Phospholipases D (PLDs) hydrolyze phospholipids to yield phosphatidic acid and the corresponding alcohol and catalyze a transesterification (transphosphatidylation) reaction when alcohol is present as a nucleophilic donor. Bacterial forms of PLDs have shown to be suitable as biocatalysts for the synthesis of phospholipid derivatives of industrial interest. Recently, PLD from Streptomyces PMF, an enzyme with a high transphosphatidylation activity, was purified and its crystallographic structure was solved at 1.4Å. A 315-bp fragment of the pld gene of Streptomyces PMF was amplified by PCR using chromosomal DNA as a template and a pair of heterologous primers based on Streptomyces antibioticus pld gene sequence. The complete pld gene was isolated by colony hybridization and sequenced. DNA sequence analysis revealed a significant similarity with known pld gene sequences and showed the presence of highly conserved sequence motifs, namely the HKD motifs, shared by other members of the PLD superfamily. In order to promote the secretion of the protein into the medium, the mature PLD gene was fused in a pET derivative with the PelB signal sequence. Expression was performed in Escherichia coli BL21(DE3)pLysE cells after induction with IPTG, yielding 0.5mg PLD/l of culture medium after protein purification to homogeneity. An inactive PLD variant, generated by site-directed mutagenesis experiments, was produced in large amounts using the same expression system, thus confirming the correlation between the effect of toxicity of PLD activity in E. coli cells and the low levels of enzyme production. Western blot analyses were used to detect PLD production in the culture supernatant, the recombinant protein was purified to homogeneity and structural and functional analyses confirmed its identity with the Streptomyces PLD.
|Titolo:||Cloning and expression in Escherichia coli of the gene encoding Streptomyces PMF PLD, a phospholipase D with high transphosphatidylation activity|
|Autori interni:||VANONI, MARIA ANTONIETTA|
|Parole Chiave:||phospholipase D; streptomyces PMF; cloning; heterologous expression|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2003|
|Digital Object Identifier (DOI):||10.1016/S0141-0229(03)00190-X|
|Appare nelle tipologie:||01 - Articolo su periodico|