In Vitro Antiviral Activity of Tyrosinase from Mushroom Agaricus bisporus against Hepatitis C Virus.

Tyrosinases from a commercial Agaricus bisporus protein extract and directly isolated from white mushrooms were purified in order to obtaining the well-known tyrosinase from A. bisporus (TyrAB) of 45 kDa and a newly discovered 50 kDa tyrosinase isoform (Tyr50 kDa), and tested showing high antiviral activity against the hepatitis C virus for the first time. Cell toxicity and antiviral activity of tyrosinases were determined in cultured Huh 5-2 liver tumor cells transfected with a replicon system (a plasmid that includes all non-structural hepatitis C virus proteins and replicates autonomously). TyrAB was able to inhibit the replication of the hepatitis C virus without inducing toxicity in liver cells. In addition, the post-translational isoform Tyr50 kDa showed higher antiviral capacity than the former (up to 10 times greater), also exhibiting 10 times higher activity than the commercial drug Ribavirin®. This antiviral activity was directly proportional to the enzymatic activity of tyrosinases, as no antiviral capacity was observed in the inactive form of the enzymes. The tyrosinases approach could represent a new antiviral inhibition mechanism, through a plausible catalytic mechanism of selective hydroxylation of the key role of tyrosine residues in viral proteases.

The enzyme-induced formation of iron hybrid nanostructures with different morphologies.

A new synthesis method for tailor-made iron-hybrid nanoparticles has been carried out for the first time using enzymes, which directly induce the formation of inorganic iron species. The role of the protein was critical for the formation and morphology of the iron nanostructures and, depending on the enzyme, by simple mixing with ammonium iron(ii) sulfate at room temperature and under air, it was possible to obtain, for the first time, well stabilized superparamagnetic iron and iron oxide nanorods, nanosheets and nanorings or even completely amorphous non-magnetic iron structures in the protein network. These iron nanostructure-enzyme hybrids showed excellent results as heterogeneous catalysts in organic chemistry (chemoselective hydrogenation and C-C bonding formation) and environmental remediation processes.

Ultra-Small Pd(0) Nanoparticles into a Designed Semisynthetic Lipase: An Efficient and Recyclable Heterogeneous Biohybrid Catalyst for the Heck Reaction under Mild Conditions.

A novel heterogeneous enzyme-palladium (Pd) (0) nanoparticles (PdNPs) bionanohybrid has been synthesized by an efficient, green, and straightforward methodology. A designed Geobacillus thermocatenulatus lipase (GTL) variant genetically and then chemically modified by the introduction of a tailor-made cysteine-containing complementary peptide- was used as the stabilizing and reducing agent for the in situ formation of ultra-small PdNPs nanoparticles embedded on the protein structure. This bionanohybrid was an excellent catalyst in the synthesis of trans-ethyl cinnamate by Heck reaction at 65 °C. It showed the best catalytic performance in dimethylformamide (DMF) containing 10⁻25% of water as a solvent but was also able to catalyze the reaction in pure DMF or with a higher amount of water as co-solvent. The recyclability and stability were excellent, maintaining more than 90% of catalytic activity after five cycles of use.

Ultra-Fast Degradation of p-Aminophenol by a Nanostructured Iron Catalyst.

Full degradation of p-aminophenol in aqueous solution at room temperature by using a heterogeneous nanostructured iron hybrid catalyst in the presence of hydrogen peroxide is described. A nanostructured iron catalyst was prepared by in situ formation of iron carbonate nanorods on the protein network using an aqueous solution of an enzyme, lipase B from Candida antarctica (CAL-B). A second kind of iron nanostructured catalyst was obtained by the sunsequent treatment of the hybrid with an aqueous liquid extract of Mentha x piperita. Remarkable differences were observed using TEM imaging. When M. piperita extract was used, nanoparticles appeared instead of nanorods. Catalytic activity of these iron nanocatalysts was studied in the degradation of the environmental pollutant p-aminophenol (pAP) under different operating parameters, such as pH, presence of buffer or hydrogen peroxide concentration. Optimal conditions were pH 4 in acetate buffer 10 mM containing 1% (v/v) H2O2 for FeCO3NRs@CALB, while for FeCO3NRs@CALB-Mentha, water containing 1% (v/v) H2O2, resulted the best. A complete degradation of 100 ppm of pAP was achieved in 2 and 3 min respectively using 1 g Fe/L. This novel nanocatalyst was recycled five times maintaining full catalytic performance.

