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UNIVERSITI PUTRA MALAYSIA
IZUDDIN ABDUL RAHMAN
IB 2013 4
FORMULATION AND EVALUATION OF AN AUTOMATIC DISHWASHING DETERGENT CONTAINING T1 LIPASE
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FORMULATION AND EVALUATION OF AN AUTOMATIC
DISHWASHING DETERGENT CONTAINING T1 LIPASE
By
IZUDDIN ABDUL RAHMAN
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in fulfilment of the Requirements for the Degree of Master of Science
January 2013
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of
the requirement for the degree of Master of Science
FORMULATION AND EVALUATION OF AN AUTOMATIC
DISHWASHING DETERGENT CONTAINING T1 LIPASE
By
IZUDDIN B. ABDUL RAHMAN
January 2013
Chairman: Professor Raja Noor Zaliha Raja Abd Rahman, PhD
Institute: Bioscience
Due to the concerns on the environment, the detergent industry is required to take
more environmentally friendly approaches in formulating detergents, which include
using biodegradable chemicals and enzymes. As a result, the use of enzymes in
detergent formulation has become a must, and enzymes of many types and functions
are being developed. Consequently, a recombinant enzyme called
T1 lipase (E.C. 3.1.1.3), which has been well-studied and successfully produced, was
`evaluated as a detergent enzyme for automatic dishwashing detergent (ADD)
formulation.
T1 lipase was mass produced at shake flask scale by growing the Escherichia coli
BL21 expression host, which carried the T1 gene and harvesting the crude T1 lipase
from the bacterial cells through sonication. The enzymatic activity was assayed, and
the presence of the enzyme was confirmed using sodium dodecyl sulfate
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polyacrylamide gel electrophoresis (SDS-PAGE). The crude enzyme was not purified
to its homogeneity, but the presence of the host proteins was assumed not to give any
significant effect to the subsequent results of this study, which was proven by the
absence of the lipases, amylases, and proteases from the host organism.
The crude T1 lipase in its free form was then checked for compatibility with the
detergent components by conducting stability tests and checking its residual
enzymatic activity. T1 lipase was mostly stable in nonionic surfactants, especially
those that are made of polyhydric alcohols. T1 lipase was also stable in a mixture of
sodium carbonate and glycine, which yielded pH 9.25 close to T1 lipase optimum pH
of 9.0. However, sodium carbonate alone destabilized T1 lipase possibly due to the
interaction between carbonates and Ca2+. These results indicated that polyhydric
alcohols and glycine had stabilizing effects on T1 lipase.
Next, the crude T1 lipase was spray-dried with the addition of wall materials, such as
gum arabic and maltodextrin. A blend consisting of gum arabic/maltodextrin/T1
lipase supernatant with a ratio of 6:12:3 produced products that were mostly spherical
when viewed under an electron microscope, and the encapsulated T1 lipase yielded a
three-fold increase in enzymatic activity compared to the free T1 lipase. The
spraydried product was also more soluble due to the hydrophilic nature of the wall
materials.
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Subsequently, the crude T1 lipase was evaluated as an auxiliary component for ADD
formulations. The dishwashing performance of the formulated ADDs was evaluated
in term of percent soil removed using the Leenert‘s Improved Detergency Tester. The
dishwashing performance of the formulated detergent was positively affected by the
increase in temperature but negatively affected by the presence of hard water,
specifically Ca2+ and Mg2+. The presence of hard water reduced the efficiency of the
formulated ADDs in removing soil by an average of 68%. However, T1 lipase was
not negatively affected by the presence of hard water, and this enzyme was enhanced
by the presence of polyacrylates. Moreover, the presence of Ca2+ improved the
structural integrity of T1 lipase, especially at elevated temperature.
Ultimately, an ADD formulation dubbed as ―U1‖ was produced based on the results
of this study. Overall, the addition of T1 lipase to the ADD formulations indicated an
improved dishwashing performance of up to 17% compared with the absence of T1
lipase, especially in the presence of hard water. Furthermore, this study will give more
insight on lipase as detergent enzyme, specifically in automatic dishwashing.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
FORMULASI DAN PENILAIAN DETERGEN UNTUK MESIN PENCUCI
PINGGAN MANGKUK AUTOMATIK YANG MENGANDUNGI LIPASE T1
Oleh
IZUDDIN B. ABDUL RAHMAN
Januari 2013
Pengerusi: Profesor Dr. Raja Noor Zaliha Raja Abd Rahman, PhD
Institut: Biosains
Disebabkan oleh keprihatinan terhadap alam sekitar, industri detergen dikehendaki
untuk mengambil pendekatan yang lebih mesra alam dalam merumuskan detergen,
termasuk menggunakan bahan kimia terbiodegradasi dan enzim. Kesannya,
penggunaan enzim dalam formulasi detergen telah menjadi satu kemestian, dan enzim
dari pelbagai jenis dan fungsi sedang dibangunkan. Dengan itu, enzim rekombinan
dipanggil lipase T1 (E.C. 3.1.1.3), yang telah dikaji secara mendalam dan berjaya
dihasilkan, telah dinilai sebagai enzim detergen untuk formulasi detergen untuk
pencuci pinggan mangkuk automatik (ADD).
