Journal of Microbiology

Kinetic Study on the Enzymatic Production of D-Alanine from D-Aspartic Acid: Jae-Heung Lee , Moon-Hee Sung , Yeong-Joong Jeon; J. Microbiol. 2002;40(1):33-37.

Abstract: 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 mM 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 decarboxylation 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 showed 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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