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Elucidation and Manipulation of the Hydantoin-Hydolysing Enzyme

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Elucidation and Manipulation of the Hydantoin- Hydrolysing Enzyme System of Agrobacterium tumefaciens RU-OR for the Biocatalytic Production of D-amino acids Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy of Rhodes University by Carol Janet Hartley July 2001 Abstract There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-p- hydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoin- hydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolase- encoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes. Contents Table of Contents List of Figures vi List of Tables x List of Abbreviations xii Acknowledgements xiv Research Outputs xv Chapter 1: General Introduction 1.1 Introduction 2 1.2 Biocatalytic production of optically active amino acids 4 1.2.1 Biocatalytic production using L-selective hydantoin hydrolysis 8 1.2.2 Biocatalytic production using D-selective hydantoin hydrolysis 8 1.3 Properties of 5-monosubstituted hydantoin and hydantoin derivatives 9 1.4 Natural distribution of hydantoin-hydrolysing activity 10 1.4.1 L-selective hydantoin-hydrolysing enzyme systems 12 1.4.2 D-selective hydantoin-hydrolysing enzyme systems 13 1.5 Hydantoinase enzymes 15 1.5.1 L-selective and non-selective hydantoinase enzymes 17 1.5.2 O-selective hydantoinase enzymes 21 1.6 N-carbamoyl amino acid amidohydrolase enzymes 27 1.6.1 L- selective N-carbamoyl amino acid amidohydrolase enzymes 28 1.6.2 D-selective N-carbamoyl amino acid amidohydrolase enzymes 32 1.7 Hydantoinase-N-carbamoyl amino acid amidohydrolase gene fusions 39 1.8 Hydantoin racemase enzymes 40 1.9 Regulation of hydantoin-hydrolysing enzyme production 43 1.9.1 Induction 43 1.9.2 Catabolite repression 45 1.10 Evolution of hydantoin-hydrolysing enzyme systems 46 1.11 Heterologous expression of hydantoin-hydrolysing enzymes 47 1.12 Research Proposal 52 Chapter 2: Isolation of Agrobacterium tumefaciens RU-OR and optimization of resting cell biocatalytic reactions 2.1 Introduction 55 2.2 Materials and Methods 2.2.1 Isolation of hydantoin-hydrolysing bacterial strains 57 2.2.2 Culture conditions 58 2.2.3 Optimization of resting cell biocatalytic reactions 58 2.2.4 Enantiomeric resolution 58 2.2.5 Statistical Analysis 59 2.2.6 Strain identification 59 2.3 Results 60 2.3.1 Isolation of hydantoin-hydrolysing bacterial strains 60 2.3.2 Optimization of resting cell biocatalytic reactions 62 2.3.3 Identification of strain RU-OR 72 2.4 Discussion 74 Chapter 3: Regulation of hydantoin-hydrolysing activity in Agrobacterium tumefaciens RU-OR 3.1 Introduction 81 3.2 Materials and Methods 83 3.2.1 Media and culture conditions 83 3.2.2 Ammonium shock assays 84 3.2.3 Ammonium shock recovery 84 3.2.4 Resting cell biocatalytic reactions 84 3.3 Results 85 3.3.1 Regulation of hydantoin-hydrolysing activity by carbon source 85 3.3.2 Regulation of hydantoin-hydrolysing activity by nitrogen source 85 3.3.3 Induction of hydantoin-hydrolysing activity in A. tumefaciens RU-OR 91 3.4 Discussion 93 ii Chapter 4: Mutational analysis of hydantoin-hydrolysing activity in Agrobacterium tumefaciens RU-OR 4.1 Introduction 97 4.2 Materials and Methods 102 4.2.1 Bacterial strains and culture conditions 102 4.2.2 Mutagenesis of A. tumefaciens RU-OR 102 4.2.3 Isolation of inducer-independent mutant strains 103 4.2.4 Isolation of mutant strains with altered nitrogen regulation 103 4.2.5 Isolation of glutamine-dependent mutant strains 103 4.2.6 Ammonia shock and glutamine shock assays 104 4.2.7 Ammonia shock recovery 104 4.2.8 Biocatalytic resting cell reactions 104 4.2.9 Enantiomeric resolution 104 4.2.10 Glutamine synthetase