35
A2.5cm ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF 2,2-DICHLOROPROPIONIC ACID UTILIZING BACTERIA WONG WEN YONG UNIVERSITI TEKNOLOGI MALAYSIA A2.5cm
A2.5cm ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF
2,2-DICHLOROPROPIONIC ACID UTILIZING BACTERIA
WONG WEN YONG
UNIVERSITI TEKNOLOGI MALAYSIA
A2.5cm
i
A2.5cm ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF
2,2-DICHLOROPROPIONIC ACID UTILIZING BACTERIA
WONG WEN YONG
A thesis submitted in fulfilment of the
requirement for the award of the degree of
Master of Science (Bioscience)
Faculty of Biosciences and Medical Engineering
Universiti Teknologi Malaysia
MARCH 2013
iii
___________ You
____________ For
___________ Your
_______ Support
______ And
__ Care
To __
My ______
Beloved _______
Family _________
Advisor ________
And _______
My ___
_ Friend
______ Thank
___________ You
____________ For
___________ Your
_______ Support
______ And
__ Care
To __
My ______
Beloved _______
Family _________
Advisor ________
And ______
_
iv
5cm ABSTRACT
2,2-dichloropropionic acid (2,2-DCP) is an artificial halogenated compound
used as herbicide. A bacterium able to utilize 2,2-DCP as sole carbon source was
isolated from soil in Melaka rubber estate. The bacterium was identified as Labrys sp.
strain Wy1 using bacterium’s 16S rRNA partial sequence. The cells doubling time
was 34.6 hours in liquid minimal media supplied with 20 mM 2,2-DCP as sole
carbon source. Utilization of 2,2-DCP was confirmed by detection of chloride ion
released at 0.27 mM. An endophytic bacterium isolated from Axonopus compressus
which was identified as Burkholderia cepacia strain Wy5 was also able to utilize 2,2-
DCP as sole carbon source. The bacterium has cells doubling time 2.7 hours and
chloride ion released was also detected at 47.28 ± 0.25 mM in minimal media
contained 20 mM 2,2-DCP. Cell free extract (CFE) of Burkholderia cepacia Wy5
was further characterized due to its higher activity towards 2,2-DCP compared to
Labrys sp. Wy1. Dehalogenase found in CFE of Burkholderia cepacia Wy5 has
optimal enzyme specific activity at pH8 (0.83 μmol [Cl-] min
-1 mg
-1) and 40
oC (0.78
μmol [Cl-] min
-1 mg
-1). The dehalogenase was also able to react with other α-
haloalkanoic acid including monochloroacetic acid, DL-2-chloropropionic acid and
DL-2-bromopropionic acid, but not 3-chloropropionic acid. “Group I” and “Group II”
dehalogenase primers were used to amplify dehalogenase gene from both strains
Wy1 and Wy5 but only Burkholderia cepacia Wy5 showed positive result. The
dehalogenase gene fragment amplified was designated “deh-wy5” and subsequent
analysis showed it belongs to Group I dehalogenase. Customized primers based on
D,L-dex gene were designed to amplify complete sequence of deh-wy5 due to high
similarity between partial sequence of deh-wy5 and D,L-dex. Complete sequence of
deh-wy5 was eventually amplified and found to be identical (100%) to D,L-dex.
v
A2.5cm ABSTRAK
Asid 2,2-dikloropropionik (2,2-DCP) merupakan bahan buatan berhalogen yang
diguna sebagai racun lalang. Sejenis bakteria yang disaring dari sampel tanah ladang getah
Melaka didapati mampu menggunakan 2,2-DCP sebagai sumber karbon tunggal. Bakteria
tersebut dikenal pasti sebagai Labrys sp. strain Wy1 hasil daripada kajian penjujukan 16S
rRNA-nya. Bakteria tersebut membiak dalam medium minima yang mengandungi 20 mM
2,2-DCP sebagai sumber karbon tunggal dengan tercatatnya masa gandaan sebanyak 34.6
jam. Pembebasan ion klorida sebanyak 0.27 mM yang dikesan dalam medium minima
mengesahkan penggunaan 2,2-DCP oleh bakteria tersebut. Satu lagi bakteria endofit juga
disaring dari kandungan daun rumput parit dengan nama saintifiknya Axonopus compressus.
Bakteria yang dikenal pasti sebagai Burkholderia cepacia strain Wy5 juga mampu
mengguna 2,2-DCP sebagai sumber karbon tunggal. Bilangan sel bakteria tersebut berganda
dalam masa 2.7 jam dan pembebasan ion klorida sebanyak 47.28 ± 0.25 mM dalam medium
minima yang mengandungi 20 mM 2,2-DCP juga dapat dikesan. Ekstrak isi sel (CFE)
bakteria Burkholderia cepacia Wy5 telah diuji secara terperinci memandangkan bakteria
tersebut mempunyai kadar penggunaan 2,2-DCP yang lebih tinggi berbanding dengan
bakteria Labrys sp. Wy1. Dehalogenase yang terdapat dalam ekstrak isi sel bakteria
mempunyai aktiviti enzim spesifik optimal yang tercatat pada pH8 (0.83 μmol [Cl-] min
-1
mg-1
) dan suhu 40oC (0.78 μmol [Cl
-] min
-1 mg
-1). Dehalogenase tersebut juga dapat
bertindak balas dengan asid α-haloalkanoik yang lain termasuk asid monokloroasetik, asid
DL-2-kloropropionik dan asid DL-2-bromopropionik, tetapi tiada tindak balas dikesan dengan
asid 3-kloropropionik. Primer dehalogenase “Group I” dan “Group II” telah diguna untuk
amplifikasi gen dehalogenase dari kedua-dua strain bakteria Wy1 dan Wy5 tetapi hanya
Wy5 memberi hasil positif. Penjujukan separa dehalogenase “Group I” tersebut diberi nama
“deh-wy5”. Disebabkan persamaan yang tinggi antara urutan separa deh-wy5 dengan gen D,L-
dex, primer yang berasaskan gen D,L-dex telah direka untuk tujuan amplifikasi gen
dehalogenase yang lengkap daripada bakteria strain Wy5. Akhirnya jujukan, lengkap gen
dehalogenase deh-wy5 dapat diamplifikasi dan gen tersebut didapati mempunyai persamaan
setinggi 100% berbanding dengan gen D,L-dex.
