Studies on alkaline serine protease produced by Bacillus clausii GMBE 22.
An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus clausii reported in the literature.
Marmara University, Faculty of Engineering, Department of Bioengineering, Goztepe Campus, Kadikoy, Istanbul, Turkey. dilek.kazan@mam.gov.tr
Dilek
D
Kazan
Hulya
H
Bal
Aziz Akin
AA
Denizci
Nurcin Celik
NC
Ozturk
Hasan Umit
HU
Ozturk
Aydan Salman
AS
Dilgimen
Dilek Coskuner
DC
Ozturk
Altan
A
Erarslan
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D001483:Base Sequence; D002413:Cations, Divalent / Q000737:chemistry; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
D002413:Cations, Divalent; D017931:DNA Primers; D013501:Surface-Active Agents; D006861:Hydrogen Peroxide; D012697:Serine Endopeptidases