Kirsten Biedermann

Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli.

The primary structure and physical chemical properties were determined of a nuclease expressed and secreted by Escherichia coli. The plasmid p403-SD2 carried a DNA sequence isolated from Serratia marcescens encoding the enzyme. During cultivation of the E. coli cells, 85% of the enzyme was released to the growth medium. The enzyme was purified and exhibited a single band with a molecular weight about 30,600 daltons on SDS-PAGE similar to nuclease isolated from S. marcescens. The amino acid composition and the amino acid sequence determined directly confirmed the primary structure of 245 amino acids predicted from the DNA sequence, and, in addition, the two disulfide bridges were assigned. Several physical chemical properties were examined. The ability of the enzyme to cross the outer membrane is proposed to depend upon the formation of the proper structures during the folding process.

Characterization of Serratia marcescens nuclease isoforms by plasma desorption mass spectrometry

Isoforms of Serratia marcescens nuclease found in the natural nuclease produced by S. marcescens and in recombinant nuclease produced by Escherichia coli were structurally characterized by peptide mapping using plasma desorption mass spectrometry. The nuclease isoforms produced and secreted from S. marcescens B10M1, which are present in much greater amounts than in S. marcescens W225 nuclease produced by E. coli, were characterized completely and the information used to facilitate characterization of the recombinant nuclease isoforms. After purification of the nuclease the isoforms were separated on a DEAE-cellulose anion-exchange column and then digested with endoproteinase Lys-C. The peptides generated were isolated by reverse-phase HPLC and their molecular masses determined by plasma desorption mass spectrometry. Comparison of the peptides from the native nuclease, Sm2, and the two isoforms, Sm1 and Sm3, revealed that they differed only in the N-terminus, the latter being found to lack three amino acids in Sm1 and one amino acid in Sm3. No interior post-translational changes were found in either of the three isoforms. Using this information we were able to confirm that Sm1, the isoform lacking three amino acids, was also present in very small amounts in recombinant S. marcescens W225 nuclease produced and excreted by E. coli.

Separation of isoforms of Serratia marcescens nuclease by capillary electrophoresis.

Three S. marcescens nuclease isoforms differing mainly in charge (native nuclease with pI 6.8 and two minor isoforms with pI 7.3 and 7.4) were separated using several different modes of high-performance capillary electrophoresis. Separation of the isoforms by free solution capillary electrophoresis was unsatisfactory. Separation by micellar electrokinetic capillary chromatography was therefore investigated in detail and the method optimized with respect to pH and sodium dodecyl sulphate concentration; in addition, the effect of adding various substances to control dispersion and avoid analyte adsorption at the capillary wall was examined. Under optimal conditions there was almost complete baseline separation of the two isoforms with basic pI whereas there was only partial separation of the native form and the isoform with pI 7.4. With capillary isoelectric focusing there was complete baseline separation of the native nuclease and the other two isoforms.

Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: Experiments and computer simulations

Overproduction of proteinase A by recombinantSaccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Memebrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric productivities were at high dilution rates just below washout and at high ratios of recycling. Cell-recycling fermentation gave much higher volumetric productivities and stable product concentrations in contrast to simple continuous fermentation.

Mathematical Modeling of Proteinase A Overproduction by Saccharomyces cerevisiae

A simple, structured model was developed to describe the growth and product formation behavior of two recombinant strains of Saccharomyces cerevisiae (JG176 and JG180), both overproducing extracellular proteinase A. The model parameters were estimated to data from continuous fermentations obtained at steady-state conditions. Model predictions show good agreement with experimental data obtained by batch fermentations. The two concerned organisms are distinguished from each other by the type of promoter on the plasmids controlling the proteinase A expression. The proteinase A transcription is controlled by the natural proteinase A promoter in JG176 and by a tpi promoter in JG180. By means of experiments and simulations, the extracellular product formation from the two strains with different promoter systems was compared in batch and continuous fermentations. The results showed that the proteinase A formation kinetic from JG176 was a combination of growth and nongrowth associated (production in the stationary growth phase), whereas the proteinase A formation from JG180 was truly growth associated (production in the exponential growth phase). In both batch and continuous cultivations JG176 gave the highest product concentrations and volumetric productivities.

Enzyme production in a cell recycle fermentation system evaluated by computer simulations

Enzyme production in a cell recycle fermentation system was studied by computer simulations, using a mathematical model of α-amylase production by Bacillus amyloliquefaciens. The model was modified so as to enable simulation of enzyme production by hypothetical organisms having different production kinetics at different fermentation conditions important for growth and production. The simulations were designed as a two-level factorial assay, the factor studied being fermentation with or without cell recycling, repression of product synthesis by glucose, kinetic production constants, product degradation by a protease, mode of fermentation, and starch versus glucose as the substrate carbon source.The main factor of importance for ensuring high enzyme production was cell recycling. Product formation kinetics related to the stationary growth phase combined with continuous fermentation with cell recycling also had a positive impact. The effect was greatest when two or more of these three factors were present in combinations, none of them alone guaranteeing a good result. Product degradation by a protease decreased the amount of product obtained; however, when combined with cell recycling, the protease effect was overshadowed by the increased production. Simulation of this type should prove a useful tool for analyzing troublesome fermentations and for identifying production organisms for further study in integrated fermentation systems.

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of α-amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased α-amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on α-amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of α-amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of α-amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h−1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.

Purification of recombinant proteins by immunoaffinity chromatography with preselected antibodies

Nuclease produced by recombinant Escherichia coli was successfully purified by immunoaffinity chromatography using an antibody preselected for the purpose on the basis of an elution assay. The elution assay was also used to optimize elution conditions. One step recovery of nuclease from crude periplasmic extract was 66%.

The Use of Antibodies for Characterization and Quantification of a Recombinant Proteina

In this study, we characterized proteinase A secreted by recombinant Saccharomyces cerevisiae bearing a multicopy plasmid containing the encoding gene (PEP4). Polyclonal and monoclonal antibodies were raised to study the product heterogeneity. Characterization of proteinase A was performed by immunoelectrophoresis and immunoblotting techniques. None of the monoclonal antibodies raised against proteinase A was found to react with the glycosyl side chains; thus cross-reaction with other glycosylated proteins (e.g. carboxypeptidase Y) was very low. This study allowed us to develop an ELISA method for the quantification of proteinase A in culture supernatants as well as the evaluation of monoclonal antibodies for their use in immunoaffinity chromatography.

α-Amylase production by Bacillus amyloliquefaciens in a membrane recycle bioreactor

A strain of Bacillus amyloliquefaciens was cultivated in a membrane recycle bioreactor for the production of an extracellular α-amylase. Continuous cultivations of B. amyloliquefaciens in the recycle fermentor led to higher cell mass and volumetric productivities than the ones obtained in batch or chemostat cultivations; the α-amylase activities were lower than in the batch mode but significantly higher than in the chemostat mode. The operation of the membrane recycle bioreactor was sometimes disturbed by high broth viscosity leading to a stronger fouling of the membrane.