Activity and enantioselectivity of wildtype and lid mutated Candida rugosa lipase isoform 1 in organic solvents

The activity and enantioselectivity of lipase 1 from Candida rugosa and of a chimera enzyme obtained by replacing the lid of isoform 1 with the lid of isoform 3 were compared in organic solvents. The alcoholysis of chloro ethyl 2-hydroxy hexanoate with methanol and of vinyl acetate with 6-methyl-5-hepten-2-ol were used as model reactions in different reaction conditions. The chimera enzyme was less active and enantioselective than the wildtype in all the conditions tested. A rationale for such decreases could be that the chimera lipase has a lower proportion of enzyme molecules in the open form. This might lead to a hindered access to the enzyme active site, thus affecting the catalytic activity.     

Mono- and disaccharides enhance the activity and enantioselectivity of Burkholderia cepacia lipase in organic solvent but do not significantly affect its conformation

Sucrose, trehalose, and mannitol were colyophilized with lipase from Burkholderia cepacia and their effects on the activity and enantioselectitivity of the enzyme evaluated using as model reactions the transesterification between n-octanol or 6-methyl-5-hepten-2-ol with vinyl acetate. The lipase co-lyophilized with sugars showed an activity which was up to 4.7-fold higher (at a sugar/lipase ratio >or= 20) than that observed without sugar. Analogously, lipase enantioselectivity, expressed as the enantiomeric ratio, increased up to 2.8-fold in the presence of sugars. The conformation of the lipase was investigated by means of Fourier transform infrared spectroscopy (FT/IR) in water and as lyophilized powder. The infrared spectra of lyophilized lipase in the presence and, even more so, in the absence of sugars were different from that of the enzyme in water. In particular, the band at around 1,654/cm, typically assigned to alpha-helix, was less intense in the lyophilized samples. Nevertheless, the enzyme in the presence of sugars showed a decrease of the bands at 1,614-1,620/cm and at 1,680-1,695/cm that indicates a lower content of intermolecular beta-sheets (typical of protein aggregates). Additionally the increase of the component at 1,546/cm in the amide II region is consistent with a hydrogen bond pattern of the enzyme more similar to that shown in water. These results suggest that although sugars are not able to fully preserve the native secondary structure, they might contribute to reduce the conformational changes caused by protein/protein interactions. These factors in combinations with others (e.g., ability to reduce deleterious interactions between the enzyme and inert supports) make sugars (both mono- and disaccharides) an interesting class of additives for improving the performance of biocatalysts in organic solvents.     

Correlated rates of synonymous site evolution across plant genomes

Synonymous substitution rates have been shown to vary among evolutionary lineages of both nuclear and organellar genes across a broad range of taxonomic groups. In animals, rate heterogeneity does not appear to be correlated across nuclear and mitochondrial genes. In this paper, we contrast substitution rates in two plant groups and show that grasses evolve more rapidly than palms at synonymous sites in a mitochondrial, a nuclear, and a plastid gene. Furthermore, we show that the relative rates of synonymous substitution between grasses and palms are similar at the three loci. The correlation in synonymous substitution rates across genes is particularly striking because the three genes evolve at very different absolute rates. In contrast, relative rates of nonsynonymous substitution are not conserved among the three genes.      

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

Cyclohexanone monooxygenase catalyzed oxidation of methyl phenyl sulfide and cyclohexanone with macromolecular NADP in a membrane reactor

Summary Methyl phenyl sulfide and cyclohexanone were oxidized to (R)- methyl phenyl sulfoxide and caprolactone by cyclohexanone monooxygenase. The reactions were carried out in a membrane reactor with the use of the macromolecular coenzyme poly (ethylene glycol)-NADP. Coenzyme regeneration was carried out with the 2-propanol/alcohol dehydrogenase system.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.     

Model based dynamics analysis in live cell microtubule images

Background

The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data.

Results

In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior.

Conclusion

Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior.