García J L, Nuñez C J, González E G, Osuna J, Soberón X, Galindo E
Department of Bioengineering, National University of Mexico, Cuernavaca, Morelos, México.
Appl Biochem Biotechnol. 1998 May-Jun;73(2-3):243-56. doi: 10.1007/BF02785659.
A protein-engineered beta-lactamase, constructed by site-directed mutagenesis in Escherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in beta-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.
一种通过在大肠杆菌中进行定点诱变构建的蛋白质工程β-内酰胺酶（E104M/G238S），具有拓宽的特异性，能够降解第一代、第二代和第三代头孢菌素。对培养条件的调控可使β-内酰胺酶的比活性提高两倍。所得细菌被用于构建一种可浸入式全细胞生物传感器，用于检测新一代头孢菌素。细胞固定在琼脂膜上，琼脂膜再附着于平面pH电极表面，从而构成一种基于检测pH变化的生物传感器。该传感器能够检测第二代和第三代头孢菌素：头孢孟多（0.4 - 4 mM）、头孢噻肟（0.4 - 3.5 mM）和头孢哌酮（0.3 - 1.85 mM）。响应时间在3.5至11分钟之间，具体取决于所测试的头孢菌素种类。该生物传感器至少稳定7天，在此期间可进行多达100次测试。
