l-Aspartate is an important chemical in the food and pharmaceutical industries. Herein, a dual-enzyme system was constructed to synthesize l-aspartate from maleic anhydride at 50 °C, which can reduce the byproduct production. Maleate transformed from maleic anhydride in the solution was converted into l-aspartate via fumarate catalyzed by maleate isomerase (MaiA) and thermostable aspartase (AspB), respectively. Because MaiA is a rate-limiting enzyme, enzyme activities of various MaiAs were compared, and the efficient and thermostable maleate isomerase