Immobilization and Characterization of D-Lactate Dehydrogenase onto 3-aminopropyl Silica Gel Support

Authors : Nazlı Ece Varan, Sevde Seyhan Tukel

Pages : 1411-1422

Doi:10.21597/jist.1683104

View : 34 | Download : 114

Publication Date : 2025-12-01

Article Type : Research Paper

Abstract :In this study, D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides was covalently immobilized onto 3-aminopropyl-functionalized silica gel using glutaraldehyde as a bifunctional crosslinker, with the aim of developing a catalytically active and thermally stable biocatalyst for D-lactic acid production. The immobilization protocol achieved an efficiency of 86% using 1 mg/mL of enzyme and 0.250 g of support material. Comparative biochemical characterization of both free and immobilized D-LDH was performed, assessing optimal pH and temperature, thermal stability and kinetic parameters. The immobilized enzyme preparation exhibited an optimal pH of 6.5 and a temperature optimum of 45 °C. These values corresponded to 7.0 and 37 °C for the free enzyme. Kinetic analysis revealed a Michaelis constant (Km) of 0.37 mM and maximum velocity (Vmax) of 86.9 U/mg protein for the free enzyme, whereas the immobilized enzyme displayed a significantly reduced Km of 0.08 mM and a lower Vmax of 19.2 U/mg protein, indicating increased substrate affinity but reduced catalytic turnover. Thermal stability assays demonstrated enhanced resistance of the immobilized D-LDH to elevated temperatures. Furthermore, reuse studies in a batch reactor showed that the immobilized enzyme preserved 38% of its original activity after 10 successive uses, underscoring its potential for repeated use in biotechnological applications.
Keywords : D-Laktat dehidrogenaz, immobilizasyon, 3-aminopropil silika jel

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