普鲁兰短梗霉N2-3菌株碱性蛋白酶的纯化及性质研究

Purification and Characters of the Protease from Marine Yeast Aureobosidium pullulans N2-3

[目的]对普鲁兰短梗霉N2-3菌株的碱性蛋白酶进行纯化,并对其性质进行研究。[方法]利用层析柱Sephadex G-75与阴离子交换柱DEAE Sepharose Fast Flow纯化菌株N2-3的胞外碱性蛋白酶,并对纯化的酶进行SDS-PAGE凝胶电泳检测,同时对纯化蛋白酶的最适反应温度、温度稳定性、最适反应pH、pH稳定性进行测定,研究金属离子及各种化合物对蛋白酶活性的影响。[结果]试验中N2-3菌株的碱性蛋白酶纯化倍数和回收率分别为2.34和25.9%;SDS-PAGE凝胶电泳显示该酶的分子量约为33 kDa;该酶的最适反应温度和pH分别为52℃和9.0;Mn2+、Mg2+、Na+离子浓度在5 mmol/L时对纯化蛋白酶的酶活有激活作用,Zn2+、Ca2+、K+、Li+对酶活的影响不大,而当存在Fe2+、Fe3+、Cu2+、Co2+、Ag+、Hg2+离子时,蛋白酶活明显降低;苯甲基硫酰氟(PMSF)强烈抑制了该蛋白酶的活性,而乙二胺四乙酸(EDTA)与碘乙酸微弱抑制了该蛋白酶的活性。[结论]为普鲁兰短梗霉N2-3菌株碱性蛋白酶的工业化生产提供了一定的理论基础。

[Objective] To purify alkaline protease of Aureobosidium pullulans N2-3,and study its properties.[Method] By using Sephadex G-75 and DEAE Sepharose Fast Flow to purify alkaline protease of Aureobosidium pullulans N2-3,and SDS-PAGE was carried out.The optimal reaction temperature,temperature stability,optimal reaction pH,pH stability were determined,effects of each metal ion and compound on protease activity were studied.[Result] The purification multiples and recovery rate were 2.34,25.9%,respectively.The molecular weight of this enzyme was about 33 kDa;the optimal temperature and pH were 52 ℃ and 9.0;the enzyme was activated by Mg2＋,Na＋ and Mn2＋ at a concentration of 5.0 mmol/L and inhibited by Fe2＋,Fe3＋,Cu2＋,Co2＋,Ag＋ and Hg2＋.Zn2＋,Ca2＋,K＋ and Li＋ have no effect on the protease activity.The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride,and weakly inhibited by EDTA and iodoacetic acid.[Conclusion] The study will provide a theoretical basis for industrialization production of protease from Aureobosidium pullulans N2-3.