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

Abstract

SummaryCharacterization 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 lllaxatase and protease A were isolated by preparative disc electrophoresis and by electrofocusing, 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 tripolyphosphat,e 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 components were found to range from pH 7 to 10.

Characterization of alkaline proteases from a novel alkali-tolerant bacterium Bacillus patagoniensis

“…The molecular mass of the protease determined by SDS-PAGE was 29.4 kDa, while an IEFzymogram performed with high pH range carrier ampholytes (Pharmalyte 8-10.5, Pharmacia) showed that its pI was >10.3 (data not shown). These properties resembled those of the highly alkaline protease group produced by alkaliphilic bacilli, which includes enzymes with a low molecular mass of 26-28 kDa and high optimum pH for activity and pI values (Zuidweg et al 1972;Betzel et al 1992;Kobayashi et al 1995). When the protease solution was analysed under non-reducing conditions, the active band showed a slightly higher apparent molecular mass.…”

Subtilopeptidase A isoenzyme system. Interaction with serum components and its importance for quantitative immunoelectrophoresis

“…Earlier work (Zuidweg et al, 1972;Verbruggen etal., 1974;Verbruggen &Baeck, 1975) demonstrated considerable electrophoretic heterogeneity in commercial crystalline subtilopeptidase A. Autodigestion and Ca2+-binding effects could be excluded as the cause of this heterogeneity. Commercial enzyme preparations were also shown to be immunochemically heterogeneous (Verbruggen et al, 1974;Belin et al, 1970).…”

Novel Oxidatively Stable Subtilisin-like Serine Proteases from Alkaliphilic Bacillus spp.: Enzymatic Properties, Sequences, and Evolutionary Relationships

“…221 (3). The high-alkaline protease group, including Savinase (4), PB92 (5), and M-protease (6) that are all produced by alkaliphilic bacilli, has a low-molecular-mass of 26 -28 kDa. They are used mainly in the detergent industry.…”