Cofactor requiring redox enzymes in organic synthesis /
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| Other Authors: | , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1992.
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| Subjects: | |
| Online Access: | Link to OAKTrust copy |
| Abstract: | New nicotinamide cofactor dependent alcohol dehydrogenases from Pseudomonas strains SBD6 and PED have been purified to homogeneity using a combination of salt precipitation, anion exchange chromatography, gel filtration chromatography, and dye matrix or affinity chromatography. The 150 fold purification of Pseudomonas sp. strain SBD6 alcohol dehydrogenase (PADH) yields approximately 10 mg of pure enzyme from 10 g of wet cells, 29% yield. The enzyme has four subunits with a total molecular weight of 162,000. Incubation with the metal chelators 1,10-phenanthroline, 2-aminoethanethiol, hydroxyquinolinesulfonic acid, N-ethylmaleimide, or potassium cyanide result in complete loss of activity. PADH is very stable (t[1/2] 7 days at pH 7 and 25°C in the absence of 2-propanol and 18 days in the presence of 10% 2-propanol, v/v). Based on NAD and 2-propanol, the kinetic parameters of PADH were determined to be V[max]= 29.9 Units mg^-1 at 25°C and pH 8.5, K[m]NAD =0.36 mM and K[m]2-propanol=0.19 mM. The alcohol dehydrogenase from strain PED (PED) was purified 257 fold from 17.1 g wet cells to give 5 mg of enzyme, 25% yield. The kinetic parameters were determined to be V[max]=36 Units mg^-1 at 23°C and pH 8.5. K[m]NAD=525 μM and K[m]2-propanol=75 μM. The kinetic mechanism of PADH and PED is ordered bi-bi with the cofactor binding first and releasing last. PADH and PED transfer the pro-(R) hydride from NADH to the si face of the carbonyl substrates to give (R) alcohols in high enantiomeric excess, a stereochemical process different from that of other known alcohol dehydrogenases. Lactobacillus kefir alcohol dehydrogenase produces the same relative stereochemistry, but requires the phosphorylated nicotinamide cofactor. The stereochemistry of the products from Thermoanaerobium brockii alcohol dehydrogenase was determined to be the same as the products from horse liver and yeast alcohol dehydrogenases with transfer of the pro-(R) hydride to the re face of carbonyls to give (S) alcohols... |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Chemistry." |
| Physical Description: | xix, 159 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |