UNIVERSITI PUTRA MALAYSIA EFFECT OF TRANSITION … · ruthenium ion (Ru3+) ... Kajian ini meliputi...
Transcript of UNIVERSITI PUTRA MALAYSIA EFFECT OF TRANSITION … · ruthenium ion (Ru3+) ... Kajian ini meliputi...
UNIVERSITI PUTRA MALAYSIA
EFFECT OF TRANSITION METAL ION SUBSTITUTION ON THERMOSTABLE T1 LIPASE ACTIVITY
HARMIZA HARUN
FS 2013 12
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EFFECT OF TRANSITION METAL ION SUBSTITUTION ON THERMOSTABLE T1 LIPASE ACTIVITY
By
HARMIZA HARUN
Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Master of Science
January 2013
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Especially dedicated to my dearly beloved:
Husband,
Mohd Hafizuddin Bin Ruslan
Son,
Muhammad Hasif Danish Bin Mohd Hafizuddin
Parents,
Hj. Harun Bin Abu
Hjh. Zainab Binti Hj. Abdullah
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of requirement for the degree of Master of Science
EFFECT OF TRANSITION METAL ION SUBSTITUTION ON THERMOSTABLE T1 LIPASE ACTIVITY
By
HARMIZA HARUN
January 2013
Chairman: Professor Mohd Basyaruddin Abdul Rahman, PhD
Faculty: Science
This study involved the effect of transition metal ion removal and substitution on
lipase activity and thermostability. The role of metal ions in the activity and stability
of the enzyme was studied using the holoenzyme, apoenzyme and metal substituted
enzyme. T1 lipase from Geobacillus zalihae contains a structural zinc ion (Zn2+) that
apparently contributes to the high temperature tolerance. This Zn2+ ion is tightly
bound to histidine (H81 and H87) in the catalytic domain and aspartate (D61 and
D238) in the extra domain of T1 lipase. Initially, Molecular Dynamics (MD)
simulation using YASARA software was performed at 343 K and pH 9 to determine
the changes in the protein structure, dynamics and flexibility of T1 lipase affected due
to Zn2+ ion depletion. Interestingly, D238, H81 and H87 which also the Zn2+ ion
binding ligand, were found to be highly fluctuated in the absence of Zn2+ ion which
may have caused weaker intramolecular interaction of zinc ion-binding coordination.
The stability of T1 lipase and apo-T1 lipase was estimated based on the Gibbs free
energy (ΔG) analysis using FoldX software. Based on the ΔG value calculated, the
stability of apo-T1 lipase was reduced to -53 kcal/mol as compared to T1 lipase, -65
kcal/mol. Hence, it is suggested that Zn2+ ion contributes to the stability of T1 lipase
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at elevated temperature. The role of the Zn2+ ion was elucidated by its elimination
from the structure of T1 lipase with two metal chelators such as, 2,6-
pyridinedicarboxylic acid (2,6-PDCA) and N,N,N’,N’-tetrakis-(2-pyridylmethyl)
ethylenediamine (TPEN). Among them, the incubation of T1 lipase with TPEN
removed almost up to 100 % of the Zn2+ ion followed by 83 % removal by 2,6-PDCA.
The specific activity of apo-T1 lipase was decreased (84 U/mg) as compared to T1
lipase (104 U/mg), thus confirming the role of Zn2+ ion in the stability of T1 lipase.
This data is supported by the details obtained in the thermostability study and half-life
study of both lipases. T1 lipase has been modified by means of transition metal ion
replacement in order to improve its enzymatic activity and protein stability at high
temperature. Three potential transition metal ions such as Mn2+ ion, Pd2+ion, and
ruthenium ion (Ru3+) has been added in apo-T1 lipase via dialysis at 4 oC. Zn2+ ion in
T1 lipase was successfully replaced by Mn2+ ion and Pd2+ ion with the ratio of 1 mol
of protein to ~1 mol of transition metal ion. The specific activity of Mn2+-T1 lipase
and Pd2+-T1 lipase was enhanced about 1.6 fold and 1.1 fold respectively. However,
the substitution of T1 lipase with Ru3+ was unsuccessful due to enzyme inactivation.
The biochemical and biophysical characterization of T1 lipase and apo-T1 lipase was
performed. Both lipases shared the same optimum temperature which is 70 oC.
However, the removal of Zn2+ has reduced the thermostability and half live (t1/2) of T1
lipase by 10 oC and 20 minutes rescpectively. The fluorescence emission spectra
showed the maximum intensity (λmax) was red-shifted from 433 nm to 511 nm due to
the elimination of Zn2+ ion. This change ascribed the decrease of the exposure of
hydrophobic cavity in apo-T1 lipase. In addition, the changes in the stability and
activity of T1 lipase and apo-T1 lipase were investigated during denaturation by urea.
