Optimization of oligomeric enzyme activity in ionic liquids using Rhodotorula glutinis yeast phenylalanine ammonia lyase.

Phenylalanine ammonia lyase (E.C.4.3.1.24, PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO4]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF6]) is comparable to that obtained in aqueous buffer medium. Different conditions were optimized for maximal PAL forward activity including time of incubation (30.0min)L-phenylalanine substrate concentration (30.0mM), nature of buffer (50.0mM Tris-HCl), pH (9.0), temperature (37°C), and speed of agitation (100 rev min-1). Under these optimized conditions, about 83% conversion of substrate to product was obtained for the PAL forward reaction that was determined using UV spectroscopy at 290nm. PAL reverse reaction in ([BMIM][PF6]) was determined spectrophotometrically at 520nm; and about 59% substrate conversion was obtained. This data provides further knowledge in enzyme biocatalysis in non-aqueous media, and may be of importance when studying the function of other oligomeric/multimeric proteins and enzymes in ionic liquids.

Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity.

Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 µM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications.

Density of river otters (Lontra canadensis) in relation to energy development in the Green River Basin, Wyoming.

Exploration and extraction of oil and natural gas have increased in recent years and are expected to expand in the future. Reduction in water quality from energy extraction may negatively affect water supply for agriculture and urban use within catchments as well as down river. We used non-invasive genetic techniques and capture-recapture modeling to estimate the abundance and density of North American river otters (Lontra canadensis), a sentinel species of aquatic ecosystems, in Southwestern Wyoming. While densities in two of three river reaches were similar to those reported in other freshwater systems in the western US (1.45-2.39 km per otter), otters appeared to avoid areas near energy development. We found no strong difference in habitat variables, such as overstory cover, at the site or reach level. Also, fish abundance was similar among the three river reaches. Otter activity in our study area could have been affected by elevated levels of disturbance surrounding the industrial gas fields, and by potential surface water contamination as indicated by patterns in water conductivity. Continued monitoring of surface water quality in Southwestern Wyoming with the aid of continuously recording devices and sentinel species is warranted.

Enzyme activity evaluation of organic solvent-treated phenylalanine ammonia lyase.

The direct one-step synthesis of L-phenylalanine methyl ester in an organic-aqueous biphasic system using phenylalanine ammonia lyase (E.C.4.3.1.5, PAL) containing Rhodotorula glutinis yeast whole cells was reported earlier. We report here further optimization of this biotransformation using isolated PAL, when the lyophilized enzyme is treated with different water miscible and water immiscible organic solvents. Use of isolated PAL enzyme is advantageous in overcoming diffusion barriers encountered when using PAL containing R.glutinis whole cells, and resulted in increased product yield due to better interaction of enzyme with the substrate. Among the water miscible solvents, ethanol treated and methanol-treated enzymes supported maximum PAL forward and reverse activities; respectively. In the water immiscible solvents category, heptane-treated enzyme exhibited maximal activity for both PAL forward and reverse reactions. PAL activity obtained with enzyme specimens treated with methanol, ethanol, and heptane varied in the range of 91­99% of that observed in aqueous buffer medium for the forward reaction; and 89­95% for the reverse reaction. n-butanol,acetone, and benzene were found to have a inhibitory effect on PAL enzyme, in that, it resulted in only 31­33% activity of that obtained with aqueous solution. Raman spectroscopy was used to monitor amide I and II bands which are sensitive to changes in the secondary structure of proteins. No changes in structure could be detected from the analyses of AI and AII bands of PAL spectra. This data obtained for PAL, a tetramer, could be significant in predicting how solvent interactions affect the structure and function of multimeric proteins and enzymes in nonaqueous media.

Purification of peroxidase from Horseradish (Armoracia rusticana) roots.

Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.

Manganese (Mn2+)-dependent storage stabilization of Rhodotorula glutinis phenylalanine ammonia-lyase activity.

Phenylalanine ammonia-lyase (PAL; E C 4.3.1.5) reverse reaction has been exploited for the commercial production of optically pure l-phenylalanine from trans-cinnamic acid. Optimal conditions for the growth and PAL activity of Rhodotorula glutinis cells and an improved method for the synthesis of l-phenylalanine have been reported. A major problem encountered during these studies was rapid loss of PAL activity during storage of the yeast cells, which were therefore unsuitable for long-term and repeated use. Enhancement of enzyme stability in the presence of various additives including polyhydric compounds and metal ions is described. Whole cells retained nearly 85% of the original enzyme activity for at least 12 weeks when a low concentration of Mn2+ (0.01%) was included in the storage buffer medium (50 mM Tris-HCl, pH 8.8). In contrast, <3.0% activity was present in the control within 4 weeks. Mn2+-dependent stabilization of PAL was also observed with an isolated enzyme preparation (73% retention in activity for 12 weeks) obtained by ultrasonication of R. glutinis whole cells. The data suggest that Mn2+ ions may be responsible for the specific stabilization of a more active conformation of the enzyme. In addition, enzyme stability as a function of temperature was studied, and the optimal temperature for maximal activity retention was 0-2 degrees C. The effects of various additives on the induction of PAL have also been examined. These results could have direct implications in studies on activity, inhibition, and reaction mechanism of this biotechnologically important enzyme.

A modern view of phenylalanine ammonia lyase.

Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure L-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (L-phenylalanyl-L-aspartyl methyl ester). The enzyme's natural ability to break down L-phenylalanine makes PAL a reliable treatment for the genetic condition phenylketonuria. In this mini-review, we discuss prominent details relating to the physiological role of PAL, the mechanism of catalysis, methods of determination and purification, enzyme kinetics, and enzyme activity in nonaqueous media. Two topics of current study on PAL, molecular biology and crystal structure, are also discussed.

Cash management and revitalization of public medical centres in Nigeria: a strategic analysis.

In times like this, when Nigeria (like many other developing countries) is bracing up to the contemporary challenges posed by adoption and advancement of globally driven millennium development goals (MDGs), public medical centers (PMCs) cannot afford to be reckoned with financial epilepsy, bankruptcy, and degeneracy. This concern informed the thrust of the study. In the process, pertinent research questions were posed which elicited corresponding hypothetical propositions. With primary data volunteered by 150 administrative officials drawn from PMCs across the country, analytical proceedings were facilitated by the application of chi-square (x2) technique. The findings brought to the fore, the general bad shape of cash management in PMCs in the country. The recommendations for urgent attention underscored the constitution of strategic budget communities (SBCs), revitalization of internal audit committees (IACs), and attraction of goodwill private-sector endowments through convincing justification of the utilization and optimization of current government logistic subventions.

A protein interaction map of Drosophila melanogaster.

Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid-based protein-interaction map of the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions. Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a starting point for a systems biology modeling of multicellular organisms, including humans.

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

Phenylpyruvic acid may be a direct precursor of mandelic acid without intermediate transamination of phenylalanine.

A series of compounds related to phenylalanine was administered to rats. Output of mandelic acid, a major but unexplained metabolite in phenylketonuria, was increased after the administration of phenylethanolamine or phenylpyruvic acid, whereas phenylethylamine or phenylalanine increased its excretion only marginally. Phenylacetic acid, previously suggested as a possible precursor in man, was almost without effect. It seems likely that mandelic acid can be formed from phenylpyruvic acid directly, without intermediate transamination to phenylalanine.