Thesis (M.Sc.)--Chulalongkorn University, 2003

Alanine dehydrogenase (EC 1.4.1.1) catalyzes the NAD[superscript +]- dependent reversible oxidative deamination of L-alanine to form ammonia, pyruvate, and NADH. The enzyme is important as a catalyst for the synthesis of amino acids. Moreover, it is therefore applicable to diagonosis of malignant hematopoietic disease. Alnine dehydrogenase from Aeromonas hydrophila has high activity and high substrate specificity, so it is suitable for L-alanine production. However, the enzyme activity lost rapidly upon the incubation at temperature above 50 ํC for 10 min. To improve the enzyme thermostability, site-directed mutagenesis was performed. Uncharged amino acids were replaced by charge residues. G38E, L58R, L101E, P168R and A231E were selected for electrostatic interaction formation based on three dimensional structure of thermophilic counterpart enzyme from Bacillus stearothermophilus. In crude enzyme, L101E and A231E showed higher stability than wild type. While G38E showed similar enzyme level with wild type, L58R and P168R showed lower. Thus, residues of Leu 58 and A231 were chosen for double mutation. Alanine dehydrogenase from L101E, A231E and L101E/Arg231 were purified for comparison of their characters with wild type. The purity of wild type and all mutant enzymes were more than 90%, estimated by native PAGE. Mobility of mutant enzymes on native gel was faster than that of wild type. Whereas, no difference was detected by SDS-PAGE. This evidence reflected the net charge on molecule of enzymes. T[subscript m] of L101E/A231E increased 3 ํC while single mutants showed 2 ํC higher than wild type. Furthermore, optimum temperature of mutants shifted up from 50 ํC in wild type to 52 ํC. The optimum pH, substrate specificity and kinetic properties (K[subscript m]) for L-alanine and NAD[superscript +] of all mutants were not differed from wild type, so no change in an active site of enzyme was occurred. Thus, replacement of Leu 101 and Ala 231 with Glu could form electrostatic interaction which could enhance thermostability.