EXPRESSION AND STRUCTURAL INVESTIGATION OF ACID TOLERANT ARSENITE OXIDASE WITH BIOSENSOR POTENTIAL
TEOH WEI KHENG
A thesis submitted in fulfilment of the requirements for the award of the degree of
Doctor of Philosophy (Bioscience)
AUGUST 2017
Provided by Universiti Teknologi Malaysia Institutional Repository
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ACKNOWLEDGEMENT
First of all, I would like to thank my supervisor Associate Professor Dr. Shafinaz Shahir. The thesis would not come to fruition without her continuous guidance and patience all along the way. Plenty of thanks to my co-supervisor, Dr. Faezah Mohd Salleh. With her encouragement and insightful comments, it really helped me a lot to refine my thought process and be more meticulous in my thesis writing.
For all the helpful discussion and knowledge sharing, I would like to thank Ummirul Mukminin bin Kahar. With the critical input and unselfishness in sharing his experience had made me learned so much. Besides that, I am also indebted to Dr. Hasmerya Maarof for her assistance in bioinformatics studies. Furthermore, special thanks to all the seniors in the faculty, Ivy Bay, Neoh Chin Hong, Lim Chi Kim, Khor Beng Hooi, Ang Siow Kuang and Lam Chee Yong. They are the motivation for me to undertake this journey until the very end.
I would not able to complete this tough journey without the support of my family and friends. Thanks to Zaratulnur Mohd Bahari, for being the best companion along all the ups and downs throughout our graduate years together. I would definitely miss the chatting during our lunch times together. For all my friends, Chai Kian Piaw, Chan Chia Sing and Chew Yue Ming, it is great to have them throughout this journey and may all of us can achieve what we dreamt for in life. I am especially grateful to have my parents and brother for being there to continuously support me no matter what happen. Last but not least, thanks to my loved one Chong Chee Kang for his patience and emotional support throughout my darkest day.
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ABSTRACT
Groundwater contamination by the two dominant toxic arsenic species, arsenite (As(III)) and arsenate (As(V)) has caused a global epidemic of arsenic poisoning effecting over 100 million people. Currently there is no commercially available As(III) biosensor to specifically detect this most toxic inorganic compound. Arsenite oxidase (Aio) catalyzes the oxidation of As(III) to As(V) by two electron transfer. As a redox enzyme, Aio is potentially suitable for construction of enzyme based electrochemical biosensor to detect As(III) specifically. A moderate acidophilic arsenite oxidizer, Thiomonas delicata DSM 16361 was used as source of Aio in this study. To obtain Aio sufficient for biosensor construction, it was first necessary to optimize the expression and purification of recombinant Aio in E. coli strain. Full length aioBA gene was isolated and deposited to GenBank under accession number KX792110. Expression of the recombinant Aio was successfully performed in ZYM-5052 autoinduction medium at 20 C for 48 hours in E. coli strain C43(DE3). The Aio was purified to homogeneity with purity > 90% and characterized. The purified Aio was found to be heterodimeric with subunits of 91 and 21 kDa (17 kDa without signal peptide) in size, respectively. Specific activity of purified Aio was 4 U/mg, with substrate Km of 14 µM. The temperature-activity profile of purified Aio was found optimum at 55 C and Aio retained nearly 45% of its initial activity after pre-incubation at 60 C for 1 hour. The enzyme was stable in acidic pH ranging from pH 2.5 to 6. Activity of Aio was retained in the presence of 10 mM metal ions (K+, Li+, Co2+, Ca2+, Ni2+, Mn2+, and Zn2+) and anions (NO2−, SO2−
4 , and Cl−). In order to elucidate the acidophilic adaptation of Aio, homology model of Aio was constructed using X-ray crystallized structure of Aio from Alcaligenes faecalis (PDB ID: 1G8K) owing to the high sequence identity of 64%. The homology model was compared to the two structures from alkaliphilic sources, A. faecalis and Rhizobium sp. NT-26. Notable difference between the structures and model was observed on the surface of the enzyme as well as the existence of a unique loop region in T. delicata Aio. Several structural features that might be implicated in the acidophilic adaptation of T. delicata Aio were: (a) a higher proline content in Aio, (b) positively charged surface protein, (c) a decreased number of salt bridges and hydrogen bonds, and (d) introduction of polar and charged residues distal to catalytic site. When Aio was incorporated in biosensor construction, the DCPIP/Aio electrochemical assay was capable of detecting 10 to 500 ppb As(III) by using carbon screen printed electrode. This revealed the potential of Aio as a biosensing material for determining safe level of As(III) in water systems. This is the first characterization study of acid tolerant Aio from T. delicata DSM 16361 with biosensor potential.
