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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