Videira P A, Fialho A M, Marques A R, Coutinho P M, Sá-Correia I
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal.
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):517-22. doi: 10.1007/s00253-003-1226-6. Epub 2003 Feb 26.
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).
对产生结冷胶的少动鞘氨醇单胞菌（Sphingomonas paucimobilis）ATCC 31461的ces10基因进行了克隆和测序。推导蛋白的多序列比对表明，Ces10属于丝氨酸水解酶家族，具有一个潜在的催化三联体，由Ser(153)（位于G-X-S-X-G共有序列内）、His(75)和Asp(125)组成。模式搜索后得到的混合区结果以及在BLAST分析中检测到的低同源性表明，Ces10与其他已鉴定的细菌酯酶/脂肪酶有显著差异。然而，Ces10氨基酸序列与红球菌属海洛因酯酶有45%的相似性，与枯草芽孢杆菌对硝基苄酯酶有48%的相似性。预测分子量为30,641 Da的Ces10在大肠杆菌中过量表达，并以带组氨酸标签的形式纯化至同质。使用不同酰基链长度的对硝基苯酯（p-NP-酯）作为底物的酶活性测定证实了预期的酯酶活性。Ces10对短链脂肪酸表现出明显的偏好，对p-NP-丙酸酯活性最高（最适pH 7.4，最适温度37℃）。
