Bioproduction of cis‐(1S,2R)‐indandiol,a chiral pharmaceutical intermediate,using a solid–liquid two‐phase partitioning bioreactor for enhanced removal of inhibitors

BACKGROUND: A solid‐liquid two‐phase partitioning bioreactor (TPPB) was used in the biotransformation of indene to cis‐(1S,2R)‐indandiol by Pseudomonas putida 421‐5 (ATCC 55687). Metered substrate feeding in single‐phase operation, or delivery from an immiscible liquid, have previously been employed to regulate the exposure of the biocatalyst to inhibitory concentrations of the substrate. In contrast, the solid‐liquid platform provided in situ substrate addition (ISSA) as well as simultaneous it in situ product removal (ISPR) as a means of overcoming substrate and product toxicity. Three different modes of operation were compared for their effects on the performance of this biotransformation: single‐phase, fed‐batch operation was carried out as a benchmark in 2.75 L aqueous medium, and subsequently with the inclusion of either 700 g liquid silicone oil or 700 g solid polymer beads. RESULTS: Biphasic modes achieved a 3‐fold productivity improvement with respect to single‐phase (30 to 90 mg L^?1 h^?1), and solid‐liquid productivity was similar to liquid‐liquid operation while achieving more extensive removal of inhibitory compounds resulting in a slightly higher product titer (1.29 vs 1.16 g L^?1). The operability of the reactor was improved by the phase stability of the solid polymer beads relative to immiscible organic solvents, preventing emulsion formation and facilitating analytics. CONCLUSION: Solid polymer beads replaced the immiscible liquid auxiliary phase for substrate delivery while performing simultaneous inhibitory molecule sequestration. Copyright © 2011 Society of Chemical Industry     

Spectroscopic and Theoretical Study on Electronically Modified Chromophores in LOV Domains: 8‐Bromo‐ and 8‐Trifluoromethyl‐Substituted Flavins

Two chemically synthesized flavin derivatives, 8‐trifluoromethyl‐ and 8‐bromoriboflavin (8‐CF₃RF and 8‐BrRF), were photochemically characterized in H₂O and studied spectroscopically after incorporation into the LOV domain of the blue light photoreceptor YtvA from Bacillus subtilis. The spectroscopic studies were paralleled by high‐level quantum chemical calculations. In solution, 8‐BrRF showed a remarkably high triplet quantum yield (0.97, parent compound riboflavin, RF: 0.6) and a small fluorescence quantum yield (0.07, RF: 0.27). For 8‐CF₃RF, the triplet yield was 0.12, and the fluorescence quantum yield was 0.7. The high triplet yield of 8‐BrRF is due to the bromine heavy atom effect causing a stronger spin–orbit coupling. Theoretical calculations reveal that the decreased triplet yield of 8‐CF₃RF is due to a smaller charge transfer and a less favorable energetic position of T₂, required for intersystem crossing from S₁ to T₁, as an effect of the electron‐withdrawing CF₃ group. The reconstitution of the LOV domain with the new flavins resulted in the typical LOV photochemistry, consisting of triplet state formation and covalent binding of the chromophore, followed by a thermal recovery of the parent state, albeit with different kinetics and photophysical properties.     

Impact of cosolvents (polyols) on globular protein functionality: Ultrasonic velocity, density, surface tension and solubility study

The objective of this study was to understand how cosolvents influence the molecular and functional properties of globular proteins in aqueous solutions. The ultrasonic velocity, density and adiabatic compressibility of cosolvent solutions (0–50 wt% sorbitol or glycerol) were measured in the absence and presence of a globular protein (1 wt% β-lactoglobulin) at 30 °C. These measurements were used to calculate the partial specific apparent volume and adiabatic compressibility of the protein. The protein's volume decreased and its compressibility increased in the presence of high cosolvent concentrations, which were attributed to changes in the properties of the protein interior and solvation layer. Sorbitol was more effective than glycerol at decreasing the protein volume at high cosolvent concentrations, which may be because glycerol has some surface activity and may therefore accumulate around hydrophobic regions on the protein surface. Our data were used to account for the observation that sorbitol is more effective than glycerol at increasing the thermal stability and self-association of the β-lactoglobulin. A better understanding of the influence of protein–cosolvent–solvent interactions on the functionality of globular proteins may facilitate the design of protein-based products.     

Predicting sales performance, job satisfaction, and depression by using the Achievement Strivings and Impatience-Irritability dimensions of Type A behavior.

