Multi-substituted styrenyl-trehalose prepared during the monomer synthesis acted as an efficient cross-linker to form a hydrogel after redoxinitiation with APS/TEMED , eliminating the need for additional cross-linkers or chromatographic purification of the monomer. After formation, the crude hydrogel was extensively washed via Soxhlet extraction in order to remove any unreacted trehalose and other residual non-polymerized components in the precipitation mixture, producing a colorless gel. Finally, the gel was lyophilized to produce a white powder. Originally, this method only produced hydrogel with an overall low yield of 17 %,which is too low to be relevant for use. Thus, we systematically optimized for the hydrogel synthesis to increase scale and sustainability. To increase the yield, we decreased the equivalents of trehalose from 5 to 3.33, effectively increasing the ratio of 4-vinylbenzyl chloride to trehalose. LC/MS of the dried solids confirmed that the product contained a mixture of mono- and di-substituted styrenyl-trehalose with various regioisomers at 94.1 % and 5.9 %, respectively . Higher order components such as tri-substituted trehalose were not observed by LC/MS either because they too dilute to detect or they were not ionizable. We then degassed the monomer/cross-linker and initiator solutions prior to gelation. This gave an overall reaction yield of 88 %. The increase in 4-vinylbenzyl chloride allowed for an increase in modified trehalose in the crude trehalose monomer/cross-linker mixture , which in turn resulted in a higher yield. When this improved synthesis was scaled 100-fold, it was carefully monitored with regard to reaction conversion, solvent removal, and drying to ensure that trehalose was sufficiently modified for gelation. With these necessary modifications, such as slow precipitation, the multi-gram reaction gave an overall yield of 75.6 %. As the goal for reaction yields in industry is above 70 %, this yield is industrially-promising.The final product demonstrated a storage modulus that was larger than the loss modulus as determined by a rheology frequency sweep with constant strain,maceta de plastico cuadrada indicating that the material passed its gel point and demonstrated solid-like behavior.
The swelling ratio was calculated as 15.63 ± 0.71 indicates that the material absorbs water many times its dry weight, as is characteristic of hydrogels.Though both a high yield and scalable synthesis were demonstrated, there are other factors that are desirable for industrial use, such as minimal use of hazardous materials, especially halogenated and toxic solvents.The precipitation step to isolate the crude reaction mixture was originally undertaken in hexanes, a neurotoxin,and DCM, a carcinogen.To replace these solvents, we tried to eliminate the precipitation step. This attempt was irreproducible and therefore not explored further. We next endeavored to replace hexanes and DCM with sustainable solvents of similar polarities. Based on waste, environmental, health, and safety issues, GlaxoSmithKline has produced a guide which scores solvents on sustainability.The solvents are assigned colors, red, amber, or green, where red is reserved for problematic solvents, such as hexanes and DCM, green is for the more sustainable solvents, and amber is for solvents in between the two extremes. Heptane and toluene are amber solvents with similar polarities as hexanes and were therefore envisioned as substitutes for hexanes. Acetone, an amber solvent, butanone, a green solvent, and ethyl acetate, a green solvent, with higher polarity, were explored as substitutes for DCM. Solvent ratios were varied to ensure miscibility of DMSO. Acetone, ethyl acetate, and butanone were too polar to sufficiently precipitate modified trehalose. Yet, when combined with heptane or toluene, gels formed after the precipitation step . The overall yields of these gels were lower because of the employed washing method. Material is lost when decanting and replacing solvent versus Soxhlet extraction; this washing method was necessary due to the small scale of the reactions.Precipitating into green/amber solvents gave similar yields , with the exception of butanone/heptane . The most sustainable precipitation that gave the highest yield, ethyl acetate/toluene , was scaled 100-fold and then used for gelation to give a 64.1 % yield. This yield can likely be optimized further with industrial equipment and shows potential as a greener alternative to the original synthesis.
