将杂优-2平菇菌丝进行液体培养,发酵液经硫酸铵分级沉淀、DEAE(diethylaminoethyl)-Sepharose fast flow层析和Superdex-200 prep grade层析等方法纯化,获得了电泳纯的杂优-2平菇漆酶,并对纯化的漆酶进行了部分酶学性质研究。结果显示,杂优-2平菇漆酶比活力为115 U/mg,分子质量约为244.0 k D,亚基分子质量约为85.6 k D。最适反应p H值和最适反应温度分别为5.0和55℃,在p H 6.0~8.0及40~55℃范围内稳定性较好;最适条件下,以2,2’-联氮-二(3-乙基苯并噻唑-6-磺酸)为底物的Km值为2.1 mmol/L,最大反应速率(vmax)为0.117μmol/(min·L)。Fe^(2+)、抗坏血酸对该酶活性具有完全抑制作用,乙二胺四乙酸、Ag^+、Mg^(2+)、Li^+对该酶活性影响较小;草酸、甲醇、正丁醇、K^+、Ca^(2+)、Ba^(2+)、Zn^(2+)、Cd^(2+)、Pb^(2+)、Mn^(2+)、Co^(2+)对该酶活性有不同程度的抑制作用;Cu^(2+)激活作用不明显;尿素、乙醇、异丙醇对该酶活性具有激活作用。
[Objective]The mechanism of alkaline phosphatase（ALP） was studied to promote rice-field eel aquaculture industry. [ Method] The effects of effectors such as multiple metal ions and organic solvents on ALP in viscera of rice-field eel. [ Result] Na^＋ and K ^＋ didn＇t generate big influences on enzyme activity;Mg^2＋ and Ca^2＋ could promote ALP while Li^＋,Cu^2＋ and Zn^2＋ could restrain ALP enzyme activity. Both HPO4^2- and WO4^2- generated by en- zyme catalyzing disodium phenyl phosphate possessed strong inhibitory effects on emzymc, and 9.5 mmol/L HPO4^2 - would make enzyme activity decline by 13% while 9.5 mmol/L WO4^3- would make enzyme decline by 34%. The inhibition types of them were both competitive inhibition on enzyme activity. The organic solvents such as methanol, ethanol,ethylene glycol,isopropannl all generated influences on ALP and the order according to their inhibitory effects was isopropanol 〉 ethanol 〉 methanol 〉 ethylene glycol. [ Conclusion] The inflncnces of various effeetors on ALP aetivity of rice-field eel were studied from dynamics perspective to provide theoretical basis for further clarifying ALP mechanism.
