Identification and Characterization of a New Alkaline Thermolysin-Like Protease, BtsTLP1, from Bacillus thuringiensis Serovar Sichuansis Strain MC28
2
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps. The mature form of BtsTLP1 has a molecule mass of 35.6 kDa as determined by mass spectrometry analyses. The biochemical characterization indicates that BtsTLP1 has an apparent Km value of 1.57 mg/ml for azocasein and is active between 20oC and 80oC. Unlike other reported neutral gram-positive thermolysin homologs with optimal pH around 7, BtsTLP1 exhibits an alkaline pH optimum around 10. The activity of BtsTLP1 is strongly inhibited by EDTA and a group of specific divalent ions, with Zn2+ and Cu2+ showing particular effects in promoting the enzyme autolysis. Furthermore, our data also indicate that BtsTLP1 has potential in cleaning applications.
Abstract
Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps. The mature form of BtsTLP1 has a molecule mass of 35.6 kDa as determined by mass spectrometry analyses. The biochemical characterization indicates that BtsTLP1 has an apparent Km value of 1.57 mg/ml for azocasein and is active between 20oC and 80oC. Unlike other reported neutral gram-positive thermolysin homologs with optimal pH around 7, BtsTLP1 exhibits an alkaline pH optimum around 10. The activity of BtsTLP1 is strongly inhibited by EDTA and a group of specific divalent ions, with Zn2+ and Cu2+ showing particular effects in promoting the enzyme autolysis. Furthermore, our data also indicate that BtsTLP1 has potential in cleaning applications.
참고문헌 (36)
- Adekoya OA, "The thermolysin family (M4) of enzymes: therapeutic and biotechnological potential", Chem. Biol. Drug Des, 73, pp.7~16 (2009)
- Beaufort N, "The thermolysin-like metalloproteinase and virulence factor LasB from pathogenic Pseudomonas aeruginosa induces anoikis of human vascular cells", Cell Microbiol, 13, pp.1149~1167 (2011)
- Chung MC, "Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors", J. Biol. Chem, 281, pp.31408~31418 (2006)
- Colman PM, "The structure of thermolysin: an electron density map at 2-3 A resolution", J. Mol. Biol, 70, pp.701~724 (1972)
- de Kreij A, "Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids", J. Biol. Chem, 275, pp.31115~31120 (2000)
- Fassina G, "Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex", Eur. J. Biochem, 156, pp.221~228 (1986)
- Fukasawa KM, "Metal preferences of zinc-binding motif on metalloproteases", J. Amino Acids, 2011, pp.574816~ (2011)
- Gao X, "Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family", Proc. Natl. Acad. Sci. USA, 107, pp.17569~17574 (2010)
- Guan P, "Complete genome sequence of Bacillus thuringiensis serovar Sichuansis strain MC28", J. Bacteriol, 194, pp.6975~ (2012)
- Gupta R, "Bacterial alkaline proteases: molecular approaches and industrial applications", Appl. Microbiol. Biotechnol, 59, pp.15~32 (2002)
- Hase CC, "Bacterial extracellular zinccontaining metalloproteases", Microbiol. Rev, 57, pp.823~837 (1993)
- Hashida Y, "Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin", J. Biochem, 141, pp.879~888 (2007)
- Hatanaka T, "Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2", Arch. Biochem. Biophys, 434, pp.289~298 (2005)
- He HL, "Structural and functional characterization of mature forms of metalloprotease E495 from Arctic sea-ice bacterium Pseudoalteromonas sp. SM495", PLoS One, 7, pp.e35442~ (2012)
- Holland DR, "Structural analysis of zinc substitutions in the active site of thermolysin", Protein Sci, 4, pp.1955~1965 (1995)
- Holmes MA, "Structure of thermolysin refined at 1.6 A resolution", J. Mol. Biol, 160, pp.623~639 (1982)
- Holmes MA, "Structural analysis of the inhibition of thermolysin by an active-sitedirected irreversible inhibitor", Biochemistry, 22, pp.236~240 (1983)
- Inouye K, "Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization", Protein Expr. Purif, 46, pp.248~255 (2006)
- Kim M, "Gene cloning and characterization of a Bacillus vietnamensis metalloprotease", Biosci. Biotechnol. Biochem, 68, pp.1533~1540 (2004)
- Kumar R, "Microbial protease and application as laundry detergent additive", Res. J. Microbiol, 3, pp.12~ (2008)
- Larkin MA, "Clustal W and Clustal X version 2.0", Bioinformatics, 23, pp.2947~2948 (2007)
- Li Q, "Commercial proteases:present and future", FEBS Lett, 587, pp.1155~1163 (2013)
- Miyamoto K, "Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7", Biosci. Biotechnol. Biochem, 66, pp.416~421 (2002)
- Miyoshi S, "An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning", Microb. Pathog, 33, pp.127~134 (2002)
- Morya VK, "In silico characterization of alkaline proteases from different species of Aspergillus", Appl. Biochem. Biotechnol, 166, pp.243~257 (2012)
- Ohta Y, "Thermostable protease from thermophilic bacteria. I. Thermostability, physiocochemical properties, and amino acid composition", J. Biol. Chem, 241, pp.5919~5925 (1966)
- Petersen TN, "SignalP 4.0: discriminating signal peptides from transmembrane regions", Nat. Methods, 8, pp.785~786 (2011)
- Rawlings ND, "MEROPS: the database of proteolytic enzymes, their substrates and inhibitors", Nucleic Acids Res, 40, pp.D343~D350 (2012)
- Tamura K, "MEGA6: molecular evolutionary genetics analysis version 6.0", Mol. Biol. Evol, 30, pp.2725~2729 (2013)
- Tan F, "Rapid cloning, identification, and application of one novel crystal protein gene cry30Fa1 from Bacillus thuringiensis", FEMS Microbiol. Lett, 302, pp.46~51 (2010)
- Tan F, "Cloning and characterization of two novel crystal protein genes, cry54Aa1 and cry30Fa1, from Bacillus thuringiensis strain BtMC28", Curr. Microbiol, 58, pp.654~659 (2009)
- Titani K, "Amino acid sequence of thermolysin. Isolation and characterization of the fragments obtained by cleavage with cyanogen bromide", Biochemistry, 11, pp.2427~2435 (1972)
- Vogtentanz G, "A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor", Protein Expr. Purif, 55, pp.40~52 (2007)
- Wu JW, "Extracellular metalloproteases from bacteria", Appl. Microbiol. Biotechnol, 92, pp.253~262 (2011)
- Xie BB, "Cold adaptation of zinc metalloproteases in the thermolysin family from deep sea and arctic sea ice bacteria revealed by catalytic and structural properties and molecular dynamics:new insights into relationship between conformational flexibility and hydrogen bonding", J. Biol. Chem, 284, pp.9257~9269 (2009)
- Yang J, "Purification, characterization, and gene cloning of a cold-adapted thermolysinlike protease from Halobacillus sp. SCSIO 20089", J. Biosci. Bioeng, 115, pp.628~632 (2013)
