Jason A. Berberich

Skin injuries, particularly severe injuries, such as full-thickness wounds or surgical sites, are susceptible to bacterial infection. Materials composed of keratin proteins have been investigated for use in wound dressings, and at least two products based on these materials have received FDA clearance for treating radiation dermatitis via the 510(k) mechanism. We hypothesized that hydrogels composed of oxidatively extracted keratins, known as keratose, could achieve sustained release of several classes of antibiotic drugs that might improve the performance of keratins as wound dressing materials. Reverse-phase high performance liquid chromatography methods were developed to quantify release of ciprofloxacin, cefazolin, and neomycin antibiotics from keratose hydrogels. Nearly 100% of ciprofloxacin and cefazolin were released from the hydrogels within 7 d, while only approximately 20% of neomycin was released. In a combination keratose hydrogel containing neomycin and cefazolin, the amount of cefazolin release decreased (compared to hydrogels with only cefazolin) to about 60% over 7 d whereas neomycin release increased (compared to hydrogels with only neomycin) to nearly 30%. The antibiotics released from the hydrogels inhibited growth of gram-positive ( S. aureus ) and gram-negative ( P. aeruginosa ) bacteria in an in vitro broth inhibition assay. Keratose hydrogels containing a combination of the first-line antibiotics neomycin and cefazolin inhibited S. aureus for 18 d and P. aeruginosa for 6 d which was comparable to or better than hydrogels containing broad-spectrum ciprofloxacin that had effect for 11 d. This work suggests that modification of keratin biomaterials with antibiotics may enhance their utility by inhibiting bacterial infections.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) persist in aquatic environments due to their extremely strong C–F bonds and high environmental stability/poor biodegradability, creating an urgent demand for high-performance and scalable adsorption treatment technologies. This study reports the synthesis, characterization, and fixed-bed column adsorption performance of a dual-polymer-modified activated carbon adsorbent engineered for enhanced PFAS capture. Activated carbon (AC) was sequentially functionalized with polyethyleneimine (PEI) and poly(chlorotrifluoroethylene–co-vinylidene fluoride) (Kel–F) to form the fluorine-rich, amine-grafted composites AC–PEI–KelF and AC–PEI–KelF–PEI. Surface-area studies of the modified AC adsorbents showed a reduction in surface area of about a factor of two, possibly due to pore blockage by the polymers. Continuous-flow column breakthrough studies demonstrated dramatic improvements in perfluorooctanoic acid (PFOA) removal efficiency, with 50% maximum breakthrough time increasing from 80 min for AC to 540 min for AC–PEI–KelF and 1500 min for the AC–PEI–KelF–PEI formulation. Thomas model adsorption capacities for AC, AC–PEI–KelF and AC–PEI–KelF–PEI were, respectively, 65, 490, and 1130 mg/g. The adsorption mechanism, using selective mobile phases, was shown to be due to a combination of electrostatic and hydrophobic/fluorophilic interactions. Kinetic analysis showed that adsorption exhibited pseudo-second-order behavior, with multi-stage intraparticle diffusion. The optimized composite also exhibited strong regeneration stability, retaining 79% performance after six adsorption–desorption cycles, and displayed high selectivity for PFOA, even in the presence of structurally related competitors such as octanoic acid. The adsorption of perfluoropentanoic acid and undecafluoro-2-methyl-3-oxahexanoic acid (GenX) was also significant, and some interaction with trifluoroacetic acid was noted.

Enzymatic depolymerization of polyethylene terephthalate (PET) is a promising alternative to traditional recycling showing promise in addressing issues with high energy demands and challenges associated with mixed-waste streams. While engineered, thermostable PETase variants exist, achieving high yields under industrially relevant process conditions remains a significant hurdle. Poor thermostability and PET crystallinity are often blamed for low conversions, but these factors do not fully account for the low productivity observed in all cases. This suggests that other poorly understood phenomena may be limiting the catalytic efficiency of PETase warranting further investigation. This work systematically explores the impact of mixing, and in particular, the presence of the air–water and the solid–liquid interfaces, on PETase stability. We found that PET degradation plateaus while soluble PETase activity is lost at increasing mixing rates when an air–water interface is present. It is hypothesized that the enzyme adsorbs to the air–water interface leading to denaturation, aggregation and precipitation. Additional studies demonstrate that the hydrophobic PET film negatively impacts enzyme stability and the rate of enzyme inactivation is also proportional to the PET surface area. Traditional approaches to mitigate enzyme inactivation at interfaces, such as using blocking agents, were effective in preserving enzyme activity, but resulted in significantly reduced PET degradation rates. In contrast, PEGylation of PETase yielded improved stability and enhanced PET degradation during mixing. These findings help explain the significant conversion discrepancies reported in previous studies and demonstrate a robust strategy for improving PETase performance under industrially relevant conditions.

