Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8
Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8
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DOI：
10.1002/bit.21856
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发表时间：
2008-08-15
影响因子：
3.8
通讯作者：
Wilson, David. B.
中科院分区：
文献类型：
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作者：
Moser, Felix;Irwin, Diana;Wilson, David. B.
对 E7（一种单结构域家族 33 纤维素结合模块 (CBM) 蛋白）和 E8（一种非催化三结构域蛋白，由家族 33 CBM、FNIII 结构域和家族 2 CBM 组成）进行了克隆、表达、纯化和表征。蛋白质印迹显示，当 Thermobifida fusca 在纤维二糖、Solka floc、柳枝稷或苜蓿以及 β-1,3 连接的葡萄糖分子（如昆布二糖或茯苓多糖）上生长时，E7 和 E8 被诱导和分泌。在所有测试的 pH 值下，E8 都能与 α-和 β-几丁质以及细菌微晶纤维素 (BMCC) 良好结合。与 E8 相比，E7 与 P-几丁质的结合较强，与 α-几丁质的结合较差，与 BMCC 的结合较弱。滤纸结合测定显示E7结合28％，E8结合39％，来自Cel6B的纯化CBM2结合结构域结合88％，并且仅结合5％的Cel5A催化结构域。 C 端 6 x His 标签影响 E7 和 E8 与这些底物的结合。滤纸活性测定显示，当 E7 或 E8 存在时，T. fusca 纤维素酶的活性增强。这种效应在纤维素酶浓度非常低或反应时间非常长的情况下观察到，并且主要与纤维素酶的类型和混合物中纤维素酶的数量无关。 E8 以及较小程度的 E7 显着增强了粘质沙雷氏菌几丁质酶 C 对 β-几丁质的活性。
E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a non-catalytic, three-domain protein consisting of a Family 33 CBM, a FNIII domain, followed by a Family 2 CBM, were cloned, expressed, purified, and characterized. Western blots showed that E7 and E8 were induced and secreted when Thermobifida fusca was grown on cellobiose, Solka floc, switchgrass, or alfalfa as well as on beta-1,3 linked glucose molecules such as laminaribiose or pachyman. E8 bound well to alpha- and beta-chitin and bacterial microcrystalline cellulose (BMCC) at all pHs tested. E7 bound strongly to P-chitin, less well to a-chitin and more weakly to BMCC than E8. Filter paper binding assays showed that E7 was 28% bound, E8 was 39% bound, a purified CBM2 binding domain from Cel6B was 88% bound, and only 5% of the Cel5A catalytic domain was bound. A C-terminal 6 x His tag influenced binding of both E7 and E8 to these substrates. Filter paper activity assays showed enhanced activity of T. fusca cellulases when E7 or E8 was present. This effect was observed at very low concentrations of cellulases or at very long times into the reaction and was mainly independent of the type of cellulase and the number of cellulases in the mixture. E8, and to a lesser extent E7, significantly enhanced the activity of Serratia marscescens Chitinase C on beta-chitin.