Purification and Biochemical Characterization of Recombinant Alanine Dehydrogenase fvom Thermus caldophilux GK24

  • Bae, Jung-Don (Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Cho, Youn-Jeung (EnzBank, Inc.) ;
  • Kim, Doo-Il (Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Lee, Dae-Sil (Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Shin, Hyun-Jae (EnzBank, Inc.)
  • 발행 : 2003.08.01

초록

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to $70^{\circ}C$. The activity of the enzyme was inhibited by 1 mM $Zn^{2+}$, 20% hexane, and 20% $CHCl_3$. However, 10 mM $Mg^{2+}$ and 40% propanol had no effect on the enzyme activity. The Michaelis constants ($K_m$) for the substrates were $50\;\mu\textrm{M}$ for NADH, 0.2 mM for pyruvate, 39.4 mM for $NH_4+$, 2.6 mM for L-alanine, and 1.8 mM for $NAD^+$.

키워드

참고문헌

  1. Infect. Immun. v.60 Structure and function of a 40,000-molecular-weight protein antigen of Mycobacterium tuberculosis Andersen,A.B.;P.Anderson;L.Ljungqvist
  2. J. Bacteriol. v.171 Purification and properties of L-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus capsulatus E1F1 Caballero,F.J.;J.Cardenas;F.Castillo
  3. Biochim. Biophys. Acta v.630 Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii Frankel,A.D.;F.R.Jones https://doi.org/10.1016/0304-4165(80)90417-1
  4. J. Microbiol. Biotechnol. v.12 Cloning expression of the gene for inoganic pyrophosphatase of Thermus caldophilus GK24 and properties of the enzyme Hoe,H.S.;I.G.Joe;H.J.Shin;H.J.Jeon;H.K.Kim;J.S.Lee;Y.S.Kim;D.S.Lee;S.T.Kwon
  5. Gene v.212 Host vector system for high-level expression and purification of recombinant, enzymatically active alanine dehydrogenase of Mycobacterium tuberculosis Hutter,B.;M.Singh https://doi.org/10.1016/S0378-1119(98)00134-6
  6. Biochem. J. v.343 Properties of the 40 kDa antigen of Mycobacterium tuberculosis, a functional L-alanine dehydrogenase Hutter,B.;M.Singh https://doi.org/10.1042/0264-6021:3430669
  7. J. Microbiol. Biotechnol. v.12 Cellulase-free thermostable alkaline xylanase from thermophilic and alkalophilic Bacillus sp. JB-99 Johnvesly,B.;S.Virupakshi;G.N.Patil;Ramalingam;G.R.Naik
  8. Bull. Korean Chem. Soc. v.21 Regulatory mechanism of L-alanine dehydrogenase from Bacillus subtilis Kim,S.J.;Y.J.Kim;M.R.Seo;B.S.Jhun
  9. Biochemistry v.29 Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: Molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD${(P)}^+$-dependent dehydrogenases Kuroda,S.;K.Tanizawa;Y.Sakamoto;H.Tananka;K.Soda https://doi.org/10.1021/bi00456a025
  10. Mol. Cell v.7 Cloning and analysis of the DNA polymerase-encoding gene from Thermus caldophilus GK24 Kwon,S.T.;J.S.Kim;J.H.Park;H.K.Kim;D.S.Lee
  11. Arch. Microbiol. v.174 Purification, properties and primary structure of alanine dehydrogenas involved in taurine metabolism in the anaerobe Bilophila wadsworthia Laue,H.;A.M.Cook https://doi.org/10.1007/s002030000190
  12. J. Microbiol. Biotechnol. v.6 Production of L-DOPA by thermostable tyrosine phenol-lyase of a thermophilic Symbiobacterium species overexpressed in recombinant Escherichia coli Lee,S.G.;H.S.Ro;S.P.Hong;E.H.Kim;M.H.Sung
  13. J. Bacteriol. v.118 Regulation of alanine dehydrogenase in Bacillus licheniformis McCowen,S.M.;P.V.Phibbs
  14. Eur. J. Biochem v.199 Thermostable alanine depydrogenase from thermophilic Bacillus sphaericus DSM 462 Ohshima,T.;M.Sakane;T.Yamazaki;K.Soda
  15. Eur. J. Biochem v.100 Purification and properties of alanine dehydrogenase from Bacillus sphaericus Ohshima,T.;K.Soda https://doi.org/10.1111/j.1432-1033.1979.tb02030.x
  16. Trend Biotechnol. v.7 Thermostable amino acid dehydrogenase: Applications and gene cloning Ohshima,T.;K.Soda https://doi.org/10.1016/0167-7799(89)90106-6
  17. Biotechnol. Lett. v.7 Screening of thermostable leucine and alanine dehydrogenases in thermophilic Bacillus strains Ohshima,T.;C.Wandrey;M.Sugiura;K.Soda https://doi.org/10.1007/BF01088008
  18. J. Biochem.(Tokyo) v.116 Purification and characterization of alanine dehydogenase from a cyanobaterium, Phormidium lapideum Sawa,Y.;M.Tani;K.Murata;H.Shibata;H.Ochiai
  19. J. Bacteriol v.175 Alanine dehydrogenase(ald) is required for normal sporulation in Bacillus subtilis Siranosian,K.J.;K.Ireton;A.D.Grossman
  20. Arch. Biochem. Biophys. v.304 Alanine dehydrogenase from soybean nodule bacteroids: Purification and properties Smith,M.T.;S.W.Emerich https://doi.org/10.1006/abbi.1993.1365
  21. Arch. Microbiol. v.145 Metabolism of L-alanine in Desulfotomaculum ruminis and two marine Desulfovibrio strains Stams,A.;T.Hansen https://doi.org/10.1007/BF00443658
  22. J. Microbiol. Biotechnol. v.11 Purification and characterization of extracellular and intracellular glutamine synthetases from Mycobacterium bovis BCG Suh,C.I.;J.M.Lim;H.C.Sung
  23. Eur. J. Biochem. v.145 Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium Tajuchi,H.;H.Matsuzawa;T.Ohta https://doi.org/10.1111/j.1432-1033.1984.tb08550.x
  24. Biochim.Biophys. Acta v.615 L-alanine dehydrogenase from Thermus thermophilus Vali,Z.;F.Kilar;S.Lakatos;S.A.Venyaminov;P.Zavodszky https://doi.org/10.1016/0005-2744(80)90006-6
  25. Arch. Microbiol. v.150 Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens G.Basarova;V.Behal https://doi.org/10.1007/BF00422283