vi
A2.5cm TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ABSTRACT iv
ABSTRAK v
TABLE OF CONTENTS vi
LIST OF TABLES x
LIST OF FIGURES xiii
LIST OF SYMBOLS xvi
LIST OF APPENDICES xvii
1 INTRODUCTION 1
1.1 Introduction 1
1.2 Rationale of Investigation 4
1.3 Objectives 5
2 LITERATURE REVIEWS 6
2.1 Xenobiotics: An Overview 6
2.2 Persistent Organic Pollutants (POPs) and Its Health
Effects
11
2.3 Persistent Organic Pollutants (POPs) Pesticide in
Malaysia
14
2.4 Bioaugmentation as a Soil Bioremediation
Approach
18
2.5 Properties of 2,2-Dichloropropionic Acid (2,2-DCP) 20
2.6 Chemistry of Halogenated Compound 22
vii
2.7 Microbial Dehalogenation 25
2.8 Dehalogenation Mechanism 28
2.9 Dehalogenation of Halogenated Alkanoic Acid 31
2.10 Classification of 2-Haloalkanoic Acid Hydrolytic
Dehalogenases
33
2.11 Biochemistry of 2-Haloalkanoic Acid Hydrolytic
Dehalogenases
38
2.12 Genetics of Haloalkanoic Acid Dehalogenase 41
3 MATERIALS AND METHODOLOGY 45
3.1 Culturing Media Composition 45
3.1.1 Minimal Media 45
3.1.2 LB (Lysogeny Broth) 47
3.1.3 Glycerol Stock Culture 47
3.2 Bacterial Isolation and Purification 48
3.2.1 Isolation and Purification of Soil Bacteria 48
3.2.2 Isolation and Purification of Endophytic
Bacteria from Leaves
48
3.3 Measurement of Microbial Growth 49
3.4 Halide Ion Assay (HIA) 49
3.4.1 HIA Reagent 49
3.4.2 HIA Standard Curve and Sample Testing 50
3.5. Cell Free Extract Preparation 51
3.6 Protein Concentration Determination 52
3.7 Enzyme Activity Assay 53
3.8 SDS-PAGE 54
3.8.1 Chemicals and Preparation 54
3.8.2 SDS-PAGE Apparatus Assembling, Gel
Loading and Sample Loading
57
3.8.3 Gel Staining, Destain and Drying 59
3.9 Molecular Analysis 60
3.9.1 DNA Extraction 60
3.9.2 Measurement of DNA Concentration 61
viii
3.9.3 Gel Electrophoresis 61
3.9.4 Polymerase Chain Reaction (PCR)
Amplification of 16S rRNA Gene
62
3.9.5 Dehalogenase Gene PCR Amplification 64
3.9.6 Phylogenetic Analysis 66
3.10 Biochemical Characterization 66
3.11 Cell Fixation for Scanning Electron Microscopy 67
4 ISOLATION AND CHARACTERIZATION OF
LABRYS SP. STRAIN WY1 ABLE TO UTILIZE 2,2-
DICHLOROPROPIONATE (2,2-DCP) AS SOLE
SOURCE OF CARBON
68
4.1 Introduction 68
4.2 Results 69
4.2.1 Isolation and Characterization of 2,2-DCP
Degrading Bacteria
69
4.2.2 Growth Profile 71
4.2.3 Halide Ion Assay 73
4.2.4 PCR Amplification of 16S rRNA gene 74
4.2.5 Sequencing and Analysis of 16S rRNA
Gene
75
4.2.6 Phylogenetic Study 79
4.2.7 Biochemical Tests 81
4.2.8 Amplification of Dehalogenase Gene 82
4.3 Discussion 83
5 ISOLATION AND CHARACTERIZATION OF
ENDOPHYTE FROM AXONOPUS COMPRESSUS
(RUMPUT PARIT) CAPABLE OF UTILIZE 2,2-DCP
AS SOLE CARBON SOURCE
86
5.1 Introduction 86
5.2 Results 87
5.2.1 Bacteria Characterization 87
ix
5.2.2 Scanning Electron Microscopy (SEM) 89
5.2.3 Growth Profile 91
5.2.4 Halide Ion Assay 92
5.2.5 PCR Amplification of 16S rRNA Gene 93
5.2.6 Sequencing and Analysis of 16S rRNA
Gene
94
5.2.7 Phylogenetic Study 96
5.2.8 Biochemical Test (API®) 98
5.2.9 Cell Free Extract (CFE) Analysis 100
5.2.9.1 CFE Enzyme Activity in Different
Buffers
100
5.2.9.2 CFE Enzyme Activity in Different
pH and Temperature
101
5.2.9.3 CFE Enzyme Activity towards
Different Substrate
102
5.2.9.4 SDS-PAGE 103
5.2.10 Amplification of Dehalogenase Gene 104
5.2.10.1 Gel Electrophoresis of PCR Product 104
5.2.10.2 Sequencing and Analysis of
Dehalogenase Gene
105
5.2.11 Amplification of Complete deh-wy5 (D,L-
dex-alike) Gene
108
5.2.11.1 Primers Design based on D,L-dex
Gene
108
5.2.11.2 Gel Electrophoresis of PCR product 111
5.2.11.3 Sequencing and Analysis of
deh-wy5 (D,L-dex-alike) Gene
112
5.3 Discussion 116
6 CONCLUSION 121
6.1 General Conclusion 121
REFERENCES 123
Appendix 136
x
A2.5cm LIST OF TABLES
TABLE TITLE PAGE
2.1 Estimated annual industrial production of chlorinated
hydrocarbons and major applications (Fetzner, 1998)
7~9
2.2 Some endocrine disrupting effects of pops and selected
other chemicals
13
2.3 Part of registered pesticide from October 2005 to
December 2011 listed by Ministry of Agriculture and
Agro-Based Industry Malaysia
15
2.4 Effect of number of chlorine on halogenated organic acid
to the pKa value
23
2.5 Effect of type of halogen on halogenated organic acid to
the pKa value
24
2.6 Effect of chlorine position on halogenated butanoic acid to
the pKa value
24
2.7 Haloalkanoate dehalogenases produced by miroorganisms
and their substrate specificity
35
2.8 Further characterization of 2-haloalkanoic acid hydrolytic
dehalogenase into discrete Class according to their
substrate specificity and product configuration (Slater et
al., 1995). Group categorizing system according to
Weightman et al. (1982) and Hardman (1991) were also
included.
