Optimization and purification of alkaline proteases produced by marine Bacillus sp. MIG newly isolated from eastern harbour of Alexandria.
A marine Bacillus strain was isolated from the eastern harbour of Alexandria and identified as Bacillus sp. MIG. Maximum activity of studied proteases was obtained when the bacterium was grown in medium with 1% wheat bran and 0.5% yeast extract in addition to the mineral salts and incubated for 48 h at 30 degrees C and 120 rpm. Two alkaline proteases (Pro 1 and Pro 2) were purified to homogeneity using cation exchange chromatography on CM-Sepharose CL-6B followed by Sephadex G-75 superfine. The optimum activities were at pH 11 or 12, and temperatures of 50 and 55 degrees C for Pro 1 and Pro 2 respectively. These two enzymes were relatively stable over pH range from 7.0- 11. Pro 2 was found to be more stable at 50 degrees C in absence of Ca2+ and retained about 47% of its activity after 3 h at this temperature, while Pro 1 lost its activity completely at the same conditions. The two enzymes were active against haemoglobin and casein; in addition, Pro 2 exhibited moderate activity against keratin. Both enzymes were partially inhibited by Ag+ and Hg2+. PMSF completely inhibited the enzymes, while dithiothreitol and 2-mercaptoethanol stimulated their activities, suggesting to be thiol-dependent serine proteases. The enzymes were stable in the presence of the surfactants and bleaching agent (H2O2) and relatively stable in presence of some commercial detergents.
Gouda
Mona K
MK
Botany Department, Faculty of Science, Alexandria University, Alexandria, Egypt. monagouda@yahoo.com
D001407:Bacillus / Q000201:enzymology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004534:Egypt; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D011480:Protease Inhibitors; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D012623:Seawater / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011480:Protease Inhibitors; D012336:RNA, Ribosomal, 16S; D010450:Endopeptidases; C087822:alkaline protease