Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency
- PMID: 18397761
- DOI: 10.1016/j.biochi.2008.03.004
Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency
Abstract
We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH2-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (kcat/Km) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H2O2, which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.
Similar articles
-
Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family.Biochimie. 2011 Apr;93(4):783-91. doi: 10.1016/j.biochi.2011.01.011. Epub 2011 Jan 31. Biochimie. 2011. PMID: 21281692
-
Purification and properties of a novel surface-active agent- and alkaline-resistant protease from Bacillus sp. Y.Agric Biol Chem. 1991 Sep;55(9):2251-8. Agric Biol Chem. 1991. PMID: 1368737
-
Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12.Int J Biol Macromol. 2019 Jan;121:1227-1239. doi: 10.1016/j.ijbiomac.2018.10.139. Epub 2018 Oct 21. Int J Biol Macromol. 2019. PMID: 30352229
-
Detergent alkaline proteases: enzymatic properties, genes, and crystal structures.J Biosci Bioeng. 2007 Jun;103(6):501-8. doi: 10.1263/jbb.103.501. J Biosci Bioeng. 2007. PMID: 17630120 Review.
-
Bacterial alkaline proteases: molecular approaches and industrial applications.Appl Microbiol Biotechnol. 2002 Jun;59(1):15-32. doi: 10.1007/s00253-002-0975-y. Epub 2002 Apr 20. Appl Microbiol Biotechnol. 2002. PMID: 12073127 Review.
Cited by
-
Statistical Approach for Optimization of Physiochemical Requirements on Alkaline Protease Production from Bacillus licheniformis NCIM 2042.Enzyme Res. 2012;2012:905804. doi: 10.1155/2012/905804. Epub 2012 Jan 5. Enzyme Res. 2012. PMID: 22347624 Free PMC article.
-
Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus.3 Biotech. 2016 Jun;6(1):53. doi: 10.1007/s13205-016-0377-y. Epub 2016 Feb 13. 3 Biotech. 2016. PMID: 28330122 Free PMC article.
-
Purification and biochemical characterization of a novel thermostable protease from the oyster mushroom Pleurotus sajor-caju strain CTM10057 with industrial interest.BMC Biotechnol. 2019 Jul 1;19(1):43. doi: 10.1186/s12896-019-0536-4. BMC Biotechnol. 2019. PMID: 31262286 Free PMC article.
-
Purification and partial characterization of a detergent and oxidizing agent stable alkaline protease from a newly isolated Bacillus subtilis VSG-4 of tropical soil.J Microbiol. 2011 Jun;49(3):455-61. doi: 10.1007/s12275-011-0427-4. Epub 2011 Jun 30. J Microbiol. 2011. PMID: 21717332
-
Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide.PLoS One. 2014 Sep 29;9(9):e108367. doi: 10.1371/journal.pone.0108367. eCollection 2014. PLoS One. 2014. PMID: 25264614 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources