Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1

Molybdenum-reducing bacteria were isolated from soil and water samples throughout Malaysia and one isolate from workshop area in Seri Kembangan, Selangor was identified to reduce molybdenum to molybdenum blue in less than 48 hours with the most intense blue-colour colony formation. Further analysis...

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Main Author:	Abu Bakar, Nurlizah
Format:	Thesis
Language:	English English
Published:	2010
Subjects:	Heavy metals Molybdenum Staphylococcus
Online Access:	http://psasir.upm.edu.my/id/eprint/26461/1/FBSB%202010%2026R.pdf
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spelling	my-upm-ir.264612013-09-23T06:31:34Z Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1 2010-10 Abu Bakar, Nurlizah Molybdenum-reducing bacteria were isolated from soil and water samples throughout Malaysia and one isolate from workshop area in Seri Kembangan, Selangor was identified to reduce molybdenum to molybdenum blue in less than 48 hours with the most intense blue-colour colony formation. Further analysis using biochemical test, 16S rRNA ribosomal gene sequence comparison and molecular phylogenetics analysis showed that isolate Point 1 is Gram-positive coccus and grouped under genus Staphylococcus and was submitted to Genbank under accession number of EU828636 as Staphylococcus sp. isolate liz1. The characterization studies showed that the bacterium gave the highest Mo-reducing activity under the combination of 1% glucose, 50 mM molybdate, 5 mM phosphate, at pH 6.0 and temperature 37°C in 48 hours of incubation time. Partial purification and characterization were conducted on crude Mo-reducing enzyme with two anion exchange chromatography columns (Q-Sepharose and Mono Q). Three bands were obtained on the Mono-Q fraction at 25, 40 and 55 kDa using reducing SDS polyacrylamide-gel electrophoresis (SDS-PAGE). Characterization of Mo-reducing enzyme kinetics gave the Km and Vmax values of 5.722 mM and 127.1 nmole Mo-blue/min/mg protein for laboratory-prepared phosphomolybdate (LPPM), and 15.58 mM and 152.8 nmole Mo-blue/min/mg protein for NADH, respectively. The optimum pH and temperature for enzyme were pH 5.0 and 37°C, respectively. The inhibition profiles of heavy metal on Mo-reducing activity were determined and IC50 values were calculated. The results showed that the enzyme has high sensitivity towards copper (0.2845 mg L-1), silver (0.2773 mg L-1), and mercury (0.4187 mg L-1). Three commercialized herbs samples were tested using the developed enzyme assay and the results were compared with that of atomic absorption spectroscopy (AAS) analysis. The results showed that one of the herb sample contained high level of mercury and copper when analysed using AAS (1.016 mg L-1 and 0.421 mg L-1, respectively). The findings were similar to the biological assay system developed for the detection of selected heavy metals, indicating the usefulness of the assay. Heavy metals Molybdenum Staphylococcus 2010-10 Thesis http://psasir.upm.edu.my/id/eprint/26461/ http://psasir.upm.edu.my/id/eprint/26461/1/FBSB%202010%2026R.pdf application/pdf en public masters Universiti Putra Malaysia Heavy metals Molybdenum Staphylococcus Faculty of Biotechnology and Biomolecular Sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Heavy metals Molybdenum Staphylococcus
spellingShingle	Heavy metals Molybdenum Staphylococcus Abu Bakar, Nurlizah Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1
description	Molybdenum-reducing bacteria were isolated from soil and water samples throughout Malaysia and one isolate from workshop area in Seri Kembangan, Selangor was identified to reduce molybdenum to molybdenum blue in less than 48 hours with the most intense blue-colour colony formation. Further analysis using biochemical test, 16S rRNA ribosomal gene sequence comparison and molecular phylogenetics analysis showed that isolate Point 1 is Gram-positive coccus and grouped under genus Staphylococcus and was submitted to Genbank under accession number of EU828636 as Staphylococcus sp. isolate liz1. The characterization studies showed that the bacterium gave the highest Mo-reducing activity under the combination of 1% glucose, 50 mM molybdate, 5 mM phosphate, at pH 6.0 and temperature 37°C in 48 hours of incubation time. Partial purification and characterization were conducted on crude Mo-reducing enzyme with two anion exchange chromatography columns (Q-Sepharose and Mono Q). Three bands were obtained on the Mono-Q fraction at 25, 40 and 55 kDa using reducing SDS polyacrylamide-gel electrophoresis (SDS-PAGE). Characterization of Mo-reducing enzyme kinetics gave the Km and Vmax values of 5.722 mM and 127.1 nmole Mo-blue/min/mg protein for laboratory-prepared phosphomolybdate (LPPM), and 15.58 mM and 152.8 nmole Mo-blue/min/mg protein for NADH, respectively. The optimum pH and temperature for enzyme were pH 5.0 and 37°C, respectively. The inhibition profiles of heavy metal on Mo-reducing activity were determined and IC50 values were calculated. The results showed that the enzyme has high sensitivity towards copper (0.2845 mg L-1), silver (0.2773 mg L-1), and mercury (0.4187 mg L-1). Three commercialized herbs samples were tested using the developed enzyme assay and the results were compared with that of atomic absorption spectroscopy (AAS) analysis. The results showed that one of the herb sample contained high level of mercury and copper when analysed using AAS (1.016 mg L-1 and 0.421 mg L-1, respectively). The findings were similar to the biological assay system developed for the detection of selected heavy metals, indicating the usefulness of the assay.
format	Thesis
qualification_level	Master's degree
author	Abu Bakar, Nurlizah
author_facet	Abu Bakar, Nurlizah
author_sort	Abu Bakar, Nurlizah
title	Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1
title_short	Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1
title_full	Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1
title_fullStr	Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1
title_full_unstemmed	Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1
title_sort	development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from staphylococcus sp. isolate liz1
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2010
url	http://psasir.upm.edu.my/id/eprint/26461/1/FBSB%202010%2026R.pdf
_version_	1747811539547586560

Development of an inhibitive enzyme assay for heavy metal detection using molybdenum-reducing enzyme from Staphylococcus sp. isolate liz1

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