enzyme assays 105 4.3 Results 105 4.3.1 Mutagenesis of A. tumefaciens RU-OR 105 4.3.2 Selection and characterization of inducer-independent mutant 106 strains 4.3.3 Selection and characterization of inducer-independent mutant 110 strains with altered nitrogen regulation 4.3.4 Hydantoinase and NCAAH activity of glutamine auxotrophic 111 mutant strains 4.3.5 Biomass yield, specific hydantoinase and specific NCAAH 116 activity in regulatory mutants during growth in (NH4)2S04 121 4.4 Discussion Chapter 5: Screening and isolation of genes encoding hydantoin­ hydrolysing activity from a genomic DNA library of A. tumefaciens RU-OR 5.1 Introduction 129 5.2 Materials and Methods 132 5.2.1 Isolation and calibration of chromosomal DNA 132 5.2.2 Construction of A. tumefaciens RU-OR genomic DNA iii library 133 5.2.3 Genomic DNA library screening (agar plate method) 134 5.2.4 Genomic DNA library screening (microtitre plate method) 135 5.2.5 Plasmid isolation and restriction fragment analysis 136 5.2.6 PCR amplification of NCAAH gene from A. tumefaciens RU-OR 136 5.2.7 Resting cell and sonicated crude extract biocatalytic reactions 137 5.2.8 Analytical methods 137 5.3 Results 138 5.3.1 ConstrucUon of a genomic library of A. tumefaciens RU-OR 138 5.3.2 Screening for genes encoding hydantoin-hydrolysing activity with MM plates containing phenol red 139 5.3.3 Screening for genes encoding hydantoin-hydrolysing activity using growth on HMM or NCMM agar plates 140 5.3.4 Screening for genes encoding hydantoin-hydrolysing activity using microtitre-plate biocatalytic assays 142 5.3.5 PCR amplification and sub-cloning of NCAAH gene from A. tumefaciens RU-OR 144 5.3.6 Comparison of the biocatalytic activity expressed by recombinant NCAAH genes 145 5.4 Discussion 148 Chapter 6: Molecular analysis of isolated clones with hydantoin­ hydrolysing activity 5.5 Introduction 153 6.2 Materials and Methods 156 6.2.1 Culture conditions 156 6.2.2 Sonicated crude extract biocatalytic reactions 156 6.2.3 Analytical methods 156 6.2.4 Molecular techniques 156 6.2.5 DNA: DNA Hybridisation 157 6.2.6 Sequence analYSis 158 6.3 Results 159 iv 6.3.1 Deletion analysis of p683-1 a 159 6.3.2 Chromosomal mapping of the nca genes from A. fumefaciens RU-OR 162 6.3.3 Molecular analysis of nca genes from A. tumefaciens RU- OR 166 6.3.4 Molecular analysis of the promotor regions upstream of NCDAAH genes 178 6.4 Discussion 180 Chapter 7: General Conclusions 7.1 The biocatalyst: selection and characterization of the hydantoin- hydrolysing enzyme system of A. fumefaciens RU-OR 187 7.2 Regulation of hydantoin-hydrolysing activity in RU-OR 189 7.3 Mutational analysis of hydantoins-hydrolysing activity in RU-OR and overproduction of hydantoin-hydrolysing enzymes 191 7.4 NCA R1 and NCA R2: Implications and Applications 192 7.5 Future Research 193 Appendices Appendix 1: List of materials 197 Appendix 2: Media 199 Appendix 3: Standard procedure for growth, harvesting and resting cell biocatalytic reactions with A. tumefapiens RU-OR 201 Appendix 4: Analytical methods 202 Appendix 5:" Alignment of 168 rRNA gene sequences 209 Appendix 6: List of primers 210 Appendix 7: Plasmids used in this study 211 Appendix 8: Sequence analysis of pGEMH1 using nested deletions, and partial DNA sequence of 5'-3' strand of pGEMH1 215 Appendix 9: Alignment of ORF nucleotide sequences encoding NCDAAH enzymes 217 References 221 v List of Figures Figure 1.1: Reaction pathway for the hydrolysis of hydantoin to form enantiomerically pure amino acids. 5 Figure 1.2: Some examples of the wide range of enantiomerically pure amino acids that can be produced by hydrolysis of hydantoin derivatives, with different R groups altering the amino acid product.
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