Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, GAC 1200, La Jolla, CA 92037, USA.
Mol Genet Metab. 2010 Jan;99(1):4-9. doi: 10.1016/j.ymgme.2009.09.002.
Phenylalanine ammonia lyase (PAL) has long been recognized as a potential enzyme replacement therapeutic for treatment of phenylketonuria. However, various strategies for the oral delivery of PAL have been complicated by the low intestinal pH, aggressive proteolytic digestion and circulation time in the GI tract. In this work, we report 3 strategies to address these challenges. First, we used site-directed mutagenesis of a chymotrypsin cleavage site to modestly improve protease resistance; second, we used silica sol-gel material as a matrix to demonstrate that a silica matrix can provide protection to entrapped PAL proteins against intestinal proteases, as well as a low pH of 3.5; finally, we demonstrated that PEGylation of AvPAL surface lysines can reduce the inactivation of the enzyme by trypsin.
苯丙氨酸解氨酶（PAL）长期以来一直被认为是治疗苯丙酮尿症的潜在酶替代治疗方法。然而，各种口服递送 PAL 的策略都受到肠道内低 pH 值、强烈的蛋白水解消化和循环时间的影响。在这项工作中，我们报告了 3 种解决这些挑战的策略。首先，我们使用糜蛋白酶切割位点的定点突变来适度提高蛋白酶抗性；其次，我们使用硅溶胶-凝胶材料作为基质，证明硅基质可以为包埋的 PAL 蛋白提供保护，防止其受到肠道蛋白酶和 3.5 的低 pH 值的影响；最后，我们证明了 AvPAL 表面赖氨酸的聚乙二醇化可以减少胰蛋白酶对酶的失活。
