Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, 21073, Hamburg, Germany.
Extremophiles. 2010 May;14(3):273-85. doi: 10.1007/s00792-010-0306-7. Epub 2010 Mar 9.
A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all alpha/beta hydrolases (G x S x G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser(106), Asp(196), and His(225). Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25 degrees C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40 degrees C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90 degrees C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C(2)-C(8)).
从耐冷菌假交替单胞菌的基因文库筛选中鉴定和测序了一个编码酯酶（estO）的基因。对 1203bp 的编码区进行分析，发现推断的肽序列由 400 个氨基酸组成，预测分子量为 44.1kDa。EstO 含有一个 N 端酯酶结构域和一个 C 端额外的 OsmC 结构域（渗透诱导蛋白家族）。从第 104 位到 108 位检测到所有α/β水解酶典型的高度保守的五残基基序（GxSxG），同时还存在一个推定的催化三联体，由 Ser（106）、Asp（196）和 His（225）组成。序列比较表明，EstO 与含有类似酯酶和 OsmC 结构域的假想蛋白具有 90%的氨基酸同一性，但与已知酯酶的氨基酸序列只有约 10%的同一性。产生了带有和不带有 OsmC 结构域的 EstO 变体，并作为 His 标签融合蛋白在大肠杆菌中进行了纯化。EstO 的最适 pH 为 7.5，最适温度为 25°C，在冰点下仍保持超过 50%的活性。EstO 的热稳定性（在 40°C 下 5 小时后有 50%的活性）在截断变体中显著增加（在 90°C 下 2.5 小时后有 50%的活性）。此外，该酯酶对短链脂肪酸（C2-C8）的酯具有广泛的底物特异性。
