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制药工业中对研究用青霉酰胺酶裂解青霉素以制备半合成新抗生素的原料6-APA 十分重视。曾报道,测定青霉素酰胺酶活力的方法有电位滴定法、光谱法、气相色谱法等等,但灵敏度与专属性均较差。作者研究了萤光法与HPLC 法相互补充的测定方法。萤光法是基于6-APA 与萤光胺反应十分敏感的特点(敏感度在10~(-9)克分子)故特别适宜于酰酶活力的动力学研究。而HPLC 法可同时对裂解液中存在的6-APA、苯醋酸和底物青霉素G 进行快速连续测定,是一种重现性、敏感性相当高的测定方法。方法:0.003～100mg 的青霉素G,溶于10ml0.4 M 磷酸缓冲液,加100μl 酶溶液于37°水解。间隔一定时间,吸取1.5 ml 的水解混合液加0.5 ml(0.015 mg/ml)萤光胺丙酮溶液,10分钟后,用萤光分光光度计,于470及390nm 处读数。另取100 μl 的该溶液,用0.9ml 冷水稀释,注2 μl 于Perkin Elmer 3B 型HPLC 仪中,层析柱Merck RP-18(10 μm),流动相为含55%甲醇的0.1 M pH 3.5磷酸盐缓冲液,流速1ml/分,温度20°,用LC-75UV 检测器。青霉素酶(青霉酰胺酶制剂中的杂质)存在时,由
The pharmaceutical industry attaches great importance to the study of 6-APA, a raw material for the cleavage of penicillin by penicillinase to prepare a new semi-synthetic antibiotic. It has been reported that the determination of penicillin amidase activity potentiometric titration, spectroscopy, gas chromatography and so on, but the sensitivity and specificity are poor. The authors studied the fluorescence and HPLC methods complement each other. Fluorescence is based on the very sensitive 6-APA reaction with the fluorescent amine (sensitivity of 10 -9 (mole)) so it is particularly suitable for kinetic studies of acylase activity. The rapid and continuous determination of 6-APA, phenylacetic acid and substrate penicillin G in the lysis solution by HPLC method is a reproducible and sensitive method. Method: 0.003 ~ 100mg penicillin G, dissolved in 10ml0.4 M phosphate buffer, add 100μl enzyme solution at 37 ° hydrolysis. At regular intervals, draw 1.5 ml of hydrolysed mixture plus 0.5 ml (0.015 mg / ml) of fluorescenamine in acetone. After 10 minutes, read at 470 and 390 nm using a fluorescence spectrophotometer. Another 100 μl of this solution was diluted with 0.9 ml of cold water and 2 μl of the solution was eluted on a Perkin Elmer model 3B HPLC column, Merck RP-18 (10 μm) with a mobile phase of 0.1 M pH 3.5 containing 55% methanol Phosphate buffer, flow rate 1 ml / min, temperature 20 °, with LC-75 UV detector. Penicillinase (penicillinase enzyme preparation impurities) in the presence of