Abstract

Abstract

Abstract

Abstract

Enoate reductases (ERs) selectively reduce carbon-carbon double bonds in α,β-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 °C. 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 α,β-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.

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 ∼3.5 days at 65 °C, in contrast to ∼20 min at 25 °C for the wild type, thus enhancing stability 10⁶-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.

Zeitweise getrennt: Ein Verfahren wurde entwickelt, das die homogene Biokatalyse in organisch-wässrigen Mischungen mit der durch CO₂ ausgelösten Trennung kombiniert. Mit dieser Methode lässt sich simultan das Produkt gewinnen und der homogene Biokatalysator für einen erneuten Einsatz zurückgewinnen.

Tamed OATS: A scheme that integrates homogeneous biocatalysis in organic–aqueous mixtures with CO₂-induced separation has been developed. This method allows for simultaneous product recovery and recycling of the homogeneous biocatalyst for reuse.

We have successfully applied the sequence comparison-based approach to develop a peroxidase (gene AhpC) and a water-forming NADH oxidase from Lactococcus lactis (L. lactis). We found a considerably lower maximum specific activity of nox-1 (AhpF) (15 U/mg) from L. lactis compared to its nox-2 counterpart (95 U/mg). Both nox-1 and nox-2 are turnover-limited, as expected for enzymes with labile, redox-active thiols in the active site. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2: the total turnover number TTN is 82,000 for nox-1 and nox-1/peroxidase vs. 39,000 for nox-2. Addition of exogenous thiols, however, increases nox-2 stability by a factor of two, up to the level of nox-1. Kinetic and stability analysis does not reveal any clear advantage for oxygen scavenging via the nox-1 or the nox-2 routes in lactic acid bacteria. Expression levels in lactic acid bacteria upon exposure to oxidative stress rather than kinetic performance more likely account for the previously observed superiority of nox-2 effectiveness over nox-1.