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http://dx.doi.org/10.4014/jmb.1501.01008

Identification and Characterization of a New Alkaline Thermolysin-Like Protease, BtsTLP1, from Bacillus thuringiensis Serovar Sichuansis Strain MC28  

Zhang, Zhenghong (China Research Center, DuPont Industrial Bioscience)
Hao, Helong (China Research Center, DuPont Industrial Bioscience)
Tang, Zhongmei (China Research Center, DuPont Industrial Bioscience)
Zou, Zhengzheng (China Research Center, DuPont Industrial Bioscience)
Zhang, Keya (China Research Center, DuPont Industrial Bioscience)
Xie, Zhiyong (China Research Center, DuPont Industrial Bioscience)
Babe, Lilia (Danisco US Inc., Genencor Division, DuPont Industrial Bioscience)
Goedegebuur, Frits (Genencor International B.V., DuPont Industrial Bioscience)
Gu, Xiaogang (China Research Center, DuPont Industrial Bioscience)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.8, 2015 , pp. 1281-1290 More about this Journal
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 20℃ and 80℃. 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.
Keywords
Bacillus thuringiensis; thermolysin-like protease; alkaline metalloprotease; thermolysin;
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1 Li Q, Yi L, Marek P, Iverson BL. 2013. Commercial proteases: present and future. FEBS Lett. 587: 1155-1163.   DOI
2 Miyamoto K, Tsujibo H, Nukui E, Itoh H, Kaidzu Y, Inamori Y. 2002. 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: 416-421.   DOI
3 Miyoshi S, Sonoda Y, Wakiyama H, Rahman MM, Tomochika K, Shinoda S, et al. 2002. An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning. Microb. Pathog. 33: 127-134.   DOI
4 Morya VK, Yadav S, Kim EK, Yadav D. 2012. In silico characterization of alkaline proteases from different species of Aspergillus. Appl. Biochem. Biotechnol. 166: 243-257.   DOI
5 Hase CC, Finkelstein RA. 1993. Bacterial extracellular zinccontaining metalloproteases. Microbiol. Rev. 57: 823-837.
6 Gao X, Wang J, Yu DQ, Bian F, Xie BB, Chen XL, et al. 2010. Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family. Proc. Natl. Acad. Sci. USA 107: 17569-17574.   DOI
7 Guan P, Ai P, Dai X, Zhang J, Xu L, Zhu J, et al. 2012. Complete genome sequence of Bacillus thuringiensis serovar Sichuansis strain MC28. J. Bacteriol. 194: 6975.   DOI
8 Gupta R, Beg QK, Lorenz P. 2002. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59: 15-32.   DOI
9 Hashida Y, Inouye K. 2007. 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: 879-888.   DOI
10 Hatanaka T, Yoshiko Uesugi JA, Iwabuchi M. 2005. Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2. Arch. Biochem. Biophys. 434: 289-298.   DOI
11 He HL, Guo J, Chen XL, Xie BB, Zhang XY, Yu Y, et al. 2012. Structural and functional characterization of mature forms of metalloprotease E495 from Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. PLoS One 7: e35442.   DOI
12 Holland DR, Hausrath AC, Juers D, Matthews BW. 1995. Structural analysis of zinc substitutions in the active site of thermolysin. Protein Sci. 4: 1955-1965.   DOI
13 Holmes MA, Matthews BW. 1982. Structure of thermolysin refined at 1.6 A resolution. J. Mol. Biol. 160: 623-639.   DOI
14 Colman PM, Jansonius JN, Matthews BW. 1972. The structure of thermolysin: an electron density map at 2-3 A resolution. J. Mol. Biol. 70: 701-724.   DOI
15 Adekoya OA, Sylte I. 2009. The thermolysin family (M4) of enzymes: therapeutic and biotechnological potential. Chem. Biol. Drug Des. 73: 7-16.   DOI
