Esterase EstA6 from Pseudomonas sp. CR-611 is a novel member in the utmost conserved cluster of family VI bacterial lipolytic enzymes

• Published in: Biochimie 2006-07, Vol.88 (7), p.859-867
• Main Authors: Prim, N., Bofill, C., Pastor, F.I.J., Diaz, P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteria; Bacterial lipase classification; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carboxylesterase; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Cloning, Molecular; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Electrophoresis, Polyacrylamide Gel - methods; Hydrogen-Ion Concentration; Isoelectric Focusing - methods; Lipolysis; Molecular Sequence Data; Multigene Family - genetics; Phylogeny; Pseudomonas; Pseudomonas - enzymology; Pseudomonas - genetics; Pseudomonas fluorescens; Sequence Alignment; Sequence Analysis, DNA; Substrate Specificity; Temperature
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2006.02.011

Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved β-/γ-protobacterial cluster in family VI lipases, to which EstA6 belongs.