蔵灵菇源克鲁维酵母M3菌株胆盐水解酶的特性研究

Studies on the Characteristics of Bile Salt Hydrolase by Kluyveromyces marxianus M3 from Tibetan Kefir

探讨蔵灵菇马克斯克鲁维酵母M3菌株胆盐水解酶作用底物的反应条件与金属离子对酶活性的影响及其发酵动力学类型。采用单因素多水平试验方法,在pH4～8、温度31～43℃、底物浓度4～8mmol/L及不同化学试剂(SDS、EDTA、尿素、Cu2+、Mg2+、Ca2+、Fe3+、Al3+、Mn2+)的条件下,胆盐水解酶与底物反应30min,检测酶活力;平板活菌计数法分析细胞生长与产酶的关系;双倒数作图法求得酶促反应动力学常数Km。结果表明,胆盐水解酶最适反应条件:pH为6.0、底物浓度为7mmol/L、温度为37℃,Mn2+、Fe3+、Ca2+金属离子对酶活性有较大提高作用,其他化学试剂对酶活性影响不大。M3菌株在18～21h进入稳定期,于18h对数生长期时酶活性达到最高,表明其胆盐水解酶发酵动力学类型为生长偶联合成型。其酶促反应动力学常数Km为2.60mmol/L,说明该酶与最适底物的亲和力较大。

The reacting condition of zymolyte, the metal ion effect on the enzyme activity and dynamic principle type of the bile salt hydrolase were investigated in Kluyveromyces marxianus M3 from Tibetan Kefir. The single factor and multi levels orthogonal experiments were applied. Enzyme activity was tested under the different contitions： pH 4-8, temperature 31-43℃, concentration of substrate 4-8 mool/L, and different chemical reagents（SDS, EDTA, carbamide, Cu^2＋ , Mg^2＋, Ca^2＋ , Fe^3＋. Al^3＋ , Mn^2＋） during 30 min reaction between bile salt hydrolase and substrate. The relationship between cell growth and enzyme was analyzed by plate count method. The enzyme kinetics constant Km was calculated by double-reciprocal plot. The experiment results indicated that optimal reaction condition of bile salt hydrolase was pH 6.0, concentration of substrate 7 mmol/L, temperature 37 ℃. Metal ion of Mn^2＋, Fe^3＋, Ca^2＋ had comparatively large elevation effect on the enzyme activity, and other chemical reagents had few effects on the enzyme activity. Strain M3 entered the stationary phases after 18- 21 h. The enzyme activity reached the maxlmum during log phase growth at 18 h. The experiments results showed the category of dynamic principle was growth couple synthesis type. The kinetic constant Km of bile salt hydrolase was 2.60 mmol/L, and indicated comparatively large affinity between bile salt hydrolase and optimization substrate.