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.
Contents
Project task
Acknowledgements
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
Project task
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.
Acknowledgements
I’m deeply grateful to all the members of the research group of Prof L. E. Macaskie, namely: Marion Paterson-Beedle, Andrea Humphries, Inma de Vargas, Ping Yong,
Vic Baxter-Plant, Iryna Mikheenko, David Penfold and Lynne E. Macaskie. I won’t forget all the funny moments!
Furthermore I want to thank Eszter Kovać for help with the HPLC equipment, An-Chi Chang for assistance regarding the SDS-PAGE as well as Hazel Jennings, Chris Hewitt and their colleagues for advice and support regarding the processing with the pilot plant.
I also would like to thank all my new fellows and friends for having a great time and lots of fun.
Abbreviations
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Introduction
Many industrial wastes and mine run-offs contain aggressive chemicals as well as toxic compounds such as heavy metals. Due to legislative restraints alternative methods to the traditional physico-chemical technologies are needed.
Bioremediation of these metals can be carried out by means of non-living (e.g. Na2S, organosulphides) or living (microorganisms) “tools” which offer the potential of metal deposition via biochemical or enzymic processes. These can be differentiated as biosorption and biomineralization. The first one only leads to an accumulation of the metal (ions) to the cell surface and/or cytoplasm without involving the organisms metabolism whereas the intrinsic meaning of the latter implies the participation of metabolization processes. Biosorption is a well-established technique but it is subject to similar constrains like ion-exchange systems. The removal of uranium by Rhizopus arrhizus (Tsezos et al., 1989) is one remarkable example. Instances for the “metabolic” approach are the deposition of insoluble metal sulphides by sulphate reducing bacteria (White et al., 1998) and the crystallization of hydroxides and carbonates by Alcaligenes eutrophus (Diels et al., 1995) as well as metal accumulation by Citrobacter sp. (see Macaskie, 1990; Macaskie et al., 1992 Macaskie et al., 1994; Macaskie et al., 1996), which was recently reassigned to the genus Serratia sp. (Pattanapipitpaisal et al., 2002). Serratia sp. produces a heavy-metal resistant phosphatase, which liberates inorganic phosphate (HPO42-) from a (organic) phosphate molecule. This leads in presence of metal ions to a formation of insoluble metal phosphate precipitate at the cell surface. With this method a variety of metals such as Cd2+, Pb2+, Sr2+ as well as uranium, plutonium and americium can be removed from solutions. By use of immobilised cells metal loads of up to 9 g metal / g of dry biomass can be achieved (Macaskie, 1990).
Phosphatases are phosphomonoesterases, which are omnipresent in fauna and flora appearing in many microorganisms, plants and animal tissues and catalyse the hydrolysis of the C-O-P linkage of a wide variety of phosphate esters. According to their pH optima they are classified as acid or alkaline phosphatases. Not only for their manifold role in the bioprocesses of prokaryotes but also for their possible – or better – already applied use in biotechnological tasks they are of interest to biologists and related scientists. These tasks are for instance markers for bacterial taxonomy and identification (Pompei et al., 1990), tools in molecular biology and immunology (Rossolini et al., 1998) and last but not least for bioremediation processes in environmental biology. Former studies with Serratia sp. have indicated the presence of two isoenzymes designated as Citrobacter Phosphatase I (CPI) and CPII (see for instance Jeong, 1992; Jeong et al., 1998).
As part of the project "Bioremediation of Nuclear Wastes by Biomineralization Processes" this study deals with the production and purification of the two phosphatase isoenzymes from Serratia sp. N14.
Aim of the study
The main focus of this study is the production of phosphatase CPI and CPII followed by purification of the two isoenzymes. To increase the removal of problematic nuclide fission products like 60Co, 90Sr and 137Cs from wastewaters it is necessary to understand the role and function of the involved phosphatase isoenzymes. Large-scale biomass production for future enzyme structural studies and to raise antibodies for use in immunogold labelling studies will take place in the School of Chemical Engineering University of Birmingham using the 600 L facility in the pilot plant.
The project was divided into 3 approaches:
- Growth of Serratia sp. N14 in glycerol and lactose based media in batches and continuous cultures;
- Purification of the phosphatase;
- Growth of Serratia sp. N14 in 600 L batch cultures.
With the help of already established methods it is tried to discover and understand the basic cause for the difference of CPI and CPII since the knowledge is incomplete.