It is important to note that although 4-vinylbenzyl chloride exhibits acute toxicity, we previously demonstrated that once reacted to form styrene-modified linear trehalose polymers, the material is cytocompatible and similar styrenyl polymers are nontoxic in vivo. 23With an optimized synthesis, the gels were tested for their efficacy in protecting animal feed enzymes during the conditions encountered in the feed pelleting process. Steam pelleting is a common practice as it improves feed efficiency by decreasing nutrient degradation.Frequently, temperatures reach 60 °C to 90 °C and most enzyme activity is lost at such temperatures. A method to stabilize these enzymes during pelleting that offers a dry, easy-to-handle final form of feed with active enzyme is essential to the livestock industry. We decided to investigate phytase, xylanase, and b-glucanase because 60 % of the global feed enzyme market is attributed to phytase, while 80 % of the global carbohydrase market is xylanase and b-glucanase.Phytase hydrolyzes phytate, which is the storage form of phosphorus in the feed, releasing digestible phosphates and chelated minerals.Xylanase degrades the main component of hemicellulose present in plant cell walls, b- 1,4-D-xylan, into xylose.b-Glucanase cleaves 1,4-b-D-glycosidic linkages in b–glucan, allowing the release of smaller saccharides from cellulose.For xylanase, the activity beyond 100 % retention invoked by the addition of trehalose hydrogel to phytase and xylanase may be attributed to the gel scaffold or trehalose moieties stabilizing or enhancing the protein/substrate binding and/or stabilizing the protein to the assay conditions that includes a lyophilization step. The former has been observed for glucose oxidase where the addition of trehalose augmented the affinity of the substrate for the enzyme,36 and for enzymes horseradish peroxidase and b-galactosidase upon interaction with trehalose polymer excipients.We investigated the activity of xylanase after incubation with hydrogels, without heat. Indeed, xylanase activity was greater than 100 % by the addition of even 1 weight equivalents of trehalose hydrogel .
Given that trehalose hydrogel alone has negligible signal from the enzyme assays, these results suggest that the trehalose hydrogel could be stabilizing the enzyme even without the addition of heat. The heated xylanase data was normalized to the results in Figure 2.7, and trehalose hydrogel still demonstrated substantial stabilization of the enzyme . We then considered the activity of xylanase when incubated with free trehalose without heat. We observed greater than 100 % original activity, albeit with no statistical difference compared to xylanase activity without additive . It follows that the gel scaffold or multi-valent effect of polymerized trehalose, beyond that of free trehalose, could be responsible for the improved activity of xylanase when incubated with hydrogel. We investigated the increase in activity of xylanase further by testing the aggregation state of xylanase subjected to heated assay conditions with or without hydrogel additives and comparing the results to a control subjected to all the assay conditions except for the heat step. An additional sample of fresh xylanase was prepared. At 1 mg/mL xylanase concentration in 0.1 M sodium acetate, all conditions were evaluated by dynamic light scattering . The results demonstrated small hydrodynamic sizes for fresh xylanase as well as xylanase subjected to heat with hydrogel additive. However, large increases in hydrodynamic size , indicative of aggregate formation, were observed for free xylanase subjected to assay conditions with or without heat. Thus,maceteros reciclados de plastico free xylanase must be sensitive to assay conditions, which includes a lyophilization step, and hydrogel addition stabilizes xylanase to these conditions, thereby likely explaining the greater activity. Trehalose polymers stabilize proteins to both lyophilization and heat by suppressing water crystallization, preventing aggregation and helping to refold proteins,and likely the hydrogels act by similar mechanisms. The release of enzymes within the gel was tested. Phytase was chosen as the model enzyme as it has the highest molecular weight compared to xylanase and b-glucanase and should therefore have the slowest diffusion rate of the three enzymes. The experiment was conducted at pH 6.3 using simulated intestinal fluid,37 and 37 °C to mimic the conditions found within the small intestine of pigs.FITC-labeled phytase demonstrated 100 % release from the trehalose hydrogels within 4 hours . It has been estimated that transit of feed through the small intestines of pigs is approximately 3 – 4 hours.Thus, the release is already within the relevant time frame and will likely be expedited with increased agitation in the gastrointestinal tract. We also demonstrated that the addition of trehalase enzyme had no effect on the release of phytase from the trehalose hydrogels . Trehalase is an enzyme found in the intestines and kidneys of many organisms, including humans and pigs,that catalyzes the conversion of trehalose into two glucose units. Therefore, we hypothesized that trehalase could potentially breakdown trehalose within the hydrogel, catalyzing its degradation and release of sequestered cargo. However, previous studies demonstrate that disaccharide mimics are completely buried within the trehalase enzyme, requiring significant conformational changes for substrate entry into the enzyme’s active site.Thus, it is likely that the interaction between trehalose linked within the hydrogel and trehalase’s active site is entropically unfavorable or sterically forbidden, preventing degradation. Note that the increase in trehalase concentration from 3.5 U/mL to 1000 U/mL in this experiment still had no effect on phytase release .Lastly, we explored whether trehalose polymers could similarly stabilize whole cells.
