Kinetic Study on the Enzymatic Production of D-Alanine from D-Aspartic Acid

  • Lee, Jae-Heung (Deparment of Food and Life Science, Faculty of Life Science and Technology, Sungkyunkwan University, 300 Chunchun-dong) ;
  • Sung, Moon-Hee (Korea Research Institute of Bioscience and Bioengineering, Yusung) ;
  • Jeon, Yeong-Joong (Amicogen Inc., 694-4 sangchon-ri, Jinsung-myon)
  • Published : 2002.03.01


An enzymatic reaction for the production of D-alanine from D-aspartic acid and pyruvate as substrates by a thermostable D-amino acid aminotransferase (D-AAT) was investigated at various conditions In the temperature range of 40-70$\^{C}$ and pH range of 6.0-9.5. The D-AAT was produced with recombinant E. coli BL21, which hosted the chimeric plasmid pTLK2 harboring the D-AAT from the novel thermophilic Bacillus sp. LK-2. The enzyme reaction was shown to follow the Ping Pong Bi Bi mechanism. The K$\_$m/ values for D-aspartic acid and pyruvate were 4.38 mar and 0.72 mM, respectively. It was observed that competitive inhibition by D-alanine, the product of this reaction, was evident with the inhibition constant K$\_$i/ value of 0.1 mM. A unique feature of this reaction scheme is that the decorboxylation of oxaloacetic acid, one of the products, spontaneously produces pyruvate. Therefore, only a catalytic amount of pyruvate is necessary for the enzyme conversion reaction to proceed. A typical time-course kinetic study skewed that D-alanine up to 88 mM could be produced from 100 mM of D-aspartic acid with a molar yield of 1.0.


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