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ABSTRAK
Pencemaran air bawah tanah oleh dua spesies arsenik yang dominan, arsenit (As(III)) dan arsenat (As(V)) telah mengakibatkan wabak keracunan arsenik global melibatkan lebih 100 juta orang. Pada masa kini tiada biosensor komersial yang dapat mengesan sebatian arsenik bukan organik yang paling tosik secara khusus. Arsenit oksidase (Aio) adalah pemangkin untuk pengoksidaan dari As(III) ke As(V) dengan dua pemindahan elektron. Sebagai enzim redoks, Aio berpotensi untuk digunakan dalam pembinaan enzim biosensor elektrokimia untuk mengesan As(III) secara khusus. Thiomonas delicata DSM 16361 yang merupakan mikrob berciri asid serdehana digunakan sebagai sumber Aio dalam kajian ini. Untuk mendapatkan Aio yang cukup untuk pembinaan biosensor, ekspresi dan penulenan Aio rekombinan optimum amat diperlukan. Jujukan penuh gen aioBA dipencilkan dan didepositkan ke GenBank bawah nombor pencapaian KX792110. Ekspresi optimum untuk Aio rekombinan dilaksanakan dalam keadaan aerobik di dalam autoinduksi media ZYM- 5052 pada suhu 20 C selama 48 jam dengan E. coli C43(DE3). Aio ditulenkan ke homogenan dengan 90% ketulenan dan dicirikan. Aio tulen adalah heterodimerik dan mengandungi subunit dengan berat molekul 91 dan 21 kDa (17 kDa tanpa isyarat peptida) masing-masing. Aktiviti spesifik Aio tulen adalah 4 U/mg, dengan Km
substrat 14 µM. Profil suhu-aktiviti Aio tulen adalah optimum pada suhu 55 C dan Aio mengekalkan 45% aktiviti asalnya setelah dieramkan pada suhu 60 C selama sejam. Enzim ini stabil dalam pH berasid dari pH 2.5 ke 6. Aktiviti Aio dapat dikekalkan dalam 10 mM ion logam (K+, Li+, Co2+, Ca2+, Ni2+, Mn2+, dan Zn2+) dan juga anion (NO2−, SO2−
4 , dan Cl−). Untuk memahami adaptasi keasidan, model homologi Aio telah dibina dengan menggunakan struktur kristal X-ray Aio daripada Alcaligenes faecalis disebabkan oleh identiti jujukan yang tinggi, 64%. Homologi model tersebut dibandingkan dengan dua struktur dari sumber alkalifilik, A. faecalis dan Rhizobium sp. NT-26. Perbezaan yang ketara antara templat dengan model diperhatikan pada permukaan enzim dan kewujudan gelung yang unik dalam Aio T. delicata. Ciri-ciri struktur tersendiri yang mungkin dikaitkan dengan adaptasi keasidan Aio T. delicata dikenalpastikan seperti berikut: (a) pertambahan bilangan proline dalam Aio, (b) permukaan protein bercaj positif, (c) pengurangan bilangan jambatan garam dan ikatan hidrogen, dan (d) penambahan bilangan asid amino berkutub dan bercaj berjauhan dengan tapak pemangkin. Assay elektrokimia DCPIP/Aio menunjukkan keputusan yang baik dalam pengesanan As(III), dan dapat mengesan daripada 10 ke 500 ppb As(III) dengan menggunakan elektrod cetakan skrin karbon. Ini mendedahkan potensi Aio untuk dijadikan sebagai pengesan biologi untuk menentukan kepekatan As(III) dalam sistem air. Ini adalah kajian pencirian pertama Aio yang toleran asid daripada T. delicata DSM 16361 dengan potensi biosensor.
