Chen K C
Antimicrob Agents Chemother. 1986 Oct;30(4):536-41. doi: 10.1128/AAC.30.4.536.
A rapid and specific procedure was developed for the simultaneous detection of bacterial acylases and beta-lactamases, using ampicillin and cephalexin as substrates. Bacterial suspensions from agar plates were incubated separately with each beta-lactam substrate for 1 h at 37 degrees C. The supernatant of the reaction mixture was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products resulting from acylase hydrolysis, including the intact beta-lactam nucleus, 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, and the acyl side chain acid, D-(-)-alpha-aminophenylacetic acid, and the end product resulting from beta-lactamase hydrolysis (D-phenylglycylpenicilloic acid or D-phenylglycyldeacetoxycephalosporoic acid) were separated from each unhydrolyzed substrate and amino acids by this procedure. The presence of the intact beta-lactam nucleus in the reaction mixture is the indication of acylase activity. This method is sensitive and reproducible and has been successfully applied to screening for acylase activity in a variety of bacteria. It may be pharmaceutically useful for identifying organisms capable of removing the acyl side chain from naturally occurring beta-lactam antibiotics such as penicillin G, penicillin V, and cephalosporin C for production of the beta-lactam nuclei which serve as the starting materials for semisynthetic beta-lactam antibiotics.
开发了一种快速且特异的方法，以氨苄青霉素和头孢氨苄为底物同时检测细菌酰基转移酶和β-内酰胺酶。将琼脂平板上的细菌悬液分别与每种β-内酰胺底物在37℃孵育1小时。将反应混合物的上清液用丹磺酰化，丹磺酰衍生物通过在聚酰胺薄板上进行二维薄层色谱分离。通过该方法可将酰基转移酶水解产生的终产物，包括完整的β-内酰胺核、6-氨基青霉烷酸或7-氨基去乙酰氧基头孢烷酸，以及酰基侧链酸D-(-)-α-氨基苯乙酸，与每种未水解的底物和氨基酸分开，同时也可将β-内酰胺酶水解产生的终产物（D-苯甘氨酰青霉酸或D-苯甘氨酰去乙酰氧基头孢菌酸）与未水解的底物和氨基酸分开。反应混合物中完整β-内酰胺核的存在表明有酰基转移酶活性。该方法灵敏且可重复，已成功应用于多种细菌中酰基转移酶活性的筛选。它在药学上可能有助于鉴定能够从天然存在的β-内酰胺抗生素如青霉素G、青霉素V和头孢菌素C上去除酰基侧链以生产作为半合成β-内酰胺抗生素起始原料的β-内酰胺核的微生物。
