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UNIVERSITI PUTRA MALAYSIA
RUDZANNA RUSLAN
FBSB 2013 27
KINETICS, CRYSTALLIZATION, AND STRUCTURAL ELUCIDATION OF THERMOSTABLE D311E T1 LIPASE VARIANT FROM Geobacillus zalihae
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KINETICS, CRYSTALLIZATION, AND
STRUCTURAL ELUCIDATION OF
THERMOSTABLE D311E T1 LIPASE VARIANT
FROM Geobacillus zalihae
RUDZANNA RUSLAN
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
2013
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KINETICS, CRYSTALLIZATION, AND STRUCTURAL ELUCIDATION OF
THERMOSTABLE D311E T1 LIPASE VARIANT FROM Geobacillus zalihae
By
RUDZANNA RUSLAN
Thesis Submitted to the School of Graduate Studies,
Universiti Putra Malaysia, in Fulfilment of the
Requirements for the Degree of Master of Science
May 2013
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COPYRIGHT
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Copyright © Universiti Putra Malaysia
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment
of the requirement for the degree of Master of Science
KINETICS, CRYSTALLIZATION, AND STRUCTURAL ELUCIDATION OF
THERMOSTABLE D311E T1 LIPASE VARIANT FROM Geobacillus zalihae
By
RUDZANNA RUSLAN
May 2013
Chair: Professor Raja Noor Zaliha Raja Abd. Rahman, D.Eng.
Faculty: Biotechnology and Biomolecular Sciences
T1 lipase from Geobacillus zalihae is a thermophilic enzyme with an optimum
temperature of 70 oC at pH 9 and stable up to 65
oC. By introducing an additional ion
pair, T1 lipase was believed to be stable at high temperature. Thus, further study of
D311E lipase variant are required to determine the effect of ion pairs on T1 stability.
The aims of this work are to analyse the kinetic properties and characterise the
D311E lipase variant, to optimise crystallisation conditions and to elucidate the 3-D
structure of crystallised D311E lipase. This involved a few purification steps,
characterisation, kinetic analysis, crystallisation and structural elucidation processes.
Wild-type T1 and variant D311E lipases were successfully purified in a series of
purification steps. The recovery of wild-type T1 was 22.37 % with the purification
fold of 5.16. Meanwhile, the final yield and fold of D311E lipase was 15.71 % and
10.56, respectively. The purification fold of D311E lipase variant was higher than
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wild-type T1 lipase due to higher specific activity of purified D311E lipase. Purified
lipases were used for characterisation and crystallisation.
The wild-type T1 and D311E lipase variant had an optimum temperature of 70 oC.
The D311E lipase variant was stable and remaining activity at 70 % with pre-
incubation at 60 oC for 75 min while the wild-type T1 lipase was stable only for 15
min. When the lipases were pre-incubated at 70 oC, the D311E lipase variant was
stable and remaining activity at 70 % for 10 h while the wild-type T1 lipase was
stable for 6 h. Kinetic studies of T1 and D311E lipase using p-nitrophenyl laurate as
substrate was conducted by using Hanes-Woolf plot. For T1, the Km, Vmax and
Vmax/Km were 39.03 mM, 5.75 mM s-1
and 0.147 mM s-1
mM-1
, respectively while for
D311E lipase, the Km, Vmax and Vmax/Km were 14.08 mM, 0.23 mM s-1
and 0.016 mM
s-1
mM-1
, respectively which shows that the variant has high substrate affinity
compared to its wild-type. The circular dichroism spectra of T1 and D311E lipase
were analysed as a function of temperature at 220 nm. Tm for T1 and D311E lipase
were approximately 68.52 °C and 70.59 °C, respectively. It showed that mutation at
D311 increases the stability of T1 lipase and exhibited higher Tm. The D311E also
exhibited higher activity and stability compared to T1 lipase. Thus, crystallisation
and X-ray diffraction were carried out to ensure the consistency of the results at
atomic level.
Crystallisation of purified D311E lipase was carried out using sitting drop vapour
diffusion and capillary gel tube counter diffusion method. Optimisation of D311E
lipase crystallisation was performed in order to see the crystal growth effect by using
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different buffer pH, protein concentration, salt concentration, and temperature using
sitting drop vapour diffusion method. For sitting drop vapour diffusion method, the
optimum crystallisation formulation was 1.5 M of NaCl, 0.1 M, MES pH 5.5, 1.35
mg/mL of D311E lipase and 20 oC for growth. The size of the crystal obtained was
0.2 mm x 0.1 mm x 0.1 mm. While in the capillary gel tube counter diffusion,
optimum conditions to grow D311E lipase crystal were using 3.0 mg/mL protein
with formulation C2-21 (Crystal Screen, Hampton Research) at 1 mm of gel tube
length. However, the size of crystal grown was approximately 50 µm x 50 µm x 30
µm which is smaller than sitting drop vapour diffusion crystallisation method and
therefore, insufficient for in-house X-ray diffraction. Thus, the crystal with optimum
condition obtained in sitting drop vapour diffusion method was used for X-ray
diffraction.
