Both developmental and metabolic signals activate the promoter of a class I patatin gene

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

Continuous microbial desulfurization of coal--application of a multistage slurry reactor and analysis of the interactions of microbial and chemical kinetics

Microbial desulfurization of coal by pyrite oxidizing bacterial enrichment cultures has been studied in air-agitated slurry reactors of 4- and 20-L volumes. Batch experiments showed that inoculation with an active bacterial culture is essential to minimize the lag phase, although a considerable number of pyrite oxidizing bacteria was found on the coal prior to desulfurization. For detailed investigations of kinetics, energy requirements, and technical applicability, a bioreactor equipment consisting of a cascade of eight stages was developed and operated continuously. Microbial desulfurization of coal-monitored by measuring the axial profile of dissolved iron concentration, real and maximum oxygen consumption rates, and cell concentration-at pulp densities to 30% was performed over a period of 200 days without any disturbances concerning the aeration system, fluidization, transport of solids and microbial growth. At a pulp density of 20%, a pyrite conversion of 68% was achieved after the third reactor stage at a total residence time of five days in the first three stages. The kinetics of pyrite degradation were found to be well described by a rate equation of first order in pyrite surface area concentration if the pyrite is directly accessible for microbial attack. Rate constants were determined to 0.48 mg pyrite/(cm(2) day) in the first and to 0.24 mg pyrite/(cm(2) day) in the following reactor stages. Kinetic models taking into account adsorption/desorption as well as growth kinetics failed to describe the observed reaction rates. However, a model treating pyrite degradation and microbial growth kinetics formalistically seems to be applicable when backmixing between the reactor stages can be avoided. The advantage of a multistage reactor in comparison to single-stage equipment was shown by calculation. To obtain a pyrite conversion of 68%, a three-stage reactor would require only 58% of the volume of single-stage equipment.Measurement of oxygen consumption rates proved to provide quickly and easily measurable parameters to observe microbial coal desulfurization in technical scale: the real oxygen consumption rate is correlated to the pyrite oxidation rate and the maximum oxygen consumption rate is correlated to the concentration of viable cells. The Y(o/s) coefficient for the amount of oxygen consumed per mass unit of pyrite oxygen was determined to approximately 0.33 in comparison to 1.0 which can be calculated from stoichiornetry. This could yet not be explained. Chemical leaching experiments as well as sulfur analyses of desulfurized coal samples showed that the microorganisms play the main role in degradation of pyrite from coal and that pyrite oxidation by ferric iron can be neglected.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Formal modeling of approximate relations in biochemical systems

Aiming to satisfy the need for the formalization of semiquantitative reasoning in the analysis of biochemical systems, the O[M] formalism for reasoning with orders of magnitude and approximate relations has been developed. It is based on seven primitive relations among quantities and compound relations which are formed as implicit disjunctions of primitives. O[M] can perform inferences by using formal approximate relations, algebraic equations, inequalities, if-then rules, assumptions, and goals. The applications discussed include Michaelis-Menten kinetics, different modes of inhibition of an enzymatic reaction, analysis of fluxes in biochemical networks, and identification of rate-limiting steps of biochemical pathways. In these applications, O[M] provides a medium for acquisition and formalization of previously informal concepts, analysis of systems at the order-of-magnitude level of detail, and automation of commonsense reasoning.     

The effect of enzyme concentration on the rate of the hydrolysis of cellulose

The relationship among extent of hydrolysis, reaction time, and enzyme dosage was investigated. For this, Sigmacell 50 and pretreated poplar wood (20 g/L) was hydrolyzed with varying dosages of cellulases from three different sources (5 to 100 FPU/g) for time periods ranging from 2 to 94 h. It was found that the formation of glucose can be described by summation of two parallel first order reactions. The extent of hydrolysis at fixed time increases with increasing enzyme dosage in a hyperbolic function. From the empirical data it is possible to calculate the fractions of easily and difficult hydrolyzable cellulose and the digestability which could maximally be obtained at infinite enzyme loadings. In the system Sigmacell 50 and Celluclast the easily and difficult hydrolyzable components are 43.0 and 57.0%, respectively, and the maximum digestability at 94 h is 82.6%. Poplar wood, steam treated at 200 degrees , 220 degrees , and 240 degrees C, showed with Celluclast at 24 h a maximum digestability (weight percentage of wood degraded to glucose) of 43.9, 64.9, and 68.0%. The relationships derived from experimental data allow one to compare objectively the effectiveness of different cellulase enzymes and different pretreatments.     

Influence of the solvent on the conformational-dependent properties of random coil polypeptides. II. Mean square optical anisotropies and Kerr constants

Mean square optical anisotropies and molar Kerr constants were calculated for homopolypeptides of the 20 natural amino acids and of several enzymes and proteins in the random-coil state. The effect of hydration was taken into account in constructing the molecular potential that gives the conformational energies as a function of the rotational angles phi and psi of the backbone and chi(1) of the side chain. The Rotational Isomeric State model was used in calculated energies, the Valence Optical Scheme and the matrix calculus technique of Flory being employed in the evaluation of the optical properties. The results are compared with calculations for the same substances that were performed without taking into account the solvent, as well as with other similar studies. The Kerr constant is confirmed as being one of the most sensitive properties of a given polypeptide to the residue class and to the sequence of those residues.