Genipin and glutaraldehyde based laccase two-layers immobilization with improved properties: New biocatalysts with high potential for enzymatic removal of trace organic contaminants.

This research proposes the preparation of a two-layer laccase biocatalyst using genipin or/and glutaraldehyde as cross-linking agents. The multilayer biocatalysts were prepared using different combinations of genipin and glutaraldehyde in the individual preparation of the first and second laccase layers. First, chitosan was treated with genipin or glutaraldehyde, followed by the immobilization of the first laccase layer to form a single-layer biocatalyst. Then, the immobilized laccases were coated once again with genipin or glutaraldehyde, and a new laccase layer was immobilized onto the system, resulting in the final two-layer biocatalyst. Compared to the single-layer biocatalysts, catalytic activity increased 1.7- and 3.4-fold when glutaraldehyde coating was used to prepare the second laccase layer. However, adding a second layer did not always produce more active biocatalysts, since the two-layer biocatalysts prepared with genipin (GenLacGenLac and GluLacGenLac) presented a decrease in activity of 65% and 28%, respectively. However, these two-layer biocatalysts prepared with genipin maintained 100% of their initial activity after 5 cycles of ABTS oxidation. Nevertheless, the two-layer, genipin-coated biocatalyst resulted in a higher removal of trace organic contaminants, since it removed 100% of mefenamic acid and 66% of acetaminophen, compared with the glutaraldehyde-coated biocatalyst, which removed 20% of mefenamic acid, and 18% of acetaminophen.

Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative Gα12 Bias Still Produce Prolonged Cardiac Effects.

We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and ß-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

Impact of Pharmaceutical Compounds in the Bioremediation of Municipal Biosolids by the White-Rot-Fungi Trametes hirsuta.

The potential of microorganisms for the treatment of municipal biosolids is continuously growing. The present studies evaluated the potency of Trametes hirsuta for the reduction in biosolid mass, production of extracellular enzymes, and removal of pharmaceutical compounds (PhACs) in biosolid slurry in the presence and absence of spiked PhACs [5 non-steroidal anti-inflammatories (NSAIs) and 2 psychoactive compounds (PACs)]. Toxicity after 35 days of fungal treatment was also assessed. Results showed that the growth of T. hirsuta is limited above 25% and wholly inhibited above 50% of biosolids in the slurry. At 12% of biosolid concentration, biosolid mass was reduced by 90%, NSAIs were entirely removed, but PACs' removal was only ~20%. Increasing biosolid content to 25% did not markedly affect biosolid reduction but significantly enhanced the removal of PACs (>50%). Results also showed that both PhACs and biosolids induced the production of oxidative enzymes. In 12% biosolids in the slurry, the oxidative potential measured by the ABTS assay (OABTS) reached 5,000 mM of OABTS in the presence of PhACs, and 2,500 mM of OABTS without PhACs, as compared to 1,200 mM of OABTS in control culture. Finally, we report that white rot fungi (WRF) treatment significantly decreased the toxicity of the biosolids.

Enzyme polymer engineered structure strategy to enhance cross-linked enzyme aggregate stability: a step forward in laccase exploitation for cannabidiol removal from wastewater.

Despite all its advantages and potential, cross-linking enzyme aggregate (CLEA) technology is still not applied at an industrial scale for enzyme insolubilization for bioremediation purposes. In this study, the enzyme polymer engineered structure (EPES) method was used to enhance CLEA stability and reuse. A crude laccase from Trametes hirsuta was successfully insolubilized to form EPES-CLEAs. The polymeric network provided excellent stability (> 90%) to CLEAs after a 24-h incubation in a non-buffered municipal wastewater effluent (WW), and the biocatalysts were recycled using a centrifugation process. While CLEAs activity dropped to 17%, EPES-CLEAs showed a laccase activity retention of 67% after five cycles of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) oxidation. After 8 h of treatment in WW, the EPES-CLEAs were equally as effective in removing cannabidiol (CBD) as the free-LAC (~ 37%). This research demonstrates that the EPES method is a promising alternative for CLEA stabilization and reuse in environmental conditions.

Constraining the Side Chain of C-Terminal Amino Acids in Apelin-13 Greatly Increases Affinity, Modulates Signaling, and Improves the Pharmacokinetic Profile.

Side-chain-constrained amino acids are useful tools to modulate the biological properties of peptides. In this study, we applied side-chain constraints to apelin-13 (Ape13) by substituting the Pro12 and Phe13 positions, affecting the binding affinity and signaling profile on the apelin receptor (APJ). The residues 1Nal, Trp, and Aia were found to be beneficial substitutions for Pro12, and the resulting analogues displayed high affinity for APJ (Ki 0.08-0.18 nM vs Ape13 Ki 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Dbzg; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, Ki 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Dbzg, Ki 0.08 nM) are the most potent Ape13 analogues activating the Gα12 pathways (53, EC50 Gα12 2.8 nM vs Ape13, EC50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro (t1/2 5.8-7.3 h in rat plasma) and in vivo.

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

Evaluation of the efficiency of Trametes hirsuta for the removal of multiple pharmaceutical compounds under low concentrations relevant to the environment.

An evaluation of the efficiency of the White-rot fungi (WRF) Trametes hirsuta to remove multi-classes pharmaceutical active compounds (17 PhACs) at low and environmentally realistic concentrations (20-500 ng L(-1)) was performed. The importance of biosorption over enzymatic activity on PhACs removal was also evaluated. Results highlight the importance to consider environmentally relevant PhACs concentrations while evaluating the removal capacities of WRF in wastewaters treatment processes, as PhACs concentration strongly influence both the enzymatic activity profile and the removal efficiency. Results also show that under tested experimental conditions, laccase was the only active extracellular lignin modifying enzyme and that biosorption and possibly intracellular enzymes also contribute to the removal of some PhACs.