Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: Sequence analysis and over-expression in Escherichia coli

Alon, R.

doi:10.1016/0378-1097(93)90612-6

Cited by 11 publications

(9 citation statements)

References 14 publications

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“…This may reflect the requirement of extracellular proteins, involved either directly in alkane degradation or indirectly via the production of bioemulsifiers during growth on hexadecane. In this respect, it is interesting that an esterase has been identified in A. lwoffii RAG-1 (formerly A. calcoaceticus), which presumably is exported and bound to the outer surface of the cell (1,61,67). The esterase is involved in release of the bioemulsifier emulsan (21,67), an anionic heteropolysaccharide which is essential for the organism's ability to grow on crude oil and other hydrocarbons (21).…”

Section: Discussionmentioning

confidence: 99%

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase

Kok¹,

Thor²,

Nugteren-Roodzant³

et al. 1995

J Bacteriol

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At the onset of the stationary phase in rich media, the gram-negative soil bacterium Acinetobacter calcoaceticus BD413 produces several lipolytic enzymes (40), which have partly overlapping substrate specificities. BD413 estA, encoding one of the cell-bound esterases (40), and lipA, encoding the extracellular lipase (43), have been cloned and characterized. The regulation of their expression is currently being investigated. In addition, we also aim at defining posttranscriptional factors involved in regulation of enzyme production. The latter aspect is especially important for the extracellular lipase of A. calcoaceticus BD413, since several components involved in export of the protein, including concurrent protein processing events, may have significant control over lipase production.The extracellular lipase of A. calcoaceticus BD413 presumably is exported from the cell via a two-step translocation process (43): across the cytoplasmic membrane, this translocation will be mediated by the Sec system (59, 66), whereas translocation through the outer membrane proceeds via a translocation complex similar to the Xcp system in Pseudomonas spp. (69) and the Pul system in Klebsiella spp. (59). These assumptions are based upon the identification of an N-terminal export signal peptide, indicative of the involvement of a Seclike system, and upon the high degree of similarity of A. calcoaceticus LipA to Pseudomonas lipases that are secreted via a two-step mechanism. Moreover, we have demonstrated that a periplasmic processing enzyme, a protein disulfide oxidoreductase, is required for high-level production of lipase in A. calcoaceticus BD413 (43).In Pseudomonas spp., production of lipases that are similar to Acinetobacter LipA requires the presence of a lipase-specific helper protein, which is N terminally anchored in the cytoplasmic membrane, with its C-terminal domain in the periplasm. Activity of this type of protein is essential for the second lipase translocation step, across the outer membrane (14, 34). In the periplasm, such helper proteins have been shown to mediate the formation of secondary structure elements in the periplasmic form of the lipase, suggesting a chaperone-like function of the helper protein. Unlike the situation in pseudomonads, as yet no essential lipase helper protein has been identified in A. calcoaceticus. In Pseudomonas species, these proteins are invariably encoded downstream of the structural lipase gene, and this is not the case in A. calcoaceticus BD413 (43).Previously, we have reported on the identification of two

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Section: Discussionmentioning

confidence: 99%

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase

Kok¹,

Thor²,

Nugteren-Roodzant³

et al. 1995

J Bacteriol

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“…The biopolymer accumulates on the cell surface of exponential-phase RAG-1 cells as a minicapsule, and is released into the medium as a protein-polysaccharide complex as the cells approach stationary phase (Goldman et al, 1982 ;Pines et al, 1983). This release from the cell surface involves the participation of an exocellular esterase which has been cloned, sequenced and overexpressed in Escherichia coli (Alon & Gutnick, 1993 ;Reddy et al, 1989 ;. Removal of the protein yields a polymer termed apoemulsan, which retains much of the emulsifying activity towards certain hydrocarbon substrates but is inactive in the emulsification of relatively non-polar, hydrophobic, aliphatic materials (Zosim et al, 1986 ;.…”

Section: Abbreviationsmentioning

confidence: 99%

Analysis of the wee gene cluster responsible for the biosynthesis of the polymeric bioemulsifier from the oil-degrading strain Acinetobacter lwoffii RAG-1 The GenBank/EMBL accession number for the sequence analysis of the eight fragments determined in this work is AJ243431.

Nakar

Gutnick

2001

Microbiology

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A cluster (27 kbp) of genes responsible for the biosynthesis of the amphipathic, polysaccharide bioemulsifier emulsan from the oil-degrading Acinetobacter lwoffii RAG-1 was isolated and characterized. The complete sequence of this cluster, termed wee, consisted of 20 ORFs. One set of 17 ORFs was transcribed in one direction, while a second set of three ORFs, 607 bp upstream of the first, was transcribed in the opposite direction. Mutations in either of the two regions caused defects in emulsan production, yielding specific activities of 5-14 % of parental emulsifying activity. Putative functions could be assigned to proteins involved in production of nucleotide amino sugar precursors, transglycosylation, transacetylation, polymerization and transport. However, no JUMPstart or ops sequences, normally found associated with some polysaccharide biosynthetic gene clusters, were identified. Evidence is presented suggesting that the bioemulsifier may be a member of the group 1 or group 4 polysaccharides.

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“…Emulsan release from the bacterial cell surface is apparently mediated by the action of a cell surface esterase, which is one of the key components in the active emulsan-protein complex (39). This esterase, encoded by the est gene (909 bp) has been cloned, sequenced, and overexpressed in Escherichia coli (1). The facts that the exocellular esterase is involved in emulsan release, has been shown to deacetylate the biopolymer (38), and has been found associated with the extracellular emulsan complex suggest that its interaction with apoemulsan might affect the emul-sifying activity of the biopolymer.…”

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confidence: 99%

An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan

Bach

Berdichevsky

Gutnick

2003

Appl Environ Microbiol

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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.

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Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: Sequence analysis and over-expression in Escherichia coli

Cited by 11 publications

References 14 publications

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase

Analysis of the wee gene cluster responsible for the biosynthesis of the polymeric bioemulsifier from the oil-degrading strain Acinetobacter lwoffii RAG-1 The GenBank/EMBL accession number for the sequence analysis of the eight fragments determined in this work is AJ243431.

An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan

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