A good quality crystal of D311E lipase was diffracted at 2.1 Å using an in-house X-
ray beam and this crystal belonged to the monoclinic space group C2 with the unit
cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. The generated model
was further built and refined. The phase problem was solved by using molecular
replacement method. T1 lipase crystal structure (PDB ID: 2DSN) was used as the
template. The quality of lipase structure was validated using Ramachandran plot and
Errat. Hence, the coordinates of D311E crystal structure was deposited to RCSB
Protein Data Bank under PDB ID code 3UMJ.
Structural analysis showed the existence of an additional ion pair around E311 in
D311E structure which led to the formation of an ion pair network comprising of five
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amino acid residues (Arg274, Thr278, Gly279, Arg303 and Glu311) connected by
seven ion pairs. In conclusion, the presence of the additional ion pair stabilised the
formation of inter-connection in D311E and regulates stability of lipase at high
temperatures.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan ijazah Master Sains
KINETIK, PENGKRISTALAN, DAN ELUSIDASI STRUKTUR BAGI
THERMOSTABIL VARIAN LIPASE D311E T1 DARIPADA Geobacillus
zalihae
Oleh
RUDZANNA RUSLAN
Mei 2013
Pengerusi: Professor Raja Noor Zaliha Raja Abd. Rahman, D.Eng.
Fakulti: Bioteknologi dan Sains Biomolekul
T1 lipase dari Geobacillus zalihae adalah enzim termofilik dengan suhu optimum 70
oC pada pH 9 dan stabil sehingga ke 65
oC. Dengan memperkenalkan pasangan ion
tambahan, T1 lipase telah dipercayai menjadi stabil pada suhu tinggi. Oleh itu,
D311E lipase telah dibina untuk menentukan kesan pasangan ion terhadap kestabilan
T1. Objektif kerja ini adalah untuk menganalisis sifat-sifat kinetik dan mencirikan
lipase varian D311E, untuk mengoptimumkan keadaan pengkristalan dan untuk
mengilusidasi struktur 3-D bagi D311E lipase. Ini melibatkan beberapa langkah
penulenan, pencirian, analisis kinetik, pengkristalan dan proses elusidasi struktur.
T1 jenis liar dan varian lipase D311E telah berjaya ditulenkan dalam satu siri
langkah penulenan. Hasil akhir lipase jenis liar T1 adalah 22.37 % dengan lipatan
penulenan sebanyak 5.16. Sementara itu, hasil akhir dan lipatan penulenan untuk
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varian lipase D311E masing-masing adalah 15.71 % dan 10.56. Lipatan penulenan
bagi varian D311E lipase adalah lebih tinggi daripada T1 lipase jenis liar disebabkan
aktiviti khusus yang lebih tinggi oleh D311E lipase. Lipase yang telah ditulenkan
digunakan untuk pencirian dan pengkristalan.
T1 lipase jenis liar dan D311E varian mempunyai suhu yang optimum pada suhu 70
oC. D311E varian stabil dan mengekalkan aktiviti sebanyak 70 % dengan pra-
pengeraman pada suhu 60 oC selama 75 min manakala T1 lipase liar jenis stabil
hanya selama 15 minit. Apabila lipases telah pra-dieram pada suhu 70 oC, D311E
lipase varian adalah stabil dan mengekalkan aktiviti sebanyak 70 % selama 10 jam
manakala T1 lipase jenis liar stabil selama 6 jam. Kajian kinetik untuk lipase T1 dan
D311E dilakukan melalui plot Hanes-Woolf. Km, Vmax dan Vmax/Km untuk T1 masing-
masing adalah 39.03 mM, 5.75 mM s-1
dan 0.147 mM s-1
mM-1
manakala bagi lipase
D311E, Km, Vmax dan Vmax/Km masing-masing adalah 14.08 mM, 0.23 mM s-1
dan
0.016 mM s-1
mM-1
. Ini menunjukkan bahawa varian D311E mempunyai afiniti
substrat yang tinggi berbanding dengan lipase jenis liar. Spektrum dikroism bulat
bagi lipase T1 dan D311E dianalisis sebagai fungsi suhu pada 220 nm. Tm untuk
lipase T1 adalah 68.52 °C dan D311E adalah 70.59 °C. Ini menunjukkan bahawa
mutasi pada D311 telah meningkatkan kestabilan dan nilai Tm lipase T1. D311E juga
menunjukkan aktiviti dan kestabilan yang lebih tinggi berbanding dengan lipase T1.