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TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii ACKNOWLEDGEMENT iii ABSTRACT iv ABSTRAK v TABLE OF CONTENTS vi LIST OF TABLES xii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xx LIST OF SYMBOLS xxii LIST OF APPENDICES xxiii
1 INTRODUCTION 1 1.1 Background of study 1 1.2 Problem statement 3 1.3 Objective of study 4 1.4 Scope of study 4 1.5 Significance of study 4
2 LITERATURE REVIEW 6 2.1 Arsenic 6
2.1.1 Sources of arsenic in the environment 7 2.1.2 Aqueous speciation of arsenic 9
2.2 Arsenic biotransformation 11 2.3 Remediation of arsenic 13 2.4 Prokaryotic arsenite oxidation 14
2.4.1 Arsenite oxidation by Thiomonas 17 2.5 Arsenite oxidase 19 2.6 Localization of arsenite oxidase 20 2.7 Biochemical characterization of Aio 23
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2.8 Recombinant expression of Aio 26 2.9 In silico analysis 26
2.9.1 Three dimensional (3D) structure of Aio 26 2.9.2 Homology modeling 27 2.9.3 SWISS-MODEL 28 2.9.4 MODELLER 29 2.9.5 SAAPdap 29
2.10 Acidophilic enzymes and their properties 30 2.11 Application of Aio 32 2.12 Electrochemical biosensor 33
2.12.1 Cyclic voltammetry 34 2.13 Fabric based electrochemical device 35
3 MATERIALS AND METHODS 36 3.1 Materials 36
3.1.1 Chemicals and biologicals 36 3.1.2 Bacterial strains 36 3.1.3 Plasmids 37 3.1.4 PCR Primers 37 3.1.5 Enzymes 37 3.1.6 Culture media 38
3.1.6.1 R2A medium 38 3.1.6.2 Luria-Bertani medium 38 3.1.6.3 Media for autoinduction 38
3.2 Molecular biology methods 39 3.2.1 Genomic DNA extraction 39 3.2.2 Preparation of competent cells 40 3.2.3 Heat-shock bacterial transformation 40 3.2.4 Plasmid DNA extraction and purification 40 3.2.5 PCR product purification 41 3.2.6 Colony PCR 41 3.2.7 Restriction enzyme digestion of DNA 41 3.2.8 Agarose gel electrophoresis 42 3.2.9 Quantification of DNA 42 3.2.10 DNA ligation 42 3.2.11 DNA sequencing 43
3.3 General protein methods 43 3.3.1 Cell lysis 43
3.3.1.1 Osmotic shock 43
3.3.2 Ammonium sulfate precipitation 44 3.3.3 Determination of protein concentration 44 3.3.4 Quantification of protein yield from
recombinant expression and sample con- centration 45
3.3.5 Denaturing protein gel electrophoresis (SDS-PAGE) 45
3.3.6 Native PAGE 45 3.3.7 Dialysis of protein samples 45
3.4 Cloning and expression of arsenite oxidase gene 46 3.4.1 Construction of expression system for Aio 46 3.4.2 Amplification of aioBA gene 47 3.4.3 Construct 1 (pET21a-aioBA): cloning of
aioBA into pET21a 48 3.4.4 Construct 2 (pET21a-aioBA-His): addi-
tion of His tag to aioBA 50 3.4.5 Construct 3 (pET21a-aioBA-HisTAT):
construction of recombinant Aio without signal peptide 52
3.4.6 Protein expression of recombinant con- structs 53 3.4.6.1 IPTG expression 53 3.4.6.2 Autoinduction 54
3.5 Purification of Aio 54 3.5.1 Large scale expression of recombinant
Aio 54 3.5.2 Purification of His-tagged recombinant
Aio using IMAC 55 3.5.3 Gel filtration chromatography 55
3.6 Characterization of Aio 56 3.6.1 Arsenite oxidase assay 56 3.6.2 Arsenite oxidase kinetic study 56 3.6.3 Effect of pH on purified Aio 56 3.6.4 Effect of pH stability on purified Aio 56 3.6.5 Effect of temperature on purified Aio 57 3.6.6 Effect of temperature on stability of
purified Aio 57
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3.6.7 Effect of metal ions and chemical reagents on purified Aio 57
3.6.8 Statistical analysis 58 3.7 In silico structural characterization of Aio 58
3.7.1 Construction of phylogenetic trees 58 3.7.2 Homology modeling and verification 59 3.7.3 Structural analysis 61
3.8 Preliminary studies of DCPIP mediated As(III) biosensor using Aio 62 3.8.1 MES/KCl buffer 62 3.8.2 Electrochemical measurement using car-
bon screen printed electrodes 63 3.8.2.1 Electrochemical behaviour of
mediator 63 3.8.2.2 Preparation of sample of
DCPIP/Aio electrochemical assay 63
3.8.3 Electrochemical measurement using fabric-based electrochemical devices 64
4 CLONING AND EXPRESSION STUDY OF ARSENITE OXIDASE GENE 67 4.1 Introduction 67 4.2 Results and discussion 68
