三色雷蘑核糖核酸酶的分离纯化和理化性质

Purification and Characterization of a Ribonuclease from Leucopaxillus tricolor Fruit Bodies

依次采用离子交换层析(DEAE-Cellulose、CM-Cellulose和SP-Sepharose)和凝胶过滤层析(Superdex 75 HR10/300 GL)从三色雷蘑(Leucopaxillus tricolor)子实体中分离纯化得到一种核糖核酸酶(Ribonuclease,RNase)。该酶比活为1406 U/mg,纯化倍数为154,通过HPLC-LTQ-Orbitrap测序得到4条肽段序列,分别为DMAVSYLSR、IGLSSIPR、ILGRFVVDTYK和SGKANAATPK。该核糖核酸酶的相对分子质量为15000,最适反应pH为 9.0,最适反应温度为40 ℃,热稳定性高,80 ℃孵育1 h后能保持90%以上的活性:Cu^2+、Ca^2+、Mn^2+、Cd^2+和Al^3+在1.25~10 mmol/L浓度下对该酶活性具有抑制作用,且抑制作用随着金属离子浓度的升高而变强;Fe^2+在1.25~5 mmol/L的浓度下,Pb^2+、Zn^2+、Mg^2+、Hg^2+、Fe^3+在1.25~2.5 mmol/L 的浓度下对该酶的活性有促进作用。

A ribonuclease (RNase) was purified from Leucopaxillus tricolor fruit bodies by ion exchange chromatography (DEAE-Cellulose,CM-Cellulose and SP-Sepharose) and gel filtration (Superdex 75 HR10/300 GL). The purification fold of this RNase was 154 and the specific activity was 1406 U/mg. Four amino acid sequences were obtained (DMAVSYLSR,IGLSSIPR,ILGRFVVDTYK and SGKANAATPK) by HPLC-LTQ-Orbitrap identification of the RNase. The relative molecular mass of the RNase was estimated to be 15000. The enzyme displayed pH and temperature optima of 9.0 and 40 ℃, respectively.The enzyme showed good thermal stability and could remain more than 90% of the activity after 1 h exposure to 80 ℃.The enzyme activity was inhibited by Cu^2+, Ca^2+, Mn^2+, Cd^2+ and Al^3+ at concentration of 1.25~10 mmol/L and the inhibition effect increased with the increase of metal ion concentration. The enzyme activity was stimulated by Fe^2+ at concentration of 1.25~5 mmol/L and Pb^2+, Zn^2+, Mg^2+, Hg^2+, Fe^3+ at concentration of 1.25~2.5 mmol/L.