Maki H, Mori A, Fujiyama K, Kinoshita S, Yoshida T
International Center of Cooperative Research in Biotechnology, Osaka, Japan.
J Gen Microbiol. 1993 May;139(5):987-93. doi: 10.1099/00221287-139-5-987.
A gene (aly) encoding alginate lyase (ALY; EC 4.2.2.3) was isolated from a library constructed with the cosmid vector pHC79 and Sau3AI-digested genomic DNA of Pseudomonas sp. OS-ALG-9. Successive subcloning of the aly-containing cosmid enabled us to locate the gene on a 2.3 kb HpaI fragment. Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) of 1365 bp. The directly determined N-terminal amino acid sequence of the ALY protein purified from Pseudomonas sp. OS-ALG-9 was found in the amino acid sequence deduced from this ORF between nucleotides 282 and 366. Expression of aly was induced by IPTG in Escherichia coli and leakage of the enzyme into the extracellular milieu was significantly enhanced by addition of glycine to the growth medium. The ALY enzyme had a greater specificity for the homopolymer of mannuronate than for that of guluronate.
从用黏粒载体pHC79和铜绿假单胞菌OS-ALG-9经Sau3AI酶切的基因组DNA构建的文库中分离出了一个编码藻酸盐裂解酶（ALY；EC 4.2.2.3）的基因（aly）。对含aly的黏粒进行连续亚克隆，使我们能够将该基因定位在一个2.3 kb的HpaI片段上。对该片段进行核苷酸测序，发现了一个1365 bp的单一开放阅读框（ORF）。从铜绿假单胞菌OS-ALG-9纯化的ALY蛋白直接测定的N端氨基酸序列，出现在该ORF推导的核苷酸282至366之间的氨基酸序列中。在大肠杆菌中，iptg可诱导aly表达，向生长培养基中添加甘氨酸可显著增强该酶向细胞外环境的渗漏。ALY酶对甘露糖醛酸同聚物的特异性比对古洛糖醛酸同聚物的特异性更高。
