STRUCTURAL IMPLICATIONS OF Pseudomonas fluorescence STRAIN

AMS8 COLD-ACTIVE LIPASE IN ORGANIC SOLVENT

NORHAYATI BINTI YAACOB

FBSB 2018 62

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STRUCTURAL IMPLICATIONS OF Pseudomonas fluorescence STRAIN AMS8

COLD-ACTIVE LIPASE IN ORGANIC SOLVENT

By

NORHAYATI BINTI YAACOB

Thesis Submitted to School of Graduate Studies,

Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of

Doctor of Philosophy

December 2018

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unless otherwise stated. Use may be made of any material contained within the thesis for

non-commercial purposes from copyright holder. Commercial use of materials may only

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Copyright © Universiti Putra Malaysia

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

the requirement for the degree of Doctor of Philosophy

STRUCTURAL IMPLICATIONS OF Pseudomonas fluorescence STRAIN AMS8

COLD-ACTIVE LIPASE IN ORGANIC SOLVENT

By

NORHAYATI BINTI YAACOB

December 2018

Chairman : Mohd Shukuri Mohamad Ali, PhD

Faculty : Biotechnology and Biomolecular Sciences

Cold-active lipases from bacterial sources could benefit the enzyme industry significantly

due to its adaptive structural features which makes it active at low temperature and

flexible at low water medium. Unfortunately, there was lack of understanding regarding

the structure adaptation of cold-active lipase in organic solvent. This study embarks on

studying structure-function relationship of family I.3 cold-active AMS8 lipase in selected

organic solvents. Cold-active AMS8 lipase was catalytically active at 25 – 45 °C and has

two alpha-helix lids, a pentapeptide motif with nucleophilic-serine and repeat-toxin

sequence motifs distribution at C-terminus.

The experiment begins with structure prediction of AMS8 lipase by Small Angle X-ray

Scatterings (SAXS) and homology modeling. AMS8 lipase ab-initio model from SAXS

was found to be similar with the homology model and MIS38 Pseudomonas lipase

structure. Following this, molecular dynamics (MD) simulations and docking analyses

were performed with homology-modelled lipase where ethanol, toluene, dimethyl

sulfoxide and 2-propanol have shown interactions with active site residues. Toluene

achieved the highest energy binding (4.92 kcal/mol) with AMS8 lipase and strongly

interacts with Ser-204, Gly-205 and His-206. Based on simulations in toluene, a strong

hydrogen bond was formed at catalytic site between Gly-210 and Ser-238, but weaker

hydrogen bond was found at lid 2 between Gly-156 and Ser-160. An increase in α-helices

structure could be subjected to enzyme surface interference by toluene. AMS8 lipase also

exhibited higher number of disallowed region when simulated in toluene (1.9 %) and

hexane (3.2 %). Methanol and toluene showed improvements in AMS8 lipase substrate

binding (p-nitrophenol palmitate) but slows down the catalytic rate, kcat. Based on these

characterizations, site-directed mutagenesis was applied on regions with high

accessibility to toluene.

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Leu-208 located next to polar Ser-207 was chosen as a mutation site because of the high

solvent-accessible surface area and was located within aggregation-prone sites.

Substitution of leucine to alanine ruined hydrogen bond that formed between Ser-238 and

Gly-210 causing the tunnel to collapse. Following this, a reduction in substrate affinity

for pNPP and pNPL was observed in 0.5 % (v/v) toluene. The enhanced stability of

L208A was contributed by increase in aggregation and denaturation points which made

it easy to adapt at slightly high temperature, 45 °C. Mutating Thr-52 and Gly-55 to

tyrosine on lid 1 area stabilizes the protein conformation and improves the surface

recognition of toluene due to the presence of aromatic side chain. Presence of tyrosine at

lid 1 did not draw electrostatic interactions on the protein surface for substrate binding

but being there, the local flexibility on both surface and catalytic site embraced positive

changes to lipase activity. Mutant T52Y favours pNPP (C16) but G55Y hydrolysed

smaller pNPC (C8) in aqueous solvent. Both lid 1 mutants favoured pNPC when reacted

in 0.5 % (v/v) toluene. Although binding improvements of long-chain substrate was

evident in T52Y, its activity in toluene remained low in comparison to the recombinant.

In characterisation study, lid 1 mutants have lower optimal temperature compared to its

recombinant and L208A. Mutant T52Y has the longest half-life in aqueous medium at 25

and 37 °C while exhibited longer half-life in 0.5 % (v/v) toluene at 25 °C. Both lid 1

mutants were stable in toluene up to 3 % (v/v) concentration. In 0.5 % (v/v) toluene, all

lipases aggregated at higher temperature but denaturation happened at lower temperature

for lid 1 mutants. Unlike others, mutant T52Y displayed increased values of enthalpy and

entropy from 0 to 5 % (v/v) toluene showing improvements of protein stability and

decline in catalytic rate. All lipases exhibited no structure loss due to the unchanged and

minimal increase of its entropy value in toluene at 25 to 35 °C. In conclusion, adaptations

of cold-active lipase AMS8 in toluene (0.5 - 5 %, v/v) at temperatures 20 - 35 °C was

factorised by lid 2 flexibility, formations of substrate-tunnel, hydrogen bond in catalytic

area, alpha-helix reduction, higher aggregation points, low enthalpy and a slight increase

in entropy.

