This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine…
This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine in various concentrations, and monitoring the output through UV-Vis spectroscopy. The reacted species was also analyzed via MALDI-TOF mass spectrometry. UV-Vis spectroscopic monitoring showed that in the presence of LAF-1 or tyrosine, the reaction between HRP and TMB occurred more quickly than the control, as well as in the highest concentration of LAF-1, the evolution of a peak at 482 nm. The analysis through MALDI-TOF spectrometry showed the development of a second peak likely due to the reaction between LAF-1 and TMB, as the Δ between the peaks is 229 Da and the size of the TMB species is 240 Da.
Phenolic polymers such as polyphenols and polyphenylenes are generated industrially for several applications but are typically associated with harsh reaction conditions and environmentally hazardous chemicals, such as formaldehyde. Additionally, hydroxycinnamic acids, such as p-coumaric acid (CA), are found in high concentrations in underutilized lignin-derived hydrolysates and represent a renewable and…
Phenolic polymers such as polyphenols and polyphenylenes are generated industrially for several applications but are typically associated with harsh reaction conditions and environmentally hazardous chemicals, such as formaldehyde. Additionally, hydroxycinnamic acids, such as p-coumaric acid (CA), are found in high concentrations in underutilized lignin-derived hydrolysates and represent a renewable and sustainable feedstock for the production of various aromatics and phenolics. To that end, recently a strain of Corynebacterium glutamicum has been developed by the Joint Bioenergy Institute to express a Phenolic Acid Decarboxylase (PAD), which can convert CA into 4-vinylphenol (4VP). 4VP is cytotoxic but can be polymerized by ligninolytic enzymes such as laccases or peroxidases into less-toxic poly(4-vinylphenol) (PVP). This work investigates the potential of polymerizing 4VP in situ by adding ligninolytic enzymes into the fermentation media to polymerize 4VP into PVP as it is produced, while reducing cellular toxicity to aid in chemical conversion.
The engineered C. glutamicum strain was cultured in the presence of CA to produce 4VP, with a maximum yield of 80.75%. Simultaneously, two ligninolytic enzymes, laccase and horseradish peroxidase (HRP), were explored in an in vitro experiment for their ability to polymerize 4VP, with laccase achieving full polymerization within 45 minutes and HRP able to polymerize 54.06% of 4VP in 24 hours. The resulting polymers were further analyzed by using gas permeation chromatography - nuclear magnetic resonance, validating the synthesis of PVP from 4VP with the addition of laccase or HRP. Finally, the C. glutamicum strain was evaluated for its ability to grow in the presence of hydrogen peroxide, which is a necessary reagent for HRP functionality, and it was able to reach an optical density of 3.69 within 36 hours. These findings suggest that in situ polymerization may be possible. Further work is underway to explore the enzyme kinetics at different pH, validate the potential of polymerization in situ, and study the fermentative benefits associated with in situ polymerization. This will be followed by additional analytical studies to characterize the resulting PVP.
