一种嗜热型碳酸酐酶基因的克隆表达及酶学性质

Cloning and expression of a thermophilic carbonic anhydrase and its enzymatic properties

本文将嗜热茵Methanocella conradii HZ254的β型碳酸酐酶基因mtc在大肠杆菌中进行了克隆和表达。将PCR扩增得到mtc基因与pET-24a（＋）载体进行连接，转入大肠杆菌JM109中，得到工程菌JM109-pET24a-mtc。诱导表达后做SDS-PAGE电泳，显示有与预期分子量大小相近的（约29kDa）产物条带。该表达产物具有催化CO2水合的活性，但没有酯酶活力。研究表明，重组酶在40℃下稳定，40℃～60℃对酶有激活作用，65℃以上会导致酶的失活。在pH7．0时，酶有最好的pH稳定性。1mmol／L的Fe2＋、Mg2＋、Mn2＋和Ca2＋均对酶有激活作用，而Cu2＋则有明显的抑制作用。1mmol／L的F-对酶活没有明显的影响，而磺胺和I-则有比较明显的抑制作用，Br-、HCO3-、Cl-、NO3-和SO42-对酶均有一的抑制作用。

Abstract The gene （mtc） of a β-class carbonic anhydrase from thermophiles Methanocella conradii HZ254 was cloned and expressed in E. coli. The gene was amplified by PCR, ligated with pET-24a （ ＋ ） vector and transformed into comptent E. coli JM109. The resulting recombinant was named JM109-pET24a-mtc. The molecular mass of the induced expression product was 29 kDa nearly identical to the expected. It could catalyze CO2 hydration, but had no esterase activity. The activity of this recombinant carbonic anhydrase was stable at 40 ℃, enhanced by the temperature of 40 ℃ - 60 ℃ and completely destroyed by above 65 ℃. It was the most stable at pH 7.0. Its activity was enhanced by 1 mmol/L of Fe2 ＋ , Mg2 ＋,Mn 2 ＋and Ca2 ＋, but inhibited by Cu2＋. 1 mmol/L of sulfanilamide, I , Br-, HCO3- , CI-, NOs and SO42- also inhibited it at different levels, but F- had no effect on it.