Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada.
Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi deII'Insubria, via J. H. Dunant 3, 21100 Varese, ltaly.
ACS Appl Bio Mater. 2021 Jul 19;4(7):5598-5604. doi: 10.1021/acsabm.1c00409. Epub 2021 Jun 28.
d-Amino acid oxidase (DAAO) enzymes bind a range of d-amino acids with variable affinity. As such, the design of selective DAAO-based enzymatic biosensors remains a challenge for real-world biosensor application. Herein, a methodology for developing biosensors with varying substrate selectivity is presented. First, we address DAAO-based biosensor selectivity toward d-serine by introducing point mutations into DAAO using rational design. Next, the wild-type yeast DAAO (DAAO WT) and variants human DAAO W209R and yeast M213G are characterized for their selectivity and activity toward d-serine and d-alanine, the preferred DAAO substrates. The DAAO enzymes have been immobilized for final biosensor design, where they demonstrate selectivity comparable to free DAAO. The cross-linking procedure impacts on DAAO structure and function and the use of a regeneration strategy allows the biosensor response to be improved.
d-氨基酸氧化酶（DAAO）酶以不同的亲和力结合多种 d-氨基酸。因此，设计基于 DAAO 的酶生物传感器的选择性仍然是实际生物传感器应用中的一个挑战。本文提出了一种用于开发具有不同底物选择性的生物传感器的方法。首先，我们通过使用合理设计对 DAAO 进行点突变，解决了基于 DAAO 的生物传感器对 d-丝氨酸的选择性问题。接下来，对野生型酵母 DAAO（DAAO WT）和变体人 DAAO W209R 和酵母 M213G 进行了表征，以确定它们对 d-丝氨酸和 d-丙氨酸（DAAO 的首选底物）的选择性和活性。最后，将 DAAO 酶进行固定化用于最终的生物传感器设计，结果表明其选择性与游离 DAAO 相当。交联过程会影响 DAAO 的结构和功能，使用再生策略可以提高生物传感器的响应。
