Fungal degradation of wood:: initial proteomic analysis of extracellular proteins of Phanerochaete chrysosporium grown on oak substrate
Fungal degradation of wood:: initial proteomic analysis of extracellular proteins of Phanerochaete chrysosporium grown on oak substrate
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DOI：
10.1007/s00294-004-0550-4
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发表时间：
2005-01-01
期刊：
影响因子：
2.5
通讯作者：
Tien, M
中科院分区：
文献类型：
--
作者：
Abbas, A;Koc, H;Tien, M
采用双向凝胶电泳技术对白腐真菌黄孢原毛平革菌胞外蛋白进行分离。红橡木木片上生长的固体基质培养物产生的细胞外蛋白制剂不适合2-D凝胶分析。然而，用水预洗涤木屑有助于减少棕色物质的量，棕色物质导致等电聚焦凝胶的酸性侧上的涂抹。这些木材培养物的二维凝胶显示了超过45个蛋白质点。用胰蛋白酶对这些斑点进行凝胶内消化，然后通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS）或通过液相色谱（LC）/MS/MS测序进行肽指纹分析。两种方法的数据通过Protein Prospector和当地P. chrysosporium注释数据库进行分析。MALDI-TOF/MS在分析的25种蛋白质中仅鉴定出2种。这很可能是由于与糖基化相关的问题。通过LC/MS/MS对相同的25种蛋白质进行蛋白质测序，鉴定出16种蛋白质。大多数鉴定的蛋白质作用于纤维素或半纤维素或其水解产物。到目前为止，还没有检测到木质素过氧化物酶、锰过氧化物酶或漆酶。
Two-dimensional (2-D) gel electrophoresis was used to separate the extracellular proteins produced by the white-rot fungus Phanerochaete chrysosporium. Solid-substrate cultures grown on red oak wood chips yielded extracellular protein preparations which were not suitable for 2-D gel analysis. However, pre-washing the wood chips with water helped decrease the amount of brown material which caused smearing on the acidic side of the isoelectric focusing gel. The 2-D gels from these wood-grown cultures revealed more than 45 protein spots. These spots were subjected to in-gel digestion with trypsin followed by either peptide fingerprint analysis by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or by liquid chromatography (LC)/MS/MS sequencing. Data from both methods were analyzed by Protein Prospector and the local P. chrysosporium annotated database. MALDI-TOF/MS only identified two proteins out of 25 analyzed. This was most likely due to problems associated with glycosylation. Protein sequencing by LC/MS/MS of the same 25 proteins resulted in identification of 16 proteins. Most of the proteins identified act on either cellulose or hemicellulose or their hydrolysis products. Thus far no lignin peroxidase, Mn peroxidase or laccases have been detected.