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Abstrak tesis yang dikemukakan kepada Senat of Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Doktor Falsafah

IMPLIKASI STRUKTUR LIPASE AKTIF-SEJUK Pseudomonas fluorescens

STRAIN AMS8 DALAM PELARUT ORGANIK

Oleh

NORHAYATI BINTI YAACOB

Disember 2018

Pengerusi : Mohd Shukuri Mohamad Ali, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Lipase aktif-sejuk dari sumber bakteria dapat memberi manfaat secara langsung kepada

industri enzim disebabkan oleh ciri-ciri penyesuaian struktural yang menjadikannya aktif

pada suhu rendah dan melentur di medium rendah air. Malangnya, terdapat kurang

pemahaman berkait dengan penyesuaian struktur lipase aktif-sejuk di dalam pelarut

organik. Kajian ini bermula dengan meneliti hubungan struktur-fungsi pada keluarga

lipase I.3 AMS8 aktif-sejuk di dalam pelarut organik terpilih. Lipase AMS8 aktif-sejuk

aktif memangkin pada 25- 45 ºC dan mempunyai dua penutup alfa helix, motif

pentapeptida berserta serina-nukleofilik dan kedudukan jujukan motif toksin-berulang di

terminal-C.

Experimen bermula dengan ramalan struktur lipase AMS8 menerusi Serakan X-ray

Sudut Kecil (SAXS) dan pemodelan homologi. Model ab-initio SAXS lipase AMS8

mempunyai persamaan dengan model homologi dan struktur lipase Pseudomonas

MIS38. Berikutan ini, simulasi dinamik molekular (MD) dan analisis mengedok

dilakukan dengan model homologi lipase di mana etanol, toluena, dimetil sulfoksida dan

2-propanol telah menunjukkan interaksi dengan residu-residu tapak aktif. Toluena

mencapai tenaga ikatan yang tertinggi (4.92 kcal/mol) dengan lipase AMS8 dan kukuh

berinteraksi dengan Ser-204, Gly-205 dan His-206. Berdasarkan simulasi dalam toluena,

ikatan hidrogen yang kuat telah terbentuk di tapak mangkin antara Gly-210 dan Ser-238,

tetapi ikatan hidrogen yang lemah telah dijumpai pada penutup 2 di antara Gly-156 dan

Ser-160. Peningkatan dalam struktur alfa-helix boleh merujuk kepada gangguan

permukaan enzim oleh toluena. Lipase AMS8 juga menunjukkan pertambahan bilangan

bagi kawasan yang tidak dibenarkan apabila disimulasi di dalam toluena (1.9 %) dan

heksana (3.2 %). Metanol dan toluena menunjukkan penambahbaikan ke atas substrat

mengikat lipase AMS8 (p-nitrofenol palmitat) tetapi rendah bagi kadar memangkin, kcat.

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Berdasarkan pencirian tersebut, mutagenesis kawasan terarah digunakan pada bahagian

yang mempunyai ketercapaian tinggi kepada toluena.

Leu-208 yang terletak bersebelahan Ser-207 yang polar dipilih sebagai kawasan mutasi

disebabkan tingginya keluasan permukaan pelarut mudah capai dan terletak dalam

kawasan cenderung pengagregatan. Pertukaran leusina kepada alanina merosakkan

ikatan hidrogen terbentuk antara Ser-238 dan Gly-210 menyebabkan keruntuhan pada

terowong. Berikutan ini, pengurangan dalam afiniti substrat untuk pNPP dan pNPL

dilihat pada 0.5 % (v/v) toluena. Peningkatan kestabilan oleh L208A disumbangkan

melalui kenaikan takat pengagregatan dan penyahaslian yang menjadikan ia mudah

menyesuai pada suhu sedikit tinggi, 45 ºC. Memutasi Thr-52 dan Gly-55 kepada tirosina

pada bahagian penutup 1 menstabilkan konformasi protein dan menambahbaik

pengecaman permukaan oleh toluena disebabkan kehadiran rantai sisi aromatik.

Kewujudan tirosina pada penutup 1 tidak mendorong interaksi-interaksi elektrostatik di

atas permukaan protein untuk mengikati substrat, namun keberadaannya di sana,

membawa kepada kelenturan setempat di kedua permukaan dan kawasan pemangkin

yang memberi perubahan positif kepada aktiviti lipase. Mutan T52Y menyukai pNPP

(C16) tetapi G55Y menghidrolisis pNPC (C8) yang bersaiz kecil di dalam larutan akueus.

Kedua-dua mutan penutup 1 memilih pNPC apabila bereaksi di dalam 0.5 % (v/v)

toluene. Walaupun peningkatan mengikat bagi substrat rantai-panjang adalah jelas dalam

T52Y, aktiviti di dalam toluena kekal rendah jika dibandingkan dengan rekombinan.

Dalam kajian pencirian, mutan-mutan penutup 1 mempunyai suhu optima yang lebih

rendah berbanding dengan rekombinan dan L208A. Mutan T52Y mempunyai separuh

hayat yang terpanjang di dalam medium akueus pada 25 dan 37 ºC sementara

menyaksikan separuh-hayat yang panjang di dalam 0.5 % (v/v) toluena pada 25 ºC.

Kedua mutan penutup 1 adalah stabil di dalam toluena sehingga kepekatan 3 % (v/v).

Dalam 0.5 % (v/v) toluena, kesemua lipase mengagregat pada suhu yang meninggi tetapi

penyahaslian berlaku pada suhu yang rendah untuk mutan-mutan penutup 1. Tidak

seperti yang lain, mutan T52Y memperlihatkan peningkatan nilai-nilai entalpi dan

entropi dari 0 sehingga 5 % (v/v) toluena sekaligus menunjukkan penambahbaikan ke

atas penstabilan protein dan penurunan dalam kadar memangkin. Semua lipase

menunjukkan tiada struktur yang hilang berdasarkan pada nilai entropi yang tidak

berubah dan sedikit tinggi di dalam toluena pada 25 hingga 35 ºC. Secara kesimpulan,

penyesuaian lipase aktif-sejuk AMS8 di dalam toluena (0.5 – 5 %, v/v) pada suhu 20 –

35 ºC dipuncakan pada pelenturan penutup 2, pembentukan terowong substrat, ikatan

hidrogen dalam kawasan pemangkin, pengurangan alfa helix, kenaikan takat

pengagregatan, penurunan entalpi dan sedikit peningkatan dalam entropi.

