樟子松外生菌根中NADP-依赖型异柠檬酸脱氢酶的酶学性质研究

摘要

The purpose of the work is to purify the isocitrate dehydrogenase(IDH)in mycorrhizal tissue ofSuillus luteus-Pinus sylvestrisvar. mongolica, root tissue ofP. sylvestrisvar. mongolica and cultured fungal mycelia ofS. luteus, and identify their enzymatic characterizations. The IDHs of 3 sources were purified by ammonium sulfate precipitation and glucan gel chromatography and tested by SDS-PAGE electrophoresis, and enzymatic characterizations were studied. The Km for NADP+ of mycorrhiza, root and cultured fungal mycelia were 10.7μmol/L, 11.4μmol/L and 22.1μmol/L, respectively;the Kmfor isocitrate were 71.7μmol/L, 79.3μmol/L and 87.8μmol/L, respectively. The optimal pH of mycorrhiza, root and cultured fungal mycelia were 8.2, 8.0 and 7.5 respectively;they were all slightly in alkaline. The optimal temperatures of the IDHs were 45℃ for mycorrhiza and root, and 42℃ for the fungus. The activities of 3 IDHs relied on the binding of divalent metal ions, the maximum activities of IDHs were observed when assayed with Mn2+ or Mg2+ as metal cofactor;however, Ca2+, Co2+, Cu2+ and Zn2+ dramatically inhibited the activity of IDHs. Conclusively, protein content and enzyme activity of mycorrhizal IDH have been increased.%对褐环乳牛肝菌-樟子松菌根组织、樟子松根组织及褐环乳牛肝菌纯培养菌丝的异柠檬酸脱氢酶（isocitrate dehydrogenase，IDH）进行纯化和酶学性质鉴定。通过硫酸铵分级沉淀及葡聚糖凝胶层析纯化后的IDH进行SDS-PAGE电泳检测，并进行3种来源酶的酶学性质鉴定。菌根组织、根组织及真菌纯培养菌丝NADP-IDH对NADP+的Km值分别为10.7μmol/L、11.4μmol/L和22.1μmol/L；对异柠檬酸的Km值分别为71.7μmol/L、79.3μmol/L和87.8μmol/L。最适pH分别为8.2、8.0和7.5，略偏碱性。菌根IDH和根IDH的最适反应温度为45℃，真菌IDH的最适反应温度为42℃。3种IDH的活性依赖于不同的二价金属阳离子的存在， Mn2+、Mg2+存在时酶活性最强，Ca2+、Co2+、Cu2+和Zn2+对酶的活性有较强抑制作用。菌根真菌与樟子松形成外生菌根之后异柠檬酸脱氢酶的蛋白含量及酶活力都得到了提高。