title: Characterization of a Novel Thermostable Oligopeptidase from Geobacillus thermoleovorans DSM 15325.
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 (His(400)-Glu(401)-X-XHis (404)). 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°C, 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) micrometer/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°C and 60°C, respectively. The recombinant oligopeptidase is one of the most thermostable M3B peptidase, retaining 71% residual activity after incubation at 60°C 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.
authors: Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, LT-03101 Vilnius, Lithuania.
authors: Andrius
authors: A
authors: Jasilionis
authors: Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, LT-03101 Vilnius, Lithuania.
authors: Nomeda
authors: N
authors: Kuisiene
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C028759:oligopeptidase
keywords: Geobacillus thermoleovorans; M3B subfamily; Zinc metallopeptidases; oligoendopeptidase F; thermostability