UNIVERSITI PUTRA MALAYSIA ENZYMATIC …psasir.upm.edu.my/31942/1/FS 2012 41R.pdfHIDROKSAMIK LEMAK DARIPADA MINYAK KANOLA DAN MINYAK SAWIT Oleh HOSSEIN JAHANGIRIAN Mei 2012 Pengerusi

UNIVERSITI PUTRA MALAYSIA

ENZYMATIC SYNTHESES, CHARACTERIZATIONS AND APPLICATIONS OF FATTY HYDROXAMIC ACID DERIVATIVES

FROM CANOLA AND PALM OILS

HOSSEIN JAHANGIRIAN

FS 2012 41

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ENZYMATIC SYNTHESES, CHARACTERIZATIONS AND

APPLICATIONS OF FATTY HYDROXAMIC ACID DERIVATIVES FROM

CANOLA AND PALM OILS

By

HOSSEIN JAHANGIRIAN

Thesis Submitted to the School Of Graduate Studies, Universiti Putra

Malaysia, in Fulfillment of the Requirements for the Degree of Doctor of

Philosophy

May 2012

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Doctor of Philosophy

ENZYMATIC SYNTHESES, CHARACTERIZATIONS AND

APPLICATIONS OF FATTY HYDROXAMIC ACID DERIVATIVES FROM

CANOLA AND PALM OILS

By

HOSSEIN JAHANGIRIAN

May 2012

Chairman: Prof. MD Jelas Haron, PhD

Faculty: Science

Fatty hydroxamic acids and derivatives have attracted many researches due to their

pharmaceutical and analytical applications. They have been widely applied as food

additives, growth factors, antibacterial agents, fungicides, tumor inhibitors, enzyme

inhibitors and metal chelators. A common method for the preparation of

hydroxamic acids and their derivatives is via reaction of hydroxylamine or their

derivatives with alkyl esters or activated carboxylic acids. However these reactions

often contain many steps and require expensive chemicals. In this study, four types

of N-substituted fatty hydroxamic acids as the following were synthesized. Phenyl

fatty hydroxamic acids (PFHAs), synthesized by reaction of canola oil, palm kernel

oil or palm stearin with N-phenyl hydroxylamine (N-PHA). Methyl fatty

hydroxamic acids (MFHAs), synthesized by reaction of palm kernel oil and N-

methyl hydroxylamine (N-MHA). Isopropyl fatty hydroxamic acids (IPFHAs),

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synthesized by reaction of palm kernel oil and N- isopropyl hydroxylamine (N-

IPHA). Benzyl fatty hydroxamic acids (BFHAs), synthesized by reaction of palm

kernel oil and N-benzyl hydroxylamine (N-BHA). The reactions were carried out

in a sealed flask, incubated in water bath shaker at 39°C for 72 hours and catalyzed

by immobilized lipase (Lipozyme TL IM or RM IM). The products were then

separated by filtration, solvent extraction followed by evaporation of the solvent.

The products were characterized by The FT-IR and 1H NMR spectroscopies, CHN

elemental analysis and qualitative color test with Cu(II), Fe(III) and V(V) ions. The

products as chelating agents were used for extraction of Cu(II) or Fe(III) ions from

samples containing mixture of metal ions in aqueous solution. The products and

their copper complexes were also evaluated for antibacterial activity on Escherichia

coli (E. Coli) and Staphylococcus aureus (S. Aureus) and antifungal activity on

Candida parapsilosis (C. parapsilosis), Candida albicans (C. albicans) and

Aspergillus fumigatus (A. fumigatus) using disk and well diffusion methods.

The conversion percentage of phenyl hydroxylaminolysis of the two different

commercial canola oils (Ladan and Krystal brands), palm stearin oil and palm

kernel oil were 55.6, 52.2, 51.4 and 49.7 %, respectively. In addition the conversion

percentage of methyl hydroxylaminolysis, isopropyl hydroxylaminolysis and benzyl

hydroxylaminolysis of the commercial palm kernel oil were 77.8, 65.4 and 61.7 %,

respectively. The FTIR and 1H NMR spectroscopic data, CHN elemental analysis

and qualitative color test on the purified products confirmed the existence of

PFHAs, MFHAs, IPFHAs and BFHAs.

