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[目的]植物真菌病害灰霉病是由灰葡萄孢菌(Botrytis cinerea)侵染引起,它在世界范围内造成了严重的经济损失。B.cinerea易对化学药剂产生抗性,寻找更多对灰霉病有特效的生物农药是目前急需解决的难题。鉴于顺丁烯二酸酐类化合物良好的生物活性,研究此类化合物对蛋白磷酸酶的抑制效果。[方法]将发酵优化后的B.cinerea菌体经超声破碎、硫酸铵分级盐析、透析浓缩、DEAE-Sepharase离子交换层析等步骤分离纯化,并以SDS-PAGE验证酶纯度。考察所得纯酶的酶学性质,研究顺丁烯二酸酐类化合物对B.cinerea蛋白磷酸酶的抑制作用。[结果]经分离纯化后,该酶SDS-PAGE为单一条带,提纯倍数达30.79倍,酶活回收率为39.26%。酶学特性研究结果表明其最适反应pH值为4.0,最适反应温度为37℃。该酶在pH值3.0~6.0,50℃以下内稳定。考察金属离子对B.cinerea蛋白磷酸酶酶活的影响,发现Mg2+、Fe2+、Ca2+、Ba2+能提高该酶活性,Zn2+、Cu2+、Mn2+对酶活有抑制作用。以p-NPP为底物的酶动力学参数Vmax和Km分别为12.71 mmol/(L.min)与12.84 mmol/L。4种顺丁烯二酸酐类化合物对B.cinerea蛋白磷酸酶的抑制作用由大到小排名为苯基马来酸酐、4,5-二氯邻苯二甲酸酐、二甲基马来酸酐、顺丁烯二酸酐。[结论]此酶分离纯化步骤少,减少了酶活损失。以纯化的蛋白磷酸酶为靶标,顺丁烯二酸酐类化合物对酶活抑制效果良好,可研究抑制灰霉病机理及作为生物源农药开发。
[Purpose] Botanical fungal disease Botrytis cinerea is a botrytis cinerea that causes serious economic losses worldwide. B.cinerea easily resistant to chemicals, looking for more effective against gray mold biological pesticides is currently an urgent need to solve the problem. In view of the good biological activity of maleic anhydride compounds, the inhibitory effect of such compounds on protein phosphatases was investigated. [Method] The fermentation-optimized B.cinerea was separated by sonication, fractionated by ammonium sulfate, concentrated by dialysis, purified by DEAE-Sepharose ion exchange chromatography. The purity of the enzyme was verified by SDS-PAGE. The enzymatic properties of pure enzyme were investigated to study the inhibitory effect of maleic anhydride on B.cinerea protein phosphatase. [Result] SDS-PAGE of this enzyme was a single band after purification. The purification fold was 30.79 times and the recovery rate of enzyme activity was 39.26%. The results of enzymology showed that the optimal reaction pH was 4.0 and the optimum reaction temperature was 37 ℃. The enzyme at pH 3.0 ~ 6.0, 50 ℃ the following stability. The effects of metal ions on phosphatase activity of B.cinerea protein were investigated. It was found that Mg2 +, Fe2 +, Ca2 + and Ba2 + could enhance the activity of the enzyme. Zn2 +, Cu2 + and Mn2 + could inhibit the enzyme activity. The kinetic parameters Vmax and Km of p-NPP were 12.71 mmol / (L.min) and 12.84 mmol / L, respectively. The inhibitory effects of four kinds of maleic anhydride compounds on B.cinerea protein phosphatase from high to low were phenyl maleic anhydride, 4,5-dichlorophthalic anhydride, dimethyl maleic anhydride, Maleic anhydride. [Conclusion] This enzyme had fewer steps of isolation and purification and reduced the loss of enzyme activity. With purified protein phosphatase as a target, maleic anhydride compounds have a good inhibitory effect on the enzyme activity and can be used to study the mechanism of inhibiting gray mold disease and develop it as a biogenic pesticide.