Nguyen-Distèche M, Leyh-Bouille M, Ghuysen J M
Biochem J. 1982 Oct 1;207(1):109-15. doi: 10.1042/bj2070109.
The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high transpeptidase activity (greater than 95%) and very low carboxy-peptidase activity (less than 5%). The penicillin-binding protein/transpeptidase can be extracted directly from the mycelium with N-cetyl-NNN-trimethylammonium bromide (Cetavlon) and subsequently obtained at 90% purity and with an 8000-fold specific enrichment (when compared with the activity of the isolated membranes) by a two-step procedure involving Sephadex filtration and affinity chromatography on ampicillin-linked CH Sepharose 4B in the presence of detergent. At saturating concentrations of the co-substrates diacetyl-L-Lys-D-Ala-D-Ala and Gly-Gly, the catalytic-centre activity is about 0.3 s-1.
链霉菌K15的膜结合型26000道尔顿青霉素结合蛋白已被分离出来，其形式为一种有效的、对青霉素敏感的D-丙氨酰-D-丙氨酸裂解肽酶，具有高转肽酶活性（大于95%）和极低的羧肽酶活性（小于5%）。青霉素结合蛋白/转肽酶可用N-十六烷基-N,N,N-三甲基溴化铵（西曲溴铵）直接从菌丝体中提取，随后通过两步法获得，纯度达90%，比活性提高8000倍（与分离膜的活性相比），该两步法包括葡聚糖凝胶过滤和在去污剂存在下于氨苄青霉素连接的CH琼脂糖4B上进行亲和层析。在共底物二乙酰-L-赖氨酸-D-丙氨酰-D-丙氨酸和甘氨酰-甘氨酸的饱和浓度下，催化中心活性约为0.3 s-1。
