Catalysts on Demand: Selective Oxidations by Laboratory-Evolved Cytochrome P450 BM3

Efficient catalysts for selective oxidation of C-H bonds using atmospheric oxygen are highly desirable to decrease the economic and environmental costs associated with conventional oxidation processes. We have used methods of directed evolution to generate variants of bacterial cytochrome P450 BM3 that catalyze hydroxylation and epoxidation of a wide range of nonnative substrates. This fatty acid hydroxylase was converted to a propane monooxygenase (PMO) capable of hydroxylating propane at rates comparable to that of BM3 on its natural substrates. Variants along the PMO evolutionary lineage showed broadened substrate scope; these became the starting points for evolution of a wide array of enzymes that can hydroxylate and derivatize organic scaffolds. This work demonstrates how a single member of enzyme family is readily converted by evolution into a whole family of catalysts for organic synthesis

Enzymatic functionalization of carbon-hydrogen bonds

The development of new catalytic methods to functionalize carbon–hydrogen (C–H) bonds continues to progress at a rapid pace due to the significant economic and environmental benefits of these transformations over traditional synthetic methods. In nature, enzymes catalyze regio- and stereoselective C–H bond functionalization using transformations ranging from hydroxylation to hydroalkylation under ambient reaction conditions. The efficiency of these enzymes relative to analogous chemical processes has led to their increased use as biocatalysts in preparative and industrial applications. Furthermore, unlike small molecule catalysts, enzymes can be systematically optimized via directed evolution for a particular application and can be expressed in vivo to augment the biosynthetic capability of living organisms. While a variety of technical challenges must still be overcome for practical application of many enzymes for C–H bond functionalization, continued research on natural enzymes and on novel artificial metalloenzymes will lead to improved synthetic processes for efficient synthesis of complex molecules. In this critical review, we discuss the most prevalent mechanistic strategies used by enzymes to functionalize non-acidic C–H bonds, the application and evolution of these enzymes for chemical synthesis, and a number of potential biosynthetic capabilities uniquely enabled by these powerful catalysts (110 references)

Measurement of temperature profiles in hot gases and flames

Computer program was written for calculation of molecular radiative transfer from hot gases. Shape of temperature profile was approximated in terms of simple geometric forms so profile could be characterized in terms of few parameters. Parameters were adjusted in calculations using appropriate radiative-transfer expression until best fit was obtained with observed spectra

Selective decay by Casimir dissipation in fluids

The problem of parameterizing the interactions of larger scales and smaller scales in fluid flows is addressed by considering a property of two-dimensional incompressible turbulence. The property we consider is selective decay, in which a Casimir of the ideal formulation (enstrophy in 2D flows, helicity in 3D flows) decays in time, while the energy stays essentially constant. This paper introduces a mechanism that produces selective decay by enforcing Casimir dissipation in fluid dynamics. This mechanism turns out to be related in certain cases to the numerical method of anticipated vorticity discussed in \cite{SaBa1981,SaBa1985}. Several examples are given and a general theory of selective decay is developed that uses the Lie-Poisson structure of the ideal theory. A scale-selection operator allows the resulting modifications of the fluid motion equations to be interpreted in several examples as parameterizing the nonlinear, dynamical interactions between disparate scales. The type of modified fluid equation systems derived here may be useful in modelling turbulent geophysical flows where it is computationally prohibitive to rely on the slower, indirect effects of a realistic viscosity, such as in large-scale, coherent, oceanic flows interacting with much smaller eddies

Spin-Correlation Coefficients and Phase-Shift Analysis for p+3^3He Elastic Scattering

Angular Distributions for the target spin-dependent observables A$_{0y}$, A$_{xx}$, and A$_{yy}$ have been measured using polarized proton beams at several energies between 2 and 6 MeV and a spin-exchange optical pumping polarized $^3$He target. These measurements have been included in a global phase-shift analysis following that of George and Knutson, who reported two best-fit phase-shift solutions to the previous global p+$^3$He elastic scattering database below 12 MeV. These new measurements, along with measurements of cross-section and beam-analyzing power made over a similar energy range by Fisher \textit{et al.}, allowed a single, unique solution to be obtained. The new measurements and phase-shifts are compared with theoretical calculations using realistic nucleon-nucleon potential models.Comment: Submitted to Phys. Rev.

Fourier transform infrared spectrometer for a single aerosol particle

A spectrometer is reported here for obtaining the infrared spectrum of a single aqueous aerosol particle by a Fourier transform technique. The particle is held in an electrodynamic balance and irradiated simultaneously by the infrared output from a Michelson interferometer and the visible light from a dye laser. The size of the particle is modulated by chopping the IR beam, and the resulting visible scattered light fluctuation is detected at 90° with a photomultiplier tube. The amplitude of the scattered light fluctuation is measured with a lock-in amplifier at each interferometer mirror position. The electronic circuitry for stepping the interferometer mirror is presented and discussed. Inverting the lock-in signal by a discrete fast Fourier transform routine (FFT) yields the particle absorption spectrum. The resulting spectrum for an (NH4)2SO4 droplet is presented

Measurement of temperature profiles in hot gases by emission-absorption spectroscopy Final report

Measurement of spectral radiances and absorptances in hot gase

Directed evolution of Vibrio fischeri LuxR for improved response to butanoyl-homoserine lactone

LuxR is the 3-oxohexanoyl-homoserine lactone (3OC6HSL) dependent transcriptional activator of the prototypical acyl-homoserine lactone (AHL) quorum sensing system of Vibrio fischeri. Wild-type LuxR exhibits no response to butanoyl-HSL (C4HSL) in quantitative bioassays at concentrations of up to 1 µM; a previously described LuxR variant (LuxR-G2E) exhibits a broadened response to diverse AHLs, including pentanoyl-HSL (C5HSL), but not to C4HSL. Here, two rounds of directed evolution of LuxR-G2E generated variants of LuxR that responded to C4HSL at concentrations as low as 10 nM. One variant, LuxR-G4E, had only one change, I45F, relative to the parent LuxR-G2E, which itself differs from wild-type at three residues. Dissection of the four mutations within LuxR-G4E demonstrated that at least three of these changes were simultaneously required to achieve any measurable C4HSL response. The four changes improved both sensitivity and specificity towards C4HSL relative to any of the other 14 possible combinations of those residues. These data confirm that LuxR is evolutionarily pliable and suggest that LuxR is not intrinsically asymmetric in its response to quorum sensing signals with different acyl-side chain lengths

Spaces of finite element differential forms

We discuss the construction of finite element spaces of differential forms which satisfy the crucial assumptions of the finite element exterior calculus, namely that they can be assembled into subcomplexes of the de Rham complex which admit commuting projections. We present two families of spaces in the case of simplicial meshes, and two other families in the case of cubical meshes. We make use of the exterior calculus and the Koszul complex to define and understand the spaces. These tools allow us to treat a wide variety of situations, which are often treated separately, in a unified fashion.Comment: To appear in: Analysis and Numerics of Partial Differential Equations, U. Gianazza, F. Brezzi, P. Colli Franzone, and G. Gilardi, eds., Springer 2013. v2: a few minor typos corrected. v3: a few more typo correction