Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1

P. Gopal Reddy, Rinat Allon, Moshe Mevarech, Simona Mendelovitz, Y. Sato, David L. Gutnick*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

A putative esterase gene (est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escherichia coli. Esterase-positive clones exhibited high levels of esterase activity even in intact cells. In addition, expression of the est gene conferred on E. coli the ability to grow on simple triglycerides such as triacetin (TAC). The original esterase-positive plasmid pRA17 carried a 2.2-kb insert from a partial MboI digest of RAG-1 DNA, which gave a single band with RAG-1 DNA following Southern hybridization. By subcloning and sequencing the est gene was found to contain a sequence of 870 bp which could be translated to yield a protein of Mr 32700. In support of the sequencing results was the finding that when pRA17 was expressed in minicells, a unique peptide of Mr 32500 was identified. This peptide was not found in minicells transformed with esterase-negative plasmids, such as pRA176, which contained a Tn5 insertion in the est gene. The fact that the production of active esterase depended on the orientation of the est gene within the vector suggested that transcription proceeded from the tet promoter in pBR322.

Original languageEnglish
Pages (from-to)145-152
Number of pages8
JournalGene
Volume76
Issue number1
DOIs
StatePublished - 15 Mar 1989

Funding

FundersFunder number
Petroleum Fermentations NV

    Keywords

    • Recombinant DNA
    • minicells
    • nucleotide sequence
    • triacetin
    • triglycerides

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