Identification of pig liver esterase variants by tandem mass spectroscopy analysis and their characterization
Identification of pig liver esterase variants by tandem mass spectroscopy analysis and their characterization
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通过串联质谱分析鉴定猪肝酯酶变体及其表征
DOI：
10.1007/s00253-007-1061-2
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发表时间：
2007
影响因子：
5
通讯作者：
U. Bornscheuer
中科院分区：
文献类型：
--
作者：
Elke Brüsehaber;D. Böttcher;Anna Musidlowska;Dirk Albrecht;Michael Hecker;K. Doderer;U. Bornscheuer
猪肝酯酶（PLE）可能是有机合成中最重要的羧基酯酶，可从动物组织中提取。然而，由于几种同工酶（α-、β-和γ-PLE）的存在，在应用中出现了问题。γ-同工酶的功能表达已被证实，并且与商业制剂相比，其对映体选择性存在差异。α-和β-PLE的氨基酸和核苷酸序列尚不清楚。在这项工作中，通过2D凝胶电泳、蛋白酶消化和基质辅助激光解吸/电离飞行时间（TOF）和电喷雾电离四极杆TOF质谱分析，从商业PLE制剂中鉴定了α-同工酶的推定序列。在此基础上，通过定点诱变将3个氨基酸交换引入γ-rPLE编码基因，并在大肠杆菌Origami （DE3）中表达。所产生的PLE突变体具有底物特异性和对映体选择性。突变体V236P/A237G对（R，S）-1-苯基-2-乙酸丁酯的对映体选择性显著提高，E = 100。
Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (α-, β- and γ-PLE). The functional expression of the γ-isoenzyme was already shown and differences in the enantioselectivity compared to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the α- and β-PLE are still unknown. In this work, putative sequences of the α-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis, digestion with proteases and analysis using Matrix-assisted laser desorption/ionization–time of flight (TOF) and electrospray ionisation quadrupole–TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene encoding γ-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity. No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G.