This study is part of the project "Bioremediation of Nuclear Wastes by Biomineralization Processes" which uses an established biomineralization process (using Serratia sp.) for the removal of uranyl ions as hydrogen uranyl phosphate (HUP). HUP will be tested as a host crystal for intercalative ion exchange or co-crystallative removal of problematic nuclide fission products (FP) like 60Co, 90Sr and 137Cs using "cold" isotopes in Birmingham in parallel to tests in Korea using real wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate, which precipitates with heavy metals as cell-bound metal phosphate, thus depositing the uranyl phosphate "host crystal" for the sequestration of the FP. The phosphatase is localised periplasmically and also within the extracellular polymeric matrix (EPM). Successful operation of the process depends on the correct localization of the enzyme into the extracellular matrix. It can be speculated that the periplasmic enzyme pool is a reservoir awaiting export and other studies have suggested the presence of two phosphatase isoenzymes, which differ in their chemical- and radiostability but are not yet assigned to either phosphatase pool or EPM since they are immunologically cross-reactive. The two phosphatases (designated CPI and CPII) are very similar but distinguished simply using cationic (CPII retained) and anionic (CPI retained) ion exchange resins.
This study will concentrate on the production of phosphatase CPI and CPII which will be differentiated by enzyme partial purification (exocellularly-localised and residual whole-cell enzymes) followed by quantification of their cation exchange (CPII) and anion exchange (CPI) resin binding. Large scale biomass preparation for bulk enzyme production for enzyme structural studies (X ray crystallography using the Korean Synchrotron Facility) will use the 600 L facility in the pilot plant in the School of Chemical Engineering University of Birmingham.
The overall objective of the project is to promote the localisation of the more radiostable isoenzyme (CPI) into the exocellular matrix for maximum production of uranyl phosphate in the presence of the high-active nuclide fission products and to understand this radiostability in terms of the associated water in the active site pocket of this novel phosphohydrolase.
Table of Contents
PROJECT TASK
ACKNOWLEDGEMENTS
CONTENTS
ABBREVIATIONS
Introduction
Aim of the study
Basics
Materials and methods
1.1 Materials
1.1.1 Chemicals
1.1.2 Organism
1.1.3 Media
1.1.3.1 Minimal Medium
1.2 General Methods
1.2.1 Growth of microorganisms
1.2.2 Enzyme purification
1.2.3 SDS – PAGE
Results and discussion
1.3 Growth of Serratia sp. N14
1.4 Purification of Serratia sp. N14 phosphatase
Summary and prospects
Bibliography
APPENDICES
Appendix I Bradford assay
Appendix II Bicinchoninic acid (BCA) protein assay
Appendix III Phosphatase activity assay
Appendix IV SDS – PAGE
Appendix V Chromatography stages
Research Objectives and Key Topics
This project aims to enhance the bioremediation of nuclear waste by optimizing the production and purification of phosphatase isoenzymes from Serratia sp. N14, which are critical for the mineralization of heavy metals and radionuclides.
- Large-scale production of phosphatase isoenzymes (CPI and CPII)
- Development of purification protocols for Serratia sp. N14 enzymes
- Investigation of enzyme behavior under varying culture conditions
- Identification of biochemical differences between CPI and CPII isoenzymes
Excerpt from the Book
1.4 Purification of Serratia sp. N14 phosphatase
The basic procedure for the separation and purification of the two isoenzymes CPI and CPII was established by Jeong (1992). The following scheme gives an overlook:
After cell disruption, centrifugation and ammonium sulphate fractionation between 0.9 g and 3.4 g of protein precipitate was gained. This was of the same colour (pink or yellow-brownish) as the culture medium before harvest. To determine the loss of active enzyme over the purification procedure, the activity of the different purification fractions of one batch is shown in table 4.
The results were comparable to those reported in the past (Jeong et al., 1998). Around 25% of phosphatase activity is associated with cell debris and membranes and behaves therefore similar to S. typhimurium acid and E. coli alkaline phosphatases.
Summary of Chapters
Introduction: Provides the context of bioremediation using phosphatase-producing bacteria to treat heavy metal and nuclear waste.
Aim of the study: Outlines the project goals, focusing on the production and purification of phosphatase isoenzymes for structural and functional analysis.
Basics: Reviews the metabolic mechanisms of Serratia sp. N14 and existing knowledge on phosphatase activity and isoenzyme behavior.
Materials and methods: Details the cultivation of Serratia sp. N14, the enzyme purification workflow, and the analytical techniques used.
Results and discussion: Presents the findings from the cultivation experiments and the purification of phosphatase isoenzymes, including SDS-PAGE and chromatography data.
Summary and prospects: Concludes the study by reviewing key results and suggesting future research directions.
Keywords
Serratia sp. N14, Phosphatase, Bioremediation, Biomineralization, CPI, CPII, Enzyme purification, Ion exchange chromatography, Heavy metals, Nuclear waste, PhoN gene, Hydroxyapatite, Protein assay, SDS-PAGE
Frequently Asked Questions
What is the core focus of this research?
The research focuses on the production and purification of two phosphatase isoenzymes (CPI and CPII) from the bacterium Serratia sp. N14 to optimize the biomineralization of heavy metals and nuclear waste products.
What are the primary thematic areas?
The main themes include microbial cultivation under various media conditions, enzyme purification strategies, biochemical characterization of phosphatase isoenzymes, and the analysis of enzyme stability and activity.
What is the overarching research goal?
The primary goal is to gain a deeper understanding of the differences between CPI and CPII to improve the efficiency of industrial bioremediation processes for radioactive fission products.
Which scientific methods are utilized?
The study employs microbiological growth techniques (batch and continuous culture), enzyme purification via ion exchange and hydroxyapatite chromatography, SDS-PAGE analysis, and protein assays like the Bradford and BCA methods.
What does the main body cover?
The main body details the experimental setups for different growth conditions, describes the complex protein purification pipeline, and discusses the resulting enzyme activities and chromatography profiles.
Which keywords define this work?
The work is defined by terms such as bioremediation, Serratia sp. N14, phosphatase isoenzymes, biomineralization, and enzyme purification.
Why was lactose used in the culture medium?
Lactose proved to be an effective, reproducible substitute for glycerol, facilitating higher phosphatase activity and better suited for industrial-scale application.
What is the significance of the "unknown fragment" observed in the absorption spectra?
The fragment associated with the CPI isoenzyme might explain its distinct ion exchange behavior compared to CPII, serving as a potential marker for differentiating between the two enzymes.
- Quote paper
- Holger Pflicke (Author), 2003, Purification of Serratia sp. phosphatase, identification/localisation of the two phosphatase isoenzymes and large scale production of the enzyme, Munich, GRIN Verlag, https://www.grin.com/document/18268
