Abstract
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 Pichia pastoris. The pPIC9K-Drosophila melanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like 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.
摘要
目的
通过比较果蝇乙酰胆碱酯酶基因剪接变种(DmAChE-like)与果蝇乙酰胆碱酯酶(DmAChE)之间的酶活差异以及对有机磷农药的敏感性差异,为酶抑制法检测农药残留提供新的酶源。
创新点
DmAChE-like 是果蝇乙酰胆碱酯酶基因发生剪接突变产生的,在此之前并未被报道过. 本文通过检测证明了DmAChE-like具有乙酰胆碱酯酶活性,并且对大多数有机磷农药敏感. 这个发现为酶抑制法检测农药残留提供了新的酶源.。这个发现为酶抑制法检测农药残留提供了新的酶源。
方法
在本研究中,我们通过构建毕赤酵母重组表达菌株GS115-pPIC9K-DmAChE-like 并进行诱导表达,利用Ellman法检测其酶活,并进行酶学性质研究。我们选取了5种常见农药进行农药敏感性试验,比较其与DmAChE之间的差异性。
结论
在本研究中,我们构建了基因工程的GS115-pPIC9K-DmAChE-like 菌株,并在实验室水平诱导了基因表达。我们测试了各个发酵上清液的酶活性,证明了DmAChE-like具有乙酰胆碱酯酶活性。通过Tricine-SDS-PAGE,我们发现特异的蛋白条带为4.3 kDa,与预期结果一致。农药敏感性试验发现DmAChE-like对大多数选择的有机磷农药敏感,表明它可以用作有机磷农药检测酶抑制方法的新酶源。
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Acknowledgments
This work was supported by the National Development and Reform Commission of China (No. 20111158). We thank Wentao JING Engineer (Biological Experiment Teaching Center, College of Life Sciences Zhejiang University Hangzhou China) for providing the RNA samples from the head of Drosophila melanogaster.
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Liujia SHI performed the experimental research and data analysis, wrote and edited the manuscript. Fangfang YANG discovered the DmAChE-like gene and verified its function. Yanyan XU provided assistance for the experiment. Shoufeng WANG contributed to the study design, data analysis, and writing and editing of the manuscript. All authors have read and approved the final manuscript and, and therefore have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Liujia SHI, Fangfang YANG, Yanyan XU, and Shoufeng WANG declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Shi, L., Yang, F., Xu, Y. et al. Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides. J. Zhejiang Univ. Sci. B 22, 204–213 (2021). https://doi.org/10.1631/jzus.B2000525
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DOI: https://doi.org/10.1631/jzus.B2000525