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OALib Journal期刊
ISSN: 2333-9721
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胆红素氧化酶及其变异体在胆红素血症评价中的应用
, PP. 1337-1340
Keywords: 胆红素,胆红素氧化酶,聚合酶链反应,高胆红素血症
Abstract:
目的探讨胆红素氧化酶中配位氨基酸对保持酶活性的重要性和酶促反应的动力学,并评价胆红素氧化酶突变体能否作为一种更好的高胆红素血症的诊断试剂。方法胆红素氧化酶突变体i402g和c457s通过聚合酶链反应获得,并经氨基酸序列测定加以证实。钌搀杂的突变体c457s通过变异体与钌配合物的直接反应得到。研究了重组胆红素氧化酶及其变异体的光谱学性质,以及重组胆红素氧化酶和变异体i402g的酶促反应的动力学。结果变异体i402g和c457s被成功的表达和纯化。变异体i402g表现出较低的活性(1.67u/mg),c457s则完全失去了活性(0u/mg),但是钌搀杂的突变体c457s表现出较高的活性(5.50和6.42u/mg)。光谱学实验结果表明i402g突变体在较低的ph时第一类铜呈+1价态,即还原态;突变体c457s中失去第一类铜,使得突变体无法保持完整的活性中心,从而失去了酶活性。酶促反应的动力学实验显示重组胆红素氧化酶和变异体i402g动力学参数kcat分别为235.8min-1和6.9min-1,说明重组胆红素氧化酶具有很高的催化能力。结论配位的氨基酸对保持重组胆红素氧化酶及其变异体活性中心的完整性和酶活性具有重要的意义。重组胆红素氧化酶远比突变体i402g和c457s的活性高,说明突变体i402g和c457s不适合作为诊断试剂。钌搀杂有助于在突变体c457s中形成新的完整的活性中心,从而产生酶活性。
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