Biomedical subjects

Phillip C Wright

Publications and source records attributed to Phillip C Wright.

26 records · Page 2Linked to original sources

PubMed2003

Alcohol dehydrogenases from thermophilic and hyperthermophilic archaea and bacteria.

Many studies have been undertaken to characterise alcohol dehydrogenases (ADHs) from thermophiles and hyperthermophiles, mainly to better understand their activities and thermostability. To date, there are 20 thermophilic archaeal and 17 thermophilic bacterial strains known to have ADHs or similar enzymes, including the hypothetical proteins. Some of these thermophiles are found to have multiple ADHs, sometimes of different types. A rigid delineation of amino acid sequences amongst currently elucidated thermophilic ADHs and similar proteins is phylogenetically apparent. All are NAD(P)-dependent, with one exception that utilises the cofactor F(420) instead. Within the NAD(P)-dependent group, the thermophilic ADHs are orderly clustered as zinc-dependent ADHs, short-chain ADHs, and iron-containing/activated ADHs. Distance matrix calculations reveal that thermophilic ADHs within one type are homologous, with those derived from a single genus often showing high similarities. Elucidation of the enzyme activity and stability, coupled with structure analysis, provides excellent information to explain the relationship between them, and thermophilic ADHs diversity.

Alcohol Dehydrogenase↗

PubMed2003

Piezotolerance as a metabolic engineering tool for the biosynthesis of natural products.

Thermodynamically, high-pressure (>10's of MPa) has a potentially vastly superior effect on reactions and their rates within metabolic processes than temperature. Thus, it might be expected that changes in the pressure experienced by living organisms would have effects on the products of their metabolism. To examine the potential for modification of metabolic pathways based on thermodynamic principles we have performed simple molecular dynamics simulations, in vacuo and in aquo on the metabolites synthesized by recombinant polyketide synthases (PKS). We were able to determine, in this in silico study, the volume changes associated with each reaction step along the parallel PKS pathways. Results indicate the importance of explicitly including the solvent in the simulations. Furthermore, the addition of solvent and high pressure reveals that high pressure may have a beneficial effect on certain pathways over others. Thus, the future looks bright for pressure driven novel secondary metabolite discoveries, and their sustained and efficient production via metabolic engineering.

Computer Simulation↗

PubMed2003

2-Propanol degradation by Sulfolobus solfataricus.

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 degrees C with or without glucose at 10 g l-1. The presence of 3.92 g 2-propanol l-1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l-1 increased 2-propanol and acetone utilization from 0.93 g l-1 to 1.77 g l-1 and from 0.11 g l-1 to 1.62 g l-1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l-1 h-1, compared to 0.0012 g l-1 h-1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.

2-Propanol↗

PubMed2002

Kinetic analysis of high-concentration isopropanol biodegradation by a solvent-tolerant mixed microbial culture.

The ability of a previously enriched microbial population to utilize isopropanol (IPA) as the sole carbon source within a minimal salts medium is studied. The advantage of prior enrichment procedures for the improvement of IPA biodegradation performance is demonstrated for an IPA concentration of up to 24 g L(-1). Results showing the interrelationship between temperature and substrate utilization and inhibition levels at temperatures of between 2 degrees C and 45 degrees C are examined. Models of inhibition based on enzyme kinetics are assessed via nonlinear analysis, in order to accurately represent the growth kinetics of this solvent-tolerant mixed culture. The model that best describes the data is the Levenspiel substrate inhibition model, which can predict the maximum substrate level above which growth is completely limited. This is the first report of IPA treatment of up to 24 g L(-1) by an aerobic solvent-tolerant population.

2-Propanol↗

PubMed2002

Biodegradation of high-concentration isopropanol by a solvent-tolerant thermophile, Bacillus pallidus.