Oleh itu, penghabluran dan pembelauan oleh sinar-X telah dijalankan bagi
memastikan ketekalan keputusan di peringkat atom.
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Lipase D311E tulen berjaya dikristalkan melalui kaedah difusi wap cara titis duduk
dan difusi kaunter cara kapilari tiub gel. Pengoptimuman pengkristalan lipase D311E
dilakukan untuk melihat kesan pertumbuhan kristal dengan menggunakan penimbal
pH yang berbeza, kepekatan protein, kepekatan garam, dan suhu menggunakan
kaedah titis gantung duduk. Bagi kaedah difusi wap cara titis duduk, formulasi
pengkristalan yang optimum adalah 1.5 M NaCl, 0.1 M, MES pH 5.5, 1.35 mg/mL
lipase D311E dan suhu 20 oC untuk pertumbuahan. Saiz kristal yang diperolehi
adalah 0.2 mm x 0.1 mm x 0.1 mm. Manakala, bagi kaedah penyebaran kaunter cara
kapilari tiub gel, keadaan optimum untuk kristal lipase D311E berkembang adalah
menggunakan 3.0 mg/mL protein dengan formulasi C2-21 (Crystal Screen Hampton
Research,) pada seluruh kepanjangan tiub (1, 5, 8 dan 10 mm). Walau
bagaimanapun, saiz kristal yang tumbuh menggunakan kaedah ini adalah lebih kecil
iaitu pada 50 μm x 50 μm x 30 μm berbanding dengan kristal kaedah difusi wap cara
titis duduk. Saiz ini tidak mencukupi untuk pembelauan sinar-X. Oleh itu, kristal
daripada kaedah difusi wap cara titis duduk dalam keadaan yang optimum telah
digunakan untuk tujuan pembelauan sinar-X.
Kristal lipase D311E yang berkualiti tinggi telah dibelaukan pada resolusi 2.1 Å
menggunakan X-ray dan kristal ini mempunyai ruang monoklinik kumpulan C2
dengan parameter sel unit a = 117.32 Å, b = 81.16 Å dan c = 100.14 Å. Model yang
dihasilkan terus dibina dan diperhalusi. Masalah fasa telah diselesaikan dengan
menggunakan kaedah penggantian molekul. Struktur kristal lipase T1 (PDB ID:
2DSN) telah digunakan sebagai templat. Struktur lipase telah disahkan dengan
menggunakan plot Ramachandran dan Errat. Kemudian, koordinat struktur kristal
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bagi D311E telah didepositkan ke RCSB Protein Data Bank di bawah kod PDB ID
3UMJ.
Analisis struktur menunjukkan kewujudan satu pasangan ion tambahan di sekitar
E311 pada struktur D311E. Pembentukan rangkaian sepasang ion terdiri daripada
lima residue asid amino (Arg274, Thr278, Gly279, Arg303 dan Glu311) yang
disambungkan oleh tujuh pasangan ion. Sebagai ringkasan, penambahan satu
pasangan ion telah menstabilkan pembentukan sambungan antara dalam D311E serta
mengawal kestabilan lipase pada suhu yang tinggi.
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ACKNOWLEDGEMENTS
I would like to express my sincere acknowledgement to my supervisor, Professor Dr.
Raja Noor Zaliha Raja Abd. Rahman for the continuous support of my Master’s
study and research, for her patience, motivation, enthusiasm and immense
knowledge. Her guidance helped me in all the time of research and writing of this
thesis.
Besides my supervisor, I would like to thank the rest of my thesis committee: Prof.
Abu Bakar Salleh, Dr. Mohd Shukuri Mohamad Ali and Dr. Adam Leow Thean
Chor for their encouragement, insightful comments and hard questions.
My sincere thanks also goes to Prof. Yoshiki Higuchi, Dr. Koji Inaka and Dr. Naoki
Shibata for training opportunities in their groups in University of Hyogo, Japan and
assist me to work on this exciting project.
I thank my fellow labmates especially in Crystal Group: Ariati, Saif, Fairol, Dina,
Dayana, Maya, Azmir, Fiza, Arilla, Dura and other members for the stimulating
discussions, for the sleepless nights we were working together and for all the fun we
had in the last three years. Also I thank my labmates in Enzyme Technology Group:
Lab 140 and IBS Lab for the knowledge and supports.
Last but not least, I would like to thank my family: my parents, Ruslan Ali and Sutini
Amat Agong, my siblings and Azinuddin for supporting me spiritually throughout
my life.
