Browse > Article
http://dx.doi.org/10.4014/jmb.1412.12049

Characterization of a Novel Thermostable Oligopeptidase from Geobacillus thermoleovorans DSM 15325  

Jasilionis, Andrius (Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University)
Kuisiene, Nomeda (Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.7, 2015 , pp. 1070-1083 More about this Journal
Abstract
A gene (GT-SM3B) encoding a thermostable secreted oligoendopeptidase (GT-SM3B) was cloned from the thermophile Geobacillus thermoleovorans DSM 15325. GT-SM3B is 1,857 bp in length and encodes a single-domain protein of 618 amino acids with a 23-residue signal peptide having a calculated mass of 67.7 kDa after signal cleavage. The deduced amino acid sequence of GT-SM3B contains a conservative zinc metallopeptidase motif (His400-Glu401-X-XHis404). The described oligopeptidase belongs to the M3B subfamily of metallopeptidases and displays the highest amino acid sequence identity (40.3%) to the oligopeptidase PepFBa from mesophilic Bacillus amyloliquefaciens 23-7A among the characterized oligopeptidases. Secretory production of GT-SM3B was used, exploiting successful oligopeptidase signal peptide recognition by Escherichia coli BL21 (DE3). The recombinant enzyme was purified from the culture fluid. Homodimerization of GT-SM3B was determined by SDS-PAGE. Both the homodimer and monomer were catalytically active within a pH range of 5.0–8.0, at pH 7.3 and 40℃, showing the Km, Vmax, and kcat values for carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala-OH peptidolysis to be 2.17 ± 0.04 × 10-6 M, 2.65 ± 0.03 × 10-3 µM/min, and 5.99 ± 0.07 s-1, respectively. Peptidase remained stable at a broad pH range of 5.0–8.0. GT-SM3B was thermoactive, demonstrating 84% and 64% of maximum activity at 50℃ and 60℃, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60℃ for 1 h. GT-SM3B was shown to hydrolyze a collagenous peptide mixture derived from various types of collagen, but less preferentially than synthetic hexapeptide. This study is the first report on an extracellular thermostable metallo-oligopeptidase.
Keywords
Zinc metallopeptidases; M3B subfamily; oligoendopeptidase F; Geobacillus thermoleovorans; thermostability;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Szeltner Z, Polgar L. 2008. Structure, function and biological relevance of prolyl oligopeptidases. Curr. Protein Pept. Sci. 9: 96-107.   DOI
2 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
3 Vimr ER, Green L, Miller CG. 1983. Oligopeptidase-deficient mutants of Salmonella typhimurium. J. Bacteriol. 153: 1259-1265.
4 Watanabe K. 2004. Collagenolytic proteases from bacteria. Appl. Microbiol. Biotechnol. 63: 520-526.   DOI
5 Yan TR, Azuma N, Kaminogawa S, Yamauchi K. 1987. Purification and characterization of a novel metalloendopeptidase from Streptococcus cremonis H61. A metalloendopeptidase that recognizes the size of its substrate. Eur. J. Biochem. 163: 259-265.   DOI
6 Yan TR, Azuma N, Kaminogawa S, Yamauchi K. 1987. Purification and characterization of substrate-size-recognizing metalloendopeptidase from Streptococcus cremoris H61. Appl. Environ. Microbiol. 53: 2296-2302.
7 Zeigler DR. 2014. The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet? Microbiology 160: 1-11.   DOI
8 Zhang Y, Fu Y, Zhou S, Kang L, Li C. 2013. A straightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry. Anal. Biochem. 437: 46-48.   DOI
9 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
10 Petrera A, Amstutz B, Gioia M, Hähnlein J, Baici A, Selchow P, et al. 2012. Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates. Biol. Chem. 393: 631-640.   DOI
11 Rawlings ND, Barrett AJ. 2013. Introduction: metallopeptidases and their clans, pp. 325-370. In Rawlings ND, Salvesen G (eds.). Handbook of Proteolytic Enzymes, 3rd Ed. Academic Press, Amsterdam.
12 Rawlings ND, Waller M, Barrett AJ, Bateman A. 2014. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 42: D503-D509.   DOI
13 Savijoki K, Ingmer H, Varmanen P. 2006. Proteolytic systems in lactic acid bacteria. Appl. Microbiol. Biotechnol. 71: 394-406.   DOI
14 Starcher B. 2001. A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal. Biochem. 292: 125-129.   DOI
15 Sugihara Y, Kawasaki A, Tsujimoto Y, Matsui H, Watanabe K. 2007. Potencies of phosphine peptide inhibitors of mammalian thimet oligopeptidase and neurolysin on two bacterial Pz peptidases. Biosci. Biotechnol. Biochem. 71: 594-597.   DOI
16 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
17 Novak P, Ray PH, Dev IK. 1986. Localization and purification of two enzymes from Escherichia coli capable of hydrolyzing a signal peptide. J. Biol. Chem. 261: 420-427.
18 Oetjen J, Fives-Taylor P, Froeliger E. 2001. Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme. Infect. Immun. 69: 58-64.   DOI
19 Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.   DOI
20 Lin B, Averett WF, Novak J, Chatham WW, Hollingshead SK, Coligan JE, et al. 1996. Characterization of PepB, a group B streptococcal oligopeptidase. Infect. Immun. 64: 3401-3406.
