The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.

Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose-galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucosegalactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment. INTRODUCTIONGlucose and fructose are preferred sugars for Saccharomyces cerevisiae. Apparently, evolution of this yeast in natural environments that are rich in these sugars (e.g. fruit and nectar) has led to a complicated, multilayered regulatory programme that only enables metabolism of alternative carbon sources (e.g. maltose, ethanol and galactose) when these preferred carbon sources are dwindling.In the case of galactose, a four-enzyme metabolic route, the Leloir pathway, has to be expressed to enable its catabolism. The structural genes for galactose permease (GAL2), galactokinase (GAL1), galactose-1-phosphate uridylyltransferase (GAL7) and uridine-diphosphoglucose 4-epimerase (GAL10) all belong to the GAL regulon, which is subject to tight transcriptional regulation. Glucose causes a nearly complete transcriptional repression of the GAL genes, mediated by the non-phosphorylated form of Mig1, and thereby effectively shuts down galactose utilization (Johnston et al., 1994). Induction of the GAL genes is initiated by interaction of galactose and ATP with Gal3, which then forms a complex with the negative regulator Gal80 (Platt & Reece, 1998). This releases the positive transcriptional regulator Gal4 from Gal80 control and allows it to activate transcription of the GAL1, GAL2, GAL7 and GAL10 genes, which contain upstream activation sequences (UAS GAL ) in their promoter regions (Leuther & Johnston, 1992; Wu et al., 1996). Consequently, S. cerevisiae cells grown in batch cultures on glucose/galactose mixtures show diauxic utilizat...

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A new method to determine the blockiness and especially the block length distribution (BLD) of copolymers is described. This Systematic Workflow to Analyze Multi-fragmented Polymers with Mass Spectrometry (SWAMP-MS) is developed to characterize the sequence distribution of synthetic polymers.Copolymerization of polyamide 4,6 and polyamide 4,10 is used as a model system. Supercharged polymer ions, generated by electrospray ionization, swamp the mass spectrum due to <1> different chemical related distributions (chain composition, end groups), <2> the number of repeating units, <3> the charge and adduct distribution, and <4> isotopes distribution. Without selecting specific substrate ions, MSMS will transform this total ion-SWAMP into chemical rich information of the polymer backbone. The generated fragments contain up to 20 monomers. The reduction of blockiness is evaluated by the decreasing abundancy of specific ions originating from the starting polymers. Moreover, evaluation of all significant fragments and applying Monte Carlo simulations can rebuild the polymer backbone and generate sequence distributions of the copolymerized samples, which is currently still a holy grail in co-chemical polymer analysis.