4.2.1 Amplification of aioBA gene 68 4.2.2 Sequence analysis of the PCR amplified
aioBA 69 4.2.3 Construct 1 (pET21a-aioBA): cloning of
aioBA to pET21a 73 4.2.4 Construct 2 (pET21a-aioBA-His): addi-
tion of His tag to aioBA 74 4.2.5 Construct 3 (pET21a-aioBA-HisTAT):
construction of recombinant without sig- nal peptide 76
4.2.6 Expression studies of Aio in E. coli
expression system 78 4.2.6.1 Screening for expression of
recombinant Aio 78
4.2.6.3 Effect of different host cell strains 81
4.2.6.4 Effect of autoinduction 87 4.2.6.5 Effect of expression without
signal peptide (Construct 3) 88 4.3 Conclusion 89
5 PURIFICATION AND CHARACTERIZATION OF AR- SENITE OXIDASE 90 5.1 Introduction 90 5.2 Results and discussion 91
5.2.1 Purification of recombinant Aio 91 5.2.2 Purification of recombinant Aio without
signal peptide 98 5.2.3 Enzyme kinetics 99 5.2.4 Determination of optimum pH 100 5.2.5 pH stability 101 5.2.6 Determination of optimum temperature
and heat stability 102 5.2.7 Effects of metal ions, anions and other
compounds on recombinant Aio activity 104 5.3 Conclusion 105
6 IN SILICO STRUCTURAL CHARACTERIZATION 107 6.1 Introduction 107 6.2 Results and discussion 108
6.2.1 Primary sequence analysis 108 6.2.2 Phylogenetic analysis 112 6.2.3 Homology modeling 114 6.2.4 Model evaluation 119 6.2.5 Structure model analysis 121 6.2.6 Structure comparison 123 6.2.7 Surface comparison 125 6.2.8 Molecular contact 128 6.2.9 SAAPdap analysis 132
6.3 Conclusion 133
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7 PRELIMINARY STUDY OF DCPIP MEDIATED AS(III) BIOSENSOR USING AIO 135 7.1 Introduction 135 7.2 Results and discussion 136
7.2.1 DCPIP as electron mediator 136 7.2.2 DCPIP/Aio electrochemical assay using
carbon screen printed electrode 138 7.2.3 Aio deposited fabric-based electrochemi-
cal device 140 7.2.3.1 Cyclic voltammetric
characterization of DCPIP/FED and DCPIP/Aio/FED 141
7.3 Conclusion 144
8 CONCLUSION AND FUTURE WORKS 145 8.1 Conclusion 145 8.2 Future works 147
REFERENCES 148
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TABLE NO. TITLE PAGE
2.1 Major arsenic minerals occurring in nature (Smedley and Kinniburgh, 2002) 9
2.2 Heterotrophic arsenic oxidizing organisms (Kumari and Jagadevan, 2016) 15
2.3 Chemolithotrophic arsenic oxidizing organisms (Kumari and Jagadevan, 2016) 16
2.4 Characteristics of Thiomonas strains 18 2.5 Localization of arsenic oxidizing organisms. 22 2.6 Comparison of purified As(III) oxidases (Adapted from Heath
et al. (2012)) 25 2.7 Recombinant expression of Aio from different bacterial
strains. 26 3.1 E. coli strains and plasmids used for cloning in this study 37 3.2 Expression constructs used in the study 46 3.3 Oligonucleotide primers used for amplification of aioBA gene 47 3.4 Oligonucleotide internal primers used for sequencing of
aioBA gene 47 3.5 Oligonucleotide primers used to develop Construct 1
(pET21a-aioBA). Restriction enzyme sites are underlined 49 3.6 Oligonucleotide primers used to develop Construct 2
(pET21a-aioBA-His) 50 3.7 Oligonucleotide primers used for amplification of pET21a-
aioBA-HisTAT (Construct 3) 52 3.8 List of software tools 61 3.9 Sample content of the DCPIP/Aio electrochemical assay 63 5.1 Purification table of T. delicata recombinant Aio 98 5.2 Purification table of T. delicata recombinant AioTAT 99 5.3 Characteristic of T. delicata Aio compared with Aio from
other mesophilic organisms 100
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5.4 Effects of metal ions, anions and chemicals on Aio activity. Values are represented by mean and standard error of at least three assays. 105
6.1 Comparison of amino acid composition of T. delicata (TDEL) Aio with other known Aio from A. faecalis (AFAE) and Rhizobium sp. NT-26 (NT26). 112
6.2 Comparison of interactions surrounding active site between T. delicata AioA (TDEL), A. faecalis (1G8K) and NT-26 (4AAY) 128
6.3 Amino acid residues in AioA from T. delicata (TDEL) that differ from conserved residues across AioA proteins from A.
faecalis, Herminiimonas arsenicoxydans, Ralstonia sp. S22, Achromobacter arsenitoxydans SY8 and Rhizobium sp. NT- 26. 130