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ACKNOWLEDGEMENTS

First and foremost, I thank the Almighty, Allah S.W.T for giving me good health,

strength, determination and patience during the candidature period. I took this

opportunity to express my profound gratitude and deep regards to my supervisor, Assoc.

Prof. Dr. Mohd Shukuri Mohamad Ali for his guidance, monitoring and constant

encouragement throughout the postgraduate program.

I also would like to express a deep sense of gratitude to fellow co-supervisors, Assoc.

Prof. Dr. Adam Leow Thean Chor, Prof. Dr. Raja Noor Zaliha Raja Abd Rahman and

Dr. Nor Hafizah Ahmad Kamarudin for his/her cordial support, valuable advices and

guidance, which helped me in completing this task through various stages. Special

gratitude went to the Enzyme and Microbial Technology (EMTech) research felo, and

academic advisor, Prof. Dato’ Dr. Abu Bakar Salleh for all the valuable inputs,

constructive criticism and suggestions.

I thank all the members of EMTech laboratories in Institute of Bioscience (IBS), Biotech

2 and Biotech 3, for the knowledge and experiences shared in their respective fields.

Many thanks to the most dedicated and helpful science officers Puan Zufliha Zakaria and

Cik Norhayati Yusuf from MOLEMED (IBS), Puan Siti Munira Abdul Razak from

Department of Cell and Molecular Biology (FBSB) and Encik Zainal Abidin Kassim

from Science Faculty whom kindly assisting and helping me with running the freeze

dryer, dynamic light scatterings (DLS) and GC-MS. Heartiest appreciations goes to the

beamline scientists Dr. Nuntaporn Kamonsuttipaijit and Dr. Siriwat Soontaranon from

Synchrotron Light Research Institute (SLRI), Thailand in providing the slot for SAXS

experiments and offering advices on the latest techniques involved in the sample

preparation as well as in data analysis. Finally, I thank my understanding parents, sister

and close relatives for their constant encouragement without which the completion of this

program would not be possible.

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I certify that a Thesis Examination Committee has met on 4 December 2018 to conduct

the final examination of Norhayati Binti Yaacob on her thesis entitled "Structural

Implications of Pseudomonas fluorescens Strain AMS8 Cold-Active Lipase in Organic

Solvent” in accordance with the Universities and University College Act 1971 and the

Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15th March 1998. The

Committee recommends that the student be awarded the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Assoc. Prof. Dr. Mohd Yunus Abd. Shukor, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Assoc. Prof. Dr. Muhajir Hamid, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Assoc. Prof. Dr. Noor Azmi Shaharuddin, PhD

Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Internal Examiner)

Prof. Dr. Syed Abid Ali, PhD

Professor

University of Karachi

Pakistan

(External Examiner)

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The

members of the Supervisory Committee were as follows:

Mohd Shukuri Mohamad Ali, PhD Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Adam Thean Chor Leow, PhD Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Raja Noor Zaliha Raja Abd Rahman, PhD Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

Nor Hafizah Ahmad Kamarudin, PhD Senior lecturer

Centre for Agricultural Science Foundation,

Universiti Putra Malaysia

(Member)

___________________________

ROBIAH BINTI YUNUS, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other

degree at any other institutions;

intellectual property from the thesis and copyright of thesis are fully-owned

by Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and Innovation) before thesis is published (in the form of

written, printed or in electronic form) including books, journals, modules,

proceedings, popular writings, seminar papers, manuscripts, posters, reports, lecture

notes, learning modules or any other materials as

stated in the Universiti Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate Studies)

Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia (Research)

Rules 2012. The thesis has undergone plagiarism detection software.

Signature: _______________________ Date: __________________

Name and Matric No.: Norhayati Binti Yaacob (GS44581)

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature:

Name of Chairman of Supervisory

Committee:

Assoc. Prof. Dr. Mohd Shukuri Mohamad Ali

Signature:

Name of Member of Supervisory

Committee:

Assoc. Prof. Dr. Adam Leow Thean Chor

Signature:

Name of Member of Supervisory

Committee:

Prof. Dr. Raja Noor Zaliha Raja Abd Rahman

Signature:

Name of Member of Supervisory

Committee:

Dr. Nor Hafizah Ahmad Kamarudin

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xix

CHAPTER

1 INTRODUCTION 1

1.1 Research Statement 2

1.2 Hypothesis 3

1.3 Objectives 3

2 LITERATURE REVIEW 5

2.1 Organic-solvent tolerance in cold-active lipases 5

2.2 Characteristics of organic solvent tolerant lipases 6

2.3 Bottlenecks in elucidating tertiary structure of cold-

active lipase

8

2.4 Small Angle X-ray Scatterings (SAXS) approach on

highly flexible protein structure

14

2.5 Molecular basis of cold-active lipase activity in the

study of structure-function relationships

17

2.6 Structural Modeling of Cold-active Lipases in Organic

Solvents

19

2.6.1 Measuring cold-adapted lipase properties based

on the changes of reaction energy from MD

19

2.6.2 Catalytic sites and tunnel 20

2.6.3 Oxyanion hole 21

2.6.4 Hydrophobic interactions 22

2.6.5 Interfacial activation 22

2.6.5.1 Lid activation in the presence of

organic solvent

23

2.6.5.2 Observation of lid-opening

mechanism via Molecular

Dynamics (MD) simulation

25

2.6.6 Folding mechanism 26

2.6.7 Protein aggregation 27

2.6.8 Calcium binding area 29

2.7 Structure and function relationship of cold-active

lipases in the presence of organic solvent

30

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2.8 Understanding of cold-active lipase in organic solvents

via protein modification approach

31

2.9 General application of cold-active lipase 35

3 MATERIALS AND METHODS 37

3.1 Sequence of experiments 37

3.2 Expression and purification of recombinant AMS8

lipase

38

3.3 Protein sequence analysis 39

3.4 Structure determination of AMS8 lipase by biophysical

approach , Small Angle X-ray Scatterings (SAXS)