16 Beaufort N, Corvazier E, Hervieu A, Choqueux C, Dussiot M, Louedec L, et al. 2011. The thermolysin-like metalloproteinase and virulence factor LasB from pathogenic Pseudomonas aeruginosa induces anoikis of human vascular cells. Cell Microbiol. 13: 1149-1167.   DOI
17 Chung MC, Popova TG, Millis BA, Mukherjee DV, Zhou W, Liotta LA, et al. 2006. Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors. J. Biol. Chem. 281: 31408-31418.   DOI
18 de Kreij A, Venema G, van den Burg B. 2000. Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids. J. Biol. Chem. 275: 31115-31120.   DOI
19 Fukasawa KM, Hata T, Ono Y, Hirose J. 2011. Metal preferences of zinc-binding motif on metalloproteases. J. Amino Acids 2011: 574816.   DOI
20 Fassina G, Vita C, Dalzoppo D, Zamai M, Zambonin M, Fontana A. 1986. Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex. Eur. J. Biochem. 156: 221-228.   DOI
21 Tan F, Zhu J, Tang J, Tang X, Wang S, Zheng A, Li P. 2009. Cloning and characterization of two novel crystal protein genes, cry54Aa1 and cry30Fa1, from Bacillus thuringiensis strain BtMC28. Curr. Microbiol. 58: 654-659.   DOI
22 Xie BB, Bian F, Chen XL, He HL, Guo J, Gao X, et al. 2009. 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: 9257-9269.   DOI
23 Yang J, Li J, Mai Z, Tian X, Zhang S. 2013. Purification, characterization, and gene cloning of a cold-adapted thermolysinlike protease from Halobacillus sp. SCSIO 20089. J. Biosci. Bioeng. 115: 628-632.   DOI
24 Ohta Y, Ogura Y, Wada A. 1966. Thermostable protease from thermophilic bacteria. I. Thermostability, physiocochemical properties, and amino acid composition. J. Biol. Chem. 241: 5919-5925.
25 Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.   DOI
26 Rawlings ND, Barrett AJ, Bateman A. 2012. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 40: D343-D350.   DOI
27 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729.   DOI
28 Vogtentanz G, Collier KD, Bodo M, Chang JH, Day AG, Estell DA, et al. 2007. A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor. Protein Expr. Purif. 55: 40-52.   DOI
29 Tan F, Zheng A, Zhu J, Wang L, Li S, Deng Q, et al. 2010. Rapid cloning, identification, and application of one novel crystal protein gene cry30Fa1 from Bacillus thuringiensis. FEMS Microbiol. Lett. 302: 46-51.   DOI
30 Titani K, Hermodson MA, Ericsson LH, Walsh KA, Neurath H. 1972. Amino acid sequence of thermolysin. Isolation and characterization of the fragments obtained by cleavage with cyanogen bromide. Biochemistry 11: 2427-2435.   DOI
31 Wu JW, Chen XL. 2011. Extracellular metalloproteases from bacteria. Appl. Microbiol. Biotechnol. 92: 253-262.   DOI
32 Holmes MA, Tronrud DE, Matthews BW. 1983. Structural analysis of the inhibition of thermolysin by an active-sitedirected irreversible inhibitor. Biochemistry 22: 236-240.   DOI
33 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948.   DOI
34 Inouye K, Minoda M, Takita T, Sakurama H, Hashida Y, Kusano M, Yasukawa K. 2006. Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization. Protein Expr. Purif. 46: 248-255.   DOI
35 Kim M, Nishiyama Y, Mura K, Tokue C, Arai S. 2004. Gene cloning and characterization of a Bacillus vietnamensis metalloprotease. Biosci. Biotechnol. Biochem. 68: 1533-1540.   DOI
36 Kumar R, Savitri, Thakur N, Verma R, Bhalla TC. 2008. Microbial protease and application as laundry detergent additive. Res. J. Microbiol. 3: 12.   DOI