36~37
3.1 Minimal media components 46
3.2 Example – Composition of minimal media 46
3.3 Preparation of chloride standard solution diluted with 50
xi
minimal media
3.4 Standard solutions of BSA 52
3.5 Testing media contained 1 mM 2,2-DCP (1 unit) 53
3.6 Chemicals required for SDS-PAGE 54
3.7 Seperating gel preparation 58
3.8 Stacking gel preparation 58
3.9 Primers designed by Weisburg et al. (1991). Only fD1 and
rP1 were used in current investigation (Weisburg et al.,
1991)
63
3.10 PCR method for amplification of 16S rRNA gene 63
3.11 Touchdown PCR method for “Group I” dehalogenase gene 64
3.12 PCR method for “Group II” dehalogenase gene 64
3.13 “Group I” deh primer sequences, showing comparisons of
conserved binding sites from various sources (Hill et al.,
1999)
65
3.14 “Group II” deh primer sequences, showing comparisons of
conserved binding sites from various sources (Hill et al.,
1999)
65
3.15 General fixation schedule for animal cell 67
4.1 Bacterial colony morphology of isolate Wy1 found on 10
mM 2,2-DCP minimal media
70
4.2 Gram stain characteristics of bacteria Wy1 70
4.3 Summary of growth properties of bacteria Wy1 in different
concentration of 2,2-DCP
72
4.4 Sequences producing significant alignments with Wy1 in
descending order (BLASTn)
76
4.5 Comparison of biochemical test result of Wy1 with related
species
81
5.1 Bacterial colony morphology of isolate Wy5 found on 10
mM 2,2-DCP minimal media
87
5.2 Gram stain characteristics of isolate Wy5 88
5.3 Summary of growth properties of isolate Wy5 in different
concentration of 2,2-DCP
91
xii
5.4 Summary of chloride ion released by isolate Wy5 92
5.5 List of sequences producing significant alignments with
Wy5 from BLASTn sorted by their “maximum identity” in
descending order
95
5.6 API® 20NE test result of isolate Wy5 99
5.7 Summary of CFE dehalogenase activity tested in different
buffers. Calculation and method as reported by Ng (2007)
100
5.8 CFE dehalogenase activity towards different substrate 102
5.9 Dehalogenase genes aligned with partial deh-wy5 sequence 105
5.10 Designed primers generated by Primer3 110
5.11 PCR method of designed primers 110
5.12 Amino acid composition of deh-wy5 115
5.13 Comparison of non-stereospecific dehalogenases’ optima
pH and temperature
120
xiii
A2.5cm LIST OF FIGURES
FIGURE TITLE PAGE
2.1 Structure of 2,2-dichloropropionic acid (2,2-DCP) 20
2.2 Dehalogenation mechanisms (Fetzner and Lingens, 1994) 30
2.3 Basic mechanism of hydrolytic dehalogenation (Slater et
al., 1996)
32
2.4 2-haloalkanoic acid halidohydrolase dehalogenation
mechanism 1 resulting in inversion of product
configuration from L-isomer to D-isomer (Little and
Williams, 1971)
38
2.5 2-haloalkanoic acid halidohydrolase dehalogenation
mechanism 2 resulting in retention of product
configuration (Weightman et al., 1982)
40
2.6 Sequence alignment of L-haloacid dehalogenases by
Janssen et al. (1994)
42
2.7 Part of alignment of deduced amino acid sequences of the
Group I deh proteins by Hill et al. (1999)
44
3.1 SDS-PAGE - stock solutions 55
3.2 SDS-PAGE - working solutions 56
3.3 SDS-PAGE - sample buffers 57
4.1 Bacterial colonies on minimal media (Photo taken after 5
days of incubation)
69
4.2 Gram stained bacteria Wy1 under microscope (1000X) 70
4.3 Growth profile of strain Wy1 in triplicate of minimal
media contained four different concentration of 2,2-DCP
71
xiv
4.4 Correlation between chloride ion (mM) released in
minimal medium containing 30 mM 2,2-DCP and growth
profile recorded at A600nm
73
4.5 Gel electrophoresis of PCR product - 16S rRNA gene
fragment of isolate Wy1
74
4.6 Partial 16S rRNA gene sequence of isolate Wy1 75
4.7 Alignment between Wy1 sequence (Query) with Labrys
neptuniae strain Liujia-146 16s rRNA sequence (Sbjct)
77
4.8 Information sheet of Labrys sp. Wy1 16S rRNA partial
gene sequence from NCBI database
(http://www.ncbi.nlm.nih.gov/nuccore/jf907580)
78
4.9 Phylogeny analysis using MEGA5 79
4.10 Neighbour-Joining phylogeny tree of Labrys sp. Wy1 80
4.11 Amplification of dehalogenase gene from Wy1 82
5.1 Gram staining reveals isolate Wy5 was Gram negative 88
5.2 SEM micrograph of isolate Wy5 visualized at 10,000X,
10kV accelerating voltage
89
5.3 SEM micrograph of isolate Wy5 showing bacterial binary
fission
90
5.4 Growth profile of isolate Wy5 in triplicate of minimal
media contained 10 mM, 20 mM and 40 mM of 2,2-DCP
91
5.5 Chloride ion released ([Cl-]) of isolate Wy5 in triplicate of
three different concentration of 2,2-DCP
92
5.6 Gel electrophoresis of PCR product - 16S rRNA gene
fragment of isolate Wy5
93
5.7 Partial 16S rRNA gene sequence of isolate Wy5 94
5.8 Neighbour-Joining phylogeny tree of isolate Wy5 97
5.9 Information sheet of API® test profile ID: 1047577 from
API-WEB
98
5.10 API® 20NE test strip of isolate Wy5 after 48 hours of
incubation at 30oC
99
5.11 Enzyme specific activity from pH5~pH10, 35oC; Buffer:
0.1 M Tris-acetate, 1 mM EDTA, 10% (w/v) glycerol
101
xv
5.12 Enzyme specific activity from 25oC~50
oC, pH 7.2; Buffer:
0.1 M Tris-acetate, 1 mM EDTA, 10% (w/v) glycerol
101
5.13 SDS-PAGE of Wy5 CFE 103
5.14 PCR product of “Group I” dehalogenase 104
5.15 ClustalW alignments of 495 bp deh-wy5 with known
complete dehalogenase genes (partly shown)
106
5.16 Alignment of deh-wy5 with other dehalogenases displayed
in codon form.
107
5.17 Data sheet generated by Primer3 109
5.18 PCR products of deh-wy5 (DL-DEX-alike) genes 111
5.19 Partial DNA fragment amplified using designed primers
Dxf1 and Dxr1
112
5.20 Chromatogram of sequenced DNA fragment using
designed primers Dxf1 and Dxr1
113
5.21 Alignments between extended sequences amplified using
primers Dxf1+Dxr1 and D,L-dex displayed in codon form
using ClustalW (version 1.6)
114
xvi
A2.5cm LIST OF SYMBOLS
(v/v) - Volume percentage per 100mL volume
(w/v) - Mass percentage per 100mL volume
2,4,5-T - 2,4,5-Trichlorophenoxyacetic acid
2,4-D - 2,4-Dichlorophenoxyacetic acid
A…nm - Absorption spectroscopy at …nm light source
BLASTn - Basic local alignment search tool – nucleotide
bp - Base pairs
CFE - Cell free extract
DDT - Dichlorodiphenyltrichloroethane
dH2O - Distilled water
HCH - Hexachlorocyclohexane
HIA - Halide ion assay
kb - Kilo bases
MWr - Relative molecular weight
OD - Optical density
PCB - Polychlorinated biphenyls
PCR - Polymerase chain reaction
rpm - Revolution per minute
RT - Room temperature
V - Volts
xvii
A2.5cm LIST OF APPENDICES
APPENDIX TITLE PAGE
A Standard Graphs 136
B Growth Profile and Doubling Time Calculation of Labrys
sp. Wy1
138
C Growth Profile and Doubling Time Calculation of
Burkholderia cepacia Wy5
140
D HIA of Labrys sp. Wy1 in 20 mM 2,2-DCP 141
E HIA of Burkholderia cepacia Wy5 142
F Enzyme Assays of Burkholderia cepacia Wy5 CFE 143
G Acid Dilution Calculations 145
1
A2.5cm CHAPTER 1
INTRODUCTION
1.1 Introduction
Halogenated compounds were used extensively as herbicide and as
intermediate chemicals in many industries. Due to their complexity, toxicity,
persistence and ubiquitous distribution of these xenobiotic compounds, they have
brought threat to the health and living quality of human and other organisms (Fetzner
and Lingens, 1994). Physiologists and biochemists have known since the beginning
of the 20th
century that halogenated compounds will affect metabolic processes as
halogenated analogues of intermediary metabolites are toxic (Slater et al., 1995).
Degradation of halogenated compound by microorganisms has been reported since
the early of 20th
century by Penfold (1913). These microorganisms are capable of
evolving new enzymes, pathways and regulatory mechanisms for the degradation of
almost all xenobiotic compounds due to their short life cycle. The evolution of
dehalogenase producing microorganisms using some of these halogenated
compounds is scientifically interesting and practically important (Penfold, 1913;
Timmis and Pieper, 1999).
