ß-Ketoadipic acid production from poly(ethylene terephthalate) waste via chemobiological upcycling.

The upcycling of poly(ethylene terephthalate) (PET) waste can simultaneously produce value-added chemicals and reduce the growing environmental impact of plastic waste. In this study, we designed a chemobiological system to convert terephthalic acid (TPA), an aromatic monomer of PET, to ß-ketoadipic acid (ßKA), a C6 keto-diacid that functions as a building block for nylon-6,6 analogs. Using microwave-assisted hydrolysis in a neutral aqueous system, PET was converted to TPA with Amberlyst-15, a conventional catalyst with high conversion efficiency and reusability. The bioconversion process of TPA into ßKA used a recombinant Escherichia coli ßKA expressing two conversion modules for TPA degradation (tphAabc and tphB) and ßKA synthesis (aroY, catABC, and pcaD). To improve bioconversion, the formation of acetic acid, a deleterious factor for TPA conversion in flask cultivation, was efficiently regulated by deleting the poxB gene along with operating the bioreactor to supply oxygen. By applying two-stage fermentation consisting of the growth phase in pH 7 followed by the production phase in pH 5.5, a total of 13.61 mM ßKA was successfully produced with 96% conversion efficiency. This efficient chemobiological PET upcycling system provides a promising approach for the circular economy to acquire various chemicals from PET waste.

Chemo-Biological Upcycling of Poly(ethylene terephthalate) to Multifunctional Coating Materials.

Chemo-biological upcycling of poly(ethylene terephthalate) (PET) developed in this study includes the following key steps: chemo-enzymatic PET depolymerization, biotransformation of terephthalic acid (TPA) into catechol, and its application as a coating agent. Monomeric units were first produced through PET glycolysis into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET), and PET oligomers, and enzymatic hydrolysis of these glycolyzed products using Bacillus subtilis esterase (Bs2Est). Bs2Est efficiently hydrolyzed glycolyzed products into TPA as a key enzyme for chemo-enzymatic depolymerization. Furthermore, catechol solution produced from TPA via a whole-cell biotransformation (Escherichia coli) could be directly used for functional coating on various substrates after simple cell removal from the culture medium without further purification and water-evaporation. This work demonstrates a proof-of-concept of a PET upcycling strategy via a combination of chemo-biological conversion of PET waste into multifunctional coating materials.

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala.

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

Efficient CO2-reducing activity of NAD-dependent formate dehydrogenase from Thiobacillus sp. KNK65MA for formate production from CO2 gas.

NAD-dependent formate dehydrogenase (FDH) from Candida boidinii (CbFDH) has been widely used in various CO2-reduction systems but its practical applications are often impeded due to low CO2-reducing activity. In this study, we demonstrated superior CO2-reducing properties of FDH from Thiobacillus sp. KNK65MA (TsFDH) for production of formate from CO2 gas. To discover more efficient CO2-reducing FDHs than a reference enzyme, i.e. CbFDH, five FDHs were selected with biochemical properties and then, their CO2-reducing activities were evaluated. All FDHs including CbFDH showed better CO2-reducing activities at acidic pHs than at neutral pHs and four FDHs were more active than CbFDH in the CO2 reduction reaction. In particular, the FDH from Thiobacillus sp. KNK65MA (TsFDH) exhibited the highest CO2-reducing activity and had a dramatic preference for the reduction reaction, i.e., a 84.2-fold higher ratio of CO2 reduction to formate oxidation in catalytic efficiency (kcat/KB) compared to CbFDH. Formate was produced from CO2 gas using TsFDH and CbFDH, and TsFDH showed a 5.8-fold higher formate production rate than CbFDH. A sequence and structural comparison showed that FDHs with relatively high CO2-reducing activities had elongated N- and C-terminal loops. The experimental results demonstrate that TsFDH can be an alternative to CbFDH as a biocatalyst in CO2 reduction systems.

Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus.

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

Biochemical and structural studies of conserved Maf proteins revealed nucleotide pyrophosphatases with a preference for modified nucleotides.

Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.

Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge.

Lipase B from Candida antarctica (CalB) is a versatile biocatalyst for various bioconversions. In this study, the thermostability of CalB was improved through the introduction of a new disulfide bridge. Analysis of the B-factors of residue pairs in CalB wild type (CalB-WT) followed by simple flexibility analysis of residues in CalB-WT and its designated mutants using FIRST server were newly proposed to enhance the selective power of two computational tools (MODIP and DbD v1.20) to predict the possible disulfide bonds in proteins for the enhancement of thermostability. Five residue pairs (A162-K308, N169-F304, Q156(-) L163, S50-A273, and S239C-D252C) were chosen and the respective amino acid residues were mutated to cysteine. In the results, CalB A162C-K308C showed greatly improved thermostability while maintaining its catalytic efficiency compared to that of CalB-WT. Remarkably, the temperature at which 50% of its activity remained after 60-min incubation (T6°50) of CalB A162C_K308C was increased by 8.5°C compared to that of CalB-WT (55 and 46.5°C, respectively). Additionally, the half-life at 50°C of CalB A162C-K308C was 4.5-fold higher than that of CalB-WT (220 and 49 min, respectively). The improvement of thermostability of CalB A162C-K308C was elucidated at the molecular level by molecular dynamics (MD) simulation.