| There is an increasing demand for environmentally friendly industrial technologies. Many current industrial chemical processes use harsh conditions (i.e., high temperature or extremes of pH), create toxic waste, and require non-renewable resources. The use of biocatalysts, such as isolated enzymes, to replace these harsh processes is a field of intense study.;Many enzymes are not well suited to industrial processes because of their lack of stability or lack of specificity. Advances in the field of enzyme engineering have allowed for the alteration of enzyme characteristics, such increased thermostablity or tailored substrate specificity, to meet the needs of a specific industrial process.;In this thesis we will describe the alteration of the substrate specificity of a secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH) by rational design to include phenyl-substituted alcohols and ketones for industrial use. The W110A mutation was introduced to increase the size of the active site. W110A TeSADH uses (S)-1-pheny1-2-propanol, (S)-4-phenyl-2-butanol, and the corresponding ketones as substrates. W110A TeSADH's kinetic parameters on these substrates are in the same range as those of TeSADH on 2-butanol, making W110A TeSADH an excellent catalyst. In particular, W110A TeSADH is twice as efficient on benzylacetone as TeSADH is on 2-butanol, and it produces (S)-4-phenyl-2-butanol from benzylacetone with an enantiomeric excess above 99%. W110A TeSADH is active on aryl derivatives of phenylacetone and benzylacetone, making this enzyme a potentially useful catalyst for the chiral synthesis of aryl derivatives of alcohols.;We also describe attempts to change the cofactor specificity of TeSADH from NADP(H) to NAD(H). NADP(H) is more unstable and more costly than NAD(H), which makes it less economically feasible to use. We use both rational design and directed evolution to accomplish our goals. We constructed a triple mutant, GCY, by site-directed mutagenesis to use as our parent for directed evolution. This mutant had a 9-fold increase in Km compared to TeSADH with NAD +, but its Vmax is comparable to TeSADH with NADP +.;Two rounds of directed evolution were performed. Kinetic parameters of the first generation mutant, OE3, were better than those of its parent, GCY, for NAD+. Its Km for NAD+ decreased nearly 2-fold and its Vmax decreased only 1.1 times compared to GCY.;The kinetic parameters of the second generation mutant, 2E, were almost identical to those of its parent, OE3, for NAD+, with a 0.92-fold decrease in Km and 1.1-fold decrease in Vmax. However, the Km of 2E for NADP+ increased by 662.5 times, and the Vmax decreased by 16 times compared to wild-type TeSADH 2E has a 7.4-fold higher Vmax with NAD+ than with NADP+, and a 7.3-fold higher catalytic efficiency with NAD+ than with NADP+. These results show that the cofactor preference of 2E TeSADH has changed from NADP+ to NAD+. |