Lipase T1 telah dihasilkan secara besar-besaran pada skala kelalang makmal dengan
membiak perumah penzahiran Escherichia coli BL21, yang membawa gen T1 dan
menyediakan enzim lipase T1 yang mentah dari sel bakteria melalui sonikasi. Aktiviti
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enzim telah diukur, dan kehadiran enzim telah disahkan menggunakan gel
elektroforesis poliakrilamida dengan natrium dodesil sulfat (SDS-PAGE). Walaupun
enzim yang mentah tidak ditulenkan, kehadiran protein perumah dianggap tidak
memberi apa-apa kesan yang ketara kepada keputusan kajian ini, dan telah dibuktikan
oleh ketiadaan enzim lipase, amilase, dan protease dari organisma perumah.
Lipase T1 yang mentah dalam bentuk bebas kemudiannya diperiksa keserasiannya
dengan komponen-komponen detergen yang lain dengan melakukan ujian kestabilan
dan memeriksa baki aktiviti yang tinggal. Lipase T1 adalah stabil dalam kebanyakan
surfaktan tidak bercas, terutama yang dibuat daripada alkohol polihidrik. Lipase T1
juga stabil dalam campuran natrium karbonat dan glycine, yang menghasilkan pH
9.25 yang juga hampir dengan pH optimum lipase T1 iaitu 9.0. Walau bagaimanapun,
natrium karbonat sahaja menyahstabilkan lipase T1 mungkin disebabkan oleh
interaksi antara karbonat dan Ca2+. Keputusan ini menunjukkan bahawa alkohol
polihidrik dan glycine memberi kesan penstabilan kepada lipase T1.
Seterusnya, lipase T1 mentah telah disembur-kering dengan penambahan bahan
dinding, seperti gam arab dan maltodekstrin. Satu campuran yang terdiri daripada
gam arabic/maltodextrin/supernatan lipase T1 dengan nisbah 6:12:3 telah
menghasilkan produk yang kebanyakannya berbentuk sfera apabila dilihat di bawah
mikroskop elektron, dan lipase T1 yang disaluti bahan dinding menghasilkan
peningkatan sebanyak tiga kali ganda dalam aktiviti enzim berbanding dengan lipase
T1 dalam bentuk bebas. Produk semburan-pengeringan juga lebih senang terlarut
kerana sifat hidrofilik bahan dinding.
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Selepas itu, lipase T1 yang mentah telah dinilai sebagai satu komponen tambahan
bagi formulasi ADD. Prestasi pencucian pinggan mangkuk oleh detergen yang
diformulasikan dinilai dengan mengira peratus kotoran yang berjaya ditanggalkan
menggunakan Penguji Keberkesanan Pencucian Rekaan Leenert yang telah
dimajukan. Prestasi pencucian pinggan mangkuk oleh detergen yang diformulasikan
meningkat dengan peningkatan suhu tetapi terjejas dengan kehadiran air yang
mengandungi garam, khususnya Ca2+ dan Mg2+. Kehadiran air yang mengandungi
garam ini mengurangkan kecekapan detergen yang diformulasikan dalam
menanggalkan kotoran sebanyak 68% secara purata. Walau bagaimanapun, lipase T1
tidak terjejas oleh kehadiran air yang mengandungi garam, dan kecekapan enzim ini
telah dipertingkatkan dengan kehadiran polyacrylates. Selain itu, kehadiran Ca2+
meningkatkan integriti struktur lipase T1, terutamanya pada suhu tinggi.
Akhirnya, formulasi ADD yang digelar sebagai "U1" telah dihasilkan berdasarkan
keputusan kajian ini. Secara keseluruhannya, penambahan lipase T1 ke dalam
formulasi ADD meningkatkan prestasi pencucian pinggan mangkuk sebanyak 17%
berbanding dengan ketiadaan lipase T1, terutamanya dalam kehadiran air yang
mengandungi garam. Tambahan pula, kajian ini akan memberi gambaran yang lebih
jelas terhadap lipase sebagai enzim detergen, khususnya dalam pencucian pinggan
mangkuk secara automatik.
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ACKNOWLEDGEMENTS
First and foremost, I would like to praise Almighty Allah and the Merciful. Without
His guidance and will, the completion of this study would not be possible.
This study was quite challenging due to lack of knowledge in the field of detergent
formulation. Nevertheless, with constant supervision, motivation, and guidance from
my supervisor, this study went through, successfully; thus, I would like to extend my
sincere gratitude to Prof. Dr. Raja Noor Zaliha Raja Abd Rahman. Her efforts and
determinations to ensure her students‘ success and adequate funding throughout our
studies should be celebrated. In addition, I would like to thank my co-supervisors,
Prof. Dr. Abu Bakar Salleh and Prof. Dr. Mahiran Basri for their constructive
criticisms and advice. Not to forget my supervisors, Dr. Mohd Shukuri Mohamad Ali
and Dr. Adam Leow Thean Chor for their ideas and suggestions given towards this
study.
