Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase.
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 Ser(153) (within the G-X-S-X-G consensus sequence), His(75) and Asp(125). 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 degrees C).
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Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D004950:Esterases