Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science, South China Agricultural University, Guangzhou, 510642, People's Republic of China.
College of Light Industry and Food, Zhongkai University of Agriculture and Engineering, Guangzhou, 510225, People's Republic of China.
Appl Biochem Biotechnol. 2021 Oct;193(10):3321-3335. doi: 10.1007/s12010-021-03597-7. Epub 2021 Jun 23.
The cholinesterase-based spectrophotometric assay, also called enzyme inhibition method, is a good choice for rapid detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs). Obviously, the cholinesterase is the core reagent in enzyme inhibition method. In our previous work, a recombinant acetylcholinesterase 2 from Bombyx mori (rBmAChE2) was expressed in yeast successfully and exhibited great sensitivity. However, the yield of rBmAChE2 is not desirable. In this study, a codon optimization strategy was employed to enhance the yield of rBmAChE2 in Pichia pastoris GS115. Results showed that by replacing 6 key rare codons and increasing the percentage of bases G and C up to 46.85%, codon adaptation index (CAI) of Bombyx mori acetylcholinesterase 2 (bmace2) gene was improved from 0.70 to 0.81. After being transformed into Pichia pastoris GS115 via electroporation, the expression transformant can produce 139.7 U/mL secretory codon-optimized rBmAChE2 (opt-rBmAChE2) in the culture supernatant, 3.62 times higher than that of strain bearing the wild-type bmace2 gene. Meanwhile, opt-rBmAChE2 displayed on the yeast surface was up to 2280.02 U/g, 2.8 times higher than wild-type displayed rBmAChE2. In addition, either secretory or surface-displayed opt-rBmAChE2 maintained the similar sensitivities to the wild-type rBmAChE2 for tested inhibitors. Furthermore, the detection limits of the opt-rBmAChE2-based enzyme inhibition method for 10 kinds of OPs or CPs (0.01-2.69 mg/kg) were lower than most of the indexes present in current standard method (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019) in China. The results might contribute to the utilization of rBmAChE2 for pesticide residue screening detection in practice.
基于胆碱酯酶的分光光度测定法，也称为酶抑制法，是快速检测有机磷农药（OPs）和氨基甲酸酯农药（CPs）的理想选择。显然，胆碱酯酶是酶抑制法的核心试剂。在我们之前的工作中，成功地在酵母中表达了重组家蚕乙酰胆碱酯酶 2（rBmAChE2），并表现出很高的灵敏度。然而，rBmAChE2 的产量并不理想。在这项研究中，采用了一种密码子优化策略来提高毕赤酵母 GS115 中 rBmAChE2 的产量。结果表明，通过替换 6 个关键稀有密码子，并将碱基 G 和 C 的比例提高到 46.85%，家蚕乙酰胆碱酯酶 2（bmace2）基因的密码子适应指数（CAI）从 0.70 提高到 0.81。通过电穿孔转化到毕赤酵母 GS115 后，表达转化体可以在培养上清液中产生 139.7 U/mL 的分泌密码子优化的 rBmAChE2（opt-rBmAChE2），比携带野生型 bmace2 基因的菌株高 3.62 倍。同时，酵母表面展示的 opt-rBmAChE2 高达 2280.02 U/g，比野生型展示的 rBmAChE2 高 2.8 倍。此外，分泌型或表面展示型 opt-rBmAChE2 对测试抑制剂的敏感性与野生型 rBmAChE2 相似。此外，基于 opt-rBmAChE2 的酶抑制法对 10 种 OPs 或 CPs（0.01-2.69 mg/kg）的检测限低于中国现行标准方法（GB/T 5009.199-2003）或最大残留限量（GB 2763-2019）中的大多数指标。这些结果可能有助于在实践中利用 rBmAChE2 进行农药残留筛选检测。