Synthesis of a superparamagnetic ultrathin FeCO3 nanorods-enzyme bionanohybrid as a novel heterogeneous catalyst.

Herein we report a straightforward synthesis of an ultrathin protein-iron(ii) carbonate nanorods (FeCO3-NRs) heterogeneous bionanohybrid at room temperature and in aqueous media. The enzyme induced the in situ formation of well-dispersed FeCO3 NRs on a protein network. The addition of NaBH4 as a reducing agent allowed us to obtain nanorods (5 × 40 nm) with superparamagnetic properties. This bionanohybrid showed excellent catalytic results in reduction, oxidation and C-C bond reactions.

Efficient purification of a highly active H-subunit of tyrosinase from Agaricus bisporus.

A highly-active tyrosinase (H subunit) isoform has been purified from a commercial crude extract of Agaricus bisporus by a specific, two step-hydrophobic chromatography cascade process based on the differential adsorption of the proteins from the extract to hydrophobic-functionalized supports. At first, commercial, crude tyrosinase from Agaricus bisporus (AbTyr) dissolved in aqueous media was added to octadecyl-Sepabeads matrix at 25 °C. Under these conditions, the support specifically adsorbed a protein with a molecular weight of 47 kDa which showed no tyrosinase activity. The known H subunit of tyrosinase from Agaricus bisporus (45 kDa, H-AbTyr) and another protein of 50 kDa were present in the supernatant. Sodium phosphate buffer was added to adjust the ionic strength of the solution up to 100 mM and Triton X-100 was added (final concentration of 0.07% v/v) to control the hydrophobicity effect for both proteins. This solution was offered again to fresh octadecyl-Sepabeads support, immobilizing selectively the H-AbTyr and leaving exclusively the 50 kDa protein as a pure sample in the supernatant. This tyrosinase isoform of 50 kDa was almost 4-fold more active than the known H-TyrAb, with a specific tyrosinase activity of more than 38,000 U/mg.

Effect of Site-Specific Peptide-Tag Labeling on the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus.

Tailor-made peptides were investigated for site-specific tag labeling of Geobacillus thermocatenulatus lipase (GTL). GTL was first genetically modified by introducing a unique cysteine on the lid site of the enzyme to produce two variants (GTLσ-A193C and GTLσ-S196C). Chemical modification was performed by using a small library of cysteine-containing peptides. The synthesized peptide-lipase biocatalysts were highly stable, more active, more specific, and more selective toward different substrates than unmodified GTL. Very high enzyme thermostability of GTLσ-A193C modified with peptides Ac-Cys-Phe-Gly-Phe-Gly-Phe-CONH2 (1) and Ac-Cys-Phe-Phe-CONH2 (2) (>95 % activity after 24 h at 60 °C) was observed. The incorporation of 1 and 2 in GTLσ-S196C improved its catalytic activity in the hydrolysis of p-nitrophenyl butyrate by factors of three and greater than five, respectively. The specificity for short-chain versus long-chain esters was also strongly improved. The diacylglycerol activity of GTLσ-S196C was enhanced more than tenfold by the incorporation of 1 and more than threefold by modification of this variant with Ac-Cys-(Arg)7 -CONH2 (6) in the hydrolysis of 1-stearoyl-2-arachidonoyl-sn-glycerol. The enantioselectivity of GTLσ-S196C increased for all formed bioconjugates, and the GTLσ-S196C-1 conjugate was the most active and selective in the hydrolysis of dimethylphenyl glutarate at pH 7 (72 % ee), also showing an inversion in the enzyme enantiopreference.