39

3.4.1 Protein preparation and thrombin cleavage 40

3.4.2 Small Angle X-ray Scatterings (SAXS)

experimental set up

3.4.3 SAXS data analysis, shape reconstruction

and modeling

41

41

3.5 Comparison of SAXS model with predicted homology

model of AMS8 lipase

42

3.6 Molecular Dynamics (MD) simulations and in-silico

analysis using homology model of AMS8 lipase

43

3.6.1 Parameterization of solvent models and

molecular dynamics (MD) simulation

43

3.6.2 Methods to evaluate the effect of solvent

based on molecular dynamics simulation

43

3.6.3 Analysis of molecular docking of a single

solvent molecule to AMS8 lipase 3D model

44

3.7 Solvation energy calculation of AMS8 lipase in organic

solvent

44

3.8 AMS8 lipase secondary structure analysis by Circular

Dichroism (CD)

45

3.9 Kinetic Properties of AMS8 Lipase in the Presence of

Organic Solvents

45

3.10

In-silico predictions of “hot-spot” area in lipase-organic

solvent interactions for mutagenesis

46

3.11 Site-Directed Mutagenesis on lid and nucleophilic

elbow

46

3.11.1 Site-Directed Mutagenesis targeting

nucleophilic elbow and lid region

46

3.11.2 Protein expression and purification from

mutant lipases

47

3.12 Molecular Dynamics Simulations and Docking of all

mutant lipases in toluene

47

3.12.1 Molecular Dynamics Simulation in different

temperatures

47

3.12.2 Mutant L208A docking with alcohol,

hexanoic acid and ethyl hexanoate

48

3.12.3 Mutant T52Y and G55Y docking with oleic

acid

49

3.13 In-silico analysis on lid 1 mutation sites 49

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3.13.1 Hydrophobic Effects and Electrostatic

Potentials on Lid 1 mutants of T52Y and

G55Y

49

3.13.2 Normal Mode Analysis (NMA) and

sequence-specific solvent accessibilities

(ProtSA) analysis of lid 1 mutants of T52Y

and G55Y

49

3.14 Enzyme kinetics of recombinant AMS8 and mutants in

toluene (pNP assay)

50

3.15 Biochemical properties of recombinant and mutant

AMS8 lipases via Kwon and Rhee method (1986).

50

3.16 Thermodynamic analysis via activation energy

determination, aggregation and denaturation point

trends of recombinant and mutant AMS8 lipases

51

4 RESULTS AND DISCUSSION 53

4.1 Expression and purification of AMS8 Lipase 53

4.2 Protein sequence analysis 57

4.3 Structure determination of AMS8 lipase via biophysical

approach, Small Angle X-ray Scatterings (SAXS)

59

4.4 Comparison of AMS8 lipase SAXS model and

predicted homology model from YASAA

66

4.4.1 Homology model of AMS8 lipase via YASARA 66

4.4.2 Beads model of AMS8 lipase from SAXS 67

4.4.3 The differences of SAXS bead-model to

homology model of AMS8 lipase

71

4.5 Molecular Dynamics Simulations and Docking of

AMS8 lipase with Polar and Non-Polar Organic

Solvents

73

4.5.1 Effects of Organic Solvents to the Root-Mean-

Square Deviations and Fluctuations and

Secondary Structure

73

4.5.2 Effects of Organic Solvent to AMS8 Lipase

Stability via Hydrogen Bond Formation and

Hydrophobic Interactions

78

4.5.3 The Docking of Solvents on the Active Site

Residues and Other Binding Sites of AMS8

Lipase

82

4.5.4 Ramachandran plot and SASA analysis of

AMS8 lipase conformational structure in

organic solvents

85

4.6 Solvation Energy and Kinetic Analysis of AMS8 lipase

in Organic Solvents

91

4.6.1 Solvation (solute-solvent) interaction 91

4.6.2 Kinetic profiles of AMS8 lipase in organic

solvents

92

4.7 Effects of toluene to cold-active AMS8 lipase structural

changes

94

4.7.1 Toluene to stimulate AMS8 lipase lid 2 activation 94

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4.7.2 Effects of calcium ion on AMS8 lid 2 activation 96

4.8 Predicting “hot-spot” area for mutagenesis based on

solvent accessibility and structure stability

99

4.9 Expression and purifications of mutant lipases T52Y,

G55Y and L208A

105

4.10 Molecular dynamics analysis of mutant lipases with and

without toluene

108

4.10.1 Molecular dynamics simulation of lid 1 and

nucleophilic site mutants in toluene (an

overview)

108

4.10.2 Effects of mutation to active site stability

(L208A)

114

4.10.2.1 Effects of temperature to lid after

mutation at nucleophilic elbow

4.10.2.2 Active site flexibility at 25 °C and

35°C

4.10.2.3 Active site-substrate/product

docking

114

122

127

4.10.3 Effects of mutation to lid 1 flexibility and

stability (T52Y and G55Y)

