Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
J Ind Microbiol Biotechnol. 2014 Nov;41(11):1617-25. doi: 10.1007/s10295-014-1501-9. Epub 2014 Sep 14.
Cephalosporin C (CPC) acylase is important for the one-step production of 7-aminocephalosporanic acid (7-ACA), a key intermediate for cephalosporin antibiotics. However, its application is hampered by the low activity, substrate inhibition, and product inhibition. In this study, two rounds of combinatorial active-site saturation testing (CASTing) were carried out on the CPC acylase acyII from Pseudomonas SE83, and one mutant H57βA/H70βY with no substrate inhibition was obtained. For further engineering to reduce the product inhibition, a quick pH indicator assay was developed, allowing for real-time monitoring of the product inhibition in the presence of added 7-ACA. The utility of the assay was demonstrated by screening six libraries of site-directed saturation mutagenesis libraries of H57βA/H70βY. A new mutant H57βA/H70βY/I176βN was obtained, which showed a k cat 3.26-fold and a K IP 3.08-fold that of the wild type, respectively. Given the commercial value of the enzyme, both this pH indicator assay and the triple mutant should be useful for further engineering of the enzyme to increase the specific activity and to decrease the product inhibition.
头孢菌素 C（CPC）酰基酶对于 7-氨基头孢烷酸（7-ACA）的一步生产非常重要，7-ACA 是头孢菌素抗生素的关键中间体。然而，其应用受到低活性、底物抑制和产物抑制的限制。在这项研究中，对假单胞菌 SE83 的 CPC 酰基酶 acyII 进行了两轮组合活性位点饱和测试（CASTing），得到了一个没有底物抑制的突变体 H57βA/H70βY。为了进一步进行工程改造以降低产物抑制，开发了一种快速 pH 指示剂测定法，可实时监测添加 7-ACA 时的产物抑制。通过筛选 H57βA/H70βY 的六个定点饱和突变文库，证明了该测定法的实用性。获得了一个新的突变体 H57βA/H70βY/I176βN，其 k cat 分别比野生型提高了 3.26 倍和 K IP 提高了 3.08 倍。鉴于该酶的商业价值，这种 pH 指示剂测定法和三突变体都应该有助于进一步对酶进行工程改造，以提高比活和降低产物抑制。