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I certify that an Examination Committee has met on date of viva to conduct the final
examination of Rudzanna Ruslan on her degree of Masters of Science thesis entitled
“Kinetics, Crystallization, and Structural Elucidation of Thermostable D311E T1
Lipase Variant from Geobacillus zalihae” in accordance with Universities and
University Collages Act 1971 and the Constitution of the Universiti Putra Malaysia
[P.U.(A) 106] 15 March 1998. The Committee recommends that the candidate be
awarded the Master of Science.
Members of the Thesis Examination Committee were as follows:
Normi binti Mohd Yahaya, PhD
Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Mohd Puad bin Abdullah, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Norazizah binti Shafee, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Farah Diba Abu Bakar, PhD
Senior Lecturer
Universiti Kebangsaan Malaysia
Malaysia
(External Examiner)
____________________________
NORITAH OMAR, PhD
Assoc. Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 16 August 2013
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This thesis submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Raja Noor Zaliha Raja Abdul Rahman, D.Eng
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)
Mohd Shukuri Mohamad Ali, PhD
Senior Lecturer
Faculty of Biotechnology and Biomolecular Sciences
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.
_____________________
RUDZANNA RUSLAN
Date: 10 May 2013
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK vi
ACKNOWLEDGEMENTS x
APPROVAL xi
DECLARATION xiii
LIST OF TABLES xvii
LIST OF FIGURES xviii
LIST OF ABBREVIATIONS xxi
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 4
2.1 Lipase 4
2.2 Geobacillus zalihae strain T1 lipase 6
2.3 Purification of lipases 8
2.3.1 Affinity chromatography 10
2.3.2 Ion exchange chromatography 11
2.3.3 Hydrophobic interaction chromatography 12
2.4 Lipase characterisation 13
2.5 Crystallisation 16
2.6 Methods of protein crystallisation 18
2.6.1 Microbatch crystallisation method 19
2.6.2 Vapour diffusion crystallisation method 21
2.6.3 Counter diffusion crystallisation method 23
2.7 Structural elucidation 24
2.8 X-ray crystallography 25
2.9 Electron density maps 27
2.10 Refinement 29
2.11 Maps 29
2.12 Validation 31
2.13 Three dimensional structures 32
2.14 Ion pair and protein stability 35
3 MATERIALS AND METHODS 38
3.1 Materials 38
3.2 Bacterial strains and plasmids 42
3.3 Detection of lipase producer 42
3.4 Inoculum preparation 43
3.5 Stock culture preparation 43
3.6 Expression of lipase 43
3.7 Lipase assay 44
3.8 Standard curve of oleic acid 45
3.9 Purification of lipase 45
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3.9.1 Preparation of crude extract 45
3.9.2 Affinity chromatography 46
3.9.3 Affinity 2 for GST-tag separation 46
3.9.4 Ion exchange chromatography 47
3.10 Polyacrylamide gel electrophoresis (PAGE) 47
3.11 Characterisation of wild-type T1 and mutant D311E lipases 48
3.11.1 Determination of protein concentration 48
3.11.2 Effect of temperature on lipase activity 48
3.11.3 Effect of temperature on lipase stability 49
3.11.4 Kinetic study on lipase 49
3.11.5 Standard curve of p-nitrophenol 50
3.11.6 Circular dichroism analysis 50
3.12 Protein crystallisation of D311E lipase 51
3.12.1 Microbatch crystallisation 51
3.12.2 Sitting drop vapour diffusion method 52
3.12.3 Capillary gel tube counter diffusion method 54
3.13 Cryoprotectant preparation 56
3.14 X-ray data collection 56
3.15 Refinement 56
3.16 Validation 57
4 RESULTS AND DISCUSSION 58
4.1 Verification of variant lipase activity 58
4.2 Purification of wild-type T1 and variant D311E lipase 59
4.2.1 Affinity chromatography 60
4.2.2 Buffer exchange 62
4.2.3 Second affinity chromatography 64
4.2.4 Ion exchange chromatography 67
4.2.5 Purification table 68
4.3 Characterisation of D311E lipase 71
4.3.1 Effect of temperature on wild-type T1 and D311E lipase
variant activity 71
4.3.2 Effect of temperature on wild-type T1 and D311E lipase
Variant stability 73
4.3.3 Kinetic study 75
4.3.4 Circular dichroism analysis 81
4.3.5 Comparison of wild-type T1 and variant D311E lipases 83
4.4 Crystallisation of D311E lipase 85
4.4.1 Microbatch crystallisation 85
4.4.2 Sitting drop vapour diffusion crystallisation method 87
4.4.3 Capillary gel tube counter diffusion crystallisation method 102
4.5 X-ray diffraction 111
4.5.1 Cryoprotectant agent and crystal mount 112
4.5.2 Data collection 112
4.6 Refinement of raw data 115
4.7 Validation 120
4.8 Structural analysis 123
4.8.1 Overall structure of D311E lipase 123
4.8.2 Ion pair analysis 125