Characterization of a novel bacterial dehalogenase from cow dung

Characterization of a novel bacterial dehalogenase from cow dung

The large quantities of the halogenated compound for example 2,2- dichloropropionic acid (2,2DCP) in the environment may lead to health problems in humans and pollution due to their toxicity and recalcitrance, respectively. Interestingly, previous studies have indicated that cow dung was proven to d...

Full description

Saved in:
Bibliographic Details
Main Author: Ismail, Siti Nurul Fasehah
Format: Thesis
Language:English
Published: 2018
Subjects:
QH Natural history
Online Access:http://eprints.utm.my/id/eprint/79531/1/SitiNurulFasehahPFBME2018.pdf
Tags: Add Tag
No Tags, Be the first to tag this record!
id my-utm-ep.79531
record_format uketd_dc
spelling my-utm-ep.795312018-10-31T12:54:46Z Characterization of a novel bacterial dehalogenase from cow dung 2018 Ismail, Siti Nurul Fasehah QH Natural history The large quantities of the halogenated compound for example 2,2- dichloropropionic acid (2,2DCP) in the environment may lead to health problems in humans and pollution due to their toxicity and recalcitrance, respectively. Interestingly, previous studies have indicated that cow dung was proven to degrade pollutants. Hence, such animals feeding on a daily diet of halogen contaminated forage may influence the microflora in their digestive tract. Bacterial species from cow dung able to utilize 2,2DCP is yet to be reported. Therefore, the purpose of this study was to isolate, identify and characterize dehalogenase bacteria from cow dung. Four bacteria were isolated which are SN1, SN2, SN3, and SN4. Strain SN1 was observed with rapid growth in 20 mM 2,2DCP liquid minimal media, and was used for further experiments such as growth in different concentration of 2,2DCP, High-performance liquid chromatography (HPLC), Biolog GENIII, 16S rRNA analysis, characterization of purified enzyme, kinetic analysis, Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and amplification of dehalogenase gene. The growth of strain SN1 in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2DCP with the highest chloride ion released (39.5 μmolCIˉ/mL) while exhibiting a remarkably short doubling time of 3.85 h. The utilization of 2,2DCP was also confirmed by detection of 20 mM 2,2DCP depletion in the growth medium containing strain SN1 measured using HPLC. The result showed 98.6 % utilization of 2,2DCP in the growth medium. Species identification via Biolog GENIII system and 16S rRNA analysis was performed and identified strain SN1 as Bacillus cereus. Further investigations on dehalogenase enzyme were done by using purified enzyme of Bacillus cereus SN1. The molecular weight of the purified enzyme was 25 kDa by SDS-PAGE. The enzyme characteristics revealed it was optimum at pH 6 and 30 °C. It also has low Km value of 0.2 mM. The dehalogenase peptide was identified by LC-MS/MS with 18% sequence coverage to haloacid dehalogenase, Bacillus cereus (strain 03BB102). Moreover, Group I and Group II dehalogenase primers were used to amplify dehalogenase gene and the band only appeared for Group I. The dehalogenase gene fragment amplified was designated “DehSN1” and belongs to Group I dehalogenase since it has 75 % similarity with Group I dehalogenase (DehE). Five conserved residues were identified as Asn33, Tyr117, Cys42, Ala120 and Asp136. As a conclusion, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2DCP. Therefore, further assessment of its ability to degrade other types of haloalkanoic acids merits special consideration. 2018 Thesis http://eprints.utm.my/id/eprint/79531/ http://eprints.utm.my/id/eprint/79531/1/SitiNurulFasehahPFBME2018.pdf application/pdf en public phd doctoral Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering Faculty of Biosciences and Medical Engineering
institution Universiti Teknologi Malaysia
collection UTM Institutional Repository
language English
topic QH Natural history
spellingShingle QH Natural history
Ismail, Siti Nurul Fasehah
Characterization of a novel bacterial dehalogenase from cow dung
description The large quantities of the halogenated compound for example 2,2- dichloropropionic acid (2,2DCP) in the environment may lead to health problems in humans and pollution due to their toxicity and recalcitrance, respectively. Interestingly, previous studies have indicated that cow dung was proven to degrade pollutants. Hence, such animals feeding on a daily diet of halogen contaminated forage may influence the microflora in their digestive tract. Bacterial species from cow dung able to utilize 2,2DCP is yet to be reported. Therefore, the purpose of this study was to isolate, identify and characterize dehalogenase bacteria from cow dung. Four bacteria were isolated which are SN1, SN2, SN3, and SN4. Strain SN1 was observed with rapid growth in 20 mM 2,2DCP liquid minimal media, and was used for further experiments such as growth in different concentration of 2,2DCP, High-performance liquid chromatography (HPLC), Biolog GENIII, 16S rRNA analysis, characterization of purified enzyme, kinetic analysis, Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and amplification of dehalogenase gene. The growth of strain SN1 in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2DCP with the highest chloride ion released (39.5 μmolCIˉ/mL) while exhibiting a remarkably short doubling time of 3.85 h. The utilization of 2,2DCP was also confirmed by detection of 20 mM 2,2DCP depletion in the growth medium containing strain SN1 measured using HPLC. The result showed 98.6 % utilization of 2,2DCP in the growth medium. Species identification via Biolog GENIII system and 16S rRNA analysis was performed and identified strain SN1 as Bacillus cereus. Further investigations on dehalogenase enzyme were done by using purified enzyme of Bacillus cereus SN1. The molecular weight of the purified enzyme was 25 kDa by SDS-PAGE. The enzyme characteristics revealed it was optimum at pH 6 and 30 °C. It also has low Km value of 0.2 mM. The dehalogenase peptide was identified by LC-MS/MS with 18% sequence coverage to haloacid dehalogenase, Bacillus cereus (strain 03BB102). Moreover, Group I and Group II dehalogenase primers were used to amplify dehalogenase gene and the band only appeared for Group I. The dehalogenase gene fragment amplified was designated “DehSN1” and belongs to Group I dehalogenase since it has 75 % similarity with Group I dehalogenase (DehE). Five conserved residues were identified as Asn33, Tyr117, Cys42, Ala120 and Asp136. As a conclusion, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2DCP. Therefore, further assessment of its ability to degrade other types of haloalkanoic acids merits special consideration.
format Thesis
qualification_name Doctor of Philosophy (PhD.)
qualification_level Doctorate
author Ismail, Siti Nurul Fasehah
author_facet Ismail, Siti Nurul Fasehah
author_sort Ismail, Siti Nurul Fasehah
title Characterization of a novel bacterial dehalogenase from cow dung
title_short Characterization of a novel bacterial dehalogenase from cow dung
title_full Characterization of a novel bacterial dehalogenase from cow dung
title_fullStr Characterization of a novel bacterial dehalogenase from cow dung
title_full_unstemmed Characterization of a novel bacterial dehalogenase from cow dung
title_sort characterization of a novel bacterial dehalogenase from cow dung
granting_institution Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering
granting_department Faculty of Biosciences and Medical Engineering
publishDate 2018
url http://eprints.utm.my/id/eprint/79531/1/SitiNurulFasehahPFBME2018.pdf
_version_ 1747818249054060544

Similar Items