, Volume 74, Issue 10, pp 1164-1172
Date: 06 Nov 2009

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

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Abstract

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.

Original Russian Text © 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, 2009, published in Biokhimiya, 2009, Vol. 74, No. 10, pp. 1427–1437.