136

4.10.3.1 Effects of temperature to lid 1

mutation

136

4.10.3.2 Electrostatic surface potential and

hydrophobic effects influencing

lid 1 interaction with toluene

140

4.10.3.3 Normal Mode Analysis (NMA)

and sequence-specific solvent

accessibilities (ProtSA) of lid 1

mutants

152

4.11 Enzyme kinetics of recombinant AMS8 and mutant

lipases in toluene

153

4.12 Biochemical and biophysical properties of AMS8 lipase

and mutants (T52Y, G55Y, L208A)

156

4.13 Folding kinetics and thermodynamic analysis of

recombinant AMS8 and mutant lipases in toluene

159

5 SUMMARY, CONCLUSION AND RECOMMENDATION

FOR FUTURE RESEARCH

168

5.1 Conclusion 168

5.2 Recommendations for future research 170

REFERENCES 171

APPENDICES 189

BIODATA OF STUDENT 219

LIST OF PUBLICATIONS 220

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LIST OF TABLES

Table

Page

2.1 Structural comparisons of lipases with and organic solvent 31

2.2 Protein Engineering Approach for Improvements of Cold-

Active Lipase

34

3.1

Primer sequences designed for site-directed mutagenesis of

targeted region

46

4.1 Purification table of AMS8/pET32b lipase 57

4.2 Top five templates showing highest rank of SAXS data

alignment in SAXSTER score.

69

4.3 Network of hydrogen bonds in the core structure of AMS8

lipase located at flexible region of lid 2 and catalytic area.

78

4.4 Protein–Ligand Attractions Investigation Numerically

(PLATINUM) analysis

81

4.5 Distance of organic solvent with interacting residues. 82

4.6 List of residues included in the outlier region obtained by

Ramachandran plot from AMS8 lipase at 20 ns of molecular

dynamic simulation with respect to AMS8 lipase stability.

85

4.7 Kinetic profiles of AMS8 lipase using substrate p-nitrophenol

palmitate (pNPP) in the presence of 0.5% (v/v) organic solvent.

93

4.8 Effect of calcium ions on the stability of AMS8 lipase simulated

with and without toluene

99

4.9 Changes in protein stability, solvent accessibility, residue depth

and occluded surface packing (OSP) residues before and after

mutation of Leu-208 to alanine

102

4.10 Predicting effects of mutation on lid 1 and nucleophilic elbow

of AMS8 lipase (In-Silico)

105

4.11 A comparison of binding energy between AMS8 lipase and

mutant L208A in complex system of lipase/toluene/substrate.

135

4.12 Michaelis Menten kinetics measuring Km and kcat of mutants

T52Y, G55Y and L208A.

154

4.13 Biochemical and biophysical properties of recombinant AMS8

and mutant lipases

157

4.14 Predicted and measured folding rate (Kf) of recombinant AMS8

and mutant lipases

161

4.15

Thermostability of AMS8 and mutant lipases through the

changes of enthalpy, entropy and melting temperature at 0 and

5 % (v/v) toluene

162

4.16 Thermodynamics parameters of recombinant AMS8 lipase and

mutant lipases, T52Y, G55Y and L208A in toluene

165

4.17 Estimated activation energy in kJ/mol derived from the slope of

Arrhenius plot.

167

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LIST OF FIGURES

Figure

Page

2.1 Advantages and disadvantages of organic solvent to cold-active

lipase structure and function (source: Kavitha. 2016).

8

2.2 Lipase structures showing differences between mesophile lipase

from Pseudomonas MIS38 (PDB: 2Z8X) of family lipase I.3 (A)

and psychrotropic Photobacterium lipolyticum (PDB: 2ORY)

(B) and SMG1 lipase from Malassezia globosa (PDB: 3UUE).

11

2.3 Range of timescale of detection required in analysing flexibility

and folding of highly flexible protein such as cold-active

enzymes aided by suitable measurement techniques (source:

Karshikoff et al., 2015).

13

2.4 Kratky plot of scattering data illustrating changes in the behavior

of the curve for folded (sphere), partially folded (sphere-random

coil) and completely unfolded particles (random coil).

15

2.5 Insights of SAXS analysis based on values used to determine the

state, structure of protein in solution and ab-initio model

resolution.

16

2.6 Structure features of cold-active lipases 18

2.7 The origin of cold-active enzymes adaptation via Arrhenius plot

analysis.

20

2.8

Flexibility of closed (A) and open (B) Pseudomonas cepacia

(Burkholderia cepacia) in water (A) and toluene (B) indicated

by calculated B-factors per residue [Å2] from the last 10 ns of

simulation.

23

2.9 The differences in the structures of lids and a flexible loop in

Pseudomonas sp. MIS38 (PML) lipase (source: Angkawidjaja et

al., 2007).

24

2.10 Lid movement in P. aeruginosa lipase: a qualitative

representation.

26

2.11 Progress to protein aggregation 28

3.1 Schematic diagram flows of experiments 37

4.1 Bacterial colonies with AMS8 lipase plasmid forming a

‘clearing’ zone which can be seen through the opaque trybutyrin

plate (A), transformants AMS8/pET32b in BL21 DE3 in LB-

amp agar (B) and SDS-PAGE showing crude protein expression

of IPTG induced AMS8 lipase (C).

