Immobilized horse liver alcohol

I.W. Immobilized horse liver alcohol dehydrogenase as an online high-performance hquid chromatographic enzyme reactor for stereoselective synthesis. Chirality 47. 1999, 11, 39 5. 

H GOrisch, M Schneider. Stabilization of soluble and immobilized horse liver alcohol dehydrogenase by adenosine 5-monophosphate. Biotechnol Bir ng 26 998-1002, 1984. 

All the enzymes used in the work described above are quite stable at room temperature and can be used in a free form. They can also be used in an immobilized form to improve the stability and to facilitate the recovery. Many immobilization techniques are available today (25). The recent procedure developed by Whitesides et al using water-insoluble, cross-linked poly(aerylamide-acryloxysuccinimide) appears to be very useful and applicable to many enzymes (37). We have found that the non-crosslinked polymer can be used directly for immobilization in the absence of the diamine cross-linking reagent. Reaction of an enzyme with the reactive polymer produces an immobilized enzyme which is soluble in aqueous solutions but insoluble in organic solvents. Many enzymes have been immobilized by this way and the stability of each enzyme is enhanced by a factor of greater than 100. Horse liver alcohol dehydrogenase and FDP aldolase, for example, have been successfully immobilized and showed a marked increase in stability. 

The use of oxidoreductases in solution clearly dominates over immobilized applications. Use of immobilized whole cells (e.g. baker s yeast) [16] is, however, well described, and reports have also appeared claiming increased stability and activity of isolated horse liver alcohol dehydrogenase and other oxidoreductases immobilized on agarose [17] or salt crystals [18] (protein-coated microcrystals, PCMC [19]). Furthermore, immobilization of oxidoreductases on surfaces has been studied more intensively for the development of biosensors. 

For example, horse liver alcohol dehydrogenase (HLADH) was noncovalently immobihzed on a membrane and packed into a PBR [74] operated in a recirculated loop mode for the reduction of racemic 2-phenyl-tetrahydropyran-4-one 1 in the presence of NADH. The HLADH-reactor coupled with an enzymic cofactor regeneration system in the mobile phase could convert the substrate to the enantiopure (S,S)- and (R,S)-2. The immobilized HLADH reactor was stable over 6 months when stored at 5 °C. 

Using two types of specially synthesized rhodium-complexes (12a/12b), pyruvate is chemically hydrogenated to produce racemic lactate. Within the mixture, both a d- and L-specific lactate dehydrogenase (d-/l-LDH) are co-immobilized, which oxidize the lactate back to pyruvate while reducing NAD+ to NADH (Scheme 43.4). The reduced cofactor is then used by the producing enzyme (ADH from horse liver, HL-ADH), to reduce a ketone to an alcohol. Two examples have been examined. The first example is the reduction of cyclohexanone to cyclohexanol, which proceeded to 100% conversion after 8 days, resulting in total TONs (TTNs) of 1500 for the Rh-complexes 12 and 50 for NAD. The second example concerns the reduction of ( )-2-norbornanone to 72% endo-norbor-nanol (38% ee) and 28% exo-norbornanol (>99% ee), which was also completed in 8 days, and resulted in the same TTNs as for the first case. 

---