Hermann Manuela, Kietzmann Martin U, Ivancić Mirela, Zenzmaier Christoph, Luiten Ruud G M, Skranc Wolfgang, Wubbolts Marcel, Winkler Margit, Birner-Gruenberger Ruth, Pichler Harald, Schwab Helmut
Applied Biocatalysis Research Centre, Graz, Austria.
J Biotechnol. 2008 Feb 1;133(3):301-10. doi: 10.1016/j.jbiotec.2007.10.010. Epub 2007 Nov 1.
Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed gamma-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E>200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36U (mg protein)(-1) towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate.
猪肝酯酶（PLE）最初是从猪肝中分离得到的一种粗制、不均一的酶组分，发现它能代谢多种底物，且常常具有高度的立体选择性。然而，这种粗制酯酶制剂含有几种同工酶，其比例因批次而异。外消旋甲基 -（4E）-5-氯-2-异丙基-4-戊烯酸酯能被粗制PLE对映选择性地裂解，但不能被重组表达的PLEγ同工型裂解。由于推断一定有另一种PLE同工酶具有相关活性，我们克隆并测序了几种PLE同工型的cDNA，并在毕赤酵母中进行了功能表达。发现一种名为替代猪肝酯酶（APLE）的新型同工型能以高度立体选择性的方式（E>200）水解甲基 -（2R，4E）-5-氯-2-异丙基-4-戊烯酸酯。当在毕赤酵母中异源表达并定向分泌时，发现APLE定位于周质。假定的C末端内质网滞留信号的存在与否既不影响功能表达也不影响细胞定位。通过离子交换色谱法纯化的重组酶对外消旋甲基 -（4E）-5-氯-2-异丙基-4-戊烯酸酯的比活性为36U（mg蛋白）-1。