54

4.2 Two steps purification of recombinant AMS8 lipase. 55

4.3 SDS-PAGE analyses showing purified AMS8 lipase via affinity

chromatography and anion-exchange chromatography

56

4.4 Protein sequence of recombinant lipase AMS8/pET32b 58

4.5 Purified AMS8 lipase observations based on SDS-PAGE

analysis

60

4.6 Subtracted AMS8 lipase scattering data for preliminary SAXS

analysis

61

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4.7 Guinier plot of In [I (q)] versus q2 to determine the reciprocal

space Rg of AMS8 lipase and the zero scattering angle intensity

I(0) for molecular weight estimation of the protein (A) and the

residual data showed a randomly distributed around zero (B).

62

4.8 Areas under the P(r) function of AMS8 lipase used to estimate

Dmax with r (in Å) represents the diameter of sphere.

63

4.9 Kratky plot analysis of AMS8 lipase 64

4.10 Ab-initio model showing an assembly of densely packed beads

from AMS8 lipase by using DAMMIN

65

4.11 Model structure of AMS8 lipase was created based on the

similarity of protein sequence and structure of type I.3 lipase,

PML

66

4.12 Ab-initio model extracted from scattering data of AMS8 lipase

calculated by 10 models from DAMMIF

68

4.13 Superimposed model from two protein structures of AMS8

lipase obtained by DAMMIF [identified in grey coil] and

homology modeling [identified in purple ribbon shaped] as the

reference target.

68

4.14 SAXS pair distance distribution function (PDDF) profile of

AMS8 lipase.

70

4.15 SREFLEX analysis showing consistency of SAXS model of

AMS8 protein from restricted (green curve) to unrestricted form

(turquoise curve).

72

4.16 Molecular dynamics simulation analysis of AMS8 lipase with

various organic solvents at 25°C.

74

4.17 Circular dichroism (CD) spectra of AMS8 lipase with and

without 0.5 % (v/v) solvent in molecular ellipticity (milli degree)

at broad wavelength 190-260 nm

76

4.18 Effects of toluene to substrate accessibility via substrate-tunnel

formation

80

4.19 A mirrored Ramachandran plot nomenclature (A) and AMS8

lipase Ramachandran plot from the final MD trajectories

simulated with hexane (B), toluene (C) and 2-propanol (D).

87

4.20 Solvent-accessible surface areas (A2) of AMS8 lipase in organic

solvents

89

4.21 Hydration patterns of AMS8 lipase after 20 ns simulation in (A)

methanol and (B) toluene

90

4.22 Solvation free energy (in kcal/mol) or stability of AMS8 lipase

prior to its interaction with polar and non-polar solvents.

92

4.23 The distance of AMS8 lipase lid 1 (via Leu-57) and lid 2 (via

Thr-149) in the presence (A-before, B-after) and absence (C-

before, D-after) of toluene.

95

4.24 Influence of toluene and Ca2+ on the flexibility and structural

stability of AMS8 lipase

97

4.25 The atomic distance of Cα backbone from Ile-155 (from lid 2) to

the nearest calcium ion in water (A) and toluene (B) after

simulation of 20 ns.

98

4.26 Locations of mutation sites ( ) in AMS8 lipase sequence. 100

4.27 Molecule visualization of targeted mutation of L208A. 103

4.28 Double digestion of DNA plasmid containing mutation site. 106

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4.29 Mutant lipases (T52Y, G55Y and L208A) expression as

compared to control AMS8 lipase

107

4.30 Effects of lid 1 and active site mutations on AMS8 lipase

structural deviations (RMSF) in 25 °C simulation with (A) and

without toluene (B)

109

4.31 Effects of lid 1 and active site mutations on solvent-accessible

surface area (SASA) in 25 °C simulation with (A) and without

toluene (B)

111

4.32 Effects of lid 1 and active site mutations on AMS8 lipase

structure compactness (R-gyration) in 25 °C simulation with (A)

and without toluene (B)

113

4.33 The radius of gyration, Rg (measure of the mass-weighted spatial

distribution of the atoms in a peptide molecule/protein and a

rough measure for its compactness) in WT AMS8 and mutant

L208A in water—(A) and toluene—(B) (at 25 ◦C and 35 ◦C)

respectively.

115

4.34 Solvent accessible surface area (SASA) analysis of WT AMS8

and mutant L208A lipases in water (A) and toluene (B) at 25 °C

and 35 °C.

117

4.35 The root-mean-square (RMSD) distance between the Cα atoms

in the simulations and the lid 2 (148–167) structure of AMS8

and its mutant L208A in water—(A) and toluene—(B) at 25 °C

and 35 °C.

119

4.36 The root-mean-square (RMSF) distance between the Cα atoms

in the simulations and the protein structure of AMS8 and its

mutant L208A in water—(A) and toluene—(B) at 25 °C and

35 °C respectively.

121

4.37 Formation of a “tunnel” in the middle of AMS8 protein’s core

providing access for substrates to bind at catalytic binding sites

(orange sticks).

122

4.38 Distance of Leu-208/Ala-208 with catalytic triad S207, D255,

H313 in 25 °C simulation.

124

4.39 Distance of L208/A208 with catalytic triad S207, D255 and

H313 in 35 °C simulation.

126

4.40 AMS8 lipase active site docking with substrates ethanol and

hexanoic acid showing the binding energy and hydrogen bond

interactions

129

4.41 L208A active site docking with substrates ethanol and hexanoic

acid showing interactions of hydrogen bond.

130

4.42 Docking of mutant L208A (A) and recombinant (B) with simple

ester, ethyl hexanoate (C8H16O2).

131

4.43 Different binding conformation of ethyl hexanoate and active

site residues (Ser-207, Asp-255 and His-313) in L208A (A) and

recombinant AMS8 lipase (B).

133

4.44 Root-mean square fluctuation of recombinant AMS8 (A), T52Y

(B) and G55Y (C) in toluene simulated at 25 °C (blue line) and

37 °C (red line).

