Zhou Xiaoxia, Xia Yuxian
Genetic Engineering Research Center, Bioengineering College, Chongqing University, Chongqing 400030, PR China.
Protein Expr Purif. 2011 May;77(1):62-7. doi: 10.1016/j.pep.2010.11.017. Epub 2010 Dec 3.
The acetylcholinesterase 1 from Locusta migratoria manilensis (LmAChE1) was successfully expressed in methylotrophic yeast Pichia pastoris KM71. The maximum expression of recombinant LmAChE1 (reLmAChE1) was achieved after 9 days of induction at 2.5% methanol. The reLmAChE1 was first precipitated with ammonium sulfate (50% saturation) and then was purified with nickel affinity chromatography. The enzyme was purified 3.2×10(3)-fold with a yield of 68% and a specific activity of 8.1 U/mg. The purified reLmAChE1 exhibited highest activity at 30°C in 100 mM phosphate buffer (pH 7.4), and its activity could be inhibited by eserine sulfate and pentan-3-one-dibromide (BW284c51). Substrate specificity analysis showed that the purified reLmAChE1 preferred acetylthiocholine (ATC) and propionylthiocholine (BTC) rather than butyrylthiocholine (BTC). When ATC was used as substrate, the K(m) and V(max) values for the reLmAChE1 were 24.8 μM and 9.5 μmol/min/mg, respectively.
东亚飞蝗的乙酰胆碱酯酶1（LmAChE1）在甲基营养型酵母毕赤酵母KM71中成功表达。在2.5%甲醇诱导9天后，重组LmAChE1（reLmAChE1）实现了最大表达。reLmAChE1首先用硫酸铵（50%饱和度）沉淀，然后用镍亲和层析法纯化。该酶纯化了3.2×10³倍，产率为68%，比活性为8.1 U/mg。纯化后的reLmAChE1在30°C、100 mM磷酸盐缓冲液（pH 7.4）中表现出最高活性，其活性可被硫酸依色林和戊-3-酮-二溴化物（BW284c51）抑制。底物特异性分析表明，纯化后的reLmAChE1更喜欢乙酰硫代胆碱（ATC）和丙酰硫代胆碱（BTC）而非丁酰硫代胆碱（BTC）。当使用ATC作为底物时，reLmAChE1的K(m)和V(max)值分别为24.8 μM和9.5 μmol/min/mg。
