Details

Autor(en) / Beteiligte

• Bakholdina, S.I
• Tischenko, N.M
• Sidorin, E.V
• Isaeva, M.P
• Likhatskaya, G.N
• Dmitrenok, P.S
• Kim, N. Yu
• Chernikov, O.V
• Soloveva, T.F

Titel

Recombinant phospholipase [A.sub.1] of the outer membrane of psychrotrophic Yersinia pseudotuberculosis: expression, purification, and characterization

Ist Teil von

• Biochemistry (Moscow), 2016-01, p.47

Ort / Verlag

Springer

Erscheinungsjahr

2016

Quelle

SpringerLink Journals

Beschreibungen/Notizen

• The pldA gene encoding membrane-bound phospholipase [A.sub.1] of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase [A.sub.1] (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase [A.sub.1] was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase [A.sub.1] were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases [A.sub.1] from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed. DOI: 10.1134/S0006297916010053 Key words: Yersinia pseudotuberculosis, phospholipase [A.sub.1], recombinant protein, spatial structure modeling