137

4.45 Root-mean square deviations of recombinant AMS8, T52Y and

G55Y in toluene simulated at 25 °C and 37°C.

138

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4.46 R-gyration of recombinant AMS8, T52Y and G55Y in toluene

simulated at 25 °C and 37°C

139

4.47 Solvent accessible surface area (SASA) of recombinant AMS8,

T52Y and G55Y in toluene simulated at 25 °C and 37 °C.

140

4.48 Surface topology of AMS8 lipase showing electrostatic surface

potential in water (A) and toluene (B).

141

4.49 Electrostatic potential molecular surface of recombinant AMS8

lipase which has undergone changes in both interior and exterior

structure while simulated in toluene with putative binding site

for oleic acid

142

4.50 Surface topology of mutant T52Y (A, B) and G55Y (C, D)

showing electrostatic surface potential.

143

4.51 Surface topology of mutant T52Y (A, B) and G55Y (C, D) in

toluene exhibiting electrostatic surface potential

144

4.52 Docking analysis of mutant T52Y and G55Y with oleic acid

showing the distributions of electrostatic potential surrounding

the active site area.

145

4.53 Hydrophobicity and repositioning of lipase Tyr-52 and Tyr-55

after mutation (in water simulation).

147

4.54 Hydrogen bond interactions happening at Thr-52 and Gly-55 in

recombinant AMS8 (A, B), mutated Tyr-52 (C, D) and Tyr-55

(E, F) which concerns the orientation of aromatic side chains

149

4.55 Hydrophobicity and formation of substrate tunnel in lipase Tyr-

52 and Tyr-55 in toluene simulation

151

4.56 Comparing one from five modes of distance fluctuation map of

recombinant AMS8 (A), mutants T52Y (B) and G55Y (C) for

Cα at the end simulation trajectories in toluene.

152

4.57 Chevron plot showing protein folding kinetic based on

increasing concentrations of toluene (A: AMS8 WT, B: T52Y,

C: G55Y, D: L208A).

160

4.58 Enthalpy changes with toluene concentration 163

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LIST OF ABBREVIATIONS

α Alpha

β Beta

°C Degree celsius

% Percentage

Å Angstrom

A600nm Optical density at wavelength 600 nanometer

µL Microlitre

µm Micrometer

µmole Micromole

bp Base pair

CaCl2 Calcium chloride

DNA Deoxyribonucleic acid

E. coli Escherichia coli

g Gram

IPTG Isopropyl β-D-1-thiogalactopyranoside

kb Kilobase

kDa Kilodalton

kcat Catalytic efficiency

Kfr Folding rate constant

Km Michaelis-Menten constant

L Litre

LB Luria-Bertani

log P logarithm of the partition coefficient between 1- octanol and

water H2O

M Molar

mA MilliAmps

µmole Micromole

mg milligram

pNP p-nitrophenol

porod particles macromolecular volume

RMSD Root-mean-square deviations

RMSF Root-mean-square fluctuations

RTX Repeat-in-toxin motifs

SASA Solvent-accessible surface area

SAXS Small Angle X-ray Scatterings

SDS-PAGE Sodium dodecyl sulphate polyacrylamide

gel electrophoresis

Tm Melting temperature/Denaturation point

U/ml Unit per millilitre

U/mg Unit per milligram

v/v volume per volume

∆Gⱡ Gibbs free energy activation

∆Hⱡ enthalpy of activation

ΔSⱡ entropy of activation

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CHAPTER 1

INTRODUCTION

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are part of hydrolases that

hydrolyzes carboxylic ester bonds and triglycerides into diglycerides, monoglycerides,

fatty acids and glycerol (Houde et al., 2004). These enzymes are gaining high importance

as a biocatalyst for several novel reactions in aqueous and non-aqueous medium.

Different lipases behave differently in different organic solvents with different level of

resistance in different reaction systems. Pseudomonas lipases are very interesting not

only that they are stable in organic solvents but also because they display special

biochemical characteristics not common among the lipases produced by other

microorganisms, such as the thermoresistance and activity at alkaline pH (Kumar et al.,

2016, Soberón-Chávez and Palmeros. 2008). Pseudomonas lipases can be classified into

three subfamilies: I.1, I.2 and I.3 (Jaeger and Eggert. 2002). Family I.3 lipases are

distinguished from other lipases by their amino acid sequences and secretion mechanism

via type I secretion system (TISS). It is through the secretion of TISS that produced the

up-stream of secretion signal termed repeat in toxin motifs (RTX) attracting Ca2+ to bind.

Eventually, Ca2+ is important for lipase activity and folding. The family I.3 lipases are

composed of two domains with distinct yet related functions (Angkawidjaja and Kanaya.

2006). Among the family I.3 lipases secreted from cold-adapted Pseudomonas sp. are

KB-Lip from Pseudomonas fluorescens strain KB700A (Rashid et al., 2001), Lip TK3

from Pseudomonas fluorescens strain TK3 (Tanaka et al., 2012) and LipS from

Pseudomonas mandelii JR1 (Kim et al., 2013). Unlike other cold-active lipases, LipS is

active and stable at 40-50 °C showing a relatively high in thermal stability which is

unique to cold-adapted enzymes. Cold-active lipases from psychotropic microorganisms

showing high catalytic activity at low temperatures can be highly expressed through

recombinant methods, making them useful for biotechnological applications.