An increased in the denaturant concentration has drastically induced inactivation and
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unfolding of both lipases. In the absence of Zn2+ ion, the maximal fluorescence
emission spectrum of T1 lipase was red shifted to a maximum value of 355 nm. The
effect of transition metal ion chelation and addition on the structure of T1 lipase was
observed using Circular Dichroism (CD) based on the changes in the secondary
structure components, such as, α-helix, β-sheet, turn and random coil. Fascinatingly,
the addition of Mn2+ ion and Pd2+ ion into the apoenzyme increased the thermal
denaturation (Tm) point based on the CD analysis. In conclusion, experimental
analysis performed in this study highlighted the understanding of holding the catalytic
and extra domains together with a metal ion can stabilize this lipase; changing the
metal ion can increase this stability. This work sets the foundation for the design of
T1 lipase as a metal ion biosensor.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
KESAN PENUKAR GANTIAN ION LOGAM PERALIHAN TERHADAP AKTIVITI T1 LIPASE TERMOSTABIL
Oleh
HARMIZA HARUN
January 2013
Pengerusi: Professor Mohd Basyaruddin Abdul Rahman, PhD
Fakulti: Sains
Kajian ini meliputi kesan penyingkiran dan penukargantian unsur-unsur peralihan
logam ion terhadap aktiviti, kestabilan terma dan struktur konformasi lipase T1.
Lipase T1 yang berasal daripada Geobacillus zalihae mempunyai satu struktur ion
zink (Zn2+) yang diperlukan untuk lipase ini bertoleransi terhadap suhu yang tinggi.
Fungsi ion logam dalam aktiviti dan kestabilan enzim ini telah dikaji dengan
menggunakan holoenzim, apoenzim dan enzim dengan ion logam yang telah
digantikan. Ion Zn2+ ini terikat dengan kuat kepada histidine (H81 dan H87) di dalam
domain katalitik dan asid aspartik (D61 dan D238) di dalam domain tambahan. Pada
mulanya, simulasi molekul dinamik (MD) telah dilakukan dengan menggunakan
perisian YASARA pada suhu 343 K dan pH 9 untuk menentukan perubahan yang
memberi kesan terhadap dinamik dan fleksibiliti protein lipase T1. Tanpa kehadiran
ion Zn2+, fluktuasi tinggi yang berlaku pada ligand Zn2+ iaitu D238, H81 dan H87
mungkin mengakibatkan interaksi intramolekul yang lemah pada koordinasi ini.
Kestabilan lipase T1 dan apo-lipase T1 telah diketahui berdasarkan analisis tenaga
bebas Gibbs (ΔG) dengan menggunakan perisian FoldX. Merujuk kepada nilai ΔG
yang telah dikira, kestabilan apo-lipase T1 telah berkurang kepada -53 kcal/mol jika
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dibandingkan dengan tenaga lipase T1 yang bernilai -65 kcal/mol. Oleh itu, ion Zn2+
telah diramalkan menjadi faktor utama dalam menentukan kestabilan lipase T1 pada
suhu tinggi. Fungsi ion Zn2+ telah dikenalpasti melalui penyisihannya daripada
struktur lipase T1 dengan menggunakan dua agen penyingkiran logam seperti 2,6-
pyridinedicarboxylic acid (2,6-PDCA) dan N,N,N’,N’-tetrakis-(2-pyridylmethyl)
ethylenediamine (TPEN). Inkubasi lipase T1 dengan TPEN telah menyebabkan
hampir 100 % ion Zn2+ tersisih diikuti dengan pengurangan sebanyak 83 % ion Zn2+
oleh 2,6-PDCA. Aktiviti spesifik bagi apo-lipase T1 telah berkurang kepada 84 U/mg
jika dibandingkan dengan lipase T1 (104 U/mg). Dengan ini, peranan ion Zn2+ di
dalam memastikan sifat kestabilan terma lipase T1 telah disahkan. Data ini telah
disokong oleh perincian yang telah dicapai daripada analisa kestabilan terma dan
kestabilan suhu bagi kedua-dua lipase tersebut. Lipase T1 telah dimodifikasi melalui
penukargantian logam ion peralihan bagi membaikpulih atau meningkatkan aktiviti
enzim dan stabiliti protein pada suhu tinggi. Tiga logam peralihan yang berpotensi
seperti ion Mn2+, ion Pd2+ dan ion Ru3+ telah dipilih untuk dimasukkan ke dalam apo-
lipase T1 melalui kaedah dialisis pada suhu 4 oC. Ion Zn2+ telah berjaya digantikan
dengan ion Mn2+ dan ion Pd2+ dengan nisbah 1 mol protein kepada 1 mol logam ion
peralihan. Lipase T1-Mn2+ dan lipase T1-Pd2+ menunjukkan peningkatan aktiviti
spesifik sebanyak 1.6 dan 1.1 kali ganda masing-masing, berbanding lipase T1.