2,2-dichloropropionic acid (2,2-DCP) or Dalapon is an odourless and
colourless 2-haloalkanoic acid herbicide used to control and regulate the growth of
certain weeds, such as quick grass, Bermuda grass and cattails. It effectively inhibits
panothenic acid production (Prasad and Blackman, 1965) and pyruvate utilization in
bacteria (Redemann and Meikle, 1955). One of the earliest event of degradation of
herbicide Dalapon was reported by Magee and Colmer (1959) after observation of
bacteria that produce dehalogenase enzyme (Magee and Colmer, 1959). Since then,
2
studies on isolation of microbes that potentially produce dehalogenases have been
undertaken (Berry et al., 1979; Hardman and Slater, 1981; Motosugi et al., 1982;
Weightman et al., 1982; Allison et al., 1983; Liu et al., 1994; Schwarze et al., 1997;
Nardi-Dei et al., 1999; Huyop et al., 2004; Jing and Huyop, 2007; Huyop et al.,
2008). 2,2-DCP is readily removed from the soil by a variety of microorganisms
including species of Pseudomonas, Agrobacterium, Nocardia, Alcaligenes,
Arthrobacter and Bacillus (Foy, 1975).
The enzymes responsible for the degradation of halogenated compound were
known as dehalogenase, discovered and firstly named by Jensen (1957).
Dehalogenases catalyse the hydrolysis of halogen-substituted alkanoic acids yielding
either hydroxyalkanoic acids from mono-halogenated acids or oxo-alkanoic acids
from di-halogenated compounds products which may be readily metabolized
(Hardman and Slater, 1981). Culturing and enrichment of microorganism that can
produce dehalogenase in the presence of halogenated compound in the environment
was the most favourable method. Jensen (1957) used soil perfusion and enrichment
technique to isolate five strains of Pseudomonas sp. which able to degrade 2,2-DCP
and other α-halogenated substrate such as dichloroacetate and 2-chloropropionate
(Jensen, 1957). Several other dehalogenase producing bacteria isolated using this
method including Methylobacterium sp. HJ1 (Jing et al., 2008), Pseudomonas putida
PP3 (Senior et al., 1976), Xanthobacter autotrophicus GJ10 (Janssen et al., 1985),
Pseudomonas B6P (Mesri et al., 2009) and Rhizobium sp. (Berry et al., 1979).
Interest in biodegradation of α-substituted halogenated alkanoic acid was increased
due to the introduction of Dalapon as herbicide and lead to the isolation of many
microorganisms able to grow on 2,2-DCP as sole carbon source (Macgregor, 1963;
Burge, 1969; Berry et al., 1979; Kearney and Kellogg, 1985; Jing et al., 2008;
Huyop and Nemati, 2010).
Currently, technological applications of bacterial transformation of
halogenated compound can be considered in two major aspects: synthesis of
chemical intermediates and degradation of xenobiotic wastes. Dehalogenase can be
used as industrial biocatalysts to produce valuable intermediates for chemical
synthesis (Huyop and Cooper, 2003). Biotransformation of organic compounds with
microbial or enzyme biocatalysts offers new chemical routes for the synthesis of
3
intermediates and novel products, since these biocatalysts possess chiral specificities
and can recognize specific area on a molecule, that are difficult and expensive to
achieve by conventional chemistry (Fetzner and Lingens, 1994). For example ICI
Biological Products (U.K.) uses Pseudomonas putida AJ1/23 to produce L-2-
monochloropropionate for use in herbicide manufacture from racemic 2-
monochloropropionate, which already reached commercial scale (Motosugi et al.,
1982). Similarly, the production of optically active 3-halolactate from 2,3-
dihalopropionate was also performed with 2-haloalkanoic acid halidohydrolase from
Pseudomonas putida (Fetzner and Lingens, 1994). In addition, dehalogenating
microorganisms were also proved to be useful in a bioremediation process and the
application of specialized strains as inocula for the bioremediation of polychlorinated
biphenyls (PCP) contaminated soil and groundwater was studied extensively. For
example, Hicky et al. (1993) used the chlorobenzoate utilizers Pseudomonas
aeruginosa JB2 and Pseudomonas putida Plll and the biphenyl utilizer Pseudomonas
sp. strain PB133 to mineralize polychlorinated biphenyls in soil (Hickey et al., 1993).
4
1.2 Rationale of Investigation
Microorganisms with dehalogenating capabilities were proven to be useful in
both chemical manufacturing industry and in situ bioremediation of contaminated
soil, especially those related to chlorinated xenobiotics. In current investigation,
isolating new bacteria with higher reactivity towards chlorinated herbicide compared
to other previous research is the main goal. Degradation of chlorinated herbicide,
especially 2,2-DCP is chosen due to its more complex structure which resistance to
enzymatic attack compared to mono-substituted haloalkane, and also its well-known
environmental impact. The source of soil and Axonopus compressus (rumput parit)
used in current research was frequently exposed to various chlorinated herbicide
including Dalapon (2,2-DCP) and this could increase the chance of isolate
dehalogenating microorganisms. Agricultural soil is a common place to find
dehalogenating bacteria, however some endophytes also reported to show resistance
to heavy metals and able to degrade organic compounds in the plant, soil or water,
and thus also play an important role in pollution control (Germaine et al., 2006),
therefore the investigation of whether there is endophyte with dehalogenating
capabilities present in Axonopus compressus’ leaves, especially those possess cryptic
dehalogenase, can be a novel approach for isolation of new dehalogenating bacteria.
Moreover, these new isolated bacteria could be used in enzymatic production of
useful chemicals or as potential bioremediation agent.
The study of 2,2-DCP degradation can also be compared to that of
degradation of other chloro-substituted alkanoates, for example, 3-chloropropionic
acid, which is an analogue and isomer of 2,2-DCP (Allison et al., 1983). Further
interest in this subject was raised when it became apparent that α-chloroalkanoate-
degrading microorganisms were unable to utilize β-substituted haloalkanoates, which
differed only in chlorine substitution. Only few isolated microorganisms can degrade
β-halocarboxylic acid (β-HA) (Mesri et al., 2009; Yusn and Huyop, 2009). Some
previous studies have suggested the production of more than one dehalogenase in a
few bacterial strains (Goldman et al., 1968; Weightman et al., 1982) and the fungus
Trichoderma viride (Jensen, 1960). The only microorganism so far reported to
produce three forms of dehalogenases which degrade D-, L- and non-stereospecific
isomer of α-haloalkanoate is Rhizobium sp. (Leigh et al., 1988).
5
A limited number of genetic studies which consider the evolutionary
mechanisms of dehalogenase have been reported. Isolation of many iso-enzymic
forms of dehalogenase from a vast variety of bacterial genera gave rise to the
question of their importance in the natural environment and the evolution-
relationship of their different forms (Murdiyatmo et al., 1992). The ubiquity of the
haloacid halidohydrolases in natural bacterial isolates has led to the suggestion that
their importance in being catabolic enzymes cleaving the halo- substituents of halo
metabolites as part of degradative pathways for the degradation of more complex
halo-organic compounds (Murdiyatmo et al., 1992). The adoption of molecular
method might provide an alternative in studying variety of dehalogenases possessed
by certain microorganism. Hill et al. (1999) described systematic approach to
amplify two different families of α-halocarboxylic acid (α-HA) dehalogenase genes
of group I and group II based on the knowledge of conserved residues among
different dehalogenases. Group I dehalogenases were non-stereospecific, whereas
group II showing stereospecificity tendency, dechlorinating only L- but not D-2-
chloropropionic acid. Current investigation adopted the molecular method described
by Hill et al. (1999) might allowed us to identify cryptic or silent, as well as active
dehalogenase genes presence in the bacteria.