In spite of these advantages, enzymes do not always meet desired levels of activity,

productivity and, most importantly, stability in organic solvents. Inactivation by organic

solvents is most likely due to conformational changes of the protein structure cause by

changes in medium hydrophobicity and non-covalent interactions between the enzyme

and solvent molecules, which leads to protein unfolding and activity loss (Dror et al.,

2014, Stepankova et al., 2013). Hydrophilic polar solvents such as DMSO, ethanol and

methanol can create homogenous systems with water, but they can easily penetrate the

enzyme surface or strip off essential water molecules from the enzyme. In contrast with

the hydrophobic solvents such as hexane and toluene (methylbenzene), the lack of water

stripping from the enzyme surface is due to the partitioning of hydration water between

enzyme and the bulk solvent, which benefits in active site hydration through mobile and

weakly bound water. The presence of water around the enzyme could maintain the three-

dimensional structure needed for the enzyme activity. Hence, active site hydration is

dependent to the enzyme activity contributed by organic solvent polarity (Yang et al.,

2004). In a reaction system with low hydration, organic solvent replaces molecular

functions of water at the protein surface. This is an alternative way to provide a medium

for diffusion of substrate and product. Because enzyme activity is possible at very low

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hydration level, enzymatic modification should be introduced to minimize the structure-

linked disorder upon contact with organic solvent (Kurkal et al., 2005).

Stability of cold-active lipase in organic solvents has been regarded essential for various

biotechnology applications that involved synthesis reactions such as esterification,

interesterification and transesterification. The stability of cold-active lipase correlates to

the distribution of surface charged residues and surface property of the enzyme

(Samantha et al., 2006). Organic solvents preferentially localize the hydrophobic patch

in the active-site vicinity and so it creates a hydration shell on residues that did not

demonstrate solvents binding preference. Those set of residues that have showed change

in chemical shifts/perturbation via [15N, 1H] - HSQC resonance in 6B lipase represents

the same large hydrophobic regions on the protein surface having good affinity towards

organic molecules (Kamal et al., 2013). The surface residues localizing protein surface

determines the hydrophobicity profile of this lipase. Other prominent features were the

extensive formation of new hydrogen bonds between surface residues directly or

mediated by structural water molecules and the stabilization of Zn and Ca binding sites

(Dror et al., 2015). Active site (Ser, Asp and His), lid region, tunnels and RTX motifs are

important structural features for regulating enzymatic functions and stability. Some of

these residues must be conserved while some of them are tandemly distributed. Site-

directed mutagenesis is commonly used to reveal insights regarding structure-stability

correlations influenced by organic solvent interactions. Generally, any alteration on this

conserved motif will generate inactive protein. However, other areas that are associated

with solvent stability often found in loops on the surface of protein and the regions near

the substrate binding site. Those residues that are far from the active site region are

predicted not to affect catalytic activity.

One experimental method that allows direct identification on these residues is theoretical

analyses using molecular dynamics (MD) simulation. Following these approach, protein

modification via site-directed mutagenesis can be done to alter protein structure, function

or stability. To date, there are few reports highlighting the significance of active site and

lid regions of cold-active lipase via mutations suggesting their potential stability in polar

and non-polar organic solvents. Hence, a collective approach that includes molecular

dynamics (MD) simulation, site-directed mutagenesis, biochemical and biophysical

characterisation in the presence of selected organic solvents would facilitate the study

related to the structure and function of this extraordinary lipase. As it is not easy to

elucidate atomic details of cold-active lipase in the presence of organic solvent via X-ray

protein crystallography, many researchers have turned to computational method and

kinetics approaches in elucidating underlying mechanisms responsible for organic

solvent effects on molecular level.

1.1 Research Statements

The influence of organic solvents on the activity of cold-active AMS8 lipase might be

the results of the conformational changes which happened around the active site or other

region. At the same time, the hydrophobicity properties of each organic solvent could

play essential role starting from the process of solvent penetration to protein structure

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via diffusion or interfacial activation to tertiary structure changes caused by the solvent

molecules intrusion. However, the effects of organic solvent to alterations in lipase

structure and function need to reflect the role of highly flexible cold-active lipase which

is supposed to be different from its mesophilic and thermophilic type. Among general

but relevant issues that deserved scholastic attentions to provide sufficient information

about the characteristics of the special lipases in organic solvent are listed as follows:

1) The associations of organic solvent hydrophobicity with cold-active lipase

destabilization.

2) Differences in lipase aggregation, folding and denaturation correspond with

changes in medium hydrophobicity.

3) Concepts of protein hydration in organic solvent and its effects on protein

dynamics and function. This topic focuses on the probability of hydration shell

formed at active site region and small structural perturbation due to water

molecules shifting.

1.2 Hypothesis

It is hypothesized that:

1. Lid regulates the access of substrate to the catalytic site in the presence of

hydrophobic organic solvent due to stronger hydrophobic interactions.

2. Active site and lid region had higher solvent accessible surface area to organic

solvents.

3. Lipases with more than one lid could have other roles than being there for surface

activation and catalytic function.

1.3 Objectives

General Objective:

To determine the role of AMS8 lipase structure for better understanding of lipase reaction

in the presence of organic solvents.

Specific Objectives:

1. To compare the tertiary structure of recombinant cold-active AMS8 lipase by

biophysical (SAXS) and computational methods.

2. To examine the interactions of AMS8 lipase in the presence of organic solvents

via molecular dynamics simulation and protein-solvent docking approaches.

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3. To determine secondary structure changes and kinetic differences of recombinant

AMS8 lipase in polar and non-polar organic solvents.

4. To determine the importance of lids and nucleophilic binding region for organic

solvent stability of cold-active lipase AMS8 by using site-directed mutagenesis

(SDM).

5. To associate the influence of toluene towards biophysicochemical and

thermodynamics of recombinant AMS8 and other mutant lipases.

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