A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources.

The realisation of a novel concept for automated on-line monitoring of enzymatic activities in water was successfully demonstrated by long-term field testing at two remote Austrian ground water resources. The ß-D-glucuronidase (GLUC) activity was selected as a representative enzymatic model parameter for the on-line determination. But the device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring, as demonstrated for the ß-D-galactosidase activity. Automated filtration of volumes up to 5 litres supports sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enabled robust enzymatic on-line monitoring during a 2-year period. A proportion of 5,313 out of 5,506 GLUC activity measurements (96.5%) could be positively verified. Hydrological (discharge, gauge, turbidity, temperature, pH, electric conductivity, spectral absorbance coefficient at 254 nm) as well as microbiological parameters (Escherichia coli, coliforms) were concurrently determined to characterise the investigated ground water resources. The enzymatic on-line measurements closely reflected the different hydrological conditions and contamination patterns of the test sites. Contrary to expectations, GLUC did not qualify as a proxy-parameter for the occurrence of cultivation-based E. coli contamination and warrants further detailed investigations on its indication capacity as a rapid means for microbial faecal pollution detection in such aquatic habitats. Microbial on-line monitoring is likely to become more important in the future, complementing existing surveillance strategies for water safety management. Further perspectives on the application of such analytical on-line technologies, such as their connection with event-triggered sampling and standardised diagnostics, are discussed.

Characterization and properties of protein kinase C from the filamentous fungus Trichoderma reesei.

The Trichoderma reesei pkc1 gene encodes a fungal homologue of the protein kinase C (PKC) family. Using antibodies directed against the nt-sequence-deduced pseudosubstrate domain for identification, Pkc1p was purified by dye-ligand affinity chromatography and Mono Q anion-exchange chromatography. Both the denatured as well as the native enzyme showed an Mr of 116-118kDa, indicating that Pkc1p is a monomer. The enzyme phosphorylates the mutated (A-->S) pseudosubstrate peptide and myelin basic protein, but not histone. Replacing three of the five basic amino acids around the serine acceptor residue resulted in a 25-fold increase in the Km. Pkc1p activity was stimulated by phospholipids, but this stimulation was counteracted by micromolar concentrations of Ca2+. Three proteins (85, 48 and 45 kDa) were identified as preferred endogenous substrates of Pkc1p in vitro. The enzyme was capable of autophosphorylation, and neither phosphorylation nor dephosphorylation in vitro affected the activity of the enzyme. A 116 kDa protein of T. reesei was demonstrated to bind to the N-terminal C2-region of Pkc1p in vitro. These data define Pkc1p as a unique member of the PKC family.

Cloning and characterisation of genes (pkc1 and pkcA) encoding protein kinase C homologues from Trichoderma reesei and Aspergillus niger.

Oligonucleotides, designed on the basis of conserved flanking amino acid sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain reactions to amplify a 427-bp chromosomal DNA fragment from the filamentous fungus Trichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues of T. reesei and Aspergillus niger (pkc1 and pkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position with those found in the corresponding Schizosaccharomyces pombe pkc1+ and Drosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T.reesei, 126 kDa) and PKCA (A. niger, 122 kDa) amino acid sequences reveal domains homologous to the C1 and C3/C4 domains of PKC-related proteins, but lack typical Ca(2+)-binding (C2) domains. Both contain a large, extended N-terminus, which shares a high degree of similarity with the corresponding regions of Saccharomyces cerevisiae PKC1 and S. pombe pkc1+ and pkc2+ proteins, but which is not present in PKCs of Dictyostelium or higher eukaryotes. This extended region can be divided into three subdomains; the N-terminal one contains a hydrophobic helix-turn-helix motif, whereas the C-terminal one contains potential targets for proteolytic processing. A polyclonal antiserum raised against the pseudosubstrate-binding domain of PKC1 recognizes in T. reesei a 115-120 kDa protein in Western blots. Expression of pkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. The T. reesei PKC1 protein was partially purified and some of its properties examined: it is stimulated about twofold by phospholipids or phorbol esters but is not stimulated by Ca2+. We conclude that these PKC proteins from filamentous fungi represent the Ca(2+)-insensitive fungal homologues of the nPKC family.

A protein kinase-encoding gene, pkt1, from Trichoderma reesei, homologous to the yeast YPK1 and YPK2 (YKR2) genes.

A gene (pkt1) was isolated from the filamentous fungus Trichoderma reesei, which exhibits high homology with the yeast YPK1 and YKR2 (YPK2) genes. It contains a 2123-bp ORF that is interrupted by two introns, and it encodes a 662-amino-acid protein with a calculated M(r) of 72,820. During active growth, pkt1 is expressed as two mRNAs of 3.1 and 2.8 kb which differ in the 3' untranslated region due to the use of two different polyadenylation sites.

Subcellular compartmentation of penicillin biosynthesis in Penicillium chrysogenum. The amino acid precursors are derived from the vacuole.

The cellular localization of the origin of alpha-aminoadipate used in penicillin biosynthesis and the first enzymic step in Penicillium chrysogenum involved, delta-(alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), has been studied. Subcellular fractions were obtained from protoplasts of a high penicillin-producing strain upon lysis by Triton X-100, and vacuoles purified from them. They were identified by the aid of alpha-mannosidase as a marker enzyme, by the presence of polyphosphate, and their ability to sequester [14C]lysin, added to the protoplasts prior to subcellular fractionation. 15.6 and 26.5%, respectively, of 6-[14C]alpha-aminoadipate, and 8.5 and 10.3%, respectively, of [14C]valine added accordingly were also found in the vacuole, and the higher proportion was found in vacuoles isolated from penicillin-producing mycelia. ACVS protein was detected in the membrane as well as the soluble fraction of the purified vacuoles. We propose therefore that ACVS is located either within or bound to the vacuolar membrane, and that the precursor amino acids for penicillin biosynthesis are withdrawn from the vacuolar amino acid pool.