Isolation and Identification of Cellulose-Binding Proteins from Sheep Rumen Contents
Isolation and Identification of Cellulose-Binding Proteins from Sheep Rumen Contents
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DOI：
10.1128/aem.01838-08
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发表时间：
2009-01
影响因子：
4.4
通讯作者：
A. Toyoda;Wataru Iio;M. Mitsumori;H. Minato
中科院分区：
文献类型：
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作者：
A. Toyoda;Wataru Iio;M. Mitsumori;H. Minato
摘要 为了加深我们对瘤胃中植物细胞壁降解机制的理解，使用宏蛋白质组学方法直接从绵羊瘤胃内容物中分离和鉴定纤维素结合蛋白（CBP）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离瘤胃CBP，一些CBP显示出对羧甲基纤维素的内切葡聚糖酶活性。通过质谱分析，四种 CBP 被鉴定并注释为来自主要瘤胃纤维素分解细菌 Fibrobacter succinogenes 的已知蛋白质：四肽重复结构域蛋白、OmpA 家族蛋白、纤维粘液结构域蛋白和纤维素结合内切葡聚糖酶 F (EGF)。另一种 CBP 被鉴定为马匹酵母的纤维素体糖基水解酶家族 6 外切葡聚糖酶 Cel6A。通过使用抗 EGF 抗体的免疫组织化学分析，发现表达 EGF 的 F. succinogenes 细胞是干草茎表面或内表面细菌群落的主要成员。从绵羊瘤胃内容物中分离和鉴定的 5 个 CBP 中有 4 个来自 F. succinogenes，这一发现表明 F. succinogenes 显着参与瘤胃中的纤维素降解。
ABSTRACT To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.