A linear trehalose glycopolymer with the same backbone as the trehalose hydrogel was evaluated for its ability to stabilize bacteria to lyophilization, which is often used to preserve and store biological samples, but is also known to negatively affect protein and cell viability .The trehalose glycopolymer was added as an excipient to a strain of BL21 E. Coli bacteria under lyophilization stress. Cell proliferation was monitored by measuring the OD600 and was used to determine E. coli survival and, therefore, stabilization. Samples lyophilized in the presence of P3 had lower OD600 measurements compared to with trehalose at an equivalent concentration , but higher OD600 measurements compared to the control . While trehalose has been shown to stabilize cells in a the dry state by maintaining the structural and functional integrity of their membranes through vitrification and bacteria synthesize extracellular polysaccharides in preparation for dehydration,cytosolic trehalose has been shown to be more effective for cell stabilization than extracellular trehalose added as an excipient.As such, it may be necessary to internalize trehalose, as the free disaccharide or the glycopolymer, within the cell to see more significant stabilization. All reagents and solvents were purchased from SigmaAldrich or Fisher Scientific and used without further purification unless otherwise noted. Trehalose was purchased from The Endowment for Medical Research and was azeotropically dried with ethanol and kept under vacuum until use. Phytase was provided by Phytex, LLC. Xylanase and trehalase werepurchased from Megazyme. b-glucanase was purchased from Sigma-Aldrich. EnzChek Ultra Xylanase Assay Kit was purchased from Fisher Scientific. b-Glucanase Assay Kit was purchased from Megazyme. Kanamycin-resistant strain of BL21 E. Coli bacteria was provided by the lab of Professor Robert Clubb. 1 H-NMR spectra were performed on an Avance DRX 400 MHz instrument. Liquid chromatography-mass spectrometry experiments were carried out on an Agilent 6350 QTOF ESI with a 1260 Infinity LC and Phenomenex Luna C18 5 µm column and were eluted with a gradient of 5 – 95% solvent B over 20 minutes. To determine hydrogel mechanical properties, an AR 2000ex rheometer in parallel plate geometry was used with an 8-mm diameter stainless steel, cross hatched upper plate and 60 mm stainless steel, cross hatched lower plate cover, at 22 °C, constant strain of 1%, and an angular frequency range of 0.1 to 10 rad s-1 . Gels were swelled for 72 hours in Milli Q water, trimmed to 8 mm diameter and approximately 1 mm thickness, and carefully blotted to removed excess water before measurements. Absorbance measurements to determine the degree of fluorescein isothiocyanate labeling of phytase and concentration of protein were conducted on a Thermo Fisher Scientific NanoDrop 2000 Spectrophotometer. Other absorbance and fluorescence measurements were taken on a Tecan Infinite M1000 plate reader. Dynamic light scattering measurements were conducted on a Malvern ZetaSizer Nano at 1 mg mL-1 protein concentration in 0.1M sodium acetate buffer.Under an inert atmosphere of argon gas, sodium hydroxide pellets were added followed by dimethylsulfoxide .