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Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement. James B. Garrett, 2004.The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus . An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance . For this study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology . Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance . Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen . The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62°C for 1 h and 27% of its initial activity after 10 min at 85°C, which was a significant improvement over the appA parental phytase . Phy9X also showed a 3.5-fold enhancement in gastric stability . Differential Expression of the Escherichia coli Autoaggregation Factor Antigen 43. Mark A. Schembri, 2003.Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains . Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation . Ag43 expression is repressed by the cellular redox sensor OxyR . Here we used mutant versions of OxyR that are locked in either the reduced or the oxidized form as well as the addition of a simple redox-changing chemical to show that the redox state of OxyR influences Ag43 expression . Furthermore, the redox state of OxyR influences the biofilm-forming potential of E . coli . Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing . Rapid Identification of Candida Species by Using Nuclear Magnetic Resonance Spectroscopy and a Statistical Classification Strategy. Uwe Himmelreich, 2003.Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification . Major metabolites were identified by using two-dimensional correlation NMR spectroscopy . One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy . Analysis of NMR spectra from 442 isolates of Candida albicans, C . glabrata, C . krusei, C . parapsilosis, and C . tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification . Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites . Isolates of C . parapsilosis and C . glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity . We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms .
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