21 Lopez-Kleine L, Monnet V, Pechoux C, Trubuil A. 2008. Role of bacterial peptidase F inferred by statistical analysis and further experimental validation. HFSP J. 2: 29-41.   DOI
22 Miyake R, Kawamoto J, Wei YL, Kitagawa M, Kato I, Kurihara T, Esaki N. 2007. Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as a host. Appl. Environ. Microbiol. 73: 4849-4856.   DOI
23 Miyake R, Shigeri Y, Tatsu Y, Yumoto N, Umekawa M, Tsujimoto Y, et al. 2005. Two thimet oligopeptidase-like Pz peptidases produced by a collagen-degrading thermophile, Geobacillus collagenovorans MO-1. J. Bacteriol. 187: 4140-4148.   DOI
24 Moitinho-Silva L, Kondo MY, Oliveira LCG, Okamoto DN, Paes JA, Machado MFM, et al. 2013. Mycoplasma hyopneumoniae in vitro peptidase activities: identification and cleavage of kallikrein-kinin system-like substrates. Vet. Microbiol. 163: 264-273.   DOI
25 Gomez-Ortiz M, Gomis-Rüth FX, Huber R, Aviles FX. 1997. Inhibition of carboxypeptidase A by excess zinc: analysis of the structural determinants by X-ray crystallography. FEBS Lett. 400: 336-340.   DOI
26 Monnet V, Nardi M, Chopin A, Chopin MC, Gripon JC. 1994. Biochemical and genetic characterization of PepF, an oligopeptidase from Lactococcus lactis. J. Biol. Chem. 269: 32070-32076.
27 Dinsdale AE, Halket G, Coorevits A, van Landschoot A, Busse HJ, de Vos P, Logan NA. 2011. Emended descriptions of Geobacillus thermoleovorans and Geobacillus thermocatenulatus. Int. J. Syst. Evol. Microbiol. 61: 1802-1810.   DOI
28 Gerdts CJ, Tereshko V, Yadav MK, Dementieva I, Collart F, Joachimiak A, et al. 2006. Time-controlled microfluidic seeding in nL-volume droplets to separate nucleation and growth stages of protein crystallization. Angew. Chem. Int. Ed. Engl. 45: 8156-8160.   DOI
29 Gomis-Rüth FX. 2008. Structure and mechanism of metallocarboxypeptidases. Crit. Rev. Biochem. Mol. Biol. 43: 319-345.   DOI
30 Jasilionis A, Kaupinis A, Ger M, Valius M, Chitavichius D, Kuisiene N. 2012. Gene expression and activity analysis of the first thermophilic U32 peptidase. Cent. Eur. J. Biol. 7: 587-595.
31 Kanamaru K, Stephenson S, Perego M. 2002. Overexpression of the PepF oligopeptidase inhibits sporulation initiation in Bacillus subtilis. J. Bacteriol. 184: 43-50.   DOI
32 Kawasaki A, Nakano H, Hosokawa A, Nakatsu T, Kato H, Watanabe K. 2010. The exquisite structure and reaction mechanism of bacterial Pz-peptidase A toward collagenous peptides. J. Biol. Chem. 285: 34972-34980.   DOI
33 Kleiner DE, Stetler-Stevenson WG. 1994. Quantitative zymography: detection of picogram quantities of gelatinases. Anal. Biochem. 218: 325-329.   DOI
34 Asdornnithee S, Himeji E, Akiyama K, Sasaki T, Takata R. 1995. Isolation and characterization of Pz-peptidase from Bacillus licheniformis N22. J. Ferment. Bioeng. 79: 200-204.   DOI
35 Kuisiene N, Raugalas J, Chitavichius D. 2004. Geobacillus lituanicus sp. nov. Int. J. Syst. Evol. Microbiol. 54: 1991-1995.   DOI
36 Akiyama K, Mori K, Takata R. 1999. Cloning and sequencing of the Pz-peptidase gene from Bacillus licheniformis N22. J. Biosci. Bioeng. 87: 231-233.   DOI
37 Ansai T, Yu W, Urnowey S, Barik S, Takehara T. 2003. Construction of a pepO gene-deficient mutant of Porphyromonas gingivalis: potential role of endopeptidase O in the invasion of host cells. Oral Microbiol. Immunol. 18: 398-400.   DOI
38 Awano S, Ansai T, Mochizuki H, Yu W, Tanzawa K, Turner AJ, Takehara T. 1999. Sequencing, expression and biochemical characterization of the Porphyromonas gingivalis pepO gene encoding a protein homologous to human endothelinconverting enzyme. FEBS Lett. 460: 139-144.   DOI
39 Baankreis R, van Schalkwijk S, Alting AC, Exterkate FA. 1995. The occurrence of two intracellular oligoendopeptidases in Lactococcus lactis and their significance for peptide conversion in cheese. Appl. Microbiol. Biotechol. 44: 386-392.   DOI
40 Barrett AJ, Rawlings ND. 1992. Oligopeptidases, and the emergence of the prolyl oligopeptidase family. Biol. Chem. Hoppe Seyler 373: 353-360.   DOI
41 Caler EV, Vaena de Avalos S, Haynes PA, Andrews NM, Burleigh BA. 1998. Oligopeptidase B-dependent signaling mediates host cell invasion in Trypanosoma cruzi. EMBO J. 17: 4975-4986.   DOI
42 Agarwal V, Kuchipudi A, Fulde M, Riesbeck K, Bergmann S, Blom AM. 2013. Streptococcus pneumoniae endopeptidase O (PepO) is a multifunctional plasminogen- and fibronectin-binding protein, facilitating evasion of innate immunity and invasion of host cells. J. Biol. Chem. 288: 6849-6863.   DOI
43 Chao SH, Cheng TH, Chaw CY, Lee MH, Hsu YH, Tsai YC. 2006. Characterization of a novel PepF-like oligopeptidase secreted by Bacillus amyloliquefaciens 23 -7A. Appl. Environ. Microbiol. 72: 968-971.   DOI