Isopropanol dehydrogenase

Hiu SF, Zhu C-X, Yan R-T, Chen J-S (1987) Butanol-ethanol dehydrogenase and butanol-ethanol-isopropanol dehydrogenase different alcohol dehydrogenases in two strains of Clostridium beijerinckii (Clostridium butylicum). Appl Environ Microbiol 53 697—703... 

The MTT method is simple, accmate and yields reproducible results. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) or MTT. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding prrrple formazan crystals insolnble in aqueous solutions. The crystals are dissolved in acidified isopropanol. The resrrlting ptuple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant... 

Cofactor regeneration, an economically essential step in the synthetic use of ADH, was accomplished within the PVA matrix using isopropanol as cosubstrate for the ADH itself or for a second alcohol dehydrogenase from Thermoanaerobium hrockii (E.C. 1.1.1.2). An overall turnover number of 10 was achieved, which is a promising magnitude for technical application. However, while the presence of the cosubstrate in the gel-stabilized two-phase system improved the solubility of substrates in the gel phase and consequently enhanced the productivity of the... 

The metabolism of isopropanol is via oxidation by aldehyde dehydrogenase (ADH) to acetone. In common with other a-substituted (secondary) alcohols, isopropanol is a relatively poor substrate for ADH (WHO, 1990 Light et al., 1992). The primary metabolite, acetone, is eliminated in the expired air and in the urine and also undergoes further oxidation to acetate, formate and, ultimately, CO,. 

The most convenient and useful enzymatic methods for the regeneration of NAD(P)H are formate/formate dehydrogenase for NADH [210, 211], iso-propanol/TBADH for NADPH [57], isopropanol/ADH (Pseudomonas sp.) for NADH [61, 212] and glucose/glucose dehydrogenase (Bacillus sp.) for NADH and NADPH [213],... 

Formate dehydrogenase in conjunction with polyethyleneglycol-immobilized nicotinamide adenine dinudeotide has been used to good effect as a cofactor recycle system (39). The alcohol dehydrogenase from Thermoanaerobium hrockii catalyzed the reduction of ketones independently when driven by the cooxidation of isopropanol (40,41). 

Isopropanol is rapidly (within 30 min) and well absorbed ( 70% bioavailability) after oral administration. The Vj of isopropanol is 0.6-0.71 kg with minimal to no protein binding. Isopropanol is rapidly metabolized by alcohol dehydrogenase in a first-order, concentration-dependent manner to acetone. This apparent first-order metabolism of isopropanol is probably a result of extensive pulmonary clearance of the acetone. Approximately 80% of systemic isopropanol is metabolized to acetone with the remainder excreted unchanged via the kidneys. A very small amount of isopropanol may be eliminated via the lungs. The presence of ethanol will competitively... 

Isopropanol has a short half-life ti ) of 1 to 6 hours, as it is rapidly metabolized by alcohol dehydrogenase to acetone, which is eliminated much more slowly (t]/2,17 to 27 hours), primarily in alveolar air and urine.Therefore concentrations of acetone in serum often exceed those of isopropanol during the elimination phase following isopropanol mgestion (Figure 34-4). Acetone has CNS depressant activity similar to that of ethanol, and because of its longer half-life, it prolongs the apparent CNS effects of isopropanol. 

AOD has been coimmobilized with alcohol dehydrogenase in order to increase the sensitivity of alcohol determination (Hopkins, 1985). In the presence of oxygen and NADH ethanol is recycled between the two enzymes. Thus more H2O2 is formed than substrate is present in the enzyme membrane, i.e., the sensitivity is enhanced (see also Section 3.2.4). A further advantage of this system is that this recycling is restricted to ethanol, because methanol is converted only by AOD but notbyADH. Conversely, isopropanol is oxidized by ADH but not by AOD. Thus, by combination of the two enzymes the selectivity of the sensor for ethanol is improved. 

Figure 1-13. Effect of pH on the activity of sec-alcohol dehydrogenase (Candida boidinii) during the oxidation of isopropanol in various buffers in 50 mM concentration O sodium citrate, potassium phosphate, Atriethanolamine/HCI, ATris/HCI, glycine.

Figure 16.2-49. Asymmetric reduction of ethyl-4-chloro-3-oxobutanoate catalyzed by an alcohol dehydrogenase (ADH) in recombinant E. coli. The necessary reduction equivalents were derived from the oxidation of isopropanol with the same enzyme.

In another application, recombinant E. coli produced 36.6 g/L ethyl-(R)-4-chloro-3-hydroxybutanoate (99% ee) from 40 g/L ethyl-4-chloro-3-oxo-butanoate[210). Here, the secondary alcohol dehydrogenase served as both synthetic (asymmetric reduction) and regenerating (NADH-regeneration via isopropanol oxidation) enzyme (Fig. 16.2-49). 

Butanol is analyzed by GC-FID using a suitable column for alcohols, such as 10% SP-1000 on Chromosorb W-HP or equivalent. It is analyzed in air by charcoal adsorption, desorbing the analyte in a CS2-isopropanol mixture (99 1) and injecting the eluant onto GC-FID (NIOSH 1984, Method 1401). Other methods, such as GC/MS, HPLC, IR, and TEC, have been employed. A fluorophotometric method using alcohol dehydrogenase may be used. 

Buk) for butyrate. The proteins EtfA and EtfB are crucial for the activity of the Bed protein [56]. The required enzymes for solvent production are coenzyme A transferase (CtfA, CtfB), acetoacetate decarboxylase (Adc) as v rell as different aldehyde and alcohol dehydrogenases (BdhA, BdhB, AdhE, AdhE2). In C. beijer-inckii [57], an additional secondary alcohol dehydrogenase allows the reduction of acetone to isopropanol. All genes and enzymes were discussed in detail by Kopke andDiirre [58]. 

---