Manakala, Ion Ru3+ tidak berjaya ditukarganti ke dalam struktur lipase T1 disebabkan
oleh penyahaktifan enzim. Pencirian sifat biokimia dan biofizik bagi lipase T1dan
apoenzim (Lipase T1 tanpa ion Zn2+) telah dikaji. Kedua-dua lipase mempunyai suhu
optima yang sama, iaitu 70 oC. Walaubagaimanapun, penyingkiran ion Zn2+ telah
menurunkan kestabilan terma dan t1/2 bagi lipase T1 sebanyak 10 oC dan 20 minit
masing-masing. Seperti yang ditunjukkan oleh pembebasan spektra fluorescence,
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penyisihan ion Zn2+ telah menyebabkan intensiti maksima (λmax) teralih dari 433 nm
ke 511 nm. Perubahan ini menunjukkan pengurangan bagi keterbukaan bahagian
hidrofobik apoenzim. Tambahan lagi, perubahan bagi kestabilan dan aktiviti lipase T1
and apo- lipase T1 telah dikenalpasti semasa denaturasi oleh urea. Peningkatan
kepekatan agen denturasi telah menyahaktif dan membuka lipatan kedua-dua lipase
dengan mendadak. Ketiadaan ion Zn2+ telah menyebabkan spektra pembebasan
pendaflour maksima bagi lipase T1 teralih kepada nilai yang tertinggi iaitu 355 nm.
Kesan penyisihan dan penambahan logam ion peralihan kepada struktur lipase T1
dapat dilihat melalui perubahan dalam komponen struktur sekunder seperti, α-helix,
β-sheet, lekukan beta (β-turn) dan gelung rawak. Analisa Circular Dichroism (CD)
telah menunjukkan peningkatan titik denaturasi termal (Tm) dan kestabilan (ΔG)
apoenzim yang disebabkan oleh penambahan ion Mn2+ dan ion Pd2+. Kesimpulannya,
analisa eksperimen yang telah dijalankan di dalam kajian ini dapat memperluaskan
lagi pemahaman terhadap penyatuan domain katalitik dan ekstra dengan ion logam
dapat menstabilkan lipase; pertukaran ion logam boleh meningkatkan kestabilan.
Kajian ini menjadi asas kepada penciptaan lipase T1 sebagain biosensor ion logam.
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ACKNOWLEDGEMENTS
Alhamdulillah, all praise to Allah S.W.T for giving me strength, patience and wisdom
to complete this study.
I am eternally grateful to my dearest husband, Mohd Hafizuddin Ruslan, parents and
family for their endless love, prayers, and constant support throughout my study.
Without my family, I would never be here and fulfilling this challenging task will not
be possible. Sincerest thanks.
My heartfelt gratitude and appreciation to my supervisory committee chairperson,
Prof. Dr. Mohd Basyaruddin Abdul Rahman for his excellent supervision, knowledge,
patience, encouragement and constructive criticisms throughout the period of this
project. His determination and enthusiasm towards scientific research have always
been my motivation.
I am also indebted to my co-supervisory committee members, Prof. Dato’ Dr. Abu
Bakar Salleh, and Dr. Adam Leow Thean Chor for their invaluable assistance, ideas,
support, and positive criticism in every way. I would like extend my gratitude to Prof.
Dr. Raja Noor Zaliha, Prof Dr. Mahiran Basri, Dr. Shukuri Mohd Ali, Dr. Bimo Ario
Tejo, and Dr. Roghayeh Abedi Karjiban for their advice and suggestion in this study.
Sincerest thanks to Prof. Dr. Romas J. Kazlauskas and Dr. Qing Jing for their
assistance, knowledge, suggestions and continuous guidance during my research
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attachment at the Department of Biochemistry, Molecular Biology and Biophysics
and the Biotechnology Institute, University of Minnesota.
Special thanks are also unlimited to all present and former members of Enzyme and
Microbial Technology Lab for the splendid time we shared and for the kind assistance
everyone gave me during my study. I am also indebted to all people who have helped
and supported me during the completion of this research.
