PMID:12732528

Citation	Bach, H, Berdichevsky, Y and Gutnick, D (2003) An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan. Appl. Environ. Microbiol. 69:2608-15
Abstract	The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.
Links	PubMed PMC154541
Keywords	Acinetobacter/genetics; Acinetobacter/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/pharmacology; Base Sequence; Biodegradation, Environmental; DNA, Bacterial/genetics; Esterases/genetics; Esterases/isolation & purification; Esterases/pharmacology; Excipients/chemistry; Excipients/isolation & purification; Excipients/pharmacology; Fuel Oils; Molecular Weight; Mutagenesis, Site-Directed; Polysaccharides, Bacterial/pharmacology; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/pharmacology
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status

References

See Help:References for how to manage references in GONUTS.