The aerobic biodegradation of high-concentration, to 24 g l(-1), 2-propanol (IPA) by a thermophilic isolate ST3, identified as Bacillus pallidus, was successfully carried out for the first time. This solvent-tolerant B. pallidus utilized IPA as the sole carbon source within a minimal salts medium. Cultivation was carried out in 100-ml shake flasks at 60 degrees C and compared with cultivation within a 1-l stirred tank reactor (STR). Specific growth rate (micro) was about 0.2 h(-1) for both systems, with a maximum cell density of 2.4 x 10(8) cells ml(-1) obtained with STR cultivation. During exponential growth and stationary phase, IPA biodegradation rates were found to be 0.14 and 0.02 g l(-1) h(-1), respectively, in shake-flask experiments, whereas corresponding values of 0.09 and 0.018 g l(-1) h(-1) were achievable in the STR. Generation of acetone, the major intermediate in aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Acetone levels reached a maximum of 2.2-2.3 g l(-1) after 72 and 58 h for 100-ml and 1-l systems, respectively. Both IPA and acetone were completely removed from the medium following 160 and 175 h, respectively, during STR growth, although this was not demonstrated within shake-flask reactions. Growth of B. pallidus on acetone or IPA alone demonstrated that the maximum growth rate ( micro ) obtainable was 0.247 h(-1) at 4 g l(-1) acetone and 0.202 h(-1) at 8 g l(-1) IPA within shake-flask cultivation. These results indicate the potential of the solvent-tolerant thermophile B. pallidus ST3 in the bioremediation of hot solvent-containing industrial waste streams.

2-Propanol↗

PubMedDate not supplied

Novel approach for peptide quantitation and sequencing based on 15N and 13C metabolic labeling.

Here we describe a method for protein identification and quantification using stable isotopes via in vivo metabolic labeling of the hyperthermophilic crenarchaeon Sulfolobus solfataricus. Stable isotope labeling for quantitative proteomics is becoming increasingly popular; however, its usefulness in protein identification has not been fully exploited. We use both 15N and 13C labeling to create three different versions of the same peptide, corresponding to the unlabeled, 15N and 13C labeled versions. The peptide then appears as three different peaks in a TOF-MS scan and three corresponding sets of MS/MS spectra are obtained. With this information, the elemental carbon and nitrogen compositions for each peptide and each fragment can be calculated. When this is used as a constraint in database searching and/or de novo sequencing, the confidence of a match is increased (for an example intact peptide from 34 choices to 1). This makes the method a useful proteomic tool for both sequenced and unsequenced organisms. Furthermore, it allows for accurate protein quantitation (standard deviations over >4 peptides per protein were within 10%) of three phenotypes in one MS experiment. Abundances for each peptide are calculated by determining the relative areas of each of the three peaks in the TOF-MS spectrum.

Amino Acid Sequence↗

PubMedDate not supplied

Identification and characterization of the Sulfolobus solfataricus P2 proteome.

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.

Archaeal Proteins↗

PubMedDate not supplied

Perturbation and interpretation of nitrogen isotope distribution patterns in proteomics.

This study provides a discussion on the applications and limitations of (15)NH(4)(+) metabolic labeling in proteomic studies. The hyperthemophilic crenarchaeon Sulfolobus solfataricus was used as a model organism throughout this study. The distribution of nitrogen was studied in four different experiments in which this distribution was manipulated in a unique way. The experiments included full adaptation to media with relative isotope abundances (RIA) of 0.36%, 50%, and >98% (15)NH(4)(+). The incorporation efficiency was calculated on the basis of a comparison between theoretical and experimental spectra. In the case of full adaptation, incorporation efficiencies reflected the RIA (0.36%, 47.5% and 99% respectively). Labeling efficiencies were calculated on the basis of peak areas in TOF-MS spectra. It is shown that in the case of full adaptation, labeling efficiencies are 100%. In addition, we demonstrate that (15)NH(4)(+) labeling can be used in protein turnover studies, even when labeling is incomplete. In this case, incorporation efficiencies of 88-93% (lower than the RIA) were measured, providing evidence for amino acid recycling. Labeling efficiencies were always between 63% and 94% providing evidence for protein degradation. Finally, it was shown that isotope distributions can be useful in peptide identification.

Ammonia↗