Park Y W, Lim S T, Cho S J, Yun H D
Department of Agricultural Chemistry, Gyeongsang National University, Chinju, Korea.
Biochem Biophys Res Commun. 1997 Dec 29;241(3):636-41. doi: 10.1006/bbrc.1997.7747.
Genomic DNA of the phytopathogenic Erwinia carotovora subsp. carotovora LY34 was partially digested with Sau3AI, ligated into the BamHI site of pBlue-script II SK+, and introduced into E. coli. Two clones that were able to hydrolyse carboxymethylcellulose were selected. 1.5 kb and 1.2 kb fragments containing the celA and celB genes, respectively, were subcloned and sequenced. The celA and celB genes had open reading frames of 1,161 bp and 792 bp encoding 487 and 264 amino acid residues with calculated molecular weights of 42,003 Da and 29,890 Da, respectively. Each, CelA and CelB, carried a typical prokaryotic signal peptide of 32 and 36 amino acid residues, respectively. The apparent molecular masses of the proteins when expressed in E. coli cells were approximately 39 kDa (CelA) and 26 kDa (CelB) as assessed by CMC-SDS-PAGE. Activity staining of CMCase in an SDS-PAGE gel containing 0.1% CMC revealed that the cloned endoglucanase isozymes comigrated with the corresponding ones present in Erwinia carotovora subsp. carotovora LY34.
植物致病软腐欧文氏菌胡萝卜软腐亚种LY34的基因组DNA用Sau3AI进行部分酶切，连接到pBlue-script II SK+的BamHI位点，然后导入大肠杆菌。挑选出两个能够水解羧甲基纤维素的克隆。分别亚克隆并测序了包含celA和celB基因的1.5 kb和1.2 kb片段。celA和celB基因的开放阅读框分别为1161 bp和792 bp，编码487和264个氨基酸残基，计算分子量分别为42,003 Da和29,890 Da。CelA和CelB分别带有一个典型的原核信号肽，分别为32和36个氨基酸残基。通过CMC-SDS-PAGE评估，在大肠杆菌细胞中表达时，这两种蛋白质的表观分子量分别约为39 kDa（CelA）和26 kDa（CelB）。在含有0.1% CMC的SDS-PAGE凝胶中对CMCase进行活性染色，结果显示克隆的内切葡聚糖酶同工酶与胡萝卜软腐欧文氏菌胡萝卜软腐亚种LY34中存在的相应同工酶迁移位置相同。
