By
Timotius William Jefferson 1150613
BACHELOR’S DEGREE in
ELETRICAL ENGINEERING – BIOMEDICAL ENGINEERING CONCENTRATION
LIFE SCIENCES AND TECHNOLOGY
SWISS GERMAN UNIVERSITY The Prominence Tower
Jalan Jalur Sutera Barat No. 15, Alam Sutera Tangerang, Banten 15143 - Indonesia
August 2019
[REVISION AFTER THESIS DEFENSE ON 18th JULY 2019]
Timotius William Jefferson STATEMENT BY THE AUTHOR
I hereby declare that this submission is my own work and to the best of my knowledge, it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at any educational institution, except where due acknowledgement is made in the thesis.
Timotius William Jefferson
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Student Date:
Approved by:
Dr.rer.nat Maruli Panjaitan
_____________________________________________
Thesis Advisor Date:
Uli Julia Nasution, MBiotechSt
_____________________________________________
Thesis Co-Advisor Date:
Dr. Dipl. -Ing. Samuel P. Kusumocahyo
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Dean
Date:
Timotius William Jefferson ABSTRACT
Cephalosporin C Acylase Characterization and Purification Using Chromatography Techniques
By
Timotius William Jefferson Dr.rer.nat Maruli Panjaitan Uli Julia Nasution, MBiotechSt
SWISS GERMAN UNIVERSITY
Cephalosporin C Acylase is an enzyme that plays an important role in the one step conversion of Cephalosporin C into 7-aminocephalosporanic acid (7-ACA), a key intermediate for the synthesis of semisynthetic cephalosporin antibiotics. Purification is a method or process of removing unwanted protein molecules or impurities from protein of interest with the aim of increasing enzyme specific activity. The purification process begins by expressing Cephalosporin C Acylase using E. coli BL21 (DE3) followed by the cell lysis process. Characterization of Cephalosporin C Acylase was being examined in this study to get a profile of the enzyme before entering purification process. This study applied chromatography techniques for enzyme purification, but mainly focusing on a specific purification method, Immobilized Metal Affinity Chromatography. It is a widely used purification method to purify histidine tagged protein. Ion-exchange Chromatography was also conducted as one of the chromatography techniques for additional purification step. Purification process in this research study is capable to increase enzyme specific activity up to 4.56-fold and recover 63.9% yield. Result of protein bands analysis using SDS-PAGE techniques shown that recombinant CCA enzyme is consist of two subunits, α (25kDA) and β (58kDa) subunit. This analysis also shown that purification process is capable in removing impurities from enzyme of interest.
Keywords: Cephalosporin C Acylase, Protein Purification, Chromatography techniques, Enzyme Specific Activity.
Timotius William Jefferson
© Copyright 2019 by Timotius William Jefferson
All rights reserved
Timotius William Jefferson DEDICATION
I dedicate this works for the future enzyme purification research.
And for the advancement of my beloved country, Indonesia.
Timotius William Jefferson ACKNOWLEDGEMENTS
First and foremost , I praise God, the almighty for providing me this opportunity and granting me the capability to proceed successfully. Thank you for the blessing throughout years, lead me into this point. You given me the power to believe in myself to accomplish this bachelor degree. I could never have done this without the faith I have in you, the Almighty.
Secondly, I would like to express my gratitude and deep thanks to my supervisor at Balai Bioteknologi, Mrs. Uli Julia Nasution, MBiotechSt for the valuable guidance and advice. I was greatful because of her being so patient and also thank for her willingness to share her knowledge and experience of laboratory skills toward me during this thesis research.
Thirdly, I would like to thank my lecturer, Dr. rer.nat Maruli Pandjaitan whom without hesitation always willing to advise with any knowledge related to this topic, thank you for the encouraging and show a believe in me in completing this research study. And thank you for being my good lecture for the past 4 years. In addition, I would also thank the authority of BPPT Balai Bioteknologi for the opportunities that they gave me to done my thesis project, thank you for providing me with a good environment and facilities to complete this study.
Lastly, an honorable mention goes to my parents, who always remains at my back to support me in completing this thesis. To all my colleagues who share the same lab, Walbert Christian and Febraska Laoditta who always help and accompany me during the whole study research. Also mentioning Agatha Hintama, thank you for the help, motivation, and encouragement in doing my Thesis.
Without helps of the particular that mentioned above, maybe I would face many difficulties while doing this thesis.
