海洋芽孢杆菌碱性酯酶BSE-1的纯化与性质研究

Purification of an alkaline esterase BSE-1 from marine Bacillus sp. and its characterization

海洋芽孢杆菌酯酶发酵液通过低温高速离心(10000r/min,30min,4℃)、乙醇-硫酸铵双水相沉淀和Q-Sepharose HR阴离子交换层析等技术进行了纯化,最终得到电泳纯的酯酶BSE-1。SDS-PAGE电泳结果表明,该酶表观分子量为30kDa。以对硝基苯磷酸酯(PNPP)为底物对BSE-1进行了理化性质研究,结果表明,BSE-1的最适反应温度为60℃,最适pH值为10.0,为嗜热碱性酯酶;该酶在60℃以下、pH 7～11范围内具有良好的热稳定性;与常见酸根离子、有机溶剂的配伍性较好,但金属离子对酶活影响较大,Li^+、Mg^(2+)、Ca^(2+)对BSE-1有激活作用,1mmol/L的Ca^(2+)使酶活提高2倍以上;Ba^(2+)、Fe^(3+)、Ag^+、Sr^(2+)对BSE-1有抑制作用,1mmol/L Ba^(2+)存在下酶活仅能保持10%左右。

A novel alkaline esterase BSE-1 was purified from a marine Bacillus sp. and its partial physical and chemical char- acteristics were studied. The SDS-PAGE homogeneity BSE-1 was purified from the fermentation liquid by high speed freezing centrifugation （10000r/min, 30min, 4℃ ）, ethanol-ammonium sulfate aqueous two-phase precipitation and Q- Sepharose HR anion exchange chromatography. The SDS-PAGE result showed its molecular weight was 30 kDa. In further studies, with PNPP as substrate, the results showed that the optimum pH of this esterase was 10.0 and its optimum reaction temperature was 50^（2. BSE-1 was stable when the temperature was below 60℃ and pH was 7 - 11. This char- acter gave BSE-1 good property to apply under higher temperature. BSE-1 also showed high activity at rather higher alka- line pH （higher than 10）. BSE-1 was compatible with some acid radical ions and showed good resistance to general orgarlic solvent. The effects of metal ions on BSE-1 showed that Li^＋ , Mg^2＋ and Ca^2＋ could increase its activity, and the activity would increase to 200% under lmmol/L Ca^2 ＋ . On the other hand, Ba^2 ＋ , Fe^3 ＋ , Ag^ ＋ and Sr^2 ＋ inhibited the activity of BSE-1, only 10% activity was remained under 1mmol/L Ba^2＋ .