6.4 Amino acid differences between T. delicata Aio and A.
faecalis Aio identified by SAAPdap analysis 133 B.1 Stock solutions 166
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FIGURE NO. TITLE PAGE
2.1 The arsenic geocycle. (Bhattacharjee and Rosen, 2007). 8 2.2 Distribution of As(III) and As(V) as a function of pH at 25 C
(Sharma and Sohn, 2009). 10 2.3 The Eh-pH diagram for arsenic in the system As-O2-H2O
at 25 C and 1 bar total pressure (Smedley and Kinniburgh, 2002). 11
2.4 Synopsis of the mechanism involved in arsenic treatment processes (Delavat et al., 2012) 13
2.5 Flow diagram for comparative modeling of proteins showing standard process (solid arrows) and feedback/refinement mechanisms (dashed arrows) (Lushington, 2008) 28
2.6 Example of SAAPdap analysis 30 3.1 Partial sequence of aioBA from T. delicata DSM 16361
(Quemeneur et al., 2008) 48 3.2 Primer binding position for primers designed to derive the
whole sequence for the PCR product of aioBA. SP indicates the signal peptide region at aioB. 48
3.3 The restriction enzymes that would digest aioBA gene internally analyzed using NEBcutter 49
3.4 Construct 1 pET21a-aioBA, SP indicates signal peptide region 50
3.5 Primer binding position (aioBAfHis and aioBArHis) for construction of pET21a-aioBA-His (Construct 2) by inverse PCR. SP indicates signal peptide region. 51
3.6 Signal peptide prediction from SignalP server shows the suitable cleavage site is located between residues 33 and 34 52
3.7 Construct pET21a-aioBA-HisTAT 53 3.8 Flow chart of the T. delicata Aio homology modeling and
characterization 58
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3.9 Schematic illustration of the fabrication process of the FED (Malon et al., 2014). 64
3.10 The setup of FED with respective electrodes. WE=carbon paste (green), CE=carbon paste (red), RE=Ag/AgCl (yellow) 65
3.11 Steps for construction of DCPIP/Aio/FED for sample detection 66
4.1 Agarose gel electrophoresis showing the genomic DNA from T. delicata. Lane M: GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific); Lanes 1 and 2: genomic DNA from T.
delicata. 68 4.2 Isolation of the aioBA from T. delicata via PCR amplification 69 4.3 NCBI Blastx analysis result of aioB. The result shows the
identity of 99% to Aio small subunit from Thiomonas strains multispecies. 70
4.4 NCBI Blastx analysis result of aioA. The result shows the identity of 99% to Aio large subunit from Thiomonas strains multispecies. 71
4.5 The nucleotide and deduced amino acid sequence of T.
delicata Aio 73 4.6 Agarose gel electrophoresis showing the size of pET21a
plasmid and aioBA gene after restriction enzyme NdeI and HindIII digestion 74
4.7 Screening of pET21a-aioBA (Construct 1) clones by colony PCR 74
4.8 Agarose gel electrophoresis showing the size of PCR amplified construct pET21a-aioBA with His tagged 75
4.9 Screening of pET21a-aioBA-His (Construct 2) clones by colony PCR 76
4.10 Agarose gel electrophoresis showing the size of pET21a plasmid and aioBA gene after restriction enzyme NdeI and HindIII digestion. 77
4.11 Screening of pET21a-aioBA-HisTAT (Construct 3) clones by colony PCR 77
4.12 Aio activity screening of Construct 1 and 2 in E. coli
BL21(DE3) 79 4.13 SDS-PAGE shows the localization of the recombinant Aio in
E. coli BL21(DE3) 80 4.14 Native PAGE of proteins from subcellular fractions of E. coli
expressing Aio 81
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4.15 SDS-PAGE of total cell proteins from different E. coli cells transformed with construct pET21a-aioBA-His. 83
4.16 SDS-PAGE of recombinant Aio in BL21(DE3) cells showing the crude lysate after varying concentration of IPTG induction at 16 C for 18 hours 84
4.17 SDS-PAGE of recombinant BL21(DE3) with construct pET21a-aioBA-His showing the soluble fraction and inclu- sion body. 85
4.18 SDS-PAGE of recombinant Aio in C43(DE3) showing the soluble fraction after varying concentration of IPTG induction at 20 C for 20 hours, 180 rpm. 86
4.19 SDS-PAGE of recombinant Aio in C43(DE3) showing the inclusion body fraction after varying concentration of IPTG induction at 20 C for 20 hours, 180 rpm. 86
4.20 Comparison of Aio activities in crude lysate of E. coli strains. Error bars represent the standard deviation of three assays, p < 0.05. 87
4.21 Comparison of Aio activities in crude lysate after IPTG induction and autoinduction at 20 C. Error bars represent the standard deviation of three assays, p < 0.05 88
5.1 Affinity IMAC purification Histrap HP 5 mL of the recombinant Aio expressed in E. coli and corresponding 12% SDS-PAGE. 92
5.2 Affinity IMAC purification Histrap HP 1 mL of the recombinant Aio expressed in E. coli and corresponding 12% SDS-PAGE. 94
