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Biomedical subjects

M R Ladisch

Publications and source records attributed to M R Ladisch.

9 recordsLinked to original sources
PubMed1995

Correlation of electrophoretic mobilities of proteins and peptides with their physicochemical properties.

Electrophoretic mobilities, mu, of nine proteins (M(r) 14,200 to 70,000) in 28 mM Tris/47 mM glycine buffer at pH 8.77 and 5 mM ionic strength were measured by laser Doppler velocimetry and correlated to ratios of charge (q) to molecular weight (M(r)) and shape factor (f/f0) by the equation mu(f/f0) = (Aq/Mpr-B). This correlation was previously reported for peptides and proteins for mu measured at 100 mM ionic strength. When A = 6.048 x 10(-3), B = 1.13 x 10(-5), and p = 2/3, the correlation fitted 51 measured and literature values over the molecular weight range of 178 to 140,000 for components whose electrophoretic mobilities ranged from +13.35 x 10(-5) to -19.7 x 10(-5) cm2/(V.s). The experimental measurements confirm the general suitability of p = 2/3 and show that the familiar charge/mass relation for electrophoresis is applicable to proteins in low-ionic-strength buffers which are typical of electrochromatography systems. Extrapolation of the correlation to different ionic strengths indicates that a low-ionic-strength buffer amplifies differences of electrophoretic mobility as a function of charge/mass, while high ionic strength diminishes such differences.

Electrophoresis↗
PubMed1991

Production of proteases by psychrotrophic microorganisms.

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 degrees C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.

Animals↗
PubMed1991

Purification and characterization of an extracellular protease produced by Pseudomonas fluorescens M3/6.

Pseudomonas fluorescens strain M3/6 was inoculated into reconstituted NDM and incubated at 7 degrees C for 46 d. A significant amount of extracellular protease was produced, mainly during the latter part of the culture's life cycle. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The isolated protease had activity on azocasein, alpha-, beta-, and kappa-caseins and a plasmin substrate but did not have plasminogen activator activity. The protease had a molecular weight of 45 kDa, an isoelectric point of pH 8.25, a broad temperature and pH range for activity, and was less heat stable in the isolated form than in the cell-free extract.

Animals↗
PubMed1977

Cellobiase from Trichoderma viride: purification, properties, kinetics, and mechanism.

Three distinct cellobiase components were isolated from a commercial Trichoderma viride cellulase preparation by repeated chromatography on DEAE cellulose eluting by a salt gradient. The purified cellobiase preparations were evaluated for physical properties, kinetics, and mechanism. Results from this work include: 1) development of one step enzyme purification procedure using DEAE-cellulose; 2) isolation of three chromatographically distinct, yet kinetically similar, cellobiase fractions of molecular weight of approximately 76,000; 3) determination of kinetics which shows that cellobiase hydrolyzes cellobiose by a noncompetitive mechanism and that the product, glucose, inhibits the enzyme, and 4) development of an equation, based on the mechanism of cellobiase action, which accurately predicts the time course of cellobiose hydrolysis over an eightfold range of substrate concentration and conversions of up to 90%. Based on the data presented in the paper, it is shown that product inhibition of cellobiase significantly retards the rate of cellobiose hydrolysis.

Disaccharides↗
PubMed1976

Studies on glucose isomerase from a Streptomyces species.

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.

Carbohydrate Epimerases↗
PubMedDate not supplied

Characterization of the swelling of a size-exclusion gel.

The swelling of a dextran gel, Sephadex G-75, was observed in an aqueous environment at room temperature by a noninvasive technique that uses light microscopy coupled to an image analysis system via a video camera. The rate of swelling was found to follow the Tanaka and Fillmore theory, from which the overall gel diffusion coefficient was estimated as 6.3 x 10(-7) cm2/s. In addition to giving a quantitative measure of gel swelling that could be useful in the mechanical design of liquid chromatography columns, this approach provides data on wet particle size and particle size range, which is needed for the modeling of diffusional and mass transfer effects in size-exclusion chromatography. In this context, key observations are that the gel particles are nearly spherical with an elliptical shape factor of 0.98 (perfect sphere = 1) and that there is little difference between sizes of particles obtained in water, 50 mM Tris-glycine buffer (pH 10.2), and buffer containing 1 mg/mL protein. The diameter of the dry material ranged from 20 to 100 microns, while the hydrated particles had diameters of 40-350 microns. The rate of swelling is rapid, with 50% swelling occurring in about 10 s and swelling to 99% of the final wet particle size being obtained in less than 90 s.

Chemical Phenomena↗
PubMedDate not supplied

Recombinant human insulin.

Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.

Amino Acid Sequence↗
PubMedDate not supplied

Simulation of diauxic production of cephalosporin C by Cephalosporium acremonium: lag model for fed-batch fermentation.

We extend a previously reported model (Chu, W.B.; Constantinides, A. Biotechnol. Bioeng. 1988, 32, 277-288) for the batch fermentation of cephalosporin C under the diauxic growth of Cephalosporium acremonium on glucose and sucrose to a fed-batch system. For this purpose, a novel lag model is proposed for diauxie, which has two functional forms, each embodying the dependence of lag on total cell mass and secondary substrate concentration. This lag model is applicable for batch simulations for arbitrary initial glucose and sucrose concentrations. We used the previously reported batch data to perform locally optimized fed-batch simulations. When applied to fed-batch fermentations, multiple lag times were accounted for. These studies showed that fed-batch fermentations (under the restriction that cell mass concentration did not exceed 25 g/L) could be more productive than simple batch runs. A representative result for a glucose-pulse fed-batch run at optimal cephalosporin production is a productivity of 4.22 mg of cephalosporin C/(L.h) and a yield of 9.25 mg of cephalosporin C/g of total sugar used.

Acremonium↗