Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis.

The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C-45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.

[Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp.]

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-ß-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

Switchable enzyme mimics based on self-assembled peptides for polyethylene terephthalate degradation.

Polyethylene terephthalate (PET), the most abundant polyester plastic, has become a global concern due to its refractoriness and accumulation in the environment. In this study, inspired by the structure and catalytic mechanism of the native enzyme, peptides, based on supramolecular self-assembly, were developed to construct enzyme mimics for PET degradation, which were achieved by combining the enzymatic active sites of serine, histidine and aspartate with the self-assembling polypeptide MAX. The two designed peptides with differences in hydrophobic residues at two positions exhibited a conformational transition from random coil to ß-sheet by changing the pH and temperature, and the catalytic activity followed the self-assembly "switch" with the fibrils formed ß-sheet, which could catalyze PET efficiently. Although the two peptides possessed same catalytic site, they showed different catalytic activities. Analysis of the structure - activity relationship of the enzyme mimics suggested that the high catalytic activity of the enzyme mimics for PET could be attributed to the formation of stable fibers of peptides and ordered arrangement of molecular conformation; in addition, hydrogen bonding and hydrophobic interactions, as the major forces, promoted effects of enzyme mimics on PET degradation. Enzyme mimics with PET-hydrolytic activity are a promising material for degrading PET and reducing environmental pollution.