5.3 Gel filtration of recombinant Aio. Eluate from Histrap HP 1 mL was concentrated and loaded onto a HiLoad 16/60 Superdex 200 gel filtration column. (a) Elution profile, (b) SDS-PAGE analysis of concentrated eluate from respective peak (P1-P4). 96
5.4 SDS-PAGE analysis of eluates from Histrap HP 1 mL under different parameter, elution at flow rate= 1 mL/min at room temperature (a) binding and elution buffer pH 8 (b) 80% ammonium sulfate precipitated crude as sample. 97
5.5 SDS-PAGE analysis of purified recombinant Aio 98 5.6 SDS-PAGE analysis of purified recombinant AioTAT. 99 5.7 Kinetic plot obtained with recombinant Aio with substrate
arsenite 100
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5.8 Effect of pH on the activity of purified recombinant Aio. The highest Aio activity was taken as 100%. Each point represents the mean ± standard error (n=3), p<0.05. 101
5.9 pH stability on the activity of purified recombinant Aio. The highest Aio activity was taken as 100%. Each point represents the mean ± standard error (n=3), p<0.05. 102
5.10 Optimum temperature of purified recombinant Aio. The highest Aio activity was taken as 100%. Each point represents the mean ± standard error (n=3), p<0.05. 103
5.11 Effect of temperature stability on purified recombinant Aio. The highest Aio activity was taken as 100%. Each point represents the mean ± standard error (n=3), p<0.05. 103
6.1 Sequence alignment of AioA and AioB from T. delicata
(TDEL). 110 6.2 Neighbour-joining (NJ) trees showing phylogenetic relation-
ships between deduced amino acid sequence of Aio from T.
delicata and other arsenite oxidases. The Aio amino acid sequence of Rhizobium sp. NT-26 was used as an out-group to root the tree. Confidence values were assessed from 1000 bootstrap replicates of the original sequence data. 113
6.3 Local quality estimates of chain A and B of primary sequence T. delicata Aio 115
6.4 Normalized QMEAN4 score of T. delicata Aio model 116 6.5 The highlighted part in yellow circle shows the loop region
of homology model of T. delicata Aio before (pink) and after (blue) loop refinement. 117
6.6 Heterodimer model of T. delicata Aio. Blue AioA, Green
AioB. 118 6.7 Ramachandran plot of T. delicata Aio 120 6.8 Domains I-IV of the large subunit AioA are drawn in orange,
yellow, green and blue, respectively. The small subunit AioB is drawn in red. (a) front view of the structure (b) sideview of the structure. 122
6.9 Space-filling homology model of T. delicata Aio. Active site of the enzyme is boxed. Conserved residues between the T.
delicata Aio and A. faecalis Aio are red; different residues are blue 124
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6.10 Superimposition of homology model T. delicata Aio with crystal structures of A. faecalis (PDB: 1G8K) and NT-26 (PDB: 4AAY) colored in pink, cyan and blue, respectively 125
6.11 Electrostatic rendering of the solvent-accessible surface of (a) T. delicata Aio, (b) A. faecalis 1G8K and (c) NT-26 4AAY. Computed at pH 7 with red (-5 kT/e) and blue (5 kT/e). Only protein atoms were considered in the calculation. The surface is colored in blue for positively charged residues and red for negatively charged residues. 127
6.12 LigPlot analysis showing the active sites interactions of bis- MGD. 129
6.13 Substituted residues in AioA within 4 A of the bis-MGD. (a) A. faecalis (b) T. delicata. 131
7.1 Cyclic voltammogram of 1 mM DCPIP in 50 mM MES, 100 mM KCl (pH 6) with increasing scan rate 10, 20, 40 and 80 mV/s. Scan range -400 to 300 mV. 136
7.2 The linear relationship between the redox peaks and the square root of the scan rate (10, 20, 40 and 80 mV/s) 137
7.3 Schematic diagram displaying the interaction between the enzyme Aio and As(III) at the electrode and the subsequent generation of current 137
7.4 Cyclic voltammogram of DCPIP/Aio electrochemical assay using carbon screen printed electrode with increasing concentration of As(III) (0-20 ppb). Scan range -400 to 300 mV. 139
7.5 Calibration plot depicting linear dose response of current to As(III) concentration (10-500 ppb) over which the calibration study was conducted with DCPIP/Aio electrochemical measurement on carbon screen printed electrode. Error bars represent standard error (n=3). 140
7.6 Cyclic voltammogram indicates higher peak current with successive DCPIP loading (5 µL with 5 mM DCPIP each time) to the same FED. Scan rate 5 mV/s, -300 to 300 mV scan range. 141
7.7 Cyclic voltammogram of DCPIP/FED (without Aio deposited at FED reaction zone) with As(III). Scan rate 5 mV/s, -300 to 300 mV scan range. 142
7.8 Cyclic voltammogram of DCPIP/Aio/FED. Scan rate 5 mV/s, -300 to 300 mV scan range. 143
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7.9 Cyclic voltammogram of As(III) to DCPIP/Aio/FED. Scan rate 5 mV/s, -800 to 400 mV scan range 144
