Alcohol dehydrogenase isolation

Alcohol dehydrogenase-catalyzed reduction of ketones is a convenient method for the production of chiral alcohols. HLAD, the most thoroughly studied enzyme, has a broad substrate specificity and accommodates a variety of substrates (Table 11). It efficiendy reduces all simple four- to nine-membered cycHc ketones and also symmetrical and racemic cis- and trans-decalindiones (167). Asymmetric reduction of aUphatic acycHc ketones (C-4—C-10) (103,104) can be efficiendy achieved by alcohol dehydrogenase isolated from Thermoanaerohium hrockii (TBADH) (168). The enzyme is remarkably stable at temperatures up to 85°C and exhibits high tolerance toward organic solvents. Alcohol dehydrogenases from horse Hver and T. hrockii... 

Other enzymes, e.g., yeast alcohol dehydrogenase and alcohol dehydrogenase isolated from horse liver, are also used for this type of reduction26, however, the former is too specific for small molecules while the latter is more useful for the reduction of cyclic ketones. 

The alcohol dehydrogenase isolated from horse fiver (LADH) [E.C.l.1.1.1.], MW 80,000, is available as a highly purified, crystalhne material composed of two (or more) isozymes (57), one of which is present in >90%. The general physical and chemical properties of this zinc enz5une have been reviewed by Sund and Theorell (35). This nicotinamide-adenine dinucleotide oxidoreductase catalyzes the aldehyde-alcohol interconversion shown in Eq. (16). 

Strict specificity toward a coenzyme regardless of the source material. Alcohol dehydrogenase isolated from yeast and horse liver reacts with DPN but not at all with TPN. Triosephosphate dehydrogenase might have been cited as another example of strict DPN specificity except that current... 

Figure 8.27 Reduction of aldehyde in SCCO2 by an isolated enzyme, horse liver alcohol dehydrogenase (HLADH) [20c] (a) Reaction scheme (b) fluorinated coenzyme soluble in CO2 and (c) effect of coenzyme on the reaction.

The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid." The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)" and alcohol dehydrogenase." A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing. 

Hummel, W., Abokitse, K., Drauz, K. et al. (2003) Towards a large-scale asymmetric reduction process with isolated enzymes Expression of an (5)-alcohol dehydrogenase in E. coli and studies on the synthetic potential of this biocatalyst. Advanced Synthesis and Catalysis, 345 (1 + 2), 153-159. 

An isolated DNA molecule comprising DNA which encodes a group III alcohol dehydrogenase and DNA which encodes a BDS-active biocatalyst via nicotinamide adenosine dinucleotide-dependent manner. 

Hydroxylation of the benzylic methyl group of tolbutamide, the preferred site of oxidative attack by CYP2C9 (22), generates hydroxytolbutamide. Hydroxytolbutamide is rapidly oxidized by other enzymes, presumably aldehyde oxidase and/or alcohol dehydrogenase (ALD), to form the major isolated metabolite, the benzoic acid analog. 

The chiral compounds (/ )- and (5)-bis(trifluoromethyl)phenylethanol are particularly useful synthetic intermediates for the pharmaceutical industry, as the alcohol functionality can be easily transformed without a loss of stereospecificity and biological activity, and the trifluoromethyl functionalities slow the degradation of the compound by human metabolism. A very efficient process was recently demonstrated for the production of the (5)-enantiomer at >99% ee through ketone reduction catalyzed by the commercially available isolated alcohol dehydrogenase enzyme from Rhodococcus erythropolis (Figure 9.1). The (7 )-enantiomer could be generated at >99% ee as well using the isolated ketone reductase enzyme KRED-101. 

Fig. 3.1.5 Cascade of membrane reactors with isolated enzymes (BFD benzoylformate decarboxylase ADH alcohol dehydrogenase FDH formate dehydrogenase).

Confirmation of this proposed pathway was established by Altenschmidt Fuchs (1991, 1992) in their study of the biochemistry of toluene decay by the denitrifying Pseudomonas sp. strain K172. These investigators confirmed the presence of benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase in cell-free extracts of this isolate. Further, [14C]benzyl... 

C. Morgan and N. Moir,/. Chem. Educ. 73, 1040-1041 (1996). Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography. ... 

Coniferaldehyde (3.76) can undergo several fates, some of which can ultimately lead to the same end product. It can be reduced to coniferyl alcohol (3.79) by the enzyme cinnamyl alcohol dehydrogenase (CAD). Alternatively, the enzyme coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (C5H), also known by its less accurate name ferulic acid 5-hydroxylase (F5H Humphreys et al., 1999) can catalyze the hydroxylation of C5 to result in 5-hydroxyconiferyl aldehyde (3.77). C5H is also able to form 5-hydroxyconiferyl alcohol (3.80) from coniferyl alcohol (3.79). This enzyme was initially identified as F5H, after analysis of the Arabidopsis ferulic acid hydroxylase 1 (fahl) mutant, which was isolated in a mutant screen based on reduced levels of the UV-fluorescent sinapoyl esters (Section 13 Chappie et al., 1992). The FAH1 gene was cloned using a T-DNA tagged mutant allele (Meyer et al., 1996), which revealed that the... 

Sibout, R., Eudes, A., Pollet, B., Goujon, T., Mila, I., Granier, F., Seguin, A., Lapierre, C., and Jouanin, L., 2003, Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants, Plant Physiol. 132 848-860. 

Jacobs, M., Dolferus, R. Van Den Bossche (1988). Isolation and biochemical analysis of ethyl methane sulfonate induced alcohol dehydrogenase null mutants of Arabidopsis thaliana (L.) Heynth. Biochemical Genetics 26, 105-12. 

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