Intercomparison of reflectances observed by GOME and SCIAMACHY in the visible wavelength range

We compare the Earth reflectances of the spectrometers Global Ozone Monitoring Experiment (GOME) and Scanning Imaging Absorption Spectrometer for Atmospheric Chartography (SCIAMACHY) over their overlapping wavelength range (240-800 nm). The goal is to investigate the quality of the radiometric calibration of SCIAMACHY using calibrated GOME data as a reference. However, severe degradation of the GOME instrument in the UV since 2001 prevents it from being a reliable reference below 500 nm. Above 500 nm, GOME is reliable and we find substantial disagreement between GOME and SCIAMACHY, of the order of 15%-20%, which we can attribute completely to the current calibration problems of SCIAMACHY. These numbers are supported by a previous study in which SCIAMACHY was compared with the imager Medium Resolution Imaging Spectrometer (MERIS) onboard the Envisat satellite.     

Using Mutability Landscapes To Guide Enzyme Thermostabilization

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature T_m (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in T_m value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes.     

In Situ Acetaldehyde Synthesis for Carboligation Reactions

The enzyme 4-oxalocrotonate tautomerase (4-OT) can promiscuously catalyze various carboligation reactions using acetaldehyde as a nucleophile. However, the highly reactive nature of acetaldehyde requires intricate handling, which can impede its usage in practical synthesis. Therefore, we investigated three enzymatic routes to synthesize acetaldehyde in situ in one-pot cascade reactions with 4-OT. Two routes afforded practical acetaldehyde concentrations, using an environmental pollutant, trans-3-chloroacrylic acid, or a bio-renewable, ethanol, as starting substrate. These routes can be combined with 4-OT catalyzed Michael-type additions and aldol condensations in one pot. This modular systems biocatalysis methodology provides a stepping stone towards the development of larger artificial metabolic networks for the practical synthesis of important chemical synthons.     

The in vivo antibody response in rat gut-associated lymphoid tissue (GALT) after immunization with bacterial polysaccharide antigen

There are major clinical observations in alcohol and other drug addicts and neurochemical studies in animals and humans that support the hypothesis for a common neurochemical basis for alcohol and other drug addiction. The common occurrence of concurrent alcohol and multiple drug dependence in clinical and general populations, family history and genetic studies, and basic and clinical research in the neurochemistry of addictive behavior provide evidence for a common genealogical vulnerability to combined alcohol and other drug addiction. Clinical neurochemical models for addictive behaviors can be derived from neurochemical pathways for the initiation and sustenance of addictive disorders. The role of tolerance and dependence is not specific to addiction but indicates a homeostatic response of the brain to the presence of a foreign substance. Animal and human studies are analyzed for clinical synthesis of a neurochemical basis for addictive disorders.     

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non-aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4-oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael-type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its “Michaelase” activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The “hotspot” position Ala33 was then randomized in a highly enantioselective, but ethanol-sensitive 4-OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ-nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability-landscape-guided enzyme engineering for efficient biocatalysis in non-aqueous solvents.     

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.     

The Generation and Exploitation of Protein Mutability Landscapes for Enzyme Engineering

The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large‐scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence–function relationships. As such, mutability landscapes are a powerful tool with which to identify functional hotspots at any place in the amino acid sequence of an enzyme. These hotspots can be used as targets for combinatorial mutagenesis to yield superior enzymes with improved catalytic properties, stability, or even new enzymatic activities. The generation of mutability landscapes for multiple properties of one enzyme provides the exciting opportunity to select mutations that are beneficial either for one or for several of these properties. This review presents an overview of the recent advances in the construction of mutability landscapes and discusses their importance for enzyme engineering.