1.3 Objectives
I. Isolate, identify and characterize soil and endophytic microorganisms capable
of utilizing 2,2-DCP as sole carbon source.
II. Characterization of 2-haloalkanoic acid dehalogenase from cell free extract
produced by isolates capable of utilize 2,2-DCP as sole carbon source.
III. Amplification and analysis of 2-haloalkanoic acid dehalogenase gene
sequence from isolated microorganisms using designed primers based on
conserved gene sequence of known dehalogenases.
123
A2.5cm REFERENCES
Abdullah, A. R. (1995). Environmental pollution in Malaysia: trends and prospects.
TrAC Trends in Analytical Chemistry, 14(5), 191-198.
Alexander, M. (1981). Biodegradation of chemicals of environmental concern.
Science, 211(4478), 132.
Allison, N., Skinner, A. and Cooper, R. (1983). The dehalogenases of a 2, 2-
dichloropropionate-degrading bacterium. Journal of General Microbiology,
129(5), 1283.
Aneck-Hahn, N. H., Schulenburg, G. W., Bornman, M. S., Farias, P. and De Jager, C.
(2007). Impaired semen quality associated with environmental DDT exposure
in young men living in a malaria area in the Limpopo Province, South Africa.
Journal of Andrology, 28(3), 423.
Asmara, W., Murdiyatmo, U., Baines, A. J., Bull, A. T. and Hardman, D. J. (1993).
Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas
cepacia MBA4. Biochemical Journal, 292(Pt 1), 69.
Balandreau, J., Viallard, V., Cournoyer, B., Coenye, T., Laevens, S. and Vandamme,
P. (2001). Burkholderia cepacia genomovar III is a common plant-associated
bacterium. Applied and environmental microbiology, 67(2), 982-985.
Bergmann, J. and Sanik Jr, J. (1957). Determination of trace amounts of chlorine in
naphtha. Analytical Chemistry, 29(2), 241-243.
Berry, E. K. M., Allison, N., Skinner, A. and Cooper, R. (1979). Degradation of the
selective herbicide 2, 2-dichloropropionate (Dalapon) by a soil bacterium.
Journal of General Microbiology, 110(1), 39.
Bogdan, A. (1977). Tropical pasture and fodder plants. Longman.
Bollag, D. M., Edelstein, S. J. and Rozycki, M. D. (1996). Protein methods. Wiley-
Liss New York.
124
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical biochemistry, 72(1-2), 248-254.
Burge, W. D. (1969). Populations of dalapon-decomposing bacteria in soil as
influenced by additions of dalapon or other carbon sources. Applied
Microbiology, 17(4), 545-550.
Busto, M. D., Smith, P. P., Perez-Mateos, M. and Burns, R. G. (1992). Degradation
of aliphatic halogen-substituted pesticides by dehalogenase isolated from
Pseudomonas alcaligenes. Identification and properties of the enzyme. The
Science of the Total Environment, 123, 267-277.
Cairns, S. S., Cornish, A. and Cooper, R. A. (1996). Cloning, sequencing and
expression in Escherichia coli of two Rhizobium sp. genes encoding
haloalkanoate dehalogenases of opposite stereospecificity. European Journal
of Biochemistry, 235(3), 744-749.
Carvalho, M. F., De Marco, P., Duque, A. F., Pacheco, C. C., Janssen, D. B. and
Castro, P. M. L. (2008). Labrys portucalensis sp. nov., a fluorobenzene-
degrading bacterium isolated from an industrially contaminated sediment in
northern Portugal. International journal of systematic and evolutionary
microbiology, 58(3), 692-698.
Chaudhry, G. R. and Chapalamadugu, S. (1991). Biodegradation of halogenated
organic compounds. Microbiology and Molecular Biology Reviews, 55(1), 59.
Chou, Y. J., Elliott, G. N., James, E. K., Lin, K. Y., Chou, J. H., Sheu, S. Y., Sheu, D.
S., Sprent, J. I. and Chen, W. M. (2007). Labrys neptuniae sp. nov., isolated
from root nodules of the aquatic legume Neptunia oleracea. International
journal of systematic and evolutionary microbiology, 57(3), 577-581.
Coenye, T. and Vandamme, P. (2003). Diversity and significance of Burkholderia
species occupying diverse ecological niches. Environmental Microbiology,
5(9), 719-729.
Coenye, T. and Vandamme, P. (2003). Intragenomic heterogeneity between multiple
16S ribosomal RNA operons in sequenced bacterial genomes. FEMS
microbiology letters, 228(1), 45-49.
125
Coleman, N. V., Mattes, T. E., Gossett, J. M. and Spain, J. C. (2002). Biodegradation
of cis-dichloroethene as the sole carbon source by a β-proteobacterium.
Applied and environmental microbiology, 68(6), 2726-2730.
Commandeur, L. and Parsons, J. (1990). Degradation of halogenated aromatic
compounds. Biodegradation, 1(2), 207-220.
Conway, G. R. (1972). Ecological aspects of pest control in Malaysia. Natural
History Press, New York, NY,
Cripps, D. J., Gocmen, A. and Peters, H. A. (1980). Porphyria turcica: twenty years
after hexachlorobenzene intoxication. Archives of Dermatology, 116(1), 46.
D.O.E, D. O. E. (2009). Contaminated Land Management and Control Guidelines No.
1: Malaysian Recommended Site Screening Levels for Contaminated Land.
IN Environment (Ed.
Doyle, R. (1984). Dalapon information sheet. Food and Drug Administration,
Bureau of Foods, HFF-420,
E.P.A., U. S. E. P. A. (1989). Drinking water health advisory: Pesticides. Dalapon,
sodium salt (CASRN 75-99-0). CRC.
Erdogan, E. E. and Karaca, A. (2011). Bioremediation of crude oil polluted soils.
Asian J. Biotechnol, 3, 206-213.
Extoxnet (1996). Pesticide Information Profile-Dalapon: A Pesticide Information
Project of Cooperative Extension. Oregon State University.
Fetzner, S. (1998). Bacterial dehalogenation. Applied microbiology and
biotechnology, 50(6), 633-657.
Fetzner, S. and Lingens, F. (1994). Bacterial dehalogenases: biochemistry, genetics,
and biotechnological applications. Microbiology and Molecular Biology
Reviews, 58(4), 641.
Foy, C. L. (1975). The chlorinated aliphatic acids. Herbicides: Chemistry,
Degradation and Mode of Action, 1
Germaine, K. J., Liu, X., Cabellos, G. G., Hogan, J. P., Ryan, D. and Dowling, D. N.
(2006). Bacterial endophyte ‐ enhanced phytoremediation of the
126
organochlorine herbicide 2, 4 ‐ dichlorophenoxyacetic acid. FEMS
microbiology ecology, 57(2), 302-310.
Goldberg, E. D. (1991). Halogenated hydrocarbons: past, present and near-future
problems. The Science of the Total Environment, 100, 17-28.
