Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC).

Background: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days.

Results: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies.

Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria.

Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e.

Software sensors for fermentation processes.

Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R(O/S) sensor which is the ratio between oxygen and energy substrate consumption. The R(O/S) variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R(O/S) sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy.

Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies.

Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed.

Confirmative electric DNA array-based test for food poisoning Bacillus cereus.

Detection of the full set of toxin encoding genes involved in gastrointestinal diseases caused by B. cereus was performed. Eight genes determining the B.

Process technology for production and recovery of heterologous proteins with Pichia pastoris.

Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes.

Interfacing Pichia pastoris cultivation with expanded bed adsorption.

For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density.

Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels.

Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth.

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP.

Gene-based identification of bacterial colonies with an electric chip.

A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method.

Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry.

Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6-48 mg PMP/g CDW) whilst highlighting population heterogeneity.

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes.

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB.

Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris.

The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam.

Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process.

The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e.

Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes.

In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u.

Control of endotoxin release in Escherichia coli fed-batch cultures.

High amounts of outer membrane (OM) components were released in glucose-limited fed-batch (GLFB) cultures at 37 degrees C at specific growth rates approaching 0.05 h(-1). Endotoxin analyses from a 20 degrees C GLFB culture gave similar results.

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli.

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales.

Analysis and control of proteolysis of recombinant proteins in Escherichia coli.

Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli. Important properties of E. coli proteases, which are relevant for the production of recombinant proteins, are reviewed.

Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip.

BACKGROUND: Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains.

Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips.

Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed.

Escherichia coli high-cell-density culture: carbon mass balances and release of outer membrane components.

Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO(2), and acetate was approximately 90% at the end of the culture (25 h, 27 g L(-1) dw).

Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures.

BACKGROUND: A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut+ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. RESULTS: A protein concentration of about 1 g L-1 was produced in the MLFB process.

Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes.

A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.

Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli.

Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process.

Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by (13)C NMR spectroscopy.

Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia coli from 0.67 to 0.