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The results of metal extraction studies showed that the MFHAs, PFHAs, IPFHAs

and BFHAs based on palm kernel oil were able to separate copper (II) ion from

mixture of Cu(II) and Mg (II), Ni (II), Al (III), Mn (II) or Co (II) in aqueous media

at pH 6.2. A good stripping recovery of Cu(II) was achieved with a single

extraction by 3M H2SO4. In addition the MFHAs and BFHAs from palm kernel oil

were found able to separate iron (III) from a mixture of Fe(III) and Mg(II), Ni(II),

Al(III), Mn(II) or Co(II) in aqueous media at pH 1.9. A good stripping recovery of

Fe(III) was obtained with a single extraction by 5M HCl.

Antimicrobial study showed that PFHAs and Cu-PFHs had stronger antibacterial

properties against E. coli compared to S. aureus. Also antibacterial property of the

PFHAs from canola oil is stronger than palm kernel oil followed by palm stearin.

Furthermore the antibacterial properties of the Cu-PFHs from canola oil, palm

kernel oil and palm stearin were stronger than antibacterial properties from their

PFHAs. Similarly the results also showed that the Cu-PFHs from canola and palm

kernel oils have stronger antifungal properties on C. parapsilosis, C. albicans and A.

fumigatus compared to their PFHAs.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

SINTESIS BERENZIM, PENCIRIAN DAN APLIKASI TERBITAN ASID

HIDROKSAMIK LEMAK DARIPADA MINYAK KANOLA DAN MINYAK

SAWIT

Oleh

HOSSEIN JAHANGIRIAN

Mei 2012

Pengerusi : Prof. MD Jelas Haron, PhD

Fakulti : Sains

Banyak penyelidikan telah dilakukan keatas asid hidroksamik lemak dan terbitannya

kerana penggunaannya dalam farmaseutikal dan analisis. Sebatian ini telah

digunakan secara meluas sebagai aditif makanan, faktor pertumbuhan, agen anti-

bakteria, racun kulat, perencat tumor, perencat enzim dan pengkelat logam. Kaedah

biasa untuk penyediaan asid hidroksamik dan terbitannya adalah melalui tindak

balas hidroksilamina atau terbitannya dengan alkil ester atau asid karboksilik

teraktif. Walau bagaimanapun, tindak balas ini sering mengandungi langkah-

langkah yang banyak dan memerlukan bahan kimia yang mahal. Dalam kajian ini,

empat jenis asid hidroksamik lemak tertukarganti seperti berikut telah disintesis.

Fenil asid hidroksamik lemak (PFHAs), disintesis melalui tindak balas minyak

kanola, minyak isirung sawit atau stearin sawit dengan N-fenil hidroksilamina

(PHA). Metil asid hidroksamik lemak (MFHAs), disintesis melalui tindak balas

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minyak isirung sawit dengan N-metil hidroksilamina (MHA). Isopropyl asid

hidroksamik lemak (IPFHAs), disintesis melalui tindak balas minyak isirung sawit

dengan N-isopropil hidroksilamina (MHA). Benzil asid hidroksamik

lemak(BFHAs), disintesis melalui tindak balas minyak isirung sawit dengan N-

benzil hidroksilamina (BHA). Tindak balas telah dijalankan dalam kelalang

bertutup yang dieram dalam kukus air berpengaduk pada suhu 39 °C selama 72 jam

dengan magkin lipase tersekatgerak (Lipozyme TL IM atau IM RM). Produk

kemudiannya dipisahkan melalui penurasan, pengekstrakan pelarut dan diikuti

dengan penyejatan pelarut.

Produk telah dicirikan dengan spektroskopi FT-IR dan 1H NMR, analisis unsur

CHN dan ujian kualitatif warna dengan ion Cu(II), Fe(III) dan V(V). Produk

sebagai agen pengkelat telah digunakan untuk pengekstrakan ion Cu(II) dan Fe(III)

daripada sampel larutan akueus yang mengandungi campuran ion logam. Produk

dan kompleksnya dengan ion logam kuprum juga dinilai untuk aktiviti anti-bakteria

keatas Escherichia coli (E. Coli) dan Staphylococcus aureus (S. aureus) dan aktiviti

antikulat keatas Candida parapsilosis (C. parapsilosis), Candida albicans (C.

albicans) dan Aspergillus fumigatus (A. fumigatus) menggunakan kaedah resapan

cakera dan takungan.

