A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

Instability under non-native processing conditions, especially at elevated temperatures, is a major factor preventing the widespread adoption of biocatalysts for industrial synthesis. A crucial distinction of many redox enzymes used to synthesize chiral compounds is the need for cofactors (e.g., NAD(P)(H)) for function. Because of the prohibitively high prices of nicotinamide cofactors, a robust cofactor-regenerating enzyme is required for the economical synthesis of fine chemicals by biocatalysis. Here we test the structure-guided consensus for the generation of a thermostable glucose dehydrogenase (GDH). The consensus sequence in combination with additional knowledge-based criteria was used to select amino acids for substitutions. Using this approach we generated 24 variants, 11 of which showed higher thermal stability than the wild-type GDH, a success rate of 46 %. Of the 24 variants, seven were located at the subunit interface-known to influence GDH stability-and six were more stable (86 % success). The best variants feature a half-life of approximately 3.5 days at 65 degrees C, in contrast to approximately 20 min at 25 degrees C for the wild type, thus enhancing stability 10(6)-fold. In addition, the three most stabilizing single mutations were transferred to two GDH homologues from Bacillus thuringiensis and Bacillus licheniformis. The thermal stability as measured by half-life and CD(222 nm) of the GDH variants was increased, as expected. The resulting stability changes provide further support for the view that these residues are critical for stability of GDHs and reinforce the success of the consensus approach for identifying stabilizing mutations.

Enoate reductases (ERs) selectively reduce carbon-carbon double bonds in a,b-unsaturated carbonyl compounds and thus can be employed to prepare enantiomerically pure aldehydes, ketones, and esters. Most known ERs, most notably Old Yellow Enzyme (OYE), are biochemically very well characterized. Some ERs have only been used in whole-cell systems, with endogenous ketoreductases often interfering with the ER activity. Not many ERs are biocatalytically characterized as to specificity and stability. Here, we cloned the genes and expressed three non-related ERs, two of them novel, in E. coli: XenA from Pseudomonas putida, KYE1 from Kluyveromyces lactis, and Yers-ER from Yersinia bercovieri. All three proteins showed broad ER specificity and broad temperature and pH optima but different specificity patterns. All three proteins prefer NADPH as cofactor over NADH and are stable up to 40 8C. By coupling Yers-ER with glucose dehydrogenase (GDH) to recycle NADP(H), conversion of > 99 % within one hour was obtained for the reduction of 2-cyclohexenone. Upon lowering the loadings of Yers-ER and GDH, we discovered rapid deactivation of either enzyme, especially of the thermostable GDH. We found that the presence of enone substrate, rather than oxygen or elevated temperature, is responsible for deactivation. In summary, we successfully demonstrate the wide specificity of enoate reductases for a range of a,b-unsaturated carbonyl compounds as well as coupling to glucose dehydrogenase for recycling of NAD(P)(H); however, the stability limitations we found need to be overcome to envision large-scale use of ERs in synthesis.

The thermostabilization of penicillin G acylase (PGA) is a difficult problem due to the large size of the protein and its complex maturation process. We developed a data-driven protein design method that requires fewer homologous sequences than the traditional consensus approach and utilizes structural information to limit the number of variants created. Approximately 50% of our 21 single-point mutants were found experimentally to be more thermostable than the wild-type PGA, two had almost threefold longer half-life at 50 degrees C, with very little effect on activity. An analysis of four programs that predict the thermostability conferred by point mutations shows little agreement between the programs and with the experimental data, emphasizing that the chosen stabilizing mutations are very difficult to predict, but that our data-driven design method should prove useful.