Institute of Pharmaceutical Biotechnology, School of Medicine, Zhejiang University, Hangzhou 310058, China.
Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering, Hangzhou 310058, China.
J Zhejiang Univ Sci B. 2021 Mar 15;22(3):204-213. doi: 10.1631/jzus.B2000525.
Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by . The pPIC9K- acetylcholinesterase () expression vector was constructed by codon optimization of this mutant strain, which was transformed into GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K- strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.
乙酰胆碱酯酶（AChE）是一种关键酶，常用于酶抑制法检测有机磷农药残留。在本实验室 AChE 表达过程中，意外发现了一种具有 AChE 活性的突变株。通过对该突变株进行密码子优化，构建了 pPIC9K-乙酰胆碱酯酶（）表达载体，将其转化至毕赤酵母 GS115 中，在含 4.0 mg/mL G418 的酵母氮源基础盐（YPD）平板上筛选阳性克隆。GS115-pPIC9K-菌株经 0.5%甲醇诱导表达 120 h 后，发酵上清液的三氯乙酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）图谱中发现 4.3 kDa 的蛋白条带。经硫酸铵沉淀初步纯化后，检测酶活为 76.9 U/（mL·min）。此外，农药敏感性测试表明，DmAChE-like 对有机磷农药具有选择性和敏感性。
