The expected product yield of a biocatalyst during its useful lifetime is an important consideration when designing a continuous biocatalytic process. One important indicator of lifetime biocatalyst productivity is the dimensionless total turnover number (TTN). Here, a method is proposed for estimating the TTN of a given biocatalyst from readily-measured biochemical quantities, namely the specific activity and the deactivation half-life, measured under identical conditions. We demonstrate that this method may be applied to any enzyme whose thermal deactivation follows first-order kinetics, regardless of the number of unfolding intermediates, and that the TTN method circumvents the potential problems associated with measuring specific catalyst output when a portion of the enzyme is already unfolded. The TTN estimation was applied to several representative biocatalysts to demonstrate its applicability in identifying the most cost-effective catalyst from a pool of engineered mutants with similar activity and thermal stability.

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 thermal deactivation of TEM-1 β-lactamase was examined using two experimental techniques: a series of isothermal batch assays and a single, continuous, non-isothermal assay in an enzyme membrane reactor (EMR). The isothermal batch-mode technique was coupled with the three-state "Equilibrium Model" of enzyme deactivation, while the results of the EMR experiment were fitted to a four-state "molten globule model". The two methods both led to the conclusions that the thermal deactivation of TEM-1 β-lactamase does not follow the Lumry-Eyring model and that the T eq of the enzyme (the point at which active and inactive states are present in equal amounts due to thermodynamic equilibrium) is at least 10 °C from the T m (melting temperature), contrary to the idea that the true temperature optimum of a biocatalyst is necessarily close to the melting temperature.

Detecting gamma-ray emission from radionuclides hidden within containers is a significant concern to national security and can be accomplished with scintillating materials such as NaI:Tl, LaBr3:Ce crystals. However, the use of these high quality crystals limits the functionality of the detectors due to their high cost and scalability issues. Therefore the development of more durable, more easily manufactured, and more cost effective scintillating materials is desired. The incorporation of nanophosphors or Quantum Dots (QDs) into a polymer matrix to produce a transparent nanocomposite could potentially provide an alternative method to fabricate scintillating detectors. Embedded in a suitable polymer matrix, nanocomposite detectors may be easily made suitably large for portal monitors. Also, preparation of suitable particle sizes and/or compositions permits selection of a photon wavelength that optimally matches the photodetector response curve to increase the number of photons collected per pulse. In this paper a series of LaF3:Ce nanophosphors with varying doping concentrations (1–30mol%Ce) were synthesized using a chemical precipitation method. Photoluminescence and photoluminescence excitation characterizations indicated that the highest luminescent intensity was obtained from the 20%Ce doped sample with a peak emission at 325 nm. The refractive indices of the nanoparticles were identified by index matching measurements. Then an index matched epoxy was selected for incorporation of these nanoparticles to prepare transparent nanocomposite scintillators. In addition, colloidal solutions of CdTe QDs with various emitting colors were synthesized and incorporated into a Polymethyl-methacrylate (PMMA) matrix to make transparent nanocomposites. An initial evaluation of the scintillation behavior of these nanocomposites was evaluated by exposure to gamma rays.