枯草芽孢杆菌(Bacillus subtilis)过氧化氢酶的分离纯化及性质

Purif ication and Characterization of Catalase from Bacillus subtilis WSHDZ-01

对产自枯草芽孢杆菌Bacillus subtilis WSHDZ-01的过氧化氢酶,通过乙醇沉淀、DEAE阴离子交换层析、疏水层析3步纯化,最终获得电泳纯的目标酶(纯化6.8倍).该过氧化氢酶的亚基相对分子质量(Mr)为63×103,在405nm处显示特征吸收峰,推测含有血红素.计算获得酶的表观米氏常数为26.87mmolL-1.该过氧化氢酶不受低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.以邻苯二胺作为电子供体测定酶活时,该酶不显示过氧化物酶活性,因此本文将纯化的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pH5～10范围内不受pH影响,此后,活性随着pH的升高而升高,并在pH11～12处有明显的酶活高峰,在pH11、25℃放置60min酶活基本不变,表明该酶在高碱条件下具有很高的活力和一定的稳定性.

A catalase from Bacillus subtilis WSHDZ-01 was purified and characterized. The purification was performed with a three-step procedure consisting of ethanol precipitation, DEAE ion exchange and hydrophobic interaction chromatography, and finally achieved a 6.8-fold purification over the crude extract. SDS-PAGE of the purified catalase revealed the final presence of a single band at an apparent molecular mass of 63x103. The native enzyme showed the typical Sort band appearing at 405 nm, which indicates that the purified catalase is a heme-containing enzyme. The apparent Km value for enzyme activity on H2O2 was calculated to be 26.87 mmol L^-1 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 to monofunctional catalase）. No peroxidase activity of this enzyme was detected when using p-phenylenediamine as an electron donor. Therefore, it was suggested that this catalase belonged to monofunctional catalases. Besides, this catalase was thermosensitive, and its activity exhibited pH-independently between pH 5-10, but showed a sharp maximum at pH 11-12. No activity was lost when the enzyme was incubated at 25 ℃ and pH 11 for 60 min. Fig 5, Tab 2, Ref 19