Pérez C, Juárez K, García-Castells E, Soberón G, Servín-González L
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F.
Gene. 1993 Jan 15;123(1):109-14. doi: 10.1016/0378-1119(93)90548-h.
A gene encoding an extracellular lipase from Streptomyces sp. M11 was cloned in the high-copy-number vector pIJ486, using S. lividans 66 as host. A 28-kDa protein was secreted by S. lividans carrying pB13, which harbors a 6-kb insert, and identified as the product of the cloned gene. Comparison of the N-terminal amino acid (aa) sequence of the purified extracellular lipase with the nucleotide (nt) sequence of the lip gene revealed the presence of a 48 aa long signal peptide. The nucleotide sequence also revealed the presence of a motif, Gly-His-Ser-Met-Gly, similar to the one found surrounding the active-site Ser in other lipases. The gene is most likely monocistronic. Subcloning experiments indicated that another gene might be required for high-level expression, since subcloning of the structural gene alone resulted in diminished extracellular lipase activity. The lipase gene promoter was identified by S1 mapping experiments, and found to be similar to other Streptomyces vegetative promoters.
利用天蓝色链霉菌66作为宿主，将来自链霉菌属M11的一种细胞外脂肪酶编码基因克隆到高拷贝数载体pIJ486中。携带pB13（含有一个6 kb插入片段）的天蓝色链霉菌分泌出一种28 kDa的蛋白质，并被鉴定为克隆基因的产物。将纯化的细胞外脂肪酶的N端氨基酸序列与脂肪酶基因的核苷酸序列进行比较，发现存在一个48个氨基酸长的信号肽。核苷酸序列还显示存在一个基序Gly-His-Ser-Met-Gly，类似于在其他脂肪酶中活性位点丝氨酸周围发现的基序。该基因很可能是单顺反子的。亚克隆实验表明，高水平表达可能还需要另一个基因，因为仅结构基因的亚克隆会导致细胞外脂肪酶活性降低。通过S1图谱实验鉴定了脂肪酶基因启动子，发现它与其他链霉菌营养型启动子相似。
