universiti putra malaysiapsasir.upm.edu.my/39950/7/fbsb 2013 27r.pdf · abstract of thesis...

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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Universiti Putra Malaysia.

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



(Internal Examiner)

Norazizah binti Shafee, PhD

Associate Professor



(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


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



(Chairman)

Abu Bakar Salleh, PhD

Professor



(Member)

Mohd Shukuri Mohamad Ali, PhD

Senior Lecturer



(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 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


3.9.3 Affinity 2 for GST-tag separation 46


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


4.2.2 Buffer exchange 62

4.2.3 Second affinity chromatography 64


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

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5 SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR

FUTURE RESEARCH 131

5.1 Conclusion 131

5.2 Recommendations 133

REFERENCES 134

APPENDICES 149

BIODATA OF STUDENT 159

LIST OF PUBLICATION/CONFERENCE 160

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