Goldman, P. (1972). Enzymology of carbon-halogen bonds. Degradation of
Synthetic Organic Molecules in the Biosphere, 147-165.
Goldman, P., Milne, G. and Keister, D. B. (1968). Carbon-halogen bond cleavage.
Journal of Biological Chemistry, 243(2), 428.
Gyorkos, J., Denomme, M., Leece, B., Homonko, K., Valli, V. and Safe, S. (1985).
Reconstituted halogenated hydrocarbon pesticide and pollutant mixtures
found in human tissues: effects on the immature male Wistar rat after short-
term exposure. Canadian journal of physiology and pharmacology, 63(1), 36-
43.
Häggblom, M. M. and Bossert, I. D. (Eds.) (2003). Dehalogenation: microbial
processes and environmental applications, Springerlink.
Hardman, D. J. (1991). Biotransformation of halogenated compounds. Critical
reviews in biotechnology, 11(1), 1-40.
Hardman, D. J. and Slater, J. H. (1981). Dehalogenases in soil bacteria. Journal of
General Microbiology, 123(1), 117.
Hickey, W., Searles, D. and Focht, D. (1993). Enhanced mineralization of
polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading
bacteria. Applied and environmental microbiology, 59(4), 1194-1200.
Higgins, T. P., Hope, S. J., Effendi, A. J., Dawson, S. and Dancer, B. N. (2005).
Biochemical and molecular characterisation of the 2, 3-dichloro-1-propanol
dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile
Agrobacterium sp. Biodegradation, 16(5), 485-492.
Hill, K. E., Marchesi, J. R. and Weightman, A. J. (1999). Investigation of two
evolutionarily unrelated halocarboxylic acid dehalogenase gene families.
Journal of bacteriology, 181(8), 2535.
Huber, H., Hohn, M. J., Rachel, R., Fuchs, T., Wimmer, V. C. and Stetter, K. O.
(2002). A new phylum of Archaea represented by a nanosized
hyperthermophilic symbiont. Nature, 417(6884), 63-67.
127
Huyop, F., Jing, N. H. and Cooper, R. A. (2008). Overexpression, Purification and
Analysis of Dehalogenase D of Rhizobium sp. Can. J. Pure Applied Sci, 2,
389-392.
Huyop, F. and Nemati, M. (2010). Properties of dehalogenase from Rhizobium sp.
RC1. African Journal of Microbiology Research, 4(25), 2836-2847.
Huyop, F., Rashid, N. A., Wahab, R. a. B. and Cooper, R. A. (2010). Purification and
properties of Rhizobial DehL expressed in Escherichia coli. African Journal
of Biotechnology, 7(12)
Huyop, F. Z. and Cooper, R. A. (2003). A potential use of Dehalogenase D (DehD)
from Rhizobium sp. for Industrial Process. Jurnal Teknologi C, (38C), 69-75.
Huyop, F. Z., Yusn, T. Y., Ismail, M., Wahab, R. and Cooper, R. A. (2004).
Overexpression and characterisation of non-stereospecific haloacid
Dehalogenase E(DehE) of Rhizobium sp. Asia-Pacific Journal of Molecular
Biology and Biotechnology, 12(1-2), 15-20.
Ibrahim, M. (2007). Persistent Organic Pollutants in Malaysia. Developments in
Environmental Sciences, 7, 629-655.
Janssen, D. B., Gerritse, J., Brackman, J., Kalk, C., Jager, D. and Witholt, B. (1988).
Purification and characterization of a bacterial dehalogenase with activity
toward halogenated alkanes, alcohols and ethers. European Journal of
Biochemistry, 171(1‐2), 67-72.
Janssen, D. B., Pries, F., Van Der Ploeg, J., Kazemier, B., Terpstra, P. and Witholt, B.
(1989). Cloning of 1, 2-dichloroethane degradation genes of Xanthobacter
autotrophicus GJ10 and expression and sequencing of the dhlA gene. Journal
of bacteriology, 171(12), 6791.
Janssen, D. B., Pries, F. and Van Der Ploeg, J. R. (1994). Genetics and biochemistry
of dehalogenating enzymes. Annual Reviews in Microbiology, 48(1), 163-191.
Janssen, D. B., Scheper, A., Dijkhuizen, L. and Witholt, B. (1985). Degradation of
halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.
Applied and environmental microbiology, 49(3), 673-677.
Jensen, H. (1957). Decomposition of chloro-substituted aliphatic acids by soil
bacteria. Canadian journal of microbiology, 3(2), 151-164.
128
Jensen, H. (1960). Decomposition of chloroacetates and chloropropionates by
bacteria. Acta agriculturae scandinavica, 10(1), 83-103.
Jing, N. H., Fatin Hanani Sulaiman1, R. A., Wahab2 Jr, R. V. P., Rashid, N. a. A.
and Huyop, F. (2008). Purification and properties of a non-stereospecific
dehalogenase enzyme E (DehE) from Methylobacterium sp. HJ1. African
Journal of Microbiology Research, 2(7), 187-191.
Jing, N. H. and Huyop, F. (2007). Dehalogenation of chlorinated aliphatic acid by
Rhodococcus sp. Asia-Pacific Journal of Molecular Biology and
Biotechnology, 15(3), 147-151.
Jing, N. H., Taha, A. M., Pakingking Jr, R. V., Wahab, R. a. B. and Huyop, F. (2008).
Dehalogenase from Methylobacterium sp. HJ1 induced by the herbicide 2, 2-
dichloropropionate (Dalapon). African Journal of Microbiology Research 2:
32-36,
Jing, N. H., Wahab, R. A., Taha, A. M., Rashid, N. a. A. and Huyop, F. (2008). A
Further Characterization of 3-Chloropropionic Acid Dehalogenase from
Rhodococcus sp. HJ 1. Research Journal of Microbiology, 3(6), 482-488.
Jones, D. H. A., Barth, P. T., Byrom, D. and Thomas, C. M. (1992). Nucleotide
sequence of the structural gene encoding a 2-haloalkanoic acid dehalogenase
of Pseudomonas putida strain AJ1 and purification of the encoded protein.
Journal of General Microbiology, 138(4), 675.
Kado, C. I. (1992). Plant pathogenic bacteria. The prokaryotes, 1, 659-674.
Kawasaki, H., Toyama, T., Maeda, T., Nishino, H. and Tonomura, K. (1994).
Cloning and sequence analysis of a plasmid-encoded 2-haloacid
dehalogenase gene from Pseudomonas putida no. 109. Bioscience,
biotechnology, and biochemistry, 58(1), 160-163.
Kawasaki, H., Tsuda, K., Matsushita, I. and Tonomura, K. (1992). Lack of homology
between two haloacetate dehalogenase genes encoded on a plasmid from
Moraxella sp. strain B. Journal of General Microbiology, 138(7), 1317.
Kearney, P. and Kellogg, S. (1985). Microbial adaptation to pesticides. Pure and
Applied Chemistry, 57, 389-403.
Kennes, C., Pries, F., Krooshof, G. H., Bokma, E., Kingma, J. and Janssen, D. B.
(1995). Replacement of tryptophan residues in haloalkane dehalogenase
129
reduces halide binding and catalytic activity. European Journal of
Biochemistry, 228(2), 403-407.
