Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus
Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus
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DOI：
10.1007/s00792-008-0139-9
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发表时间：
2008-05-01
期刊：
影响因子：
2.9
通讯作者：
Antranikian, Garabed
中科院分区：
文献类型：
--
作者：
Hess, Matthias;Katzer, Moritz;Antranikian, Garabed
通过基因组分析，鉴定了肉状小蠊酯酶EstA和酯酶EstB的两个基因，并在大肠杆菌中进行了克隆。编码EstA的PTO 0988包含579 bp，编码EstB的PTO 1141包含696 bp，分别对应192 aa和231 aa。序列比较显示，与先前表征的酶相比，这两种生物催化剂具有较低的序列一致性（14%和16%）。详细分析表明EstA和EstB是第一批来自热嗜酸菌的酯酶，不属于HSL家族成员。此外，同种三聚体EstA和EstB的表观分子质量分别为22和27 kDa的亚基代表了迄今为止报道的嗜热微生物中最小的酯酶亚基。重组酯酶通过Ni2+亲和层析纯化，并在较宽的pH （pH 4.5-8.5）和温度（10-90℃）范围内测定酯酶的活性。在70℃（EstA）和55℃（EstB）条件下，以短pnp酯为首选底物，测定了酯酶的最高活性。此外，非甾体抗炎药萘普生、酮洛芬和布洛芬的酯类均可被EstA和EstB水解。在高达90℃的温度下，这两种酶都具有极高的热稳定性。在90℃下，测定的EstA和EstB的半衰期（t(1/2)）分别为21和10 h。在洗涤剂、尿素和常用有机溶剂的存在下，酯酶活性的显著保存完成了EstA和EstB的特殊表型。
Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni2+ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5-8.5) and temperature range (10-90 degrees C). Highest activity of the esterases was measured at 70 degrees C (EstA) and 55 degrees C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90 degrees C. The determined half-life (t(1/2)) at 90 degrees C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.