Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus.
Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus.
复制标题
使用重组杆状病毒对金孢原毛平革菌活性木质素过氧化物酶进行异源表达和表征。
DOI：
10.1016/0003-9861(91)90148-c
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发表时间：
1991
影响因子：
3.9
通讯作者：
Li,JK
中科院分区：
文献类型：
--
作者：
Johnson,TM;Li,JK
利用重组杆状病毒系统异源表达了白色腐真菌黄孢原毛平革菌胞外木质素过氧化物酶基因λML-1。重组蛋白的糖基化胞外形式含有铁原卟啉IX部分，并且能够氧化碘化物和模型木质素化合物藜芦醇。在使用愈创木酚和Mn（II）的比较过氧化物酶测定中，重组木质素过氧化物酶似乎不依赖于Mn（II）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，异源表达的过氧化物酶的表观分子量相似的天然真菌同工酶H8。通过离子交换层析和使用抗H8单克隆抗体的免疫印迹分析得到的活性重组酶的洗脱曲线提供了λML-1 DNA编码木质素过氧化物酶H8的进一步证据。
The cDNA clone λML-1 encoding one of the extracellular lignin peroxidases from the white rot fungus,Phanerochaete chrysosporium, was heterologously expressed in an active form using a recombinant baculovirus system. The glycosylated extracellular form of the recombinant protein contained the ferriprotoporphyrin IX moiety and was capable of oxidizing both iodide and the model lignin compound, veratryl alcohol. In comparative peroxidase assays using guaiacol and Mn(II), the recombinant lignin peroxidase did not appear to be Mn(II) dependent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the heterologously expressed peroxidase had an apparent molecular weight similar to that of the native fungal isozyme H8. The elution profile of the active recombinant enzyme derived by ion-exchange chromatography and immunoblot analysis using an anti-H8 monoclonal antibody provided further evidence that the λML-1 DNA encodes the lignin peroxidase H8.