Covalent immobilization and characterization ofd-lactate dehydrogenase

In this thesis, the covalent immobilization of D-Lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides onto 3-aminopropyl silica gel via the glutaraldehyde spacer arm was optimized to have an active and stable preparation which is able to produce D-Lactic acid. The immobilization yield of 86 % was achieved using 1 mg/mL protein for 0.250 g support. The free and immobilized D-LDHs were characterized and their optimum pH and temperature values and kinetic parameters were determined. In addition, the storage and thermal stability of free and immobilized D-LDHs were investigated. The reuse stability of immobilized D-LDH was also determined in a batch reactor. The optimum pH for free D-LDH was determined as 7.0, however, it was 6.0 for immobilized D-LDH.The optimum temperatures for free and immobilized D-LDHs were found to be 37 C and 45 C, respectively. The Km and Vmax values for free D-LDH were determined as 0.365 mM, 86.95 U/mg protein, and the corresponding values were determined as 0.077 mM and 19.16 U/mg protein, respectively for immobilized D-LDH. The free D-LDH retained 85 % of its initial activity after 30 days of storage at 4 C, however it lost 97 % of its initial activity after 10 days of storage at 25 C. The immobilized D-LDH retained 82 % and 3 % of its initial activity after 30 days of storage at 4 C and 25 C, respectively. The free D-LDH preserved 86 % of its initial activity after 4 hours of incubation at 45 C, while immobilized D-LDH preserved 82 % under the same conditions. The immobilized D-LDH retained 38 % of its initial activity after 10 uses in a batch reactor. Keywords: D-lactate dehydrogenase, immobilization, 3-aminopropyl silica gel