Understanding the complexity of metabolic regulatory systems an investigation into the regulation of hydantoin-hydrolysis in Pseudomonas putida RU-KM3s
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
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Characterization of amide bond hydrolysis in novel hydantoinase-producing bacteria
- Authors: Skepu, Zoleka G
- Date: 2000
- Subjects: Amides , Hydrolysis , Hydantoin , Imides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3911 , http://hdl.handle.net/10962/d1003970 , Amides , Hydrolysis , Hydantoin , Imides
- Description: This thesis describes a series of investigations into the amide bond-hydrolyzing activity of bacterial strains RU-KM1, RU-KM3L, RU-KM3S, and RU-OR, which were previously isolated for their ability to hydrolyze hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids and related compounds. Several compounds may be used as substrates by biocatalysts for the production of amino acids, such as hydantoins, amino nitriles, and amides. These compounds are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as 2- arylpropionic acids, which are non-steroidal anti-inflammatory drugs. Thus, the ability of the above-mentioned strains to hydrolyze these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. The compounds used as substrates in the investigation are all essentially amides. Thus, the ability of the strains to hydrolyze imides, hydantoins, and amides, was investigated. In particular, imides have a structure which is very similar to that of hydantoins, and thus it was an objective of the study to determine whether these strains could hydrolyze imides. Imidehydrolyzing activity has only recently been discovered in microorganisms. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an Ncarbamylamino acid intermediate, and subsequently, an "-amino acid. The hydantoinhydrolyzing enzymes of a Pseudomonas putida strain, RU-KM3S, were characterized in a crude extract preparation and reaction conditions for its biocatalytic application were optimized. The optimum conditions for conversion of 5-methylhydantoin were found to be 3 hours at 40°C, with conversion yields greater than 50% achieved. The enzymes of RU-KM3S demonstrated considerable stability, retaining 80% of their activity after incubation at 40°C for 3 hours. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamylase of RUKM3S suggested that these enzymes required metal ions for activity, with metal ions such as Mg²⁺, Mn²⁺, Zn²⁺, and Co²⁺ resulting in activation of the enzymes. However, Cu²⁺ and Fe²⁺ caused inactivation of these enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was non-selective, whereas the N-carbamylase was L-selective. The hydantoin substrate selectivity of RU-KM3S was compared to that of three other hydantoinase-producing bacteria, RU-KM1, RU-KM3L, and RU-OR. The four strains were able to hydrolyze all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing higher activity than whole cells, except in the case of RU-KM1. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolyzed p-hydroxyphenylhydantoin. RU-KM3L whole cells achieved a higher conversion yield with isobutylhydantoin, whereas the crude extract achieved a higher yield with 5-t-butylhydantoin. RU-KM3S whole cells and crude extract preferentially hydrolyzed 5-n-butylhydantoin, although the yield was greater with the crude extract. The highest conversion yields were observed with RU-KM3S crude extract, with conversion yields of 71.6% and 100% for n-butylhydantoin and phydroxyphenylhydantoin, respectively.The ability of RU-KM1, RU-KM3L, and RU-KM3S to hydrolyze nitriles, initially to amides and subsequently to carboxylic acids, was investigated. These strains were demonstrated to be unable to utilize acrylonitrile, propionitrile and benzonitrile as nitrogen sources, but were able to hydrolyze acrylonitrile, propionitrile and acetonitrile, in resting cell reactions. Nitrile hydrolysis was demonstrated to be inducible in all three strains, and the enzyme system responsible for nitrile hydrolysis was proposed to be a nitrile hydratase-amidase system. Amidase activity in the four bacterial strains was investigated. The ability of RU-KM1, RUKM3L, RU-KM3S, and RU-OR to utilize amides as a nitrogen source was investigated, and the results showed that propionamide was a good nitrogen source for all four of the strains. Amide-hydrolyzing activity, by resting cells, was shown to be inducible by propionamide in all four strains. RU-KM3S demonstrated superior amide-hydrolyzing ability in that it hydrolyzed propionamide, acetamide, and acrylamide to a greater extent than the other strains. Resting cells of RU-KM1 and RU-OR were demonstrated to have the ability to hydrolyze the imide substrate, succinimide, and this imidase activity was found to be inducible. These strains were also able to utilize this imide as the sole source of nitrogen for growth, which is a novel finding, as to date, bacteria have only be reported to utilize imides as a carbon source. The identity of the enzyme system responsible for succinimide hydrolysis is not yet clear. In conclusion, the hydantoin-hydrolyzing enzymes of RU-KM3S have been shown to be possibly membrane associated, which is a novel finding that has also been proposed in three other hydantoinase-producing strains in our laboratory. This study has shown that the Ncarbamylase of RU-KM3S is L-stereoselective, which, to our knowledge, is the first report of an L-stereospecific N-carbamylase in a Pseudomonas putida. Publication of these findings is already in progress. This is the first report on the study of imide hydrolysis in either an Agrobacterium tumefaciens or a Pseudomonas sp., and publications reporting these results are in preparation.
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The isolation and characterisation of thermostable hydantoinases from hydantoinase-producing bacteria
- Authors: Phehane, Vuyisile Ntosi
- Date: 1999
- Subjects: Hydantoin , Bacteria -- Physiology , Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3998 , http://hdl.handle.net/10962/d1004058 , Hydantoin , Bacteria -- Physiology , Enzymes
- Description: In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.
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