CAJ | 학술논문

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408 nm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(protohemeⅨ)作为辅基.计算获得酶的表观米氏常数为32.5 mmol/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pH 5～9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,0.5 mol/L NaCl、pH 10.5条件下的酶活半衰期达90 h.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.
종일주저도기염、겸성기감아포간균Bacillus sp.F26중순화득도일충감성과양화경매,병대해매진행료성질연구.순화과정경류산안침정、음리자교환층석、응효과려층석급소수층석사보최종획득전영순적목표매(순화58.5배).해과양화경매적분자량위140kD,유량개대소상동적아기조성.천연매분자재408 nm처현시특정흡수봉(Soret band).필정혈색소광보현시료매분자이원계람Ⅸ(protohemeⅨ)작위보기.계산획득매적표관미씨상수위32.5 mmol/L.해과양화경매불수련이아류산납적환원작용영향,단피청화물、첩담화물화3-안기-1,2,4-삼서(단공능과양화경매적전일억제제)강렬억제.이린련회향알、린분이알화이안기련분알작위전자공체측정매활시,해매불현시과양화물매활성.동시,매적N-단서렬비대결과설명,해과양화경매여단공능과양화경매아군유일정적상사성,이여쌍공능과양화경매아군급맹과양화경매아군균몰유동원성.인차,본문장순화적감성과양화경매정성위단공능과양화경매.차외,해매구유열민감적특점,차매활재pH 5～9적범위내불수pH영향,차후,활성수착pH적승고이승고,병재pH 11처유명현적매활고봉.20℃、pH 11조건하적매활반쇠기체49h.재pH 11적고감조건하표현출최고활력화일정적은정성,저재이보도적과양화경매중환미견묘술.동시,해매야현시료량호적염감은정성,0.5 mol/L NaCl、pH 10.5조건하적매활반쇠기체90 h.령일방면,본문소연구적과양화경매시제일개래원우기감미생물적동원이취체단공능과양화경매,야시제일개래원우천연감호적단공능과양화경매,타능부분지반영출세포항양화체계대상응배경적괄응정황.
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 thetypical 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 H2 O2 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 ℃ 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.