No abstract

Growth cessation of Escherichia coli in nondialysis fedbatch culture at biomass densities over 30-40 g/L has been shown to be the result of the accumulation of acetate, lactate, pyruvate, succinate, propionate, and isobutyrate in the culture broth.' This conclusion was supported by the fact that high cell concentrations (140 g/L) and cellular yields were achieved in dialysis fed-batch culture of E. coli.According to our results acid fermentation products do not accumulate up to the dilution rate D = pclit = 0.35 h-' in the chemostat culture of E . coli K12 W3350 on glucosesalts medium. But at higher growth rates acid products were formed in substantial amounts (not published). In the literature3 this phenomenon is known as "aerobic growth with byproduct formation." Taking into account these experimental data, we assumed that it could be possible to reach high cell densities also in nondialysis fed-batch culture of E. coli if the byproduct formation could be avoided, i.e., if the growth rate p could be constantly kept below the pCrit,In fed-batch cultivation of microorganisms different strategies could be used to keep the growth rate of the culture below the pent.The physiological models (stoichiometric equations of growth) together with indirect on-line measurements (usually the gas analysis data) could be used to control the growth rate and product formation. The respiratory quotient (RQ) value has been used for monitoring the growth and ethanol production of yeasts .7.8 The simultaneous use of anticipatory and feedback control was shown to be very efficient if there were problems with oxygen starvation, molasses quality, and variable amounts of i n o c~l u m .~ In this article the results of fed-batch cultivation of E. coli K12 W3350 with computer-controlled growth rate p < pmax are presented. The control law was developed assuming that the glucose-limited (s = const = 0) quasisteady-state growth was maintained during the fed-batch phase of the cultivation. The current pumping rate of the fresh glucose-salts medium was calculated from the amount of glucose fed into the fermentor up to the current moment. MATERIALS AND METHODSThe accumulation of byproducts could be constantly monitored and, according to this, the growth rate changed. The monitoring of the level of volatile products was initially used in the development of control laws of that type.4 Later Mathers et al? used a computer-linked highperformance liquid chromatography (HPLC) system for The Control LawTaking into account the material balance of growthlimiting substrate during fed-batch cultivation of cells, the change in the amount of growth-limiting substrate in the fermentor can be described by the equation monitoring the formation of nonvolatile fermentation products of yeasts. The latter system could be used forthe direct on-line control of acid byproduct formation also in E. coli.The accumulation of byproducts could also be prevented by direct measurement of the level of growth-limiting substrate(s) and keeping it at the necessary (low) level6 s...

Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein–protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein–protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that 1H and 15N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein–protein interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.

Complementary techniques elucidate water–framework interactions in different states of dehydration, two of which have not been observed before.

Some applications of NMR and of neutron scattering require fully deuterated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since full deuteration is achieved easily only if cells are grown in minimal media. This condition used in standard batch fermentation results in both low yields and reduced activities of the biological mass. Here we report a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appearance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE600rif (resistance against high doses of rifampicin is used as selection marker) on partially deuterated media (76% and 84% D,O) with glucose as carbon source and on deuterated acetate and succinate with 100% D,O when full deuteration was to be achieved. The essential point for preserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growth rate at low levels (for glucose the growth rate, p 5 0.35 h-', for acetatelsuccinate p 5 0.1 h-') which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular component for accompanying or triggering the transition from logarithmic growth to stationary phase of E. coli cells cultivated on glucose as carbon source.The cells were first grown in fed-batch to high cell densities (above 50 g wet cells/l) under conditions of substrate limitations. A steady-flow fermentation followed keeping the growth rate at about p of 0.1 h-'. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rRNA and were fully active. The ribosomal protein and rRNA fractions could be efficiently reconstituted to highly active particles. In the case of full deuteration a matching point of 120% (tentative D,O scale) was achieved. The reported method facilitates the preparation of deuterated biological material for applications in NMR and neutron scattering analysis.