Peratus hasil fenil-hidroksilaminolilis minyak kanola (jenama Ladan dan Krystal),

stearin sawit dan minyak isirung sawit masing-masing adalah 55.6, 52.2, 51.4 dan

49.7%. Di samping itu, peratus hasil metil-hidroksilaminolisis, isopropil-

hidroksilaminolisis dan benzil-hidroksilaminolisis minyak isirung sawit komersial

masing-masing adalah 77.8, 65.4 dan 61.7%. Data spektroskopi FTIR dan 1H NMR

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, analisis unsur CHN dan ujian kualitatif warna keatas produk yang telah ditulenkan

mengesahkan kewujudan PFHAs, MFHAs, IPFHAs dan BFHAs.

Keputusan kajian pengekstrakan ion logam menunjukkan bahawa MFHAs, PFHAs,

IPFHAs dan BFHAs daripada minyak isirung kelapa sawit dapat memisahkan ion

Cu(II) dari campuran Cu(II) dengan Mg(II), Ni(II), Al(III), Mn(II) atau Co (II)

dalam larutan akueus pada pH 6.2. Pelucutan Cu(II) telah dicapai degan sekali

pengekstrakan semula menggunakan H2SO4 3M. Di samping itu MFHAs dan

BFHAs daripada minyak isirong sawit didapati mampu untuk memisahkan Fe(III)

daripad larutan akueus yang mengandungi campuran Fe (III) dengan Mg(II), Ni(II),

Al(III), Mn(II) atau Co(II) pada pH 1.9. Pelucutan Fe(III) telah dicapai melalui

pengekstrakan semula dengan HCl 5M.

Kajian antimikrobial menunjukkan bahawa PFHAs dan Cu-PFHs mempunyai sifat

antibakteria yang kuat terhadap E. coli berbanding S. aureus. Juga sifat antibakteria

PFHAs daripada minyak kanola adalah lebih kuat daripada PFHAs minyak isirung

sawit diikuti oleh PFHAs stearin sawit. Sebagai tambahan, sifat antibakteria Cu-

PFHs daripada minyak kanola, minyak isirung sawit dan stearin sawit adalah lebih

kuat berbanding PFHAs masing-masing. Keputusan kajian juga menunjukkan

bahawa Cu-PFHs daripada minyak kanola dan isirong sawit mempunyai sifat

antikulat yang kuat keatas C. parapsilosis, C. albicans dan A. fumigatus berbanding

PFHAs masing-masing.

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ACKNOWLEDGEMENTS

I offer most sincere gratitude to my supervisor, Professor Dr. Md Jelas Haron who

has supported me to perform and writing my thesis modestly and sagaciously. I also

thank to my co-supervisors Associate Professor Dr. Sidik Silong and Associate

Professor Dr. Nor Azah Yusof for their assistances.

I owe special thanks to my dear’s wife Dr. Roshanak Rafiee Moghaddam and my

lovely daughters Rojin & Ronak for all their help and patience during my study.

I owe great thanks to my gentle parents, my darling father, Mr. Hassan Jahangirian

and my dearest mother, Mrs. Fatemeh Ghiasi, and my kind parents-in-law, my

darling father-in-law, Mr. Mohammad Rafiee Moghaddam and my dearest mother-

in-law, Mrs. Najmeh Jahandoost for all things that they taught me and for guiding

me to wisdom and sageness way in life.

I owe honest thanks to my dear brothers and sisters, Mehran, Shokofeh, Siavosh,

Arash, Azar and Narges and dear brothers-in-law, Roozbeh and Arash for

supporting and encouraging me.

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I certify that Examination Committee has met on date of viva voce to conduct the

final examination of Hossein Jahangirian on his Degree of Doctor of Philosophy

thesis entitled “Enzymatic syntheses, characterizations and applications of fatty

hydroxamic acid derivatives based on canola and palm oils” in accordance with

University and University Colleges Act 1971 and the Constitution of the Universiti

Putra Malaysia [P.U. (A) 106] 15 March 1998. The Committee recommends that

the student be awarded the Degree of Doctor of Philosophy.

Member of the Examination committee were as follows

Zulkarnain B. Zainal, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Mohd Aspollah B. HJ. MD Sukari, PhD

Professor

Faculty of Science


(Internal Examiner)

Abdul Halim BIN Abdullah, PhD

Associate Professor

Faculty of Science


(Internal Examiner)

Neeraj Sharma, PhD

Professor

Department of Chemistry

Himachal Pradesh University, India

(External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


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 Doctor of Philosophy.

