Papain-catalyzed oligomerization of diethyl L-glutamate hydrochloride was conducted in phosphate buffer at 40 °C. Because of rapid oligomerization kinetics, high substrate concentrations were not needed to shift the equilibrium for oligomer synthesis. For example, at 0.03 M diethyl L-glutamate hydrochloride, oligo(γ-ethyl L-glutamate) synthesis and precipitation from solution occurred in 55% yield. MALDI-TOF spectra of precipitated products showed two series of ion peaks separated by 157 m/z units, the mass of oligo(γ-ethyl-L-glutamate) repeat units. The most abundant signals were at DP 8 and 9, in excellent agreement with DP avg values determined by 1 H NMR. Lower intensity peaks with m/z less by 28 correspond to hydrolysis of one ester group either at a chain end or a pendant group along chains. Oligo(γ-ethyl-L-glutamate) synthesis at 40 °C in phosphate buffer (0.9 M, pH 7) occurred rapidly so that by 5, 10, and 20 min the yield reached 70 ( 4%, 78 ( 4% and 81 ( 5%, respectively. High product yields were observed over a broad range of pH values. As long as the pH was maintained from 5.5 to 8.5, the product yield was g60%. Ionic strength had no significant effect on oligopeptide yield. The dominant role of phosphate buffer in reactions was its control of pH. Other influences of phosphate ions on papain, such as nonspecific salt interactions or a "salting out" of product, appear to be of little or no importance. Loss in protein concentration and activity in the supernatant was observed after one reaction. A second reaction cycle performed using recovered supernatants resulted in a decrease in oligo(γ-ethyl-L-glutamate) yield from about 75% to 20%.
Mapping the location of bound cellulase enzymes provides information on the micro-scale distribution of amenable and recalcitrant sites in pretreated woody biomass for biofuel applications. The interaction of a fluorescently labelled cellulase enzyme cocktail with steam-exploded pine (SEW) was quantified using confocal microscopy. The spatial distribution of Dylight labelled cellulase was quantified relative to lignin (autofluorescence) and cellulose (Congo red staining) by measuring their colocalisation using Pearson correlations. Correlations were greater in cellulose-rich secondary cell walls compared to lignin-rich middle lamella but with significant variations among individual biomass particles. The distribution of cellulose in the pretreated biomass accounted for 30% of the variation in the distribution of enzyme after correcting for the correlation between lignin and cellulose. For the first time, colocalisation analysis was able to quantify the spatial distribution of amenable and recalcitrant sites in relation to the histochemistry of cellulose and lignin. This study will contribute to understanding the role of pretreatment in enzymatic hydrolysis of recalcitrant softwood biomass.
Non-productive adsorption of cellulose degrading enzymes on lignin is a likely reason for reduced rate and extent of enzymatic conversion of lignocellulosic substrate to sugars. Additives such as polyethyleneglycol (PEG) may act as blocking agents in this non-productive interaction. However, the exact molecular level interactions of PEG with lignin in pre-treated lignocellulosic substrates are not known. We have used confocal fluorescence microscopy combined with Förster resonance energy transfer (FRET) to reveal molecular level interactions between lignin present in thermo-mechanically pre-treated Pinus radiata substrate, and fluorescently labeled PEG. It is demonstrated that PEG interaction with lignin is mainly associated with particles derived from secondary walls, with little or no penetration into fragments derived from the middle lamella. This nanoscale information on the PEG-substrate interaction will assist in rationalizing pre-treatment methods to reduce the recalcitrance of softwood biofuel substrates.
A simple
and environmentally friendly approach toward the thermoplastic
processing of rapidly degradable plastic-enzyme composites using three-dimensional
(3D) printing techniques is described. Polycaprolactone/Amano lipase
(PCL/AL) composite films (10 mm × 10 mm; height [h] = ∼400 μm) with an AL loading of 0.1, 1.0, and 5.0%
were prepared via 3D printing techniques that entail direct mixing
in the solid state and thermal layer-by-layer extrusion. It was found
that AL can tolerate in situ processing temperatures
up to 130 °C in the solid-state for 60 min without loss of enzymatic
activity. The composites were degraded in phosphate buffer (8 mg/mL,
composite to buffer) for 7 days at 37 °C and the resulting average
percent total weight loss (WLavg %) was found to
be 5.2, 92.9, and 100%, for the 0.1, 1.0, and 5.0% films, respectively.
The degradation rates of PCL/AL composites were found to be faster
than AL applied externally in the buffer. Thicker PCL/AL 1.0% films
(10 mm × 10 mm; h = ∼500 μm) were
also degraded over a 7 day period to examine how the weight loss occurs
over time with 3.0, 18.1, 36.4, 46.4, and 70.2% weight loss for days
1, 2, 3, 4, and 7, respectively. Differential scanning calorimetry
(DSC) analysis shows that the film’s percent crystallinity
(D
xtal%) increases over time with D
xtal% = 46.5 for day 0 and 53.1% for day 7.
Scanning electron microscopy (SEM) analysis found that film erosion
begins at the surface and that water can penetrate the interior via
surface pores activating the enzymes embedded in the film. Controlled
release experiments utilizing dye-loaded PCL/AL/dye (AL = 1.0%; dye
= 0.1%) composites were degraded over a 7 day period with the bulk
of the dye released by the fourth day. The PCL/AL multimaterial objects
containing AL-resistant polylactic acid (PLA) were also printed and
degraded to demonstrate the application of this material on more complex
structures.
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