Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5

The threat of heavy metal pollution to public health and wildlife has led to an increased in developing systems that can remove or neutralise its toxic effect in soil, sediments and wastewater. In this work, a local molybdenum reducing bacterium was isolated. This bacterium is Gram negative and ide...

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Main Author:	Abdul Rahman, Mohd Fadhil
Format:	Thesis
Language:	English English
Published:	2007
Subjects:	Serratia marcescens
Online Access:	http://psasir.upm.edu.my/id/eprint/4918/1/FBSB_2007_21.pdf
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id	my-upm-ir.4918
record_format	uketd_dc
spelling	my-upm-ir.49182013-05-27T07:19:07Z Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5 2007 Abdul Rahman, Mohd Fadhil The threat of heavy metal pollution to public health and wildlife has led to an increased in developing systems that can remove or neutralise its toxic effect in soil, sediments and wastewater. In this work, a local molybdenum reducing bacterium was isolated. This bacterium is Gram negative and identified as Serratia marcescens Strain Dr.Y5 based on Biolog ID system and 16s rRNA molecular phylogenetics studies matched 99.96% to Serratia marcescens. The isolate was originally isolated from the grounds of King Edward VII (2) primary school in Taiping, Perak. The optimum carbon source for Mo-blue production was sucrose at 1.0% concentration and optimally grown at 40 °C. While the optimum concentration for nitrogen source was 0.2(w/v) % and optimum yeast concentration was 0.05(w/v) %. The Mo-blue production were optimum at pH 6.0 with the best ratio of phosphate to molybdate giving optimum reduction was 2.9 mM to 20 mM, respectively. Molybdenum reducing activity of the enzyme extract was assayed at 865 nm using 20 mM 10:4 molybdophosphoric acid and 2 mM NADH at room temperature. Purification of phosphomolybdate reductase was done by using anion exchange on Macro Prep High Q and gel filration on Zorbax GFX-250. The enzyme was assayed using NADH or NADPH as the electron donor and phosphomolybdate as the electron acceptor. The assay was completed in less than 5 minutes and produced an intense blue color with a wavelength maximum at 865 nm. The best electron donor for the enzyme is NADH (12-MP as electon acceptor) with a maximum initial velocity, Vmax of 25.07 nmole molybdenum blue produced/min/mg/protein and a Michaelis constant, Km at 0.44 mM. The best electron acceptor substrate is 10:4 molybdophosphate, with a Km of 3.87 mM and a Vmax of 24.18 nmole molybdenum blue/min (NADH as electron donor at saturated concentrations). The phosphomolybdate reductase activity has an optimum temperature at 30 °C. At 40 °C of incubation for a period of one hour, the residual phosphomolybdate reductase activity remains 80% of the control, indicating that the enzyme is stable below 40 °C. The enzyme was inactivated rapidly at temperatures higher than 54 °C and was inactivated totally at 70 °C within 30 minutes of incubation. Serratia marcescens 2007 Thesis http://psasir.upm.edu.my/id/eprint/4918/ http://psasir.upm.edu.my/id/eprint/4918/1/FBSB_2007_21.pdf application/pdf en public masters Universiti Putra Malaysia Serratia marcescens Faculty of Biotechnology and Biomolecular sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Serratia marcescens
spellingShingle	Serratia marcescens Abdul Rahman, Mohd Fadhil Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5
description	The threat of heavy metal pollution to public health and wildlife has led to an increased in developing systems that can remove or neutralise its toxic effect in soil, sediments and wastewater. In this work, a local molybdenum reducing bacterium was isolated. This bacterium is Gram negative and identified as Serratia marcescens Strain Dr.Y5 based on Biolog ID system and 16s rRNA molecular phylogenetics studies matched 99.96% to Serratia marcescens. The isolate was originally isolated from the grounds of King Edward VII (2) primary school in Taiping, Perak. The optimum carbon source for Mo-blue production was sucrose at 1.0% concentration and optimally grown at 40 °C. While the optimum concentration for nitrogen source was 0.2(w/v) % and optimum yeast concentration was 0.05(w/v) %. The Mo-blue production were optimum at pH 6.0 with the best ratio of phosphate to molybdate giving optimum reduction was 2.9 mM to 20 mM, respectively. Molybdenum reducing activity of the enzyme extract was assayed at 865 nm using 20 mM 10:4 molybdophosphoric acid and 2 mM NADH at room temperature. Purification of phosphomolybdate reductase was done by using anion exchange on Macro Prep High Q and gel filration on Zorbax GFX-250. The enzyme was assayed using NADH or NADPH as the electron donor and phosphomolybdate as the electron acceptor. The assay was completed in less than 5 minutes and produced an intense blue color with a wavelength maximum at 865 nm. The best electron donor for the enzyme is NADH (12-MP as electon acceptor) with a maximum initial velocity, Vmax of 25.07 nmole molybdenum blue produced/min/mg/protein and a Michaelis constant, Km at 0.44 mM. The best electron acceptor substrate is 10:4 molybdophosphate, with a Km of 3.87 mM and a Vmax of 24.18 nmole molybdenum blue/min (NADH as electron donor at saturated concentrations). The phosphomolybdate reductase activity has an optimum temperature at 30 °C. At 40 °C of incubation for a period of one hour, the residual phosphomolybdate reductase activity remains 80% of the control, indicating that the enzyme is stable below 40 °C. The enzyme was inactivated rapidly at temperatures higher than 54 °C and was inactivated totally at 70 °C within 30 minutes of incubation.
format	Thesis
qualification_level	Master's degree
author	Abdul Rahman, Mohd Fadhil
author_facet	Abdul Rahman, Mohd Fadhil
author_sort	Abdul Rahman, Mohd Fadhil
title	Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5
title_short	Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5
title_full	Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5
title_fullStr	Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5
title_full_unstemmed	Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5
title_sort	purification and characterization of phospomolybdate reductase produced by locally isolated serratia marcescens strain dr.y5
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular sciences
publishDate	2007
url	http://psasir.upm.edu.my/id/eprint/4918/1/FBSB_2007_21.pdf
_version_	1747810310166675456

Purification And Characterization Of Phospomolybdate Reductase Produced By Locally Isolated Serratia Marcescens Strain Dr.Y5

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