黑腹果蝇羧酸酯酶 LmCesA1 的表达与动力学分析。
Expression and kinetic analysis of carboxylesterase LmCesA1 from Locusta migratoria.
机构信息
Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan, 030006, China.
College of Plant Protection, Shanxi Agricultural University, Taiyuan, 030031, China.
出版信息
Biotechnol Lett. 2021 May;43(5):995-1004. doi: 10.1007/s10529-021-03086-1. Epub 2021 Jan 29.
OBJECTIVE
To investigate the biochemical characterization of the carboxylesterase LmCesA1 from Locusta migratoria.
RESULTS
We expressed recombinant LmCesA1 in Sf9 cells by using the Bac-to-bac baculovirus expression system. Enzyme kinetic assays showed that the K values of LmCesA1 for α-naphthyl acetate (α-NA) and β-naphthyl acetate (β-NA) were 0.08 ± 0.01 mM and 0.22 ± 0.03 mM, respectively, suggesting that LmCesA1 has a higher affinity for α-NA. LmCesA1 retained its enzymatic activity during incubations at pH 7-10 and at 10-30 °C. In an inhibition experiment, two organophosphate pesticides (malaoxon and malathion) and one pyrethroid pesticide (deltamethrin) showed different inhibition profiles against purified LmCesA1. Recombinant LmCesA1 activity was significantly inhibited by malaoxon in vitro. UPLC analysis showed that no metabolites were detected.
CONCLUSIONS
These results suggest that overexpression of LmCesA1 enhances malathion sequestration to confer malathion tolerance in L. migratoria.
目的
研究中华稻蝗羧酸酯酶 LmCesA1 的生化特性。
结果
我们利用 Bac-to-bac 杆状病毒表达系统在 Sf9 细胞中表达了重组 LmCesA1。酶动力学分析表明，LmCesA1 对α-萘乙酸酯（α-NA）和β-萘乙酸酯（β-NA）的 K 值分别为 0.08±0.01 mM 和 0.22±0.03 mM，表明 LmCesA1 对α-NA 具有更高的亲和力。LmCesA1 在 pH 值为 7-10 和 10-30°C 的孵育条件下保持酶活性。在抑制实验中，两种有机磷农药（马拉硫磷和马拉氧磷）和一种拟除虫菊酯农药（溴氰菊酯）对纯化的 LmCesA1 表现出不同的抑制谱。重组 LmCesA1 的活性在体外明显被马拉硫磷抑制。UPLC 分析表明未检测到代谢物。
结论
这些结果表明，LmCesA1 的过表达增强了马拉硫磷的隔离作用，从而赋予了中华稻蝗对马拉硫磷的耐受性。
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