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BSA - bovine serum albumin
E. coli - Escherichia coli
EDTA - ethylene diamine teraacetate
IPTG - isopropyl-β-D-thiogalactopyranoside
kDa - kilo-Dalton
N-terminus - amino terminus
Ni-NTA - nickel-nitrilotriacetic acid
RMSD - root mean square deviation
rpm - revolutions per minute
SDS - sodium dodecyl sulfate
TAE buffer - tris-acetate-EDTA buffer
α - alpha
β - beta
Cα - carbon alpha
ppb - part per billion
ppm - part per million
v/v - volume per volume
w/v - weight per volume
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Vector map 165 B Stock solutions 166 C Arsenite oxidase activity calculation 167 D Statistical analysis 171 E List of publications 182
CHAPTER 1
1.1 Background of study
Arsenic is classified as one of the notorious carcinogen by the United States Environmental Protection Agency (EPA). Arising from both natural and anthropogenic causes, arsenic is widely spread in the environment. Changes in the oxidation state of arsenic have implications for its mobility and toxicity to living cells. The toxicity of arsenic follows the order: Inorganic As(III) species > Organic As(III) species > Inorganic As(V) species>Organic As(V) species> Elemental Arsenic (Akter and Ali, 2011). The two arsenic species which generally exposed to human and microorganisms are arsenite As(III) and arsenate As(V), with As(III) is considered 100 times more toxic (Rosen, 2002).
As(III) and As(V) species can interchange oxidation state depending on redox potential, pH and biological processes from the microbial activities. Various groups of As(III) oxidizing bacteria have been isolated from diverse arsenic contaminated environments, such as cattle-dipping fluids, hot springs, mine tailing and drainage water. The As(III)-oxidizing bacteria isolated to date can be divided into two groups: (i) chemolithoautotrophs (utilize As(III) as the electron donor) or (ii) heterotrophs (growth in the presence of organic matter).
Arsenite oxidase (Aio) catalyzes the oxidation of As(III) to As(V) by a two- electron transfer. Belonging to a member of the dimethylsulfoxide (DMSO) reductase family of molybdenum-containing enzymes, Aio consists of two heterodimeric subunits. The large subunit (AioA) shares similarities to the α subunit of the formate dehydrogenase and assimilatory nitrate reductase. The small subunit (AioB) on the other hand belongs to the structural class of the Rieske subunit of the cytochrome bc1 complex. Both the subunits have homologues in enzyme families differing
2
significantly from each other with respect to both redox cofactors and function. Hence, Lebrun et al. (2003) suggests Aio to be the ancient bioenergetic enzyme before the divergent of Bacteria and Archaea. As an ancient enzyme which utilizes As(III), a highly toxic metalloid as substrate, characterization of Aio is of special interest for bioremediation and biosensor construction. To date, two Aio have been extensively characterized which were originated from a chemolithoautotrophic Alphaproteobacteria Rhizobium sp. NT-26 (Santini and vanden Hoven, 2004) and heterotrophic Betaproteobacteria Alcaligenes faecalis (Anderson et al., 1992). Both bacteria grow optimally at slightly alkaline pH 8, which can be categorized as alkaline- tolerant bacteria.
In mining environments, particularly acid mine drainage (AMD), inorganic As(III) is widespread as a result of the bioleaching of arsenic-bearing minerals. Under low pH condition (pH <2), As(III) oxidizers from these extreme environment are commonly isolated with acid-tolerant properties with an optimum growth pH near neutrality with the exception of Thiomonas strains (Hallberg and Johnson, 2003; Battaglia-Brunet et al., 2006; Katayama et al., 2006; Duquesne et al., 2008; Bryan et al., 2009; Arsene-Ploetze et al., 2010), that grow optimally around pH 5. Microbial metabolism in AMD causes natural attenuation process to occur whereby Thiomonas
strains are suggested to have acted as the As(III) oxidizer (Casiot et al., 2003; Duquesne et al., 2003; Morin et al., 2003; Bruneel et al., 2006; Battaglia-Brunet et al., 2011; Bertin et al., 2011). Oxidation of As(III) plays a pivotal role in the natural bioremediation since it could contribute to an improved immobilization of arsenic in helping to mitigate arsenic contamination. Thiomonas strains are therefore essential for maintaining the arsenic biogeochemical cycle in AMD. Freel et al. (2015) showed that several phylogenetic groups of Thiomonas strains populated along Regious creek AMD for more than a decade even though in low abundance with differences in the As(III) oxidation capabilities. Accumulation of evolutionary changes in the harsh environment of AMD may promote unique adaptive phenotypes especially in the Thiomonas Aio.
