Imanaka T, Himeno T, Aiba S
J Gen Microbiol. 1985 Jul;131(7):1753-63. doi: 10.1099/00221287-131-7-1753.
We have constructed secretion vector plasmids that have unique BglII sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exo-large and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined. The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.
我们构建了分泌型载体质粒，这些质粒在地衣芽孢杆菌青霉素酶信号序列内部或附近具有独特的BglII位点，并且还构建了青霉素酶盒，这些酶盒缺少膜结合酶、外切大酶和外切小酶的一个、两个或三个加工位点。将这些青霉素酶盒中的每一个克隆到枯草芽孢杆菌的分泌载体上，并检测酶的产生。为了在枯草芽孢杆菌中有效表达该酶，分泌载体上需要同时存在信号序列和三个宿主特异性加工位点。酶盒上任何一个加工位点的存在都会减少青霉素酶在培养基中的积累。当使用缺少信号序列部分疏水区域且缺少三个加工位点的载体质粒时，尽管酶盒上加工位点完全或不完全存在，总青霉素酶产量仍会下降，且培养基中酶的积累极低。对这些细胞外青霉素酶的分子量测定表明该酶存在一个新的切割位点。
