Activation of Immobilized Lipase in Non-Aqueous Systems by Hydrophobic Poly-DL-Tryptophan Tethers

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule (“tether”) between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most “long” linkers (e.g. polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-μm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n ≈ 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against p-nitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed.Keywords: Immobilization, Enzyme-catalyzed organic synthesis, Candida antarctica lipase B, Hydrophobic tethers, Silica microspheres, Poly-DL-tryptopha

Hot gas and dust in a protostellar cluster near W3(OH

We used the IRAM Interferometer to obtain sub-arcsecond resolution observations of the high-mass star-forming region W3(OH) and its surroundings at a frequency of 220 GHz. With the improved angular resolution, we distinguish 3 peaks in the thermal dust continuum emission originating from the hot core region about 6 arcsec (0.06 pc) east of W3(OH). The dust emission peaks are coincident with known radio continuum sources, one of which is of non-thermal nature. The latter source is also at the center of expansion of a powerful bipolar outflow observed in water maser emission. We determine the hot core mass to be 15 solar masses based on the integrated dust continuum emission. Simultaneously many molecular lines are detected allowing the analysis of the temperature structure and the distribution of complex organic molecules in the hot core. From HNCO lines, spanning a wide range of excitation, two 200 K temperature peaks are found coincident with dust continuum emission peaks suggesting embedded heating sources within them.Comment: 12 pages, 3 figure

Forming an Early O-type Star Through Gas Accretion?

We present high angular resolution ($\sim$ 3$''$) and sensitive 1.3 mm continuum, cyanogen (CN) and vinyl cyanide (C$_2$H$_3$CN) line observations made with the Submillimeter Array (SMA) toward one of most highly obscured objects of the W51 IRS2 region, W51 North. We find that the CN line exhibits a pronounced inverse P-Cygni profile indicating that the molecular gas is infalling inwards this object with a mass accretion rate between 4 and 7 $\times$ 10$^{-2}$ M$_\odot$ yr$^{-1}$. The C$_2$H$_3$CN traces an east-west rotating molecular envelope that surrounds either a single obscured (proto)star with a kinematic mass of 40 M$_{\odot}$ or a small central cluster of B-type stars and that is associated with a compact high velocity bipolar outflow traced by H$_2$O masers and SiO molecular emission. We thus confirm that the W51 North region is part of the growing list of young massive star forming regions that have been associated with infalling motions and with large mass accretion rates ($\sim$ 10$^{-2}$ -- 10$^{-4}$), strengthening the evidence for massive stars forming with very high accretion rates sufficient to quench the formation of an UCHII region.Comment: Accepted by Astronomy and Astrophysics Letter

Concentration effects on peptide elution from pendant PEO layers

In earlier work, we have provided direction for development of responsive drug delivery systems based on modulation of structure and amphiphilicity of bioactive peptides entrapped within pendant polyethylene oxide (PEO) brush layers. Amphiphilicity promotes retention of the peptides within the hydrophobic inner region of the PEO brush layer. In this work, we describe the effects of peptide surface density on the conformational changes caused by peptide-peptide interactions, and show that this phenomenon substantially affects the rate and extent of peptide elution from PEO brush layers. Three cationic peptides were used in this study: the arginine-rich amphiphilic peptide WLBU2, the chemically identical but scrambled peptide S-WLBU2, and the non-amphiphilic homopolymer poly-L-arginine (PLR). Circular dichroism (CD) was used to evaluate surface density effects on the structure of these peptides at uncoated (hydrophobic) and PEO-coated silica nanoparticles. UV spectroscopy and a quartz crystal microbalance with dissipation monitoring (QCM-D) were used to quantify changes in the extent of peptide elution caused by those conformational changes. For amphiphilic peptides at sufficiently high surface density, peptide-peptide interactions result in conformational changes which compromise their resistance to elution. In contrast, elution of a non-amphiphilic peptide is substantially independent of its surface density, presumably due to the absence of peptide-peptide interactions. The results presented here provide a strategy to control the rate and extent of release of bioactive peptides from PEO layers, based on modulation of their amphiphilicity and surface density.Keywords: α-Helix, PEO brush, Cationic amphiphilic peptides, Polyarginine, WLBU2, Coiled-coils, Circular dichroism (CD), Peptide elutio

