Enzyme-Mimic Peptide Assembly To Achieve Amidolytic Activity.

Amyloid fibers are classified as a new generation of tunable bionanomaterials that exhibit new functions related to their distinctive characteristics, such as their universality, tunability, and stiffness. Here, we introduce the catalytic residues of serine protease into a peptide catalyst (PC) via an enzyme-mimic approach. The rational design of a repeating pattern of polar and nonpolar amino acids favors the conversion of the peptides into amyloid-like fibrils via self-assembly. Distinct fibrous morphologies have been observed at different pH values and temperatures, which indicates that different fibril packing schemes can be designed; hence, fibrillar peptides can be used to generate efficient artificial catalysts for amidolytic activities at mild pH values. The results of atomic force microscopy, Raman spectroscopy, and wide-angle X-ray scattering analyses are used to discuss and compare the fibril structure of a fibrillar PC with its amidolytic activity. The pH of the fibrillation reaction crucially affects the pKa of the side chains of the catalytic triads and is important for stable fibril formation. Temperature is another important parameter that controls the self-assembly of peptides into highly stacked and laminated morphologies. The morphology and stability of fibrils are crucial and represent important factors for demonstrating the capability of the peptides to exert amidolytic activity. The observed amidolytic activity of PC4, one of the PCs, was validated using an inhibition assay, which revealed that PC4 can perform enzyme-like amidolytic catalysis. These results provide insights into the potential use of designed peptides in the generation of efficient artificial enzymes.

Optimization of poly(N-isopropylacrylamide) as an artificial amidase.

Poly(N-isopropylacrylamide) microgel (NMG) has been developed by adding various functional groups to control surface charges, hydrophobicity, pKa and protein adsorption capacity. Here, we developed and optimized NMG anchored with three types of functional groups as a polymeric catalyst to hydrolyze amide bonds under optimized mild conditions. Various optimization strategies were evaluated for efficient hydrolysis activity on a p-nitroaniline-based substrate by using a colorimetric assay. Based on the results, we propose a mechanism to hydrolyze amide bonds and determine the theoretical average distance, using NMG bearing functional group of 1-vinylimidazole as the study model. The hydrolysis of amide bonds was inhibited by a transition-state protease inhibitor, which also confirmed the proposed reaction model for NMG. These results provide an insight into the strategies developed to functionalize hydrogels through an enzyme-mimic approach for future robust bio- and chemical conversions as well as therapeutic utilities.

Biosynthesis and characterization of polyhydroxyalkanoate containing high 3-hydroxyhexanoate monomer fraction from crude palm kernel oil by recombinant Cupriavidus necator.

The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has been investigated. Various types and concentrations of plant oils were evaluated for efficient conversion of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5 g/L was found to be most suitable for production of copolymer with a 3HHx content of approximately 70 mol%. The time profile of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA production and PHA synthase activity. (1)H NMR and (13)C NMR analyses confirmed the presence of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and flexible mechanical properties.