Timotius William Jefferson Page
STATEMENT BY THE AUTHOR ... 2
ABSTRACT ... 3
DEDICATION ... 5
ACKNOWLEDGEMENTS ... 6
TABLE OF CONTENTS ... 7
LIST OF FIGURES ... 9
LIST OF TABLES ... 11
LIST OF EQUATION ... 12
CHAPTER 1 - INTRODUCTION ... 13
1.1 Background ... 13
1.2 Research Problem ... 14
1.3 Objectives ... 14
1.4 Significance of Study ... 15
1.5 Research Question ... 15
1.6 Hypothesis ... 15
CHAPTER 2 - LITERATURE REVIEW ... 16
2.1 Cephalosporin ... 16
2.2 Cephalosporin C Acylase ... 17
2.2.1 Cephalosporin C Acylase Classification ... 17
2.2.2 Cephalosporin C conversion into 7-ACA ... 19
2.3 Cephalosporin C Acylase Expression Using Escherichia coli As A Host ... 20
2.3.1 Bacterial Culture ... 20
2.4 Enzyme Purification ... 20
2.4.1 Protein Extraction Using Cell Lysis ... 21
2.4.2 Histidine-Tagged (His-Tag) Protein Purification ... 21
2.5 Enzyme Analysis Methods ... 22
2.5.1 Bradford Assay ... 22
2.5.2 SDS- PAGE ... 22
2.5.3 High Performance Liquid Chromatography ... 23
CHAPTER 3 – RESEARCH METHOD ... 24
3.1 Venue and Time... 24
3.2 Materials and Equipment ... 24
3.2.1 Bacterial Sample ... 24
3.2.2 Chemical Materials ... 24
Timotius William Jefferson
3.3 Experiment Design ... 27
3.4 Method ... 28
3.4.1 Cephalosporin C Acylase Expression... 28
3.4.2 Cephalosporin C Acylase Purification ... 30
3.4.3 Purified enzyme analysis ... 34
CHAPTER 4 – RESULT AND DISSCUSION ... 39
4.1 Preliminary Research ... 39
4.2 Study Research Results ... 40
4.2.1 Enzyme Characterization ... 40
4.2.2 Purification of Cephalosporin C Acylase using IMAC ... 45
4.2.3 Purification of Cephalosporin C Acylase using IEC and IMAC ... 52
4.3 Discussion ... 55
4.3.1 Cephalosporin C Acylase Expression in E.coli BL21(DE3) ... 55
4.3.2 Analysis of Cephalosporin C Acylase Characterization ... 56
4.3.3 Analysis of enzyme purification using IMAC method ... 57
4.3.4 Analysis of Enzyme Purification Result under different condition ... 58
4.3.5 Analysis of enzyme purification using IEC method ... 60
4.3.6 Analysis of two step purification using IEC and IMAC ... 60
4.3.7 Enzyme analysis methods ... 63
CHAPTER 5 – CONCLUSION ... 66
5.1 Conclusion ... 66
5.2 Recommendation ... 67
APPENDICES ... 68
Appendix 1 Composition of Solutions ... 68
1.1 Solution of bacteria fermentation ... 68
1.2 Solutions for Lysis ... 68
1.3 Solutions for purification ... 68
1.4 Solutions for Bradford test ... 68
1.5 Solutions for SDS-PAGE ... 69
1.6 Solutions for HPLC ... 69
Appendix 2 Raw Data ... 70
2.1 HPLC and Bradford Standard ... 70
2.2 Enzyme Stability and Protease test ... 71
2.3 NaCl effect on enzyme activity ... 72
2.4 Purification Using IMAC ... 72
2.6 Purification Using IEC + IMAC ... 75
Appendix 3 Supporting Data ... 78
REFERENCE ... 79
Timotius William Jefferson LIST OF FIGURES
Figures Page
Figure 1 Cephalosporin C Acylase structure. ... 17
Figure 2 Derivation scheme from CPC to 7-ACA (Pollegioni et al., 2013). ... 19
Figure 3 Chemical Structure of CPC and 7-ACA (Pollegioni et al., 2013) ... 19
Figure 4 Protein Purification using His-Tag Purification ... 22
Figure 5 Enzyme Analysis Instrument. ... 26
Figure 6 Schematic of Study Experiment ... 27
Figure 7 E.coli regeneration in agar media ... 28
Figure 8 Figure of slant media ... 29
Figure 9 Incubation of production media ... 30
Figure 10 Cell Pellet (red arrow) ... 30
Figure 11 Cell Lysis Process ... 31
Figure 12 Enzyme Purification process ... 33
Figure 13 Dialysis Process ... 34
Figure 14 Figure of schematic diagram of running SDS-PAGE (Wikipedia) ... 36
Figure 15 Protein bands analysis of preliminary purification ... 39
Figure 16 Enzyme stability in different temperature ... 41
Figure 17 Enzyme Protein Bands analysis in NaCl treatment. ... 42
Figure 18 Enzyme activity comparison before and after NaCl treatment... 43
Figure 19 Chromatograms of protein binding test ... 44
Figure 20 Comparison of enzyme activity between different buffer environment ... 45
Figure 21 Sample A protein bands analysis ... 46
Figure 22 Sample B protein bands analysis ... 47
Figure 23 Sample C protein bands analysis ... 47
Figure 24 Enzyme Specific Activity among different buffer lysis ... 48
Figure 25 Purification level of purification among different lysis buffer ... 48
Figure 26 Yield recovery of purification among different lysis buffer... 49
