A rapid, accurate method with high sensitivity and reproducibility, and having the advantage of a short incubation period under constant pH, has been developed for routine measurement of microbial lipase. Assembled from readily available and economical instrumental components, the apparatus includes a pH meter, a thermoelectric heating and stirring device, a motor-driven burette, and an automatic recorder. The reaction mixture, consisting of 5 ml of a 10% olive oil-gum arabic emulsion, 2 ml of 3 m NaCl, 2 ml of sodium taurocholate (15 mg/ml) of 0.075 m CaCl(2), 5 ml of water, and 1 ml of enzyme solution, was adjusted to pH 8.0 and 37 C. The pH was maintained at a constant value by automatic addition of 0.01 n NaOH during the incubation period, which usually lasted 5 min. A lipase unit, derived from the use of this technique, may be defined as the number of microequivalents of acid liberated per minute under the specified conditions. The method was sensitive to 0.01 units. Various organisms tested produced 0.17 to 1.32 units per ml of the cell filtrate. An Arrhenius plot for staphylococcal lipase yielded 14,500 cal for function A (energy of activation).

Alcaligenes↗

PubMed1967

Isolation and purification of staphylococcal lipase.

An extracellular lipase was isolated from the cell-free supernatant fluid of a 24-hr culture of Staphylococcus aureus grown in Trypticase Soy Broth at 37 C with continuous agitation. The purification was achieved by precipitation with alcohol followed by differential precipitation at pH 8.6 and 4.3. Subsequent purification with Sephadex G 200 and BioGel 300 yielded a preparation which showed a 350- to 450-fold increase in specific activity over the original cell-free supernatant fluid. The purified lipase was homogeneous over a BioGel 300 column and showed a single peak on electrophoresis in a Veronal buffer (pH 8.6, Gamma/2 = 0.1). The electrophoretic mobility was -7.78 x 10(-5) cm(2) per v per sec.

Centrifugation↗

PubMed1967

Characterization of purified staphylococcal lipase.

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the alpha, beta, and alpha' positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme.

Fats↗

PubMed1966

Nephelometric assay of staphylococal coagulase.

Stutzenberger, Fred J. (Michigan State University, East Lansing), Charles L. San Clemente, and Dharam V. Vadehra. Nephelometric assay of staphylococcal coagulase. J. Bacteriol. 92:1005-1009. 1966.-Clotting of fibrinogen by staphylococcal coagulase was accompanied by an increase in light scattering; this property was used as a basis for a new nephelometric method. Reaction rates, which were now easily and precisely measured, were found to be directly proportional to coagulase concentration, when optimal conditions were maintained. These conditions included pH and concentrations of fibrinogen, coagulase-reacting factor, and sodium chloride in the reaction mixture. A standardized procedure for the assay is outlined, and a unit for the expression of activity is proposed.

Coagulase↗

PubMed1965

Comparison of methods of extracting intracellular proteases from bacteria.

Five commonly used methods of disintegrating bacterial cells were compared by use of Bacillus subtilis, Pseudomonas putrefaciens, and Streptococcus durans as the test organisms. These methods were: (i) sonic treatment, (ii) grinding, (iii) freezing and thawing, (iv) acetone-powder, and (v) toluene. Sonic treatment and grinding yielded more protein in the cell-free extracts than did the other methods. Likewise, the protease activities (micrograms of tyrosine liberated per milliliter of extract) of sonically disrupted and ground cell extracts on casein substrates were far greater than those in extracts from cells disintegrated by the other methods. When the specific activity was based on the amount of tyrosine liberated per milligram of protein in the extract, the acetone-powder method yielded the most active protease extract, whereas the extract obtained by sonic treatment was least active. Other methods yielded extracts with intermediate specific activity.

Bacillus subtilis↗