Growth conditions for the production of NAD-independent dehydrogenase in Acetobacter strains isolated from industrial vinegar
Todaro, A.R.1; Guedes, G.S.1; Silva, P.F.1; Freitas, J.D.1; Santanna, A.E.G.1; Jongejan, J.A.2; Machado, S.S.1
1Chemistry Department, Federal University of Alagoas-UFAL, Maceió, Brazil
2Department of Microbiology and Enzymology, Delft University of Technology, Delft, The Netherlands
Because acetic acid bacteria are able to rapidly catalyze incomplete oxidation of substrates, both regioselectively and enantioselectively, they are used in various biotechnological processes. Several enzymes from acetic acid bacteria involved in these processes have been isolated, and characterized as membrane-bound dehydrogenases. Dye-linked alcohol dehydrogenase from acetic acid bacteria has been characterized as a quinohaemprotein alcohol dehydrogenase (QH-ADH). In previous work, two Acetobacter strains, LBVE-1 and LBVE-4, were isolated from industrially produced vinegar. To evaluate the dye-linked alcohol dehydrogenase in these strains, assays for biomass production were performed in shaker flasks. Three ethanol concentrations were tested as carbon source (10, 15 e 20 g/L) incubated at three temperatures (30, 35 e 37ºC). Although highest biomass production for the two strains was obtained in the assays with 20 g/L ethanol, 30 ºC, both strains presented different growth conditions for the production of biomass highly active in NAD-independent ADH. LBVE1 crude extract from biomass grown in 10 g/L ethanol, 30ºC, had highest specific activity, 10.44 U/mg protein, while highest specific activity for LBVE4, 8.04 U/mg protein, was obtained for biomass grown in 15 g/L ethanol, 30ºC. Kinetic assays were performed to calculate apparent kinetics constants of NAD-independent ADH present in the crude extract from both strains grown under different conditions. LBVE1 crude extracts from cells grown in 10 and 20 g/L ethanol showed comparable values for Knapp and VMaxapp (Knapp =2.064±0.346 mM, VMaxapp= 13.226±0.715 mmol.min-1 and Knapp = 1.501±0.250 mM, VMaxapp= 14.516±0.641 mmol.min-1 respectively). However, LBVE4 crude extracts from cells grown in 10 g/L showed different Knapp values (Knapp=0.881±0.139 mM) from cells grown in 20 g/L ethanol (Knapp=1.818±0.0.428 mM). NAD-independent ADH activity for selected primary and secondary alcohols in the extracts from both strains grown in ethanol 20 g/L showed a decrease in the activity values with increasing carbon chain length.
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