Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680.
A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.
Wang
Zong-Da
ZD
Key Laboratory of Molecular Evolution and Biodiversity and Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, 241000 Anhui, People's Republic of China.
Wang
Bao-Juan
BJ
Ge
Ya-Dong
YD
Pan
Wei
W
Wang
Jie
J
Xu
Lei
L
Liu
Ai-Min
AM
Zhu
Guo-Ping
GP
D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D062907:Oxaloacetic Acid / Q000378:metabolism; D011387:Prokaryotic Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D013696:Temperature
D007477:Ions; D008293:Malates; D008670:Metals; D011994:Recombinant Proteins; D062907:Oxaloacetic Acid; C030298:malic acid; D008291:Malate Dehydrogenase