CHARACTERIZATION AND COMPARISON OF CLOSTRIDIUM-CELLULOVORANS ENDOGLUCANASES-XYLANASES ENGB AND ENGD HYPEREXPRESSED IN ESCHERICHIA-COLI
CHARACTERIZATION AND COMPARISON OF CLOSTRIDIUM-CELLULOVORANS ENDOGLUCANASES-XYLANASES ENGB AND ENGD HYPEREXPRESSED IN ESCHERICHIA-COLI
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DOI：
10.1128/jb.174.4.1403-1409.1992
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发表时间：
1992-02-01
影响因子：
3.2
通讯作者：
DOI, RH
中科院分区：
文献类型：
--
作者：
FOONG, FCF;DOI, RH
通过使用T7表达系统，从大肠杆菌中超表达和纯化来自Clostridium celluvorans的内切葡聚糖酶-木聚糖酶EngB和EngD。这两种酶表现出内切葡聚糖酶和木聚糖酶活性。两种内切葡聚糖酶的底物特异性相似，除了EngD具有四倍更高的对硝基苯基β-1，4-纤维二糖苷酶活性。这两种蛋白质在Pro-Thr-Thr区域的同源性高达80%，该区域将蛋白质分为-NH 2-和-COOH-末端。EngB的-COOH-区域与来自热纤梭菌的内切葡聚糖酶和木聚糖酶以及来自解纤维梭菌的内切葡聚糖酶具有高度同源性，并且不显示与纤维素（Avicel）的强结合。然而，-COOH-区域的EngD，它具有同源性的纤维素结合结构域的纤维素单胞菌fimi外切-和内切葡聚糖酶和荧光假单胞菌内切葡聚糖酶，表现出结合能力的纤维素，即使当该结构域融合的N-末端结构域的EngB。通过用抗EngB和抗EngD抗体探测Avicel纯化的纤维素酶复合物（F8），EngB和EngD均显示存在于C.食纤维菌。与EngB和EngD同源的许多蛋白质也存在于复合物上。
By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium cellulovorans were hyperexpressed and purified from Escherichia coli. The two enzymes demonstrated both endoglucanase and xylanase activities. The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl beta-1,4-cellobiosidase activity. The two proteins were very homologous (80%) up to the Pro-Thr-Thr region which divided the protein into -NH2- and -COOH- terminals. The -COOH- region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocellum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel). However, the -COOH- region of EngD, which had homology to the cellulose-binding domains of Cellulomonas fimi exo- and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB. By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C. cellulovorans. Many proteins homologous to EngB and EngD were also present on the complex.