Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit.

D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, LDH) coding sequence derived from was obtained from the NCBI database by gene mining. LDH was expressed and purified in , and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of LDH during freeze-drying and storage. Finally, a kit based on LDH was tested to determine whether the enzyme had potential clinical applications. The results showed that LDH had a specific activity of 1,864 U/mg, value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried LDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that LDH possesses high activity and stability. Test results using the ALT assay kit prepared with LDH were consistent with those of commercial kits, with a relative deviation <5%. These results indicated that LDH met the primary requirements for ALT assays, laying a foundation for the development of new ALT kits with potential clinical applications.