Evaluation of multiwavelength culture fluorescence for monitoring the aroma compound 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) production.

Fluorescence spectra of a 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) fermentation culture broth were combined with measurable process variables for off-line and on-line process monitoring. Culture broth fluorescence in UV and visible ranges was acquired by a fiber optic LCD array spectrometer. Process dynamics was followed on-line using a fiber optic probe attached to an external recirculation loop of the bioreactor. Partial least squares and stepwise regression methods were used to correlate measurable process parameters with the components of the fluorescence spectra. Both methods provided adequate approximation of yeast density, HEMF, glucose, and ethanol concentrations from fluorescence spectra. HEMF production was observed during the oxido-reductive growth phase when there was a lack of measurable oxygen in the culture broth and an excess of glucose. The addition of glucose resulted in the rapid production of HEMF and other metabolite intermediates such as ethanol, acetate, and glycerol.

NMR structural studies of human cystatin C dimers and monomers.

Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, "trapped" dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H -15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae.

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45 degrees to 47 degrees C, maximum protein yield at 45 degrees C, and no growth at 25 degrees C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45 degrees C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10 to 10 per animal) injected by various routes into normal and gamma-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.

Sequential cold-sensitive mutations in Aspergillus fumigatus. II. Analysis by the parasexual cycle.

From Aspergillus fumigatus I-21 (ATCC 32722), which grows at temperatures from 12 to 50 degrees C, three multistep, independently derived, cold-sensitive mutants unable to grow at 37 degrees C or below (Cs-37) were obtained by sequential exposure to ethylmethane sulfonate (strain AT2) or N-methyl-N'-nitro-N-nitrosoguanidine (AT1 and AT3). These mutants and ON5, a five-step Cs-37 mutant, were marked by mutations affecting spore color and nutritional requirements and crossed in four combinations by classical parasexual means. The heterokaryons demonstrated partial complementation with respect to auxotrophic requirements (suboptimal growth on minimal medium) and cold sensitivity (growth at 37 degrees C but not at 25 degrees C). Most presumed diploids, formed by exposure of the heterokaryons to d-camphor vapors, showed complete complementation but were unstable, as demonstrated by variations in spore sizes and markedly different ratios of segregant classes derived from different clones. Analysis of the segregants of the diploids or aneuploids, induced by Benomyl, indicated that multiple genes were responsible for cold sensitivity in each Cs-37 mutant, since segregants with various levels of cold sensitivity were obtained. The higher than predicted frequency of reversion to temperatures two or more steps back in the sequence of cold sensitivity mutations suggested that these genes or their products interacted. No Cs-37 segregant yielding a consistently lower frequency of revertants than the original mutants was obtained.