Release of periplasmic enzymes and other physiological effects of beta-lactamase overproduction in Escherichia coli

When Escherichia coli containing the plasmid ptac11 is induced with 10(-4) M isopropyl-beta-thiogalactopyranoside (IPTG), 90% of the beta-lactamase activity of an overnight culture is present in the medium. The high extracellular activity of beta-lactamase does not result from cell lysis but from an increase in the permeability of the outer membrane. The excreting cells release several other periplasmic enzymes into the extracellular fluid and are more sensitive to lysis by detergents. It was also shown that in these cells the level of two membrane proteins, OmpA and OmpC, is decreased. None of these phenomena were observed with the plasmid pDW17, which has a mutation in the tac promoter that reduces its activity to one fourth of the tac promoter.     

Improved performance in viscous mycelial fermentations by agitator retrofitting

For viscous mycelial fermentations it was demonstrated at the pilot-plant scale that the replacement of standard radial flow Rushton turbines with larger diameter axial-flow Prochem hydrofoil impellers significantly improved oxygen transfer efficiency. It was also determined that the Streptomyces broth under evaluation is highly shear thinning. Separate experiments using a Norcardia broth with similar Theological properties demonstrated that the oxygen transfer coefficient, K(L)a, can be greatly increased by use of water additions to reduce broth viscosity. These observations are consistent with the hypothesis that the improvement in oxygen transfer by changing agitator types is primarily due to an improvement in bulk mixing. A model is presented, based on the concepts of Bajpai and Reuss, which explains this improvement in performance in terms of enlargement of the well mixed micromixer region for viscous mycelial broths.     

New instrument for on-line viscosity measurement of fermentation media

In an attempt to resolve the difficult problem of on-line determination of the viscosity of non-Newtonian fermentation media, the authors have used a vibrating rod sensor mounted on a bioreactor. The sensor signal decreases nonlinearly with increased apparent viscosity. Electronic filtering of the signal damps the interfering effect of aeration and mechanical agitation. Sensor drift is very low (0.03% of measured value per hour).On the rheological level the sensor is primarily an empirical tool that must be specifically calibarated for each fermentation process. Once this is accomplished, it becomes possible to establish linear or second-degree correlations between the electrical signal from the sensor and the essential parameters of the ferementation process in question (pH of a feremented milk during acidification, concentration of extra cellular polysaccharide). In addition, by applying the power law to describe the rheological behavior of fermentation media, we observe a second-order polynomial correlation between the sensor signal and the behavior index (n).     

Effect of cycling on the stability of plasmid-bearing microorganisms in continuous culture

The implication of the possible existence of differences in the times required for plasmid-bearing and non-plasmid-bearing microorganisms to adjust their metabolic activities to step changes in their environment is examined. This adaptability difference suggests the possibility of maintaining an engineered strain in continuous culture by transient operation. It is shown for the case where adaptability is neglected that no cycling strategy will prevent the washout of the engineered strain, but the addition to the model of a time delay in substrate utilization can result in coexistence upon cycling. Numerical simulations of cycling in feed substrate concentration are carried out to illustrate the concept Operating diagrams are also constructed to indicate the conditions under which washout of the plasmid bearing strain can be prevented.     

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.     

Coexistence of S. cerevisiae and E. coli in chemostat under substrate competition and product inhibition

A mixed culture of Saccharomyces cerevisiae and Escherichia coli was established in a stable coexistence steady state in a chemostat under constant operating conditions. The species competed for glucose, the growth-limiting resource, and produced acetate and ethanol. The acetic acid was shown to be very inhibitory to E. coli in pure culture at pH 5 while ethanol inhibition was only marginal. No significant inhibition of S. cerevisiae growth was observed by either acetate or ethanol. Pure culture parameters were measured and used in the analysis. Linearized stability analysis for the case when both organisms produce the inhibitor showed that a transition through three stable outcomes was possible as the feed concentration is lowered. Experimental studies verified these predictions, and successive transitions from a yeast growth steady state, to a coexistence steady state, and to an E. coli growth steady state were obtained by lowering the glucose concentration in the feed from 10 to 5 to 2.5 g/L, respectively. This dynamic behavior is distinct from the outcomes of other competition-inhibition combinations and experimentally demonstrates for the first time that coexistence is possible due to substrate competition and product inhibition.     

Effect of SO(2) and O(3) on Production of Antioxidants in Conifers

Production of antioxidants was investigated in needles of fir (Abies alba Mill.) and spruce (Picea abies (L.) Karst) after exposure to low concentrations of SO₂, O₃, and a combination of both pollutants. Glutathione reacted most sensitively to pollutants followed by vitamin E and vitamin C. In spruce needles, the overall increase of antioxidants after exposure to air pollutants was lower than in needles of fir. SO₂ was more potent than O₃. Maximum increase of antioxidants was found in needles after exposure of trees to SO₂ + O₃.     

Purification and measurement of abscisic Acid and indoleacetic Acid by high performance liquid chromatography

A procedure was selected for the simultaneous extraction and purification of abscisic acid (ABA) and indoleacetic acid (IAA). Unnecessary steps were eliminated and an accumulation of aqueous phase was avoided. The superior performance of diethyl ether (compared to ethyl acetate) for bulk purification and the superior resolution provided by 250 millimeter columns packed with 5-micrometer spherical particles of strong anion exchanger and octadecylsilane (C18) greatly facilitated the purification of samples. A fixed-wavelength (254 nanometer) ultraviolet detector and a fluorescence detector connected in series on a high performance liquid chromatograph permitted nondestructive monitoring and measurement of ABA and IAA. Derivatization was not necessary for chromatography or for detection. Isocratic elution with simple mobile phases gave sharp peaks. A few simple precautions minimized losses. Recoveries through the entire procedure averaged about 75% for ABA and about 50% for IAA. Purified ABA and IAA fractions were usually free of interfering contaminants. Identities were confirmed by gas chromatography-mass spectrometry.