Biomedical subjects

Karl-Heinz Maurer

Publications and source records attributed to Karl-Heinz Maurer.

6 recordsLinked to original sources

PubMed2006

Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases.

Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

Peptide Hydrolases↗

PubMed2006

The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions.

The now finished genome sequence of Bacillus licheniformis DSM 13 allows the prediction of the genes involved in protein secretion into the extracellular environment as well as the prediction of the proteins which are translocated. From the sequence 296 proteins were predicted to contain an N-terminal signal peptide directing most of them to the Sec system, the main transport system in Gram-positive bacteria. Using 2-DE the extracellular proteome of B. licheniformis grown in different media was studied. From the approximately 200 spots visible on the gels, 89 were identified that either contain an N-terminal signal sequence or are known to be secreted by other mechanisms than the Sec pathway. The extracellular proteome of B. licheniformis includes proteins from different functional classes, like enzymes for the degradation of various macromolecules, proteins involved in cell wall turnover, flagellum- and phage-related proteins and some proteins of yet unknown function. Protein secretion is highest during stationary growth phase. Furthermore, cells grown in complex medium secrete considerably higher protein amounts than cells grown in minimal medium. Limitation of phosphate, carbon and nitrogen sources results in the secretion of specific proteins that may be involved in counteracting the starvation.

Amino Acid Sequence↗

PubMed2006

The phosphate-starvation response of Bacillus licheniformis.

The phosphate-starvation stimulon of Bacillus licheniformis was analyzed at the transcriptional and translational level. The comparison of the transcriptome and the proteome demonstrated that this specific starvation response of B. licheniformis is partially similar to that of B. subtilis. However, it is also shown that B. licheniformis has evolved its own strategies to cope with this nutrient limitation. By means of the secretome analysis the phytase was identified as the most abundant protein under phosphate-starvation conditions. Data of this study indicate that, unlike in B. subtilis, phosphate starvation in B. licheniformis does not induce the SigmaB-dependent general stress response.

6-Phytase↗

PubMed2004

A proteomic view of cell physiology of Bacillus licheniformis.

The still ongoing sequencing of Bacillus licheniformis at the Göttingen Sequencing Laboratory provides the basis for proteome studies of the bacterium. By using two-dimensional (2-D) electrophoresis and protein identification by mass spectrometry, we were able to create master gels for B. licheniformis cells grown either in minimal medium or in complex medium containing about 300 and 180 entries, respectively. With the DECODON Delta 2D software we identified the most abundant protein spots on the gels, which were shown to perform mainly basic metabolic functions in the cell such as translation, amino acid metabolism, glycolysis, and tricarboxylic acid (TCA) cycle. Based on the master gels, we were able to study the regulation of metabolic pathways such as glycolysis and TCA cycle. In cells grown in the presence of glucose a significant increase of the amount of some glycolytic enzymes (TpiA, GapA, Pgk, Pgm, Eno, Pyk) and of the pyruvate dehydrogenase (PdhA-D) was found. At the same time, there is a strong repression of almost all TCA cycle enzymes and of the ATP synthase. Glucose also stimulates the acetate kinase (AckA) and the phosphotransacetylase (Pta) which are known to be involved in the overflow metabolism in B. subtilis. Furthermore, we began developing proteomic signatures for growth of B. licheniformis in complex medium. For this purpose, we compared the proteome pattern of exponentially growing cells with that of cells in different stages during stationary phase. The most obvious proteomic signature indicates that cells during stationary phase are subjected to a severe oxidative stress and a resulting protein stress. Furthermore, the level of many vegetative proteins is strongly reduced when the growth is arrested after entry into stationary phase. The data indicate that proteomics can be a valuable tool to describe the physiological state of B. licheniformis cell populations, e.g., of cells growing in a bioreactor.

Bacillus↗

PubMed2004

Detergent proteases.

Over the past 20 years, the development of subtilisins as typical detergent proteases has employed all the tools of enzyme technology, resulting in a constant flow of new and improved enzymes. The number of molecules identified and characterized, however, is in clear opposition to the number of molecules that are entering the market. Will the next-generation detergent proteases be based on new backbones different from subtilisins, or will the use of all available technologies (rational design, directed evolution and exploitation of natural diversity) yield improved subtilisins, ending the current era dominated by high alkaline subtilisins? These questions will have to be answered not only by the performance of the molecules themselves, but also by their yield in fermentation and their compatibility with existing production technologies.

Bacteria↗

PubMed2003

Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity.

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.

Amino Acid Sequence↗