Recent research suggested that two dimensions of Type A behavior, namely, Achievement Strivings (AS) and Impatience-Irritability (II), differentially predict physical health and performance outcomes. The present study extends this research and examines whether AS and II differentially predict work performance (number of insurance policies sold), work attitudes (job satisfaction), and depression in a sample of 117 life insurance salespersons. As hypothesized, after statistically controlling for relevant biographical variables and II, AS predicted the number of policies sold and job satisfaction but was not related to depression. After partialing out the effect of relevant biographical variables and AS, II was associated with depression but not with the number of policies sold. In addition, controlling for the same variables, II was negatively associated with job satisfaction. Implications for the prediction of sales performance, job satisfaction, and depression, and interventions designed to decrease the negative consequences of Type A by reducing II but not AS, are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Application of multi-component biopolymer layers to improve the freeze–thaw stability of oil-in-water emulsions: β-Lactoglobulin–ι-carrageenan–gelatin

This study examines the influence of interfacial composition on the freeze–thaw stability of oil-in-water emulsions. Three 5% w/w oil-in-water emulsions (5 mM phosphate buffer, pH 6.0) were prepared using the layer-by-layer electrostatic deposition method that had different interfacial compositions: (i) primary emulsion (β-Lg); secondary emulsion (β-Lg–ι-carrageenan); (iii) tertiary emulsion (β-Lg–ι-carrageenan–gelatin). The primary, secondary and tertiary emulsions were subjected to from one to three freeze–thaw cycles (−20 °C for 22 h, +40 °C for 2 h) in the absence or presence of sucrose (10% w/w), and then their stability was assessed by ζ-potential, particle size, microstructure and creaming stability measurements. In the absence of sucrose, the primary and secondary emulsions were highly unstable to droplet aggregation and creaming after three freeze–thaw cycles, whereas the tertiary emulsion was stable, which was attributed to the relatively thick biopolymer layer surrounding the oil droplets. In the presence of 10% w/w sucrose, all of the emulsions were much more stable, which can be attributed to the ability of sucrose to increase the amount of non-frozen aqueous phase in the emulsions. The interfacial engineering technology used in the study could therefore lead to the creation of food emulsions with improved stability to freezing and thawing.     

Comparison of blockage factors in modelling the resistance of channels containing submerged macrophytes

The proportion of a river channel containing vegetation (termed the blockage factor) has historically been determined in three ways: cross‐sectional, surface area and volumetric. The first two versions are two‐dimensional measures. Meanwhile, the three‐dimensional volumetric version is biased towards deeper sections of a reach. A fourth version of the blockage factor is proposed that does not have such limitations: the multi‐cross‐sectional blockage factor. Between five and nine cross‐sections were sampled to determine the four blockage‐factor versions for 35 river sites containing the clumped submergent macrophyte, Ranunculus subgenus Batrachium (water‐crowfoot). The ability of these four measures to act as predictors of vegetative channel resistance was then assessed. The vegetated proportion of individual cross‐sections was poorly related to the vegetation resistance of a channel reach, primarily due to the high spatial variability of patch‐forming macrophytes. The weighted median of all cross‐sectional blockage factors measured at each site produced the strongest relationship with vegetation resistance, though this was not significantly better than the volumetric or surface area versions. The resistance model using the surface‐area blockage factor gave a very high residual between predicted and calculated resistance for a mat‐forming macrophyte species, and this model is unlikely to hold for conditions other than baseflow. Likewise, the volumetric version is not expected to hold for sites that have more depth variability than those measured in this study. Copyright © 2005 John Wiley & Sons, Ltd.     

Modulation of thermal stability and heat-induced gelation of β-lactoglobulin by high glycerol and sorbitol levels

The influence of glycerol and sorbitol on the thermal stability and heat-induced gelation of β-lactoglobulin (β-lg) in aqueous solutions was investigated. The thermal stability of β-lg was characterized by measuring the thermal denaturation temperature (T_m) using differential scanning calorimetry, while its gelation properties were characterized by measuring the gelation temperature (T_gel) and final gel rigidity (G^∗) using dynamic shear rheology. All experiments were carried out using aqueous solutions containing 10% (w/w) β-lg, glycerol (0–70% w/w) or sorbitol (0–55% w/w), and 5 mM phosphate buffer (pH 7.0). No salt was added to these solutions so that there was a relatively strong electrostatic repulsion between the protein molecules, which usually prevents gelation. When the cosolvent concentration was increased from 0% to 50%, T_m increased from 74 to 86 °C for sorbitol, but only from 74 to 76 °C for glycerol, which indicated that sorbitol was much more effective at stabilizing the native state of the globular protein than glycerol. Protein solutions containing sorbitol (0–55%) did not form a gel after heating, but those containing glycerol formed gels when the cosolvent concentration exceeded about 10%, with G^∗ increasing with increasing glycerol concentration. We attribute these effects to differences in the preferential interactions of polyols and water with the surfaces of native and heat-denatured proteins, and their influence on the protein–protein collision frequency.     

Selection of catalytically active biotin ligase and trypsin mutants by phage display

Phage display has been shown to facilitate greatly the selectionof polypeptides with desired properties by establishing a directlink between the polypeptide and the gene that encodes it. However,selection for catalytic activities displayed on phage remainsa challenge, since reaction products diffuse away from the enzymeand make it difficult to recover catalytically active phage–enzymes.We have recently described a selection methodology in whichthe reaction substrate (and eventually the reaction product)is anchored on calmodulin-tagged phage–enzymes by meansof a calmodulin binding peptide. Phage displaying a catalyticactivity are physically isolated by means of affinity reagentsspecific for the product of reaction. In this study, we investigatedthe efficiency of selection for catalysis by phage display,using a ligase (the Escherichia coli biotin ligase BirA) andan endopeptidase (the rat trypsin His57