Proteolytic components of alkaline proteases of Bacillus strains. Zymograms and electrophoretic isolation

• Published in: Biotechnology and bioengineering 1972-09, Vol.14 (5), p.685-714
• Main Authors: Zuidweg, M. H. J., Bos, C. J. K., Van Welzen, H.
• Format: Article
• Language: English
• Subjects: Alkalies; Bacillus - enzymology; Bacillus subtilis - enzymology; Buffers; Edetic Acid; Electrophoresis, Disc; Hot Temperature; Hydrogen-Ion Concentration; Isoelectric Focusing; Isoenzymes - isolation & purification; Methods; Peptide Hydrolases - isolation & purification
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.260140502

Characterization of proteolytic activity in preparations or fermentation broth is accomplished by methods based on a combination of disc electrophoresis and visualization of the activity. The methods permit a direct, comparison with reference preparations. All alkaline proteases studied, including the three subtilisins, were found to consist of several proteolytic components. The zymograms of the subtilisin type preparations show an irregular pattern. At least 14 different components may be observed, belonging to 6 types of mobility pattern, 3 of which could be assigned to the subtilisins. None of the components belongs to the group of metalloproteases. A quite regular pattern is shown by the zymogram of protease preparations produced by the alkalophilic Bacillus strains. A few of the components of the subtilisin preparations Maxatase and protease A were isolated by preparative disc electrophoresis and by disc electrofococusing, allowing a further characterization. Special attention was directed to the determination of properties specific for the application of subtilisins as additives in household detergents. Thermostability in sodium tripolyhosphate solution was found to range from about 10% for one of the minor components to 80% for the main component of Maxatase. Three types of curves representing the effect of pH on the activity were observed. The curve of the main component of Maxatase shows a characteristic shape with a maximum at pH 10.3: with other components lower pH optima were observed. Isoelectric points of the component were found to range from pH 7 to 10.