Biocatalytic properties of quinohemoprotein alcohol dehydrogenase IIG from Pseudomonas putida HK5 (original) (raw)
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European Journal of Biochemistry, 1994
Initial rate studies were performed on the oxidation of (racemic) alcohols as well as aldehydes by quinohaemoprotein ethanol dehydrogenase, type 1, from Comamonas testosteroni with potassium ferricyanide as electron acceptor. The data could be fitted with an equation derived for a mechanism (hexa-uni ping-pong) in which alcohols are oxidized to the corresponding carboxylic acids and the intermediate aldehyde becomes released from the enzyme. However, for some substrates it was necessary to assume that they exert uncompetitive inhibition. The same model was used to fit the data of conversion processes. Reversible inactivation of the enzyme takes place during the conversion, the extent being inversely proportional to the concentration of ferricyanide present at the start. From the values of the kinetic parameters obtained for (R)-and (S)-solketal [2,2-dimethyl-4-(hydroxymethyl)-l,3-dioxolane] and their corresponding aldehydes, it appeared that the second step in (S)-solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. Since the initial rate measurements as well as the progress curve analysis gave similar kinetic parameter values and product analysis revealed intermediates in the amounts predicted, it is concluded that the kinetic and enantioselective behaviour of the enzyme is adequately described by the model presented here. Finally, the results indicate that both kinetic approaches should be used in conversions with consecutive reactions since they provide complementary information.
Journal of Molecular Catalysis B: Enzymatic, 2000
Quinohaemoprotein alcohol dehydrogenases, QH-ADHs, isolated from Acetobacter, Gluconobacter and Comamonas species show appreciable enantioselectivity in the oxidation of chiral primary and secondary alcohols. Current views of the structural and mechanistic factors of importance for the understanding of the enantioselective performance of these enzymes are reviewed. Structural properties of QH-ADH, Type I, from C. testosteroni, and QH-ADH, Type II, from A. pasteurianus have been deduced from homology modeling studies based on the X-ray crystallographic data available for PQQ-containing Ž. quinoprotein methanol dehydrogenases, MDHs. Mechanisms that have been proposed for quino haemo protein alcohol dehydrogenase-catalyzed substrate oxidation are discussed in relation to the constraints set by the observed enantioselectivity.
2013
The NAD + regeneration system present in E. coli cells was exploited for the oxidation and deracemisation of secondary alcohols with the over-expressed alcohol dehydrogenase from Rhodococcus ruber DSM 44541 (E. coli/ADH-A). Thus, various racemic alcohols were selectively oxidised employing lyophilised or resting E. coli/ADH-A cells, without requiring external cofactor or co-substrate. Simple addition of these substrates to the E. coli/ADH-A cells in buffer afforded the corresponding ketones and the remaining enantioenriched (R)-alcohols. This methodology was applied for the desymmetrisation of a meso-diol, and for the synthesis of the highly valuable raspberry ketone. Moreover, a biocatalytic concurrent process was developed with resting cells of E. coli/ADH-A, ADH from Lactobacillus brevis (LBADH) and glucose dehydrogenase (GDH), for the deracemisation of various secondary alcohols, producing the desired enantiopure alcohols in up to >99% ee starting from the racemic mixture. The reaction time of deracemisation for 1phenylethanol was estimated to be less than 30 min. The stereoinversion of (S)-1-phenylethanol to its pure (R)-enantiomer was also successfully achieved, thus providing a biocatalytic alternative to the chemical Mitsunobu inversion reaction.
Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose
Journal of Biotechnology, 2006
Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20 h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD +. In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD +-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.
Applied Biochemistry and Biotechnology, 2011
Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo-and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K m value of 0.2 mM.
Biotechnology Journal, 2006
Using the organic solvents acetonitrile and 1,4-dioxane as water-miscible additives for the alcohol dehydrogenase (ADH)-catalyzed reduction of butan-2-one, we investigated the influence of the solvents on enzyme reaction behavior and enantioselectivity. The NADP + -dependent (R)-selective ADH from Lactobacillus brevis (ADH-LB) was chosen as biocatalyst. For cofactor regeneration, the substrate-coupled approach using propan-2-ol as co-substrate was applied. Acetonitrile and 1,4dioxane were tested from mole fraction 0.015 up to 0.1. Initial rate experiments revealed a complex kinetic behavior with enzyme activation caused by the substrate butan-2-one, and increasing K M values with increasing solvent concentration. Furthermore, these experiments showed an enhancement of the enantioselectivity for (R)-butan-2-ol from 37% enantiomeric excess (ee) in pure phosphate buffer up to 43% ee in the presence of 0.1 mol fraction acetonitrile. Finally, the influence of the co-solvents on water activity of the reaction mixture and on enzyme stability was investigated.