Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy.

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t 1/2) inactivation at 65 °C, 18 °C increase in apparent T m value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

Stable ultrathin partially oxidized copper film electrode for highly efficient flexible solar cells.

Advances in flexible optoelectronic devices have led to an increasing need for developing highly efficient, low-cost, flexible transparent conducting electrodes. Copper-based electrodes have been unattainable due to the relatively low optical transmission and poor oxidation resistance of copper. Here, we report the synthesis of a completely continuous, smooth copper ultra-thin film via limited copper oxidation with a trace amount of oxygen. The weakly oxidized copper thin film sandwiched between zinc oxide films exhibits good optoelectrical performance (an average transmittance of 83% over the visible spectral range of 400-800 nm and a sheet resistance of 9 Ω sq(-1)) and strong oxidation resistance. These values surpass those previously reported for copper-based electrodes; further, the record power conversion efficiency of 7.5% makes it clear that the use of an oxidized copper-based transparent electrode on a polymer substrate can provide an effective solution for the fabrication of flexible organic solar cells.