来源于豆豉的枯草芽孢杆菌MX-6所产纳豆激酶的稳定性研究

以中国豆豉中筛选到的1株纤溶酶高产菌株——枯草芽孢杆菌MX-6为研究对象,应用聚合酶链式反应(PCR)成功扩增到1473bp的纤溶酶编码基因aprN,系统进化树结果显示该纤溶酶为纳豆激酶。研究了纳豆激酶对热、pH、金属离子、化学物质、抑制剂和变性剂的稳定性及传代稳定性。结果表明:纳豆激酶在-20,4,30~50℃的热稳定性较好,pH在3~11之间酶活性相对稳定,Mg2+、Ca2+对纳豆激酶具有显著的激活作用,K+、Fe2+对纳豆激酶的稳定性基本无影响,而Mn2+、Zn2+、Cu2+、Al3+、Ba2+有显著的抑制作用,Hg2+导致纳豆激酶完全失活,牛血清蛋白、明胶、蛋白胨能够显著提高纳豆激酶的稳定性,丙二醇、海藻酸钠对纳豆激酶的稳定性基本无影响,而乙醇显著降低纳豆激酶的稳定性。PMSF导致纳豆激酶的活性完全丧失,EDAT和DTT对纳豆激酶的活性基本无影响,10mmol/L的SDS显著降低纳豆激酶的稳定性,说明此纳豆激酶是一种丝氨酸蛋白酶,不是金属酶,不含二硫键,且具有良好的传代稳定性。结果显示该酶的稳定性较好,具有开发为溶栓药物的应用价值,有利于纳豆激酶的工业化应用。

Study on Stability of Nattokinase Produced by Bacillus subtilis MX-6from Fermented Soya Beans

Bacillus subtilis MX-6, a high-yield strain of fibrinolytic enzyme, is screened from Chinese fermented soya beans. The fibrinolytic gene aprN is successfully amplified to 1473 bp by polymerase chain reaction (PCR). The phylogenetic tree results show that the fibrinolytic enzyme is nattokinase. The stability and generation stability of nattokinase to heat, pH, metal ions, chemicals, inhibitors and modifiers are studied. The results show that the thermal stability of nattokinase is good at -20,4,30~ 50 ℃, and the activity of nattokinase is relatively stable at pH 3~11. Mg 2+, Ca 2+ have significant activation effect on nattokinase. K +, Fe 2+ have little effect on the stability of nattokinase, while Mn 2+, Zn 2+, Cu 2+, Al 3+, Ba 2+ have significant inhibition effect. Hg 2+ leads to complete inactivation of nattokinase, bovine serum albumin, gelatin and peptone can significantly improve the stability of nattokinase, propylene glycol and sodium alginate have little effect on the stability of nattokinase, while ethanol significantly reduces the stability of nattokinase. PMSF results in the complete loss of nattokinase activity. EDAT and DTT have little effect on the activity of nattokinase. 10 mmol/L SDS significantly reduces the stability of nattokinase, which indicates that the nattokinase is a serine protease, not a metalloenzyme, does not contain disulfide bond, and has good generation stability. The results show that the enzyme has good stability and could be used as a thrombolytic agent, which is beneficial to the industrial application of nattokinase.