Biochemistry (Moscow)

, Volume 74, Issue 10, pp 1164–1172 | Cite as

Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants

  • R. F. Khairullin
  • A. G. Mikhailova
  • T. Yu. Sebyakina
  • N. L. Lubenets
  • R. H. Ziganshin
  • I. V. Demidyuk
  • T. Yu. Gromova
  • S. V. Kostrov
  • L. D. Rumsh


A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1–10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His6-PSP was 1.5 mg/g biomass.

Key words

oligopeptidase B trypsin Serratia proteamaculans GroEL fusion expression 



Nα-benzoyl-D,L-arginine p-nitro-anilide


basic bovine pancreatic trypsin inhibitor

buffer A

0.1 M Tris-HCl, pH 8.0, containing 50 mM CaCl2 and 1 mM MgCl2

buffer B

10 mM Hepes-KOH, pH 7.5, containing 1 mM MgCl2

buffer C

20 mM potassium phosphate, pH 7.4, containing 0.5 M NaCl, 10 mM imidazole, 0.1% 2-mercaptoethanol, and 5% glycerol


dimethyl sulfoxide




nominal molecular weight limit


oligopeptidase B


proteinase from Serratia proteamaculans.


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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • R. F. Khairullin
    • 1
  • A. G. Mikhailova
    • 1
  • T. Yu. Sebyakina
    • 1
  • N. L. Lubenets
    • 1
  • R. H. Ziganshin
    • 1
  • I. V. Demidyuk
    • 2
  • T. Yu. Gromova
    • 2
  • S. V. Kostrov
    • 2
  • L. D. Rumsh
    • 1
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Institute of Molecular GeneticsRussian Academy of SciencesMoscowRussia

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