Alcohol dehydrogenase (E. C. 1.1.1.1) from Thermoanaerobium brockii at 25 degrees C and at 65 degrees C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V(m)) for secondary alcohols at 65 degrees C are 10 to 100 times higher than those at 25 degrees C, whereas the K(m) values are more comparable.At both 25 degrees C and 65 degrees C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.At 25 degrees C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.At 65 degrees C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant-variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme-NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex.
The conversion of both xylose and xylitol to ethanol has been demonstrated in cell-free extracts of Pachysolen tannophilus. The facts that xylitol is metabolized in the extracts but not in whole cells in the absence of nystatin demonstrate that transport across the cell membrane limits its metabolism in whole cells. The metabolism of both xylose and xylitol requires NAD and ADP, but the metabolism of xylose requires and NADPH-generating system in addition. Xylose isomerase increases the rate of ethanol formation from xylose, but some xylitol accumulates nevertheless. Therefore, the cell-free system metabolizes xylose as two independent, sequential pathways, one to synthesize xylitol and one to convert it to ethanol. The consequences for process -improvement strategies are discussed.With the increased awareness of the limitations in the world supplies of petroleum came an increased interest in gasoline extenders and additives. The oxygenated additives tert-butyl ether, methanol and ethanol were found to have the additional advantages of octane enhancement and lower rates of air pollution. The use of ethanol on a national scale is associated with further advantages (1,2): the security of fuel sources; a more favorable foreign trade balance; and the renewability of the raw materials for production. Although ethanol can be produced either from a petroleum component or by fermentation, the latter source is the only one associated with all of the advantages above. The most attractive raw materials for ethanol fermentation are cane sugar, corn sugar, and sugar derived from wood and crop residues (lignocellulosic materials). In this country cane sugar is too expensive to be a source of fuel ethanol and com ($98-$104 per ton) (2), can be competitive only in special circumstances, such as government subsidy. However, lignocellulosic material ($20-$70 per ton) could be competitive, if the technical limitations associated with processing and fermentation could be overcome. Although the approximate price of
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