Thiomonas delicata DSM 16361, isolated from Cheni gold mine, shows high As(III) oxidizing capability of 4 mg As(III) l−1 hr−1 which grows optimally at pH 4 to 7 as a moderate acidophile (Battaglia-Brunet et al., 2006). Most interestingly, purification and detailed characterization of Aio from facultative chemolithoautrophic As(III) oxidizing Thiomonas strains have yet to be described. The characterization of the Aio is of great importance as we could further explore the enzymatic features that contribute to the irreplaceable role of Thiomonas strains as an As(III) oxidizer in AMD.
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This could facilitate the engineering of enzymes and aid in construction of robust biosensor for monitoring arsenic in acid mining effluent. Therefore, in this study, by performing heterologous expression and purification of the Aio from T. delicata DSM 16361 strain deposited in German Collection of Microorganisms and Cell Cultures (DSMZ), we characterized the Aio in terms of its biochemical properties. Homology modeling of the enzyme was constructed to support the experimental findings. In order to explore the potential of Aio as biosensing material for As(III) detection, Aio was applied to the electrochemical device in this study. Since cyclic voltammetry is the most commonly used and versatile electroanalytical technique for the study of redox enzyme, thus it was employed to characterize the mediated Aio for biosensing ability. To the best of our knowledge, this is the first characterization study of Aio from a moderate acidophilic chemolithoautotrophic betaproteobacterium.
1.2 Problem statement
To date, there is no commercially available biosensor to detect As(III) which is the most toxic inorganic arsenic. Only one study of enzyme based As(III) biosensor has been reported using Rhizobium sp. NT-26 Aio (Male et al., 2007). Currently, most of the arsenic test strips detect total arsenic content are expensive, low specificity and reproducibility. In arsenic remediation process, first stage pre-oxidation of As(III) is preferential to transform the arsenic species to the less soluble form As(V) for precipitation. Therefore, assessment of efficient conversion of As(III) is crucial. Moreover, As(III) is the prevalent species in anoxic drinking groundwater, hence specific detection of this bioavailable arsenic species is critical.
Additionally, molybdenum containing enzymes represent an essential group of enzyme in biology, Aio is one of it. Due to the difficulty to purify and express this exotic enzyme, to date only Aio from Alcaligenes faecalis and Rhizobium sp. NT-26 have been purified and characterized in details (Anderson et al., 1992; Ellis et al., 2001; Santini et al., 2004). Biochemical characterization has been performed to Aio from Arthrobacter sp. 15b. However, no detailed studies has been presented for biochemical characterization of purified Aio from T. delicata, which is a dominant As(III) oxidizing genus found at AMD (Freel et al., 2015). The absence of the three-dimensional structure for T. delicata Aio in PDB prompted us to construct its homology model for the acidophilic adaptation. By using in silico approach, a more comprehensive study about the structural characteristic of Aio can be obtained to further improve our knowledge concerning the adaptation of Aio from an acidophilic bacteria.
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1.3 Objective of study
This study was carried out to investigate the characterization of Aio from Thiomonas delicata DSM 16361. The specific objectives were as following:
(i) to clone and express of arsenite oxidase gene aioBA in E. coli
(ii) to purify and characterize recombinant Aio
(iii) to model the recombinant Aio structure and elucidate its acidophilic adaptation through in silico analysis
(iv) to apply Aio for the construction of As(III) biosensor
1.4 Scope of study
In this study, T. delicata strain was purchased from DSMZ under DSM number 16361. The aioBA gene was amplified from genomic DNA of T. delicata by PCR to obtain full length gene sequence. After verification of the sequence, the aioBA was cloned to pET21a expression vector and transformed into E. coli cells. Placement of polyhistidine tag to aioB was then performed for the aid of affinity purification system. Optimization of expression was performed. After that, purification of recombinant Aio was carried out, which was affinity and gel filtration chromatography. Purity of recombinant Aio was verified using SDS-PAGE. Characterization of Aio was performed in terms of biochemical studies and in silico studies. Homology modeling of Aio was constructed to allow comparison with other crystallized Aio structure. Finally, application of Aio to biosensor construct was attempted to reveal the potential of Aio as a biosensing element.
1.5 Significance of study
In the present study, aioBA from T. delicata DSM 16361 was successfully amplified and cloned to E. coli. Expression of functionally active recombinant Aio allowed two-step purification of Aio. Biochemical properties of recombinant Aio were elucidated, which filled the research gap about the potential inhibitors of Aio. New insights of T. delicata Aio adaptation to low pH were revealed from the structural information of Aio through homology model. Comparison of T. delicata Aio
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