Basics
The strain Serratia sp. N14 was originally isolated from heavy metal contaminated soil (Macaskie & Dean 1982). It removes heavy metals from solution in form of metal phosphate deposits like CdHPO4 and HUO2PO4 for the removal of cadmium and uranyl respectively via enzymatically-mediated liberation of inorganic phosphate. The deposition of the metal phosphate crystals starts at the cell periphery. The need for phosphomonoesters like glycerol 2-phosphate (G2P), the minor metal uptake of a phosphatase deficient mutant while a recombinant strain of Escherichia coli possessing cloned phoN obtained the ability to accumulate uranyl phosphate (Basnakova et al., 1998) and the link between the rate of metal accumulation and phosphatase activity (Macaskie 1990, Macaskie et al. 1994, Jeong et al. 1997) implied a phosphatase dependent metal deposition process. The function of this phosphatase is to cleave organic phosphates (e.g. G2P) to give a phosphate group:
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Then the liberated phosphate can form a precipitate with the metal ions:
illustration not visible in this excerpt
The cell-bound HUO2PO4·4H2O can itself form a convenient tool for subsequent remediation of nickel which is considered difficult to remove from waste waters by biosorption or bioprecipitation (Tsezos et al., 1995) via intercalative ion exchange of Ni2+ into the interlamellar spaces of this crystal (Basnakova & Macaskie, 1997).
Initial experiments were done with glycerol and G2P as carbon and phosphate sources, respectively (see for instance Jeong, 1992; Jeong & Macaskie, 1999). After isolation of a phosphatase constitutive variant of Serratia sp. N14, G2P was replaced by ammonium phosphate since it disables the investigation of the effect of the carbon source on the growth (cleavage of G2P in phosphate and additional glycerol).
After several tests lactose proved to be a good substitute for glycerol. With the help of such a lactose-based minimal medium and a fed-batch culture system a reproducible product of high phosphatase activity can be achieved which is necessary to make the process efficient and economic for industrial application (Macaskie et al., 1995).
The enzyme behaviour varies according to the culture condition used to grow the cells. Whereas the phosphatase activity is ~300 units (nmol product/[min·mg protein]) in glycerol-based batch cultures with predominantly CPI – production the activity is increased up to ca. 2000 units in lactose batch and over 3000 units in carbon limited cultures with a majority of the isoenzyme CPII (Macaskie et al., 1995; Jeong & Macaskie, 1999). The use of immobilised cells, e.g. polyurethane foam, ceramic or raschig rings, results in good biofilm formation (Fig. 1) and also very high phosphates activities (up to 3500 units) (Finlay et al., 1998). But it has to be noted that a characteristic feature of Serratia sp. phosphatase activity is its variability from one batch to another.
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Figure 1 Biofilm formation by Serratia cells (ESEM)
Acid phosphatase production is induced under stress conditions via the PhoP/PhoQ sensor/regulator system. But in contrast to the PhoA (alkaline phosphatase) the role of PhoN remains mainly unclear. Shift into anaerobiosis and osmotic stress (Hallett et al., 1991) as well as carbon starvation (Kasahara et al., 1991) result in upregulation. The same effect is likely to happen at low pH (Hohmann & Miller, 1994).
As mentioned above the highest phosphatase activities and biomass yield are achieved with continuous cultures. The level of phosphatase activity is linked to the proportion of the two isozymes in the culture: the more activity the higher the amount of CPII (Jeong & Macaskie, 1999). Also the ratio between CPI and CPII changes during growth – CPI is always the predominant enzyme in batch cultures but its amount goes down from a 20-fold excess in early growth stages to about 2-fold in the late exponential phase. In contrast the amounts of both isoenzymes are equal in steady-state during continuous growth – but CPI is also predominant in earlier stages. So for the production of the more radiostable CPI cultures should be harvested in the exponential phase whereas the enrichment of CPII requires a harvest in steady-state. The lower specific phosphatase activity in this stage could be compensated by the higher biomass yield (Jeong & Macaskie, 1999).
As above mentioned, phosphatases are a widespread group of enzymes, which are responsible for most of the dephosphorylating reactions (e.g. hydrolysis of phosphoester and phosphoanhydrid bonds) taking place in prokaryotic cells and have been known for a relatively long time. Enzymes with a wide substrate range are termed as nonspecific phosphohydrolases and those with an acidic pH optimum are known as nonspecific acid phosphohydrolases (NSAPs). According to their primary structure they are classified as phosphatase class A, B or C depending on coincident sections in the amino acid sequence (Rossolini et al., 1998).
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