A significant part of this study would be impossible without the help of the people
from the Advanced Oleochemical Technology Division (AOTD) and Malaysia Palm
Oil Board (MPOB), especially the director, Dr. Hazimah Abu Hassan for allowing the
dishwashing tests to be done, and Ms. Zamiah Hasman and Dr. Lim Wen Huei for
their assistance in running the tests and motivation when I was at wit‘s end.
I would like to thank my more experienced colleagues, Noor Hidayah Shahidan, Siti
Nurbaya Oslan, Zulhilmi Abdul Rahman, and Ropaning Sulong for sharing their
knowledge and giving assistance in doing lab work. Also, to the rest of my colleagues
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in Enzyme Technology group (EMTECH) for their collective contributions towards
the progress of this study.
Last but not least, my deepest gratitude to my dearest family, especially my parents,
Abdul Rahman and Rose Miniah for their constant pressure in completing my study,
prayers, and unconditional love.
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I certify that an Examination Committee has met on date of viva voce to conduct the
final examination of Izuddin b. Abdul Rahman on his Master of Science thesis
entitled "Formulation of an automatic dishwashing detergent containing locally
produced enzymes" in accordance with Universiti Pertanian Malaysia (Higher
Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations
1981. The Committee recommends that the student be awarded the Master of Science
Members of the Examination Committee were as follows:
Janna Ong Abdullah, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Rosfarizan Mohamad, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Umi Kalsom Md Shah, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Fahrul Zaman Huyop, PhD
Associate Professor
Faculty of Bioscience and Bioengineering
Universiti Teknologi Malaysia
Malaysia
(External Examiner)
_____________________
ZULKARNAIN ZAINAL, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Raja Noor Zaliha Raja Abd Rahman, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Abu Bakar Salleh, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
Mahiran Basri, PhD
Professor
Faculty of Science
Universiti Putra Malaysia
(Member)
______________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously, and is not
concurrently, submitted for any other degree at Universiti Putra Malaysia or at any
other institution.
______________________
IZUDDIN B. ABDUL RAHMAN
Date: 7 January 2013
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK v
ACKNOWLEDGEMENTS viii
APPROVAL x
DECLARATION xii
LIST OF TABLES xv
LIST OF FIGURES xvi
LIST OF ABBREVIATIONS xvii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 5
2.1 Detergent Components 5
2.1.1 Surfactants 5
2.1.2 Water Softeners 7
2.1.3 Bleaching Agents 9
2.1.4 Dispersants and Polymers 11
2.1.5 Other Components 12
2.2 Detergent Enzymes 12
2.2.1 Thermostable T1 Lipase 14
2.2.2 Enzyme Activity Assay 16
2.2.3 Enzyme Purification 16
2.2.4 Enzyme Encapsulation 17
2.3 Detergent Formulations 20
2.4 Detergent Performance 22
2.4.1 Water Hardness Effect 23
2.4.2 Temperature Effect 24
2.4.3 pH Effect 25
3 MATERIALS AND METHODS 27
3.1 Materials 27
3.2 Enzyme Production 28
3.3 Enzyme Analysis 29
3.4 Determination of the Presence of Host Enzymes 30
3.5 T1 Lipase Compatibility Tests 31
3.6 Enzyme Encapsulation 31
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3.7 Detergent Formulation 32
3.8 Hard Water Preparation 33
3.9 Dishwashing Tests 33
3.10 Statistical Analysis 36
4 RESULTS AND DISCUSSION 37
4.1 Production of Detergent Enzyme 37
4.1.1 Analysis of the Crude T1 Lipase 37
4.1.2 Presence of Host Enzymes 38
4.2 Compatibility of Detergent Enzyme 40
4.2.1 Stability of T1 Lipase in Detergent
Components 40
4.2.2 Stability of T1 Lipase in Various Alkalinity
Agents 43
4.3 Spray-drying of Detergent Enzyme 44
4.3.1 Enzyme Residual Activity 45
4.3.2 Particle Analysis 47
4.4 Performance of Detergent Formulations 50
4.4.1 Soft and Hard Water 50
4.4.2 Dispersing Agent 53
4.4.3 Surfactant Concentration 56
4.4.4 Amount of T1 Lipase 58
4.5 Summary of Detergent Formulations 60
5 CONCLUSION AND RECOMMENDATIONS FOR
FUTURE RESEARCH 62
REFERENCES 65
APPENDICES 70
Appendix A: SDS-PAGE chemical compositions 70
Appendix B: Lipase assay standard curve 71
Appendix C: Protein assay standard curve 72
Appendix D: Statistical analyses 73
LIST OF PUBLICATIONS 78
BIODATA OF STUDENT 79
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