Fujiyama K, Maki H, Kinoshita S, Yoshida T
International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, Japan.
FEMS Microbiol Lett. 1995 Feb 1;126(1):13-7. doi: 10.1111/j.1574-6968.1995.tb07383.x.
Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene (aly) from an alginate-degrading strain, Pseudomonas sp. OS-ALG-9. The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E. coli cells. A 8-fold increase in the alginate lyase (Aly) activity in E. coli JM109 (pAL205) was induced with isopropyl-beta-D-thiogalactoside, which was 210 times higher than that in E. coli LE392 (pAL28). The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N-terminal sequence of the purified enzyme. It was found that the Aly expressed in E. coli (pAL205) was a fused protein containing 7 residues from the N-terminus of beta-galactosidase alpha-peptide and the mature protein found in the Pseudomonas sp. except for three residues in the N-terminal.
大肠杆菌LE392 (pAL28) 先前作为一个阳性克隆从一株海藻酸盐降解菌株假单胞菌属OS-ALG-9中分离得到，该阳性克隆含有海藻酸裂解酶基因（aly）。质粒pAL205是pAL28连续亚克隆后得到的构建体之一，在大肠杆菌细胞中能使aly表达量最高。用异丙基-β-D-硫代半乳糖苷诱导大肠杆菌JM109 (pAL205) 中海藻酸裂解酶（Aly）活性提高了8倍，这比大肠杆菌LE392 (pAL28) 中的活性高210倍。通过aly 5'区域周围的核苷酸序列以及纯化酶的N端序列，研究了pAL205对Aly酶表达的显著提高作用。结果发现，在大肠杆菌 (pAL205) 中表达的Aly是一种融合蛋白，其N端包含来自β-半乳糖苷酶α-肽N端的7个残基以及假单胞菌属中发现的成熟蛋白，但N端有三个残基除外。
