嗜碱芽孢杆菌Bacillus sp.F26过氧化氢酶的分离纯化及性质研究

从一株低度嗜盐、兼性嗜碱芽孢杆菌Bacillus sp．F26中纯化得到一种碱性过氧化氢酶，并对该酶进行了性质研究。纯化过程经硫酸铵沉淀、阴离子交换层析、凝胶过滤层析及疏水层析四步最终获得电泳纯的目标酶(纯化58．5倍)。该过氧化氢酶的分子量为140kD，由两个大小相同的亚基组成。天然酶分子在408nm处显示特征吸收峰(Soret band)。吡啶血色素光谱显示了酶分子以原卟啉Ⅸ(protoheme Ⅸ)作为辅基。计算获得酶的表观米氏常数为32．5mmol／L。该过氧化氢酶不受连二亚硫酸钠的还原作用影响，但被氰化物、叠氮化物和3．氨基．1，2，4-三唑(单功能过氧化氢酶的专一抑制剂)强烈抑制。以邻联茴香胺、邻苯二胺和二氨基联苯胺作为电子供体测定酶活时，该酶不显示过氧化物酶活性。同时，酶的N-端序列比对结果说明，该过氧化氢酶与单功能过氧化氢酶亚群有一定的相似性，而与双功能过氧化氢酶亚群及猛过氧化氢酶亚群均没有同源性。因此，本文将纯化的碱性过氧化氢酶定性为单功能过氧化氢酶。此外，该酶具有热敏感的特点，且酶活在pH5～9的范围内不受pH影响，此后，活性随着pH的升高而升高，并在pH 11处有明显的酶活高峰。20℃、pH 11条件下的酶活半衰期达49h。在pH 11的高碱条件下表现出最高活力和一定的稳定性，这在已报道的过氧化氢酶中还未见描述。同时，该酶也显示了良好的盐碱稳定性，0．5mol／L NaCl、pH 10．5条件下的酶活半衰期达90h。另一方面，本文所研究的过氧化氢酶是第一个来源于嗜碱微生物的同源二聚体单功能过氧化氢酶，也是第一个来源于天然碱湖的单功能过氧化氢酶，它能部分地反映出细胞抗氧化体系对相应环境的适应情况。

Purification and Characterization of a Monofunctional Catalase from an Alkaliphilic Bacillus sp. F26

An alkaline catalase has been purified and characterized from a slightly halophilic and alkaliphilic bacterium Bacillus sp. F26. The purification was performed with a four step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography, and finally achieved a 58.5-fold-purifying over the crude extract. The purified catalase was composed of two identical subunits with a native molecular mass of 140 kD. The native enzyme showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide, and 3-amino-1,2,4-triazole (the specific inhibitor of monofunctional catalase). No peroxidase activity of this enzyme was detected when using o-dianisidine, diaminobenzidine (DAB) and p-phenylenediamine as electron donor. Moreover, the N-terminal sequence of this catalase exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase one. Therefore, we characterize the purified catalase as a monofunctional catalase. Besides, this monofunctional catalase was thermosensitive and its activity exhibited pH-independent over pH 5-9 but showed a sharp maximum at pH 11. An activity half-life of approximately 49 h was measured when the enzyme was incubated at 20 degrees C and pH 11. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now. Furthermore, this monofunctional catalase also showed excellent halo-alkali-stability with a half-life of approximately 90 h at 0.5 mol/L NaCl and pH 10.5. On the other hand, so far as we know, the characterized catalase is the first dimeric monofunctional catalase from alkaliphiles and is also the first monofunctional catalase derived from a natural soda lake, which could partially reflect the oxidative stress response in the corresponding environment.