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I certify that an Examination Committee met on 4 January 2013 to conduct the final examination of Harmiza Harun on her Master of Science thesis entitled “The Effect of Transition Metal Ions Substitution on The Activity of Thermostable T1 Lipase” In accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the candidate be awarded the Master of Science. Members of the Examination Committee are as follows: Nor Azowa Ibrahim, PhD Lecturer Faculty of Science Universiti Putra Malaysia (Chairman) Tan Wee Tee, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Mohd. Yunus Abd. Shukor, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Awang Ahmad Sallehin Awang Husaini, PhD Associate Professor Faculty of Resource Science Technology Universiti Malaysia Sarawak (External Examiner) __________________________ SEOW HENG FONG
Professor/Deputy Dean School of Graduate Studies University Putra Malaysia
Date:
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This thesis 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: Mohd Basyaruddin Abdul Rahman, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman) Abu Bakar Salleh, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Adam Leow Thean Chor, PhD Senior Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Romas J. Kazlauskas, PhD Professor Biochemistry, Molecular Biology and Biophysics University of Minnesota (Member)
___________________________ BUJANG BIN KIM HUAT, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia
Date:
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DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at Universiti Putra Malaysia or other institutions.
_________________ HARMIZA HARUN
Date: 4 January 2013
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TABLE OF CONTENT
Page ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii TABLE OF CONTENTS xiv LIST OF TABLES xvii LIST OF FIGURES xviii LIST OF ABBREVIATIONS xxi CHAPTER
1 INTRODUCTION 1 1.1 Research Background 1
1.2 Research Problem 3 1.3 Objectives of the Study 5 2 LITERATURE REVIEW 6
2.1 Lipase 6 2.2 Thermostable enzymes 7
2.2.1 T1 lipase 9 2.2.2 P1 lipase 10 2.2.3 L1 lipase 11
2.3 Artificial Metalloenzymes 13 2.4 Transition Metal Ions 16 2.5 Enzyme promiscuity 19 2.6 Molecular Dynamics (MD) simulations 22 2.7 Fluorescence Spectroscopy 23
2.8 Circular Dichroism (CD) analysis 24 3 MATERIALS AND METHODS 27
3.1 Materials 27 3.2 Computational analysis 30
3.2.1 Molecular dynamics (MD) simulation 31 i) Molecular dynamics procedure 31 ii) Analysis procedure 32
3.2.2 Analysis of Packing in Protein Structure (VORONOIA) 33 3.2.3 Protein stability measurement (FoldX) 33
3.2.4 Secondary structure analysis (POLYVIEW) 34 3.3 Lipase induction and expression 34 3.4 Quantitative determination of lipase activity 35
3.4.1 Standard curve of oleic acid 35 3.4.2 Lipase assay 35
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3.5 Determination of protein content 36 3.6 Purification of recombinant T1 lipase 37 3.7 SDS-PAGE electrophoresis 37 3.8 Native-PAGE electrophoresis 38 3.9 Preparation of apo-T1 lipase 38 3.10 Biochemical characterization of lipase 39
3.10.1 Effect of temperature on lipase activity 39 3.10.2 Effect of temperature on lipase stability 39 3.10.3 Effect of urea on lipase stability 40
3.11 Transition metal ion substitution of T1 lipase 41 3.12 Determination of metal ion concentration using ICP-MS 41 3.12.1 Preparation of calibration standard 41 3.12.2 Preparation of protein sample 42 3.13 Biophysical characterization of lipase 42
3.13.1 Intrinsic fluorescence 42 3.13.2 Extrinsic fluorescence 43 3.13.3 Circular Dichroism (CD) analysis 43
3.13.2.1 Estimation of protein secondary 43 structure
3.13.2.2 Thermal denaturation analysis 44 4 RESULTS AND DISCUSSION 45 4.1 Molecular dynamics simulation 45 4.1.1 T1 lipase and apo-T1 lipase dynamics 46
4.1.2 T1 lipase and apo-T1 lipase flexibility 50 4.1.3 Radius of gyration (Rg) 52 4.1.4 Solvent Acessible Surface Area (SASA) 54 4.1.5 Hydrogen Bond 56
4.2 FoldX analysis of on protein stability 59 4.3 Secondary structure prediction via POLYVIEW 60 4.4 Conclusion on MD simulations and computational 62
analysis 4.5 Expression of Geobacillus zalihae strain T1 64 4.6 Purification of recombinant T1 lipase 65 4.7 Zinc ion removal 69 4.8 Biochemical characterization 75
4.8.1 Effect of temperature on lipase activity 75 4.8.2 Effect of temperature on lipase stability 77
4.9 Effect on urea on lipase stability 82 4.10 Transition metal ion substitution of T1 lipase 86 4.11 Biophysical characterization of lipases 96
4.11.1 Fluorescence spectroscopy 96 4.11.2 Secondary structure analysis 100 4.11.3 Thermal denaturation analysis 103
4.12 Integration of computational and experimental result 107 5 CONCLUSIONS AND RECOMMENDATIONS 109