celA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-beta-1,4-glucanase
celA from Bacillus lautus PL236 encodes a novel cellulose-binding endo-beta-1,4-glucanase
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DOI：
10.1128/jb.174.11.3522-3531.1992
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发表时间：
1992-06
影响因子：
3.2
通讯作者：
Christian K. Hansen;N. R. A. Meinke;Gilkes;D. Kilburn;R. C. Miller
中科院分区：
文献类型：
--
作者：
Christian K. Hansen;N. R. A. Meinke;Gilkes;D. Kilburn;R. C. Miller
来自纤维素分解细菌灿烂芽孢杆菌PL 236的celA编码EG-A，一种内切-β-1，4-葡聚糖酶。一个2,100 bp的开放阅读框，前面是一个核糖体结合位点，编码一个分子量为76,863 Da的蛋白质，具有典型的信号序列。EG-A的NH 2-末端活性结构域与任何已报道的纤维素酶或木聚糖酶都不同源，可能代表了此类酶的一个新家族。一个150个氨基酸的羧基末端肽与其他几种纤维素酶的非催化结构域同源（A. Meinke，N.R. Gilkes，D.G. R.C.基尔伯恩小米勒，和R.A.J. Warren，J. Bacteriol. 173：7126-7135，1991）。celA上游的部分开放阅读框编码一个145个氨基酸的肽，该肽也属于上述家族。大肠杆菌提取物及枯草芽孢杆菌和B.巨大芽孢杆菌，包括其表达celA的蛋白酶缺陷突变体，揭示了两种活性蛋白，EG-A-L和EG-A-S，分别具有74，000和57，000的Mrs。EG-A-L与EG-A-S的比例取决于宿主生物体的胞外蛋白水解活性，表明EG-A-S来自EG-A-L的翻译后蛋白水解修饰。由于EG-A-S具有对应于预测的EG-A的NH 2-末端序列的NH 2-末端，加工似乎发生在所述的催化和非催化结构域之间。EG-A-L和EG-A-S被纯化至均一，并且显示出在对可溶性底物的活性以及pH和温度依赖性方面具有几乎相同的特性。与EG-A-S相反，EG-A-L与纤维素强烈结合，并且对不溶性底物的活性高于后者。我们得出结论，羧基端17,000-Mr肽EG-A-L构成纤维素结合域。
celA from the cellulolytic bacterium Bacillus lautus PL236 encodes EG-A, an endo-beta-1,4-glucanase. An open reading frame of 2,100 bp preceded by a ribosome-binding site encodes a protein with a molecular mass of 76,863 Da with a typical signal sequence. The NH2-terminal active domain of EG-A is not homologous to any reported cellulase or xylanase and may represent a new family of such enzymes. A 150-amino-acid COOH-terminal peptide is homologous to noncatalytic domains in several other cellulases (A. Meinke, N.R. Gilkes, D.G. Kilburn, R.C. Miller, Jr., and R.A.J. Warren, J. Bacteriol. 173:7126-7135, 1991). Upstream of celA, a partial open reading frame encodes a 145-amino-acid peptide which also belongs to the family mentioned. Zymogram analysis of extracts from Escherichia coli and supernatants of Bacillus subtilis and B. megaterium, including protease-deficient mutants thereof, which express celA, revealed two active proteins, EG-A-L and EG-A-S, with Mrs of 74,000 and 57,000, respectively. The proportion of EG-A-L to EG-A-S depends on the extracellular proteolytic activity of the host organism, indicating that EG-A-S arises from posttranslational proteolytic modification of EG-A-L. Since EG-A-S has an NH2 terminus corresponding to the predicted NH2-terminal sequence of EG-A, processing appears to take place between the catalytic and noncatalytic domains described. EG-A-L and EG-A-S were purified to homogeneity and shown to have almost identical characteristics with respect to activity against soluble substrates and pH and temperature dependency. EG-A-L binds strongly to cellulose, in contrast to EG-A-S, and has higher activity against insoluble substrates than the latter. We conclude that the COOH-terminal 17,000-Mr peptide of EG-A-L constitutes a cellulose-binding domain.