The members of Supervisory committee were as follows:

MD Jelas Haron, PhD

Professor

Faculty of Science


(Chairman)

Sidik Silong, PhD

Associate Professor

Faculty of Science


(Member)

Nor Azah Yusof, PhD

Associate Professor

Faculty of Science


(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


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 it is not concurrently, submitted for any other degree at University Putra

Malaysia or at any institutions.

HOSSEIN JAHANGIRIAN

Date: 25 May 2012

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xvii

LIST OF FIGUERS xix

LIST OF ABBREVIATIONS xxii

CHAPTER

1 INTRODUCTION 1

1.1 Background of study 1

1.1.1 Enzymatic reaction as desired method for hydroxamic

acids synthesis 2

1.1.2 Hydroxamic acid as antibacterial agent 3

1.1.3 Hydroxamic acid as antifungal agent 3

1.1.4 Hydroxamic acid as chelating agent 4

1.2 Hypothesis 5

1.3 Objectives 6

1.4 Justifications of the research 7

1.5 Outline of the thesis 7

2 LITERATURE REVIEW 9

2.1 Hydroxamic acids and their derivatives 9

2.1.1 History and structure 9

2.1.2 Synthesis of hydroxamic acids 11

2.1.3 Enzymatic synthesis of fatty hydroxamic acids 14

2.1.4 Metal complexes of hydroxamic acids 17

2.1.5 Copper (II) complexes of hydroxamic acids 20

2.1.6 Iron (III) complexes of hydroxamic acids 21

2.2 Canola oil 24

2.2.1 History of canola oil 24

2.2.2 Growth and development of canola 26

2.2.3 Chemistry of canola oil 27

2.2.4 Industrial applications of canola oil 29

2.3 Palm oils 30

2.3.1 History of the oil palm plant 30

2.3.2 Type of palm oil 33

2.3.3 Chemistry of palm oil 34

2.3.4 Industrial and oleo chemistry application of palm oil 35

2.4 Lipase as catalyst 37

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2.4.1 Structure and sources of lipase 37

2.4.2 Effective factors on the activity of lipase catalyst 41

2.4.3 Industrial applications of lipase 43

2.5 Solvent extraction 46

2.5.1 Definition of solvent extraction 46

2.5.2 Theory of solvent extraction 47

2.5.3 Metal separation by solvent extraction 48

2.5.4 Copper (II) separation by solvent extraction 50

2.5.5 Iron (III) separation by solvent extraction 54

2.6 Antimicrobial Susceptibility Testing (AST) 58

2.6.1 Introducing of methods 58

2.6.2 Bactericidal and bacteriostatic agents 60

2.6.3 Fungicidal and fungistatic agents 64

2.7 Biochemical processes of hydroxamic acids 66

3 ENZYMATIC SYNTHESIS OF PHENYL FATTY

HYDROXAMIC ACIDS FROM CANOLA AND PALM OILS 69

3.1 Introduction 69

3.2 Materials and methods 71

3.2.1 Materials 71

3.2.2 Synthesis of PFHAs 71

3.2.3 Characterization 72

3.3 Results and discussion 72

3.3.1 Synthesis 72

3.3.2 Effect of Organic Solvent 73

3.3.3 Effect of Reaction Time 74

3.3.4 Effect of the Type and Amount of Enzyme 75

3.3.5 Effect of the Mol Ratio of PHA to oil 77

3.3.6 Effect of Temperature 78

3.3.7 Phenyl hydroxylaminolysis of Different Oils 79

3.3.8 Characterization of PFHAs 81

3.3.8.1 Elemental Analysis 81

3.3.8.2 Fourier Transform Infrared Spectroscopy

(FTIR) 81

3.3.8.3 1H Nuclear Magnetic Resonance (

1H NMR) 82

3.4 Conclusion 84

4 ENZYMATIC SYNTHESIS OF FATTY HYDROXAMIC ACID

DERIVATIVES BASED ON PALM KERNEL OIL 86

4.1 Introduction 86

4.2 Material and method 88

4.2.1 Materials 88

4.2.2 Synthesis of MFHAs, IPFHAs and BFHAs 89


4.3 Results and Discussion 91

4.3.1 Synthesis 91

4.3.2 Effect of mol ratio of reactants 91

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4.3.3 Qualitative test of MFHAs, IPFHAs and BFHAs 94