Adsorption, structural alteration and elution of peptides at pendant PEO layers

An experimentally based, quantitative understanding of the entrapment and function of small peptides within PEO brush layers does not currently exist. Earlier work provided a rationale for expecting that an ordered, compact peptide will enter the PEO phase more readily than a peptide of similar size that adopts a less ordered, less compact form, and that amphiphilicity will promote peptide retention within the hydrophobic region of the PEO brush. Here we more deliberately describe criteria for peptide integration and structural change within the PEO brush, and discuss the reversibility of peptide entrapment with changing solvent conditions. For this purpose, circular dichroism (CD) was used to record the adsorption and conformational changes of (amphiphilic) WLBU2 and (non-amphiphilic) polyarginine peptides at uncoated (hydrophobic) and PEO-coated silica nanoparticles. Peptide conformation was controlled between disordered and α-helical forms by varying the concentration of perchlorate ion. We show an initially more ordered (α-helical) structure promotes peptide adsorption into the PEO layer. Further, a partially helical peptide undergoes an increase in helicity after entry, likely due to concomitant loss of capacity for peptide-solvent hydrogen bonding. Peptide interaction with the PEO chains resulted in entrapment and conformational change that was irreversible to elution with changing solution conditions in the case of the amphiphilic peptide. In contrast, the adsorption and conformational change of the non-amphiphilic peptide was reversible. These results indicate that responsive drug delivery systems based on peptide-loaded PEO layers can be controlled by modulation of solution conditions and peptide amphiphilicity.Keywords: circular dichroism (CD), cationic amphiphilic peptides (CAPs), polyarginine, PEO brush, peptide integration, WLBU

A ring/disk/outflow system associated with W51 North: a very massive star in the making

Sensitive and high angular resolution ($\sim$ 0.4\arcsec) SO$_2$[22$_{2,20}$ $\to$ 22$_{1,21}$] and SiO[5$\to$4] line and 1.3 and 7 mm continuum observations made with the Submillimeter Array (SMA) and the Very Large Array (VLA) towards the young massive cluster W51 IRS2 are presented. We report the presence of a large (of about 3000 AU) and massive (40 M$_\odot$) dusty circumstellar disk and a hot gas molecular ring around a high-mass protostar or a compact small stellar system associated with W51 North. The simultaneous observations of the silicon monoxide molecule, an outflow gas tracer, further revealed a massive (200 M$_\odot$) and collimated ($\sim14^\circ$) outflow nearly perpendicular to the dusty and molecular structures suggesting thus the presence of a single very massive protostar with a bolometric luminosity of more than 10$^5$ L$_\odot$. A molecular hybrid LTE model of a Keplerian and infalling ring with an inner cavity and a central stellar mass of more than 60 M$_\odot$ agrees well with the SO$_2$[22$_{2,20}$ $\to$ 22$_{1,21}$] line observations. Finally, these results suggest that mechanisms, such as mergers of low- and intermediate- mass stars, might be not necessary for forming very massive stars.Comment: Accepted by The Astrophysical Journa

Functional polymers and proteins at interfaces

Functional polymers and proteins at interfaces and surfaces are of fundamental importance for biomaterials, biocatalysis and microreactor applications. Candida antarctica lipase B immobilized on poly-DL-tryptophan tethers showed 30x higher specific activity in aqueous and non-aqueous media than the same enzyme adsorbed or covalently bound directly to the surface. An excess of enzyme vs. tethers was observed, indicating multilayer binding. This was attributed to covalent binding of the enzyme to the surface, as well as to the ends of the tethers, and adsorption of the lipophilic enzyme to the hydrophobic polymers. Aspergillus oryzae ß-galactosidase was immobilized on a novel silicon oxide nanospring mat in a microreactor by covalent disulfide bonds. Nanosprings offer high accessible surface area with very low flow resistance and excellent mechanical properties, and can be patterned onto existing devices. Continuous reactions were performed at different flowrates and substrate concentrations. The enzyme activity was stable for over 26 days, and high steady-state conversions (X > 0.7) were achieved with average residence times of less than one minute. The catalyst was regenerated in situ by reduction of the disulfide bonds and subsequent immobilization of fresh enzyme. Numerical simulation and sensitivity analysis indicate that the characteristic high permeability of the nanosprings is critical to reactor performance. In another study, a method was developed to directly image the surface distribution of small polyethylene oxide-polybutadiene-polyethylene oxide (PEO-PBD-PEO) triblock copolymer surfactants using atomic force microscopy (AFM). Triblocks with cleavable ester bonds of various PEO:PBD ratios and molecular weights were synthesized. The triblocks were self-assembled onto hydrophobic silicon wafers, and the adsorbed vinyl-containing PBD blocks immobilized by γ-irradiation. Acid hydrolysis of the cleavable linker released the PEO side-chains. The remaining immobilized PBD was labeled with β-cyclodextrins, and imaged using standard AFM "tapping mode" techniques. Triblocks deposited from dilute (1 mg/mL) solutions formed very sparse, non-uniform layers. At 10 mg/mL, regular patterns consistent with deposition of worm-like or cylindrical micelles were observed. Long PEO chains produced sparse layers, presumably by facilitating desorption of the triblocks. These studies demonstrate novel methods for solid-supported enzymatic reactions and production and characterization of surface coatings, with diverse applications in bioengineering and biomaterials