Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

Abstract

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphatepolyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co2+ and Hg2+, whereas Mg2+, Fe2+, Cu2+, Zn2+ and Sr2+ had little or no inhibitory effect. However, Mn2+ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t 1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca2+.

Production and properties of an extracellular protease from thermophilic Bacillus sp

“…These results suggest that these metal ions apparently protected the enzyme against thermal denaturation and played a vital role in maintaining the active conformation of the enzyme at higher temperatures (4). Similar effects of Mn 2+ on the activity of proteases were also observed by Rahman et al (18), and by Manachini et al (10).…”

“…Proteases secreted from thermophilic bacteria are thus of particular interest and have become increasingly useful in a range of commercial applications (1,18,19,21,23).…”

Partial purification and properties of a laundry detergent compatible alkaline protease from a newly isolated Bacillus species Y

“…In view of other investigators, 0.1% (w/v) gelatin was the best substrate concentration for thermostable protease activity produced by B. brevis geltinoamylolyticus [20]. Many of the previous studies revealed that alkaline proteases showed highest activity towards casein relative to other proteins including BSA, hemoglobin, keratin and azocasein [21,22]. The ability of the protease produced by the test organism to hydrolyze a wide range of proteins substrate may be advantageous for its use in detergents against a wide variety of stains.…”

Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

“…The activity of the alkaline protease was also observed by zymogram. The molecular weights of the alkaline proteases generally ranging from 15 to 30 kDa reported by Kumar and Takagi (20), with few reports of higher molecular weights of 32.0 kDa, 33.5 kDa determined (21,22) respectively. Other investigators reported the molecular masses of alkaline proteases from various Bacillus species range between 17-40 kDa (5, 7, 23, 24).…”