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

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 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.

References

  1. Appl. Environ. Microbiol v.63 Synthesis of optivally active amino acids from α-keto acids with Escherichia coli cells expressing heterologous genes Galkin,A;L. Kulakova;T. Yoshimura;K. Soda;N. Esaki
  2. J. Org. Chem. v.54 Biosynthesis of pradimicin A Kakushima,M;Y.Sawada;M.Nishio;T.Tsuno;T.Oki https://doi.org/10.1021/jo00272a014
  3. Adv. Biochem. Eng. Biotechnol. v.42 New developments in the chemo-enzymatic production of amino acids Kamphuis,J;W.H.Boesten;Q.B.Broxterman;H.F.M.Hermes;J.A.M.van Balken;E.M.Meiher;H.E.Shoemaker
  4. Kor. J. App. Microbiol. Biotechnol v.27 Screening and taxonomic characterization of D-amino acid aminotransferase-producing thermophiles Kwak,M.S;S.G.Lee;S.C.Jeong;S.H.Suh;J.H.Lee;Y.J.Jeon;M.H.Sung
  5. J. Biotechnol v.8 Enantioselective synthesis of various D-amino acids by a multi-enzyme system Nakajima,N;K.Tanizawa;H.Tanaka;K.Soda https://doi.org/10.1016/0168-1656(88)90006-5
  6. Biosci. Biotechnol. Biochem. v.56 Enzymatic production of D-alanine from DL-alaninamide by novel D-alaninamide specific amide hydrolase Ozaki,A;H.Kawasaki;M.Yagasaki;Y.Hashimoto https://doi.org/10.1271/bbb.56.1980
  7. Enzyme kinetics Segel,I.H
  8. FEBS Lett v.36 Chemical-enzymatic incorporation of D-amino acids into peptides:Synthesis of diaste-reomeric (D-Ala²,D-Leu 5) enkephalinamides Stoineva,I.B;D.D.Petkov
  9. Biochemistry v.34 Crystal structure of D-amino acid transferase: How the protein controls steroselectivity Sugio,S;G.A. Petsko;J.M. Manning;K. Soda;D. Ringe https://doi.org/10.1021/bi00030a002
  10. Biotechnology of amino acid production Microbial production of D-p-hydroxyphenylglycin Takahashi,S;K.Aida(ed);I.Chibata(ed);N.Nakayama(ed);K.Takinami(ed);H.Yamada(ed.)
  11. J. Biol. Chem. v.264 Thermostable D-amion acid aminotransferase from a thermostable Bacillus species Tanizawa,K;Y.Masu;S.Asano;H.Tanaka;K.Soda
  12. Appl. Microbiol. Biotechnol. v.36 D-Alanine production from DL-alanine by Candida maltosa with asymmetric degrading activity Umemura,I;K.Yamagiya;S.Komatsubara;T.Sato;T.Tosa