A new method for pH determination in isoelectric focusing experiments

A rapid and reproducible method for the pH measurement in the effluent from density gradient electrofocusing is described. By this procedure, after preparative isoelectric focusing, the detection of protein zones and pH measurement can be accomplished simultaneously, by serially coupling a uv flow cell with a pH flow cell. This last one is connected to the recorder by a control unit, which allows the simultaneous printing of pH and uv absorption on the same chart.     

A new method to determine optimum temperature and activation energies for enzymatic reactions

A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.     

A mathematical method for temperature compensation of pH measurements.

A continuous recording viscometer using oscillating capillary tubes has been developed. The basic principles, general design, and construction details of the viscosity sensing device are given. Two such devices, mechanically coupled, comprise a bridge viscometer with which two fluids can be compared directly. A bridge viscometer complete with the electronic amplifier circuits is described. The general design is readily adaptable to automatic systems.The instrument has a wide dynamic range. The fluid under test may be allowed to flow through the system. The capacity of each arm of the viscometer bridge is less than 100 μl.     

A new method for ethanol productivity determination

Summary A simple and easily applicable method for determination of the ethanol fermentation rate is described. The formulas to be used for the ethanol productivity calculation in the case of free and agar immobilized cells are given.     

A new and simplified method for the determination of ergot alkaloids

Summary The conventional method for determining ergot alkaloids in fermentation media is laborious and time consuming, largely because of the chloroform extraction procedure employed. A new and simplified method, without chloroform extraction, for measuring alkaloid in culture liquid and in colonies has been developed. The details of the procedure and its application are described. Estimates of alkaloid by the new method were about 6% higher than by the conventional method. The advantages of the new method are that it is accurate, rapid, safe and versatile in use, especially in the large scale screening of strains of the ergot fungus.

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Resumen El método tradicional para la determinación de los alcaloides del cornezuelo en el medio de fermentación es laborioso y lento, debido principalmente a la extracción con cloroformo. Se ha desarrollado un método simplificado, sin extracción con cloroformo, para medir los alcaloides presentes en cultivos líquidos y en colonias. Se describen los detalles del proceso y sus aplicaciones, Las estimaciones de alcaloides realizadas mediante este nuevo sistema eran un 6% superiores a las realizadas siguiendo el método convencional. Las ventajas de este nuevo método son su precisión, rapidez, seguridad y versatilidad especialmente en los muestreos a gran escala de cepas del hongo del cornezuelo.

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Résumé La méthode usuelle pour doser les alcaloides de l'ergot est longue et laborieuse, surtout parcequ'elle a recours à une extraction par le chloroforme. Une nouvelle méthode simplifiée, ne comportant pas d'extraction chloroformique, a été mise au point. Ce procédé et ses applications sont décrits de façon détaillée. Les dosages d'alcaloides obtenus par cetee méthode sont supérieus d'environ 6% à ceux de la méthode usuelle. La nouvelle méthode a l'avantage d'être précise, rapide, sûre et commode, en particulier pour le criblage d'un grand nombre de souches.

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Paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16 August 1985     

Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum

beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 A resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.     

A convenient pH-gradient method for the determination of the maximum and minimum pH for microbial growth

Previously existing methods for determining the pH limits for the growth of microorganisms have involved (1), the setting up of individual cultures, each having a specific pH; (2), the pH gradient plate technique devised by Sacks (1956) in which a continuous pH gradient is established in a Petri dish by means of a buffer system; and (3), the pH gradient plate technique of Zak (unpublished), in which a continuous pH gradient is established by means of an electric current. The discontinuous pH gradient technique described here provides a convenient method of determining the maximum and minimum pH at which a microorganism can grow. The technique can be used aerobically or anaerobically, and has a precision of about ± 0.1 pH unit. Data are given for several yeasts and forSerratia marcescens. In all cases, the organisms tested continued to metabolize at pH values beyond those representing the limits for growth, sometimes by as much as 0.5 pH unit. The results suggest that pH limits are unsuitable criteria in microbial classification.