Kerr, L. M. and Marchesi, J. R. (2006). Isolation of novel bacteria able to degrade
[alpha]-halocarboxylic acids by enrichment from environmental samples.
chemosphere, 64(5), 848-855.
Kobayashi, D. and Palumbo, J. (2000). Bacterial endophytes and their effects on
plants and uses in agriculture. The Biology of Microbial Endophytes (Bacon
CW, White JF Jr, eds). New York: Marcel Dekker, 199-1233.
Kok, L. (1972). Toxicity of insecticides used for Asiatic rice borer control to tropical
fish in rice paddies.
Koonin, E. V., Makarova, K. S. and Aravind, L. (2001). Horizontal Gene Transfer in
Prokaryotes: Quantification and Classification 1. Annual Reviews in
Microbiology, 55(1), 709-742.
Lamar, R. T., Evans, J. W. and Glaser, J. A. (1993). Solid-phase treatment of a
pentachlorophenol-contaminated soil using lignin-degrading fungi.
Environmental science & technology, 27(12), 2566-2571.
Lamb, T. G., Tonkyn, D. W. and Kluepfel, D. A. (1996). Movement of Pseudomonas
aureofaciens from the rhizosphere to aerial plant tissue. Canadian journal of
microbiology, 42(11), 1112-1120.
Leigh, J., Skinner, A. and Cooper, R. (1988). Partial purification, stereospecificity
and stoichiometry of three dehalogenases from a Rhizobium species. FEMS
microbiology letters, 49(3), 353-356.
Li, R., Zheng, J. W., Ni, B., Chen, K., Yang, X. J., Li, S. P. and Jiang, J. D. (2011).
Biodegradation of Pentachloronitrobenzene by< i> Labrys portucalensis</i>
pcnb-21 Isolated from Polluted Soil. Pedosphere, 21(1), 31-36.
Little, M. and Williams, P. A. (1971). A bacterial halidohydrolase. European Journal
of Biochemistry, 21(1), 99-109.
Liu, J. Q., Kurihara, T., Hasan, A., Nardi-Dei, V., Koshikawa, H., Esaki, N. and
Soda, K. (1994). Purification and characterization of thermostable and
nonthermostable 2-haloacid dehalogenases with different stereospecificities
from Pseudomonas sp. strain YL. Applied and environmental microbiology,
60(7), 2389.
130
Locher, H. H., Poolman, B., Cook, A. M. and Konings, W. N. (1993). Uptake of 4-
toluene sulfonate by Comamonas testosteroni T-2. Journal of bacteriology,
175(4), 1075.
Longnecker, M. P. and Rogan, W. J. (2001). Persistent Organic Pollutants in
Children: Commentary on the article by Karmaus et al. on page 331.
Pediatric research, 50(3), 322.
Macgregor, A. (1963). The decomposition of dichloropropionate by soil micro-
organisms. Journal of General Microbiology, 30(3), 497.
Magee, L. A. and Colmer, A. R. (1959). Decomposition of 2, 2-dichloropropionic
acid by soil bacteria. Canadian journal of microbiology, 5(3), 255-260.
Mcgowan, C., Fulthorpe, R., Wright, A. and Tiedje, J. (1998). Evidence for
interspecies gene transfer in the evolution of 2, 4-dichlorophenoxyacetic acid
degraders. Applied and environmental microbiology, 64(10), 4089-4092.
Mesri, S., Wahab, R. A. and Huyop, F. (2009). Degradation of 3-chloropropionic
acid (3CP) by Pseudomonas sp. B6P isolated from a rice paddy field. Annals
of microbiology, 59(3), 447-451.
Miller, J. A., Kalyuzhnaya, M. G., Noyes, E., Lara, J. C., Lidstrom, M. E. and
Chistoserdova, L. (2005). Labrys methylaminiphilus sp. nov., a novel
facultatively methylotrophic bacterium from a freshwater lake sediment.
International journal of systematic and evolutionary microbiology, 55(3),
1247-1253.
Motosugi, K., Esaki, N. and Soda, K. (1982). Bacterial assimilation of D-and L-2-
chloropropionates and occurrence of a new dehalogenase. Archives of
microbiology, 131(3), 179-183.
Motosugi, K., Esaki, N. and Soda, K. (1982). Purification and properties of a new
enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp. Journal of
bacteriology, 150(2), 522.
Muhammad, S. A. (2006). Analysis of persistent organic pollutants in fish: health
risk assessment through dietary intake (Thesis). Universiti Sains Malaysia.
Murdiyatmo, U., Asmara, W., Tsang, J., Baines, A. J., Bull, A. T. and Hardman, D. J.
(1992). Molecular biology of the 2-haloacid halidohydrolase IVa from
Pseudomonas cepacia MBA4. Biochemical Journal, 284(Pt 1), 87.
131
Nardi-Dei, V., Kurihara, T., Okamura, T., Liu, J. Q., Koshikawa, H., Ozaki, H.,
Terashima, Y., Esaki, N. and Soda, K. (1994). Comparative studies of genes
encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp.
strain YL, other dehalogenases, and two related hypothetical proteins from
Escherichia coli. Applied and environmental microbiology, 60(9), 3375.
Nardi-Dei, V., Kurihara, T., Park, C., Esaki, N. and Soda, K. (1997). Bacterial DL-2-
haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and
structural comparison with D-and L-2-haloacid dehalogenases. Journal of
bacteriology, 179(13), 4232.
Nardi-Dei, V., Kurihara, T., Park, C., Miyagi, M., Tsunasawa, S., Soda, K. and Esaki,
N. (1999). dl-2-Haloacid Dehalogenase from Pseudomonas sp. 113 Is a New
Class of Dehalogenase Catalyzing Hydrolytic Dehalogenation Not Involving
Enzyme-Substrate Ester Intermediate. Journal of Biological Chemistry,
274(30), 20977.
Neilson, A. H. (1996). An environmental perspective on the biodegradation of
organochlorine xenobiotics. International biodeterioration & biodegradation,
37(1-2), 3-21.
Ng, H. J. (2007). Isolation of Local Bacteria Capable of Degrading Halogenated
Compounds and Analysis of Putative Haloacid Permease Gene (Thesis).
Universiti Teknologi Malaysia.
Ng, H. J. (2007). Isolation of local bacterial capable of degrading halogenated
compounds and analysis of putative haloacid permease gene (Thesis).
Universiti Teknologi Malaysia.
Olaniran, A., Pillay, D. and Pillay, B. (2004). Haloalkane and haloacid
dehalogenases from aerobic bacterial isolates indigenous to contaminated
sites in Africa demonstrate diverse substrate specificities. chemosphere, 55(1),
27-33.
Penfold, W. (1913). The inhibitory selective action on bacteria of bodies related to
monochloroacetic acid. J. Hyg., Camb, 13, 35-48.
Peters, R., Wakelin, R. and Buffa, P. (1953). Biochemistry of fluoroacetate poisoning
the isolation and some properties of the fluorotricarboxylic acid inhibitor of
citrate metabolism. Proceedings of the Royal Society of London. Series B-
Biological Sciences, 140(901), 497-506.
132
Prasad, R. and Blackman, G. (1965). Studies in the Physiological Action of 2, 2-
Dichloropropionic Acid. Journal of Experimental Botany, 16(3), 545-568.
