Botrytis cinerea蛋白磷酸酶的分离纯化及酶学性质  

Purification and Characterization of Phosphatase from Botrytis cinerea

作　　者：方夏[1] 李琳[1] 韩君芳[1] 吴芳[1] 毛雪萍[1] 陈小龙[1] 

机构地区：[1]浙江工业大学生物与环境工程学院发酵工程研究所,杭州310014

出　　处：《农药》2011年第11期795-798,共4页Agrochemicals

基　　金：国家重点基础研究发展计划(973计划)(2010CB126101);浙江省科技厅重大专项(2007C12088)

摘　　要：【目的】植物真菌病害灰霉病是由灰葡萄孢菌（Uotrytis 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12．71mmol／（L·min）与12．84mmol／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。Botrytis cinerea is an ubiquitous fungus which causes grey mould on many economically important crops. The rapid development of tolerance to commercial fungicides against B. cinerea has led to an increase in the quantities of these compounds. Due to the excellent biological activities of compounds with maleic anhydride structure, we can study their inhibition against protein phosphatase. [Methods] The protein phosphatase from 13. cinerea was purified through ultrasonication, centrifugation, ammonium sulfate precipitation and ion-exchange chromatography. The enzyme purity was tested by SDS-PAGE. Then, we studied its enzymatic properties and the inhibition of compounds with maleic anhydride structure. [Results] The purified enzyme had only a single band on SDS-PAGE. The purification fold and yield were 30.79 and 39.26%. Its optimum pH and temperature for reaction were pH 4.0 and 37℃. The enzyme was stable at pH 3.0- 6.0 below 50℃. A survey of effects of metal ions on the enzyme showed that the metal ions such as Fe2＋,Ca2＋,Ba2＋,Mg2＋ activated the enzyme and Zn2＋,Cu2＋,Mn2＋ inhibited the enzyme. The Vmax and Km values of protein phosphatase for p-NPP were 12.71 mmol/（L.min） and 12.84 mmol/L. The results indicated that the effects on the phosphatase depended on the metal ions. In the end, we compared with the inhibition of kinds of maleic anhydride structure compounds on the enzyme： Phenylmaleic anhydride 〉 4,5-dichlorophthalic anhydride 〉 dimethyl maleic anhydride 〉 maleic anhydride. [Conclusions] In sum, the enzyme activity loss reduced through fewer purification steps. Some of the compounds with maleic anhydride investigated in the study have good inhibition activity against protein phosphatase from 13. cinerea.

关 键 词：BOTRYTIS CINEREA 蛋白磷酸酶 分离纯化 抑制 

分 类 号：TQ450.1[化学工程—农药化工]