Please use this identifier to cite or link to this item: http://hdl.handle.net/2381/29622
Title: Study of an Arthrobacter sp. esterase able to hydrolyse malathion
Authors: Heddle, Catherine.
Award date: 1997
Presented at: University of Leicester
Abstract: A bacterial isolate tentatively identified as an Arthrobacter sp. was isolated from soil using diethylsuccinate as a carbon and energy source. This bacterium was found to have two esterases that were active against diethylsuccinate, malathion and p-nitrophenyl acetate. One of the esterase genes was studied further by shotgun cloning the gene into pUC18, transforming Escherichia coli and identifying the transformant through phenotypic selection. Subcloning identified the region of DNA responsible for encoding the esterase which led to the partial purification of the protein and the determination of its N-terminal sequence. Information from the N-terminal sequence was then used to construct a degenerate oligodeoxyribonucleotide primer to start the sequencing of the gene. Sequencing identified the gene encoding the esterase as 1140bp encoding a protein of 380 amino acids which showed sequence identity of between 34-38% with other carboxyl- and aryl-esterases in the databases. These comparisons also identified a putative active site serine within the motif Ser-X-X-Lys and also the partial motif Gly-X-Ser-X-X. Sequence information facilitated the overexpression of the esterase gene in vector pT7-7 which enabled the protein to be purified. The purified esterase was found to be a monomeric protein of approx. 40kDa. The esterase was found to be inhibited by heavy metals but was stable at pHs in the range 7.4-11 and when stored for long periods at 0 and -20°C. The esterase was found to be exclusively a carboxylesterase and preferentially acted on uncharged esters such as diethylsuccinate rather than the charged monoethylsuccinate the degradation of which appeared to be the rate limiting step in the complete hydrolysis of diethylsuccinate to succinate.
Links: http://hdl.handle.net/2381/29622
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Biochemistry
Leicester Theses

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