4.3.4 Elemental analysis 95

4.3.5 Fourier Transform Infrared Spectroscopy (FTIR) 95

4.3.6 1H Nuclear Magnetic Resonance (

1H-NMR) 98

4.4 Conclusions 104

5 COPPER EXTRACTION BY FATTY HYDROXAMIC ACIDS

DERIVATIVES SYNTHESIZED BASED ON PALM KERNEL

OIL 106

5.1 Introduction 106


5.2.1 Material and apparatus 108

5.2.2 Synthesis of fatty hydroxamic acid derivatives 109

5.2.3 General copper extraction process 109

5.2.4 Copper stripping study 110

5.3 Result and discussion 111

5.3.1 Organic solvent selection and volume ratio of organic to

aqueous phase determination 111

5.3.2 Effect of pH on copper extraction 113

5.3.3 Effect of MFHAs concentration on copper extraction 114

5.3.4 Determination of mole ratio MFHAs / Cu2+

in Cu-MFHAs

115

5.3.5 Comparison of copper extraction by different fatty

hydroxamic acid derivatives 117

5.3.6 Copper separation by fatty hydroxamic acids derivatives

117

5.3.7 Copper stripping 119

5.4 Conclusion 121

6 BENZYL AND METHYL FATTY HYDROXAMIC ACIDS

BASED ON PALM KERNEL OIL AS CHELATING AGENT

FOR LIQUID-LIQUID IRON (III) EXTRACTION 122



6.2.1 Material and apparatus 124

6.2.2 Synthesize of chelating agent 124

6.2.3 General Iron(III) extraction procedure 125

6.2.4 Iron(III) stripping study 126

6.3 Result and discussion 127

6.3.1 Organic solvent selection and volume ratio org/aq phases

determination 127

6.3.2 Effect of pH on Iron (III) extraction 129

6.3.3 Effect of chelate concentration 130

6.3.4 Determination of mole ratio MFHs / Fe3+

in Fe-MFHs 131

6.3.5 Comparison of iron extraction by MFHAs and BFHAs 133

6.3.6 Iron (III) separation by MFHAs and BFHAs 133

6.3.7 Iron (III) stripping and preconcentration 134

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6.4 Conclusion 137

7 ANTIBACTERIAL EFFECT OF PHENYL FATTY

HYDROXAMIC ACIDS SYNTHESIZED FROM CANOLA OIL 138



7.2.1 Materials 140


7.2.3 Evaluation of antibacterial activity 141

7.2.4 Quantification and characterization of PFHAs 142


7.3.1 Antibacterial activity 142


7.4 Conclusion 146

8 ANTIBACTERIAL PROPERTIES OF COPPER PHENYL

FATTY HYDROXAMATE BASED ON CANOLA AND PALM

KERNEL OILS 147



8.2.1 Chemicals and bacteria 150

8.2.2 Preparation of Cu-PFHs 150

8.2.3 Evaluation of antibacterial activity 151



8.3.1 Antibacterial activity 152




(FTIR) 155

8.3.2.3 UV-Vis Spectroscopy 157

8.4 Conclusion 159

9 ANTIFUNGAL PROPERTIES OF PHENYL FATTY

HYDROXAMIC ACIDS AND THEIR COPPER COMPLEXES

SYNTHESIZED BASED ON CANOLA AND PALM KERNEL

OILS 161



9.2.1 Material and microorganisms 163


9.2.3 Preparation of Cu-PFHs 164

9.2.4 Evaluation of Antifungal Activity 164



9.3.1 Antifungal activity 166


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(FTIR) 169

9.3.2.3 UV-Vis Spectroscopy 169

9.3.2.4 1H Nuclear Magnetic Resonance (

1H NMR) 169

9.4 Conclusions 171

10 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH 173

10.1 Summary and general conclusion 173

10.2 Recommendation for future research 177

REFERENCES 179

APPENDICES 194

BIODATA OF STUDENT 196

LIST OF PUBLICATIONS 197

Documents

UNIVERSITI PUTRA MALAYSIA ENZYMATIC …psasir.upm.edu.my/31942/1/FS 2012 41R.pdfHIDROKSAMIK LEMAK DARIPADA MINYAK KANOLA DAN MINYAK SAWIT Oleh HOSSEIN JAHANGIRIAN Mei 2012 Pengerusi