Redemann, C. T. and Meikle, R. W. (1955). The inhibition of several enzyme
systems by 2, 2-dichloropropionate. Archives of biochemistry and biophysics,
59(1), 106-112.
Rieger, P. G., Meier, H. M., Gerle, M., Vogt, U., Groth, T. and Knackmuss, H. J.
(2002). Xenobiotics in the environment: present and future strategies to
obviate the problem of biological persistence. Journal of biotechnology, 94(1),
101-123.
Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Molecular biology and evolution, 4(4),
406-425.
Schneider, B., Muller, R., Frank, R. and Lingens, F. (1991). Complete nucleotide
sequences and comparison of the structural genes of two 2-haloalkanoic acid
dehalogenases from Pseudomonas sp. strain CBS3. Journal of bacteriology,
173(4), 1530.
Schwarze, R., Brokamp, A. and Schmidt, F. R. J. (1997). Isolation and
characterization of dehalogenases from 2, 2-dichloropropionate-degrading
soil bacteria. Current microbiology, 34(2), 103-109.
Senior, E., Bull, A. and Slater, J. (1976). Enzyme evolution in a microbial
community growing on the herbicide Dalapon. Nature, 263(5577), 476.
Slater, J., Bull, A. and Kuenen, J. (1982). Environmental Microbiology:
Biodegradation [and Discussion]. Philosophical Transactions of the Royal
Society of London. B, Biological Sciences, 297(1088), 575-597.
Slater, J. H., Bull, A. T. and Hardman, D. J. (1995). Microbial dehalogenation.
Biodegradation, 6(3), 181-189.
Slater, J. H., Bull, A. T. and Hardman, D. J. (1996). Microbial dehalogenation of
halogenated alkanoic acids, alcohols and alkanes. Advances in Microbial
Physiology, 38, 133-176.
Slater, J. H., Lovatt, D., Weightman, A. J., Senior, E. and Bull, A. T. (1979). The
growth of Pseudomonas putida on chlorinated aliphatic acids and its
dehalogenase activity. Journal of General Microbiology, 114(1), 125.
133
Smith, J. M., Harrison, K. and Colby, J. (1990). Purification and characterization of
D-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23. Journal
of General Microbiology, 136(5), 881.
Stoyanova, M., Pavlina, I., Moncheva, P. and Bogatzevska, N. (2007). Biodiversity
and incidence of Burkholderia species. Biotechnology and Biotechnological
Equipment, 21(3), 306.
Stringfellow, J. M., Cairns, S. S., Cornish, A. and Cooper, R. A. (1997).
Haloalkanoate dehalogenase II (DehE) of a Rhizobium sp.—molecular
analysis of the gene and formation of carbon monoxide from trihaloacetate by
the enzyme. European Journal of Biochemistry, 250(3), 789-793.
Struthers, J., Jayachandran, K. and Moorman, T. (1998). Biodegradation of atrazine
by Agrobacterium radiobacter J14a and use of this strain in bioremediation of
contaminated soil. Applied and environmental microbiology, 64(9), 3368-
3375.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011).
MEGA5: molecular evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods.
Molecular biology and evolution, 28(10), 2731-2739.
Timmis, K., Rojo, F. and Ramos, J. (1988). Prospects for laboratory engineering of
bacteria to degrade pollutants. Basic life sciences, 45, 61.
Timmis, K. N. and Pieper, D. H. (1999). Bacteria designed for bioremediation.
Trends in biotechnology, 17(5), 200-204.
Topping, A. (1992). An investigation into the transposition and dehalogenase
functions of DEH, a mobile genetic element from Pseudomonas putida strain
PP3 (Thesis). Ph. D. thesis. University of Wales, Cardiff, United Kingdom.
Tsang, J. S. H., Sallis, P. J., Bull, A. T. and Hardman, D. J. (1988). A
monobromoacetate dehalogenase from Pseudomonas cepacia MBA4.
Archives of microbiology, 150(5), 441-446.
Tsang, J. S. H. and Sam, L. (1999). Cloning and characterization of a cryptic
haloacid dehalogenase from Burkholderia cepacia MBA4. Journal of
bacteriology, 181(19), 6003.
134
U.N.E.P., U. N. E. P. (2005). Ridding the World of POPs: A Guide to The
Stockholm Convention on Persistent Organic Pollutants
http://www.pops.int/documents/guidance/begguide.pdf.
Umland, J. B. and Bellama, J. M. (1999). General chemistry. Brooks/Cole Publishing
Company.
Van Der Meer, J. R., Roelofsen, W., Schraa, G. and Zehnder, A. J. B. (1987).
Degradation of low concentrations of dichlorobenzenes and 1, 2, 4-
trichlorobenzene by< i> Pseudomonas</i> sp. strain P51 in nonsterile soil
columns. FEMS microbiology letters, 45(6), 333-341.
Van Der Ploeg, J., Van Hall, G. and Janssen, D. B. (1991). Characterization of the
haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and
sequencing of the dhlB gene. Journal of bacteriology, 173(24), 7925.
Van Hylckama Vlieg, J. E. T., Poelarends, G. J., Mars, A. E. and Janssen, D. (2000).
Detoxification of reactive intermediates during microbial metabolism of
halogenated compounds. Current opinion in microbiology, 3(3), 257-262.
Vogel, T. M. (1996). Bioaugmentation as a soil bioremediation approach. Current
opinion in biotechnology, 7(3), 311-316.
W.H.O., W. H. O. (2010). Persistent Organic Pollutants: Impact on Child Health.
Geneva, Switzerland, WHO Document Production Service.
Wade, L. G. (2009). Organic Chemistry, 7/E. Prentice Hall.
Weightman, A. J., Weightman, A. L. and Slater, J. H. (1982). Stereospecificity of 2-
monochloropropionate dehalogenation by the two dehalogenases of
Pseudomonas putida PP3: evidence for two different dehalogenation
mechanisms. Journal of General Microbiology, 128(8), 1755.
Weisburg, W. G., Barns, S. M., Pelletier, D. A. and Lane, D. J. (1991). 16S
ribosomal DNA amplification for phylogenetic study. Journal of bacteriology,
173(2), 697-703.
Wong, C. (1991). Shade tolerance of tropical forages: a review. Proc. ACIAR, (32),
64-69.
135
Yang, C. J., Zhang, X. G., Shi, G. Y., Zhao, H. Y., Chen, L., Tao, K. and Hou, T. P.
(2011). Isolation and identification of endophytic bacterium W4 against
tomato Botrytis cinerea and antagonistic activity stability. African Journal of
Microbiology Research, 5(2), 131-136.
Young, R. and Gossett, J. (1997). Effect of environmental parameters and
concentration on dechlorination of chloroethenes.
Yusn, T. Y. and Huyop, F. (2009). Degradation of 3-Chloropropionic Acid by
Escherichia coli JM 109 Expressing Dehalogenase(deh) Gene used as
Selection Marker. Biotechnology, 8(3), 385-388.
Zinniel, D. K., Lambrecht, P., Harris, N. B., Feng, Z., Kuczmarski, D., Higley, P.,
Ishimaru, C. A., Arunakumari, A., Barletta, R. G. and Vidaver, A. K. (2002).
Isolation and characterization of endophytic colonizing bacteria from
agronomic crops and prairie plants. Applied and environmental microbiology,
68(5), 2198-2208.