Background: Depolymerizing polyethylene terephthalate (PET) plastics using enzymes, such as PETase, offers a sustainable chemical recycling route. To enhance degradation, many groups have sought to engineer PETase for faster catalysis on PET and elevated stability. Considerably less effort has been focused toward expressing large quantities of the enzyme, which is necessary for large-scale application and widespread use. In this work, we evaluated several E. coli strains for their potential to produce soluble, folded, and active IsPETase, and moved the production to a benchtop bioreactor. As PETase is known to require disulfide bonds to be functional, we screened several disulfide-bond promoting strains of E. coli to produce IsPETase, FAST-PETase and Hot-PETase. Results: We found expression in SHuffle T7 Express results in higher active expression of IsPETase compared to standard E. coli production strains such as BL21(DE3), reaching a purified titer of 20 mg enzyme per L of culture from shake flasks using 2xLB medium. We characterized purified IsPETase on 4-nitrophenyl acetate and PET microplastics, showing the enzyme produced in the disulfide-bond promoting host has high activity. Using a complex medium with glycerol and a controlled bioreactor, IsPETase titer reached 104 mg per L for a 46-h culture. FAST-PETase was found to be produced at similar levels in BL21(DE3) or SHuffle T7 Express, with purified production reaching 65 mg per L culture when made in BL21(DE3). Hot-PETase titers were greatest in BL21(DE3) reaching 77 mg per L culture. Conclusions: We provide protein expression methods to produce three important PETase variants. Importantly, for IsPETase, changing expression host, medium optimization and movement to a bioreactor resulted in a 50-fold improvement in production amount with a per cell dry weight productivity of 0.45 mg_PETase g_CDW⁻¹ h⁻¹, which is tenfold greater than that for K. pastoris. We show that the benefit of using SHuffle T7 Express for expression only extends to IsPETase, with FAST-PETase and Hot-PETase better produced and purified from BL21(DE3), which is unexpected given the number of cysteines present. This work represents a systematic evaluation of protein expression and purification conditions for PETase variants to permit further study of these important enzymes.

Tissue-engineering and cell-based strategies provide an intriguing approach to treat complex conditions such as those of the endocrine system. We have previously developed a cell-based hormone therapy (cHT) to address hormonal insufficiency associated with the loss of ovarian function. To assess how the cHT strategy may achieve its efficacy, we developed a mathematical model to determine if known autocrine, paracrine, and endocrine effects of the native hypothalamus-pituitary-ovary (HPO) axis could explain our previously observed effects in ovariectomized rats following treatment with cHT. Our model suggests that cHT constructs participate in the complex machinery of the HPO axis. We were able to describe the in vivo behaviors of estrogen, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin, and androgen with good accuracy. A sensitivity analysis indicated that some parameters impact the broader HPO system more than others, but that most changes in model parameters led to proportional changes in the system. We also conducted a predictive analysis on the effect of cHT dose on HPO axis hormones and found that, with the exception of estrogen, the other HPO hormones analyzed reach a saturation level within the physically possible number of constructs.

Understanding the interactions between surfactants and proteins is important for the formulation of consumer products as surfactant binding can alter protein activity and stability. Additionally, the structure of the protein–surfactant complex can influence surface activity, which is important for emulsion and foam development. N,N-Dimethyldodecylamine N-oxide (DDAO) is an amphoteric surfactant that is nonionic at high pH. It is often used as a foam booster in detergent formulations and for the extraction of membrane proteins. In this study, a variety of biophysical characterization methods was used to investigate the impact of DDAO at pH 8 on the structure of the globular protein β-lactoglobulin (βLG). Pyrene fluorescence and surface tension studies show that βLG had minimal impact on the critical micelle concentration (CMC) of DDAO, while fluorescence and circular dichroism spectroscopy found unfolding of βLG at concentrations of DDAO greater than the CMC. Small-angle X-ray scattering results confirm changes in the structure of βLG at DDAO concentrations above the CMC. Taken together, DDAO behaves like nonionic and zwitterionic surfactants below its CMC with limited interaction with βLG, while it induces protein unfolding at concentrations higher than the CMC, resulting in a protein–surfactant complex structure that resembles a protein-decorated micelle.

A detailed mechanistic and kinetic study of enzymatically initiated RAFT polymerization is performed by combining enzymatic assays and polymerization kinetics analysis. Horseradish peroxidase (HRP) initiated RAFT polymerization of dimethylacrylamide (DMAm) was studied. This polymerization was controlled by 2-(propionic acid)ylethyl trithiocarbonate (PAETC) in the presence of H₂O₂ as a substrate and acetylacetone (ACAC) as a mediator. In general, well controlled polymers with narrow molecular weight distributions and good agreement between theoretical and measured molecular weights are consistently obtained by this method. Kinetic and enzymatic assay analyses show that HRP loading accelerates the reaction, with a critical concentration of ACAC needed to effectively generate polymerization initiating radicals. The PAETC RAFT agent is required to control the reaction, although the RAFT agent also has an inhibitory effect on enzymatic performance and polymerization. Interestingly, although H₂O₂ is the substrate for HRP there is an optimal concentration near 1 mM, under the conditions studies, with higher or lower concentrations leading to lower polymerization rates and poorer enzymatic activity. This is explained through a competition between the H₂O₂ acting as a substrate, but also an inhibitor of HRP at high concentrations.