Dave K I, Phillips L, Luckow V A, Wild J R
Department of Biochemistry, Texas A&M University System, College Station 77843.
Biotechnol Appl Biochem. 1994 Jun;19(3):271-84.
A recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been utilized to express the opd (organophosphate-degrading) gene from Pseudomonas diminuta in insect tissue-culture cells (Sf9) of the fall armyworm (Spodoptera frugiperda). The broad-spectrum organophosphate hydrolase (EC 3.1.8.1) encoded by this gene is a member of a general class of enzymes [organophosphate (OP) anhydrolases] that include parathion hydrolases, di-isopropyl-fluorophosphatases (DFPases), somanases, and OP phosphotriesterases. This particular enzyme possesses the ability to hydrolyse paraoxon (P-O bond), DFP, sarin (P-F bond), VX (P-S bond) and tabun (P-CN bond), as well as a number of other extensively used organophosphorus pesticides. The enzyme produced in infected Sf9 cells is post-translationally processed and resembles the mature form of the enzyme expressed in various bacterial cells as identified by immunoprecipitation on Western blots. N-terminal sequence analysis of enzyme expressed in insect cells revealed Gly-29 as the terminal residue, whereas expression in Escherichia coli removes this residue, exposing Ser-30 at the N-terminus. Conditions for optimal expression of the enzyme in this system are described. Furthermore, hydrolytic efficiency of some OPs with purified enzyme from this system is discussed in relation to the in situ activity of Pseudomonas diminuta MG cells.
一种重组杆状病毒，苜蓿银纹夜蛾核型多角体病毒（AcNPV），已被用于在秋粘虫（草地贪夜蛾）的昆虫组织培养细胞（Sf9）中表达来自微小假单胞菌的opd（有机磷降解）基因。该基因编码的广谱有机磷水解酶（EC 3.1.8.1）是一类通用酶[有机磷（OP）水解酶]的成员，这类酶包括对硫磷水解酶、二异丙基氟磷酸酶（DFP酶）、梭曼酶和OP磷酸三酯酶。这种特殊的酶具有水解对氧磷（P-O键）、DFP、沙林（P-F键）、VX（P-S键）和塔崩（P-CN键）以及许多其他广泛使用的有机磷农药的能力。在感染的Sf9细胞中产生的酶经过翻译后加工，并且通过蛋白质免疫印迹法进行免疫沉淀鉴定，其与在各种细菌细胞中表达的酶的成熟形式相似。对昆虫细胞中表达的酶进行N端序列分析，结果显示Gly-29为末端残基，而在大肠杆菌中表达时会去除该残基，使Ser-30暴露于N端。本文描述了该系统中酶的最佳表达条件。此外，还讨论了用该系统纯化的酶对一些有机磷农药的水解效率与微小假单胞菌MG细胞的原位活性之间的关系。
