. Recent statistics on industrial enzyme sales
worldwide in 1995 noted that $75 millions of total sales came from pectinase enzyme
(Kashyap et al., 2001). Meanwhile, according to Jayani et al. (2005), almost 25% of
global food enzyme sales was contributed by pectinase enzyme currently.
24
In nature, pectinase is produced by plants, insects, nematodes, protozoan
(Pedrolli et al., 2009) and also by saprophytic microorganism including bacteria,
fungi, actinomycetes and yeast (Jacob, 2009; Bhardwaj, 2010). Inside the plant
tissue, this pectinolytic enzyme plays an essential role in supporting the extension of
cell wall, facilitating the penetration of pollen tube (Blanco et al., 1999) and
softening the plant tissue in mature or ripening fruits through the alteration of pectin
backbone chain (Jayani et al., 2005). Oranges and tomatoes are believed as plants
that possess considerable amount of pectinase enzyme inside the plant tissues.
Besides, pectinase is also found in insect such as the rice weevil (Sitophilus
oryzae). The endosymbiotic bacteria inside the body of this insect are assumed as the
genetic source of pectinase which plays a significant role in plant – insect interaction.
The rice weevil feeds on seeds of cereals containing pectin. Thus, pectinase in this
insect is functioning as an exclusive digestive system to degrade the pectin (Shen et
al., 1996).
In the other hand, microbial pectinase yielded by bacteria, fungi and yeast is
also important especially during the phytopathologic process, plant and microbe
symbiosis interaction and also in the natural decomposition process of decaying plant
residues. Ultimately, the decomposition of these plant materials, catalyzed by
microbial pectinase contributes to the natural carbon cycle in the ecosystem (Pedrolli
et al., 2009).