Erarslan A, Güray A
Scientific and Technical Research Council of Turkey, Marmara Scientific and Industrial Research Center, Gebze-Kocaell.
J Chem Technol Biotechnol. 1991;51(2):181-95. doi: 10.1002/jctb.280510205.
Highly purified penicillin G acylase from a mutant derivative of Escherichia coli ATCC 11105 was immobilized on oxirane-acrylic beads by covalent binding via oxirane groups. The highest specific activity, (322 U g-1 dry matrix at 40 degrees C and at pH 8.0) was obtained by using an enzyme solution having 13.8 U mg-1 specific activity and 72.73 mg total protein. The efficiency of immobilization was 95.8%. Kinetic parameters of immobilized penicillin G acylase were determined at the same pH and temperature by a preparation having 8.1 mg bound protein. The Km value and substrate inhibition constant of the enzyme were found to be 11.36 mmol dm-3 and 680 mmol dm-3 penicillin G respectively. Phenylacetic acid and 6-aminopenicillanic acid were estimated as the competitive and non-competitive inhibitors of the enzyme and their inhibition constants were found to be 90 mmol dm-3 phenylacetic acid and 76.1 mmol dm-3 for 6-aminopenicillanic acid. The activation energy of the hydrolytic reaction was calculated to be 7.75 kcal mol-1. The immobilized enzyme showed highest activity at pH 8.0 and at 55 degrees C. The enzyme was stable when incubated at 4 degrees C for one day at a pH range of 5.0 to 9.0. Thermal stability (over 30 min) was observed up to 40 degrees C but decreased at higher temperatures and was almost absent at 60 degrees C. A 95% conversion rate was observed at 28 degrees C and at 40 degrees C with 60 and 30 min operation times respectively. Operational stability of the enzyme was improved further with dithiothreitol treatment. Activity loss was around 5% following 20 cycles of repeated use of the enzyme at 40 degrees C. No significant loss of activity was observed at 28 degrees C when the enzyme was used for 20 cycles. 6-Aminopenicillanic acid in the reaction mixture was observed to be stable during conversion reactions which were carried out at both temperatures.
从大肠杆菌ATCC 11105的突变衍生物中获得的高度纯化的青霉素G酰化酶，通过环氧乙烷基团共价结合固定在环氧乙烷 - 丙烯酸珠上。使用比活性为13.8 U mg-1且总蛋白为72.73 mg的酶溶液，在40℃和pH 8.0条件下获得了最高比活性（322 U g-1干基质）。固定化效率为95.8%。通过含有8.1 mg结合蛋白的制剂，在相同的pH和温度下测定固定化青霉素G酰化酶的动力学参数。发现该酶的Km值和底物抑制常数分别为11.36 mmol dm-3和680 mmol dm-3青霉素G。苯乙酸和6-氨基青霉烷酸被估计为该酶的竞争性和非竞争性抑制剂，它们的抑制常数分别为90 mmol dm-3苯乙酸和76.1 mmol dm-3 6-氨基青霉烷酸。水解反应的活化能经计算为7.75 kcal mol-1。固定化酶在pH 8.0和55℃时表现出最高活性。当在pH 5.0至9.0范围内于4℃孵育一天时，该酶是稳定的。在高达40℃时观察到热稳定性（超过30分钟），但在较高温度下降低，在60℃时几乎不存在。在28℃和40℃下分别以60分钟和30分钟的操作时间观察到95%的转化率。用二硫苏糖醇处理进一步提高了酶的操作稳定性。在40℃重复使用该酶20个循环后，活性损失约为5%。当在28℃使用该酶20个循环时，未观察到明显的活性损失。在两个温度下进行的转化反应过程中，观察到反应混合物中的6-氨基青霉烷酸是稳定的。
