Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52056 Aachen, Germany.
Chembiochem. 2010 Mar 22;11(5):691-7. doi: 10.1002/cbic.200900717.
Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH-dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed-evolution protocol based on an adapted citrulline-screening protocol in microtiter-plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild-type PpADI and retains approximately 50 % of its activity relative to its pH optimum, compared to approximately 10 % in the case of the wild-type PpADI.
精氨酸脱亚氨酶（ADI；EC 3.5.3.6）已被研究作为一种治疗精氨酸营养缺陷型肿瘤（如肝癌和黑色素瘤）的潜在抗肿瘤药物。用人淋巴白血病细胞系进行的研究证实，ADI 是一种治疗白血病的抗血管生成剂。来自假单胞菌（PpADI）的 ADI 的主要限制在于其 pH 依赖性活性谱，其最适 pH 值为 6.5。从 6.5 到 7.5 的 pH 变化导致活性下降约 80%。（人血浆的 pH 值为 7.35 至 7.45。）为了改变 PpADI 的 pH 最适值，开发并验证了一种基于已适应的瓜氨酸筛选方案的定向进化方案。ADI 工程的概念验证导致 pH 最适值为 pH 7.0，并在生理和略碱性条件下提高了抗性。在 pH 7.4 下，变体 M2（K5T/D44E/H404R）比野生型 PpADI 快四倍，与野生型 PpADI 相比，其相对于 pH 最适值保留约 50%的活性，而野生型 PpADI 保留约 10%的活性。
