Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase
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The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser153 (within the G-X-S-X-G consensus sequence), His75 and Asp125. The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641 Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pH 7.4, optimal temperature 37 °C).
KeywordsLipolytic Enzyme Triacetine Ces10 Gene Sphingomonas Paucimobilis Ces10 Protein
This work was supported by the Fundação para a Ciência e Tecnologia, Portugal (project POCTI/35733/1999) and by post-doctoral and PhD grants to P.A.V. (SFRH/BPD/5710/2001) and A.R.M. (PRAXIS XXI/BD/18146/98)].
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