David Lopez-Tejedor

Three novel glycosidases produced from Lactobacillus plantarum, so called Lp_0440, Lp_2777, and Lp_3525, were isolated and overexpressed on Escherichia coli containing a His-tag for specific purification. Their specific activity was evaluated against the hydrolysis of p-nitrophenylglycosides and p-nitrophenyl-6-phosphate glycosides (glucose and galactose) at pH 7. All three were modified with hyaluronic acid (HA) following two strategies: A simple coating by direct incubation at alkaline pH or direct chemical modification at pH 6.8 through preactivation of HA with carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at pH 4.8. The modifications exhibited important effect on enzyme activity and specificity against different glycopyranosides in the three cases. Physical modification showed a radical decrease in specific activity on all glycosidases, without any significant change in enzyme specificity toward monosaccharide (glucose or galactose) or glycoside (C-6 position free or phosphorylated). However, the surface covalent modification of the enzymes showed very interesting results. The glycosidase Lp_0440 showed low glycoside specificity at 25 °C, showing the same activity against p-nitrophenyl-glucopyranoside (pNP-Glu) or p-nitrophenyl-6-phosphate glucopyranoside (pNP-6P-Glu). However, the conjugated cHA-Lp_0440 showed a clear increase in the specificity towards the pNP-Glu and no activity against pNP-6P-Glu. The other two glycosidases (Lp_2777 and Lp_3525) showed high specificity towards pNP-6P-glycosides, especially to the glucose derivative. The HA covalent modification of Lp_3525 (cHA-Lp_3525) generated an enzyme completely specific against the pNP-6P-Glu (phosphoglycosidase) maintaining more than 80% of the activity after chemical modification. When the temperature was increased, an alteration of selectivity was observed. Lp_0440 and cHA-Lp_0440 only showed activity against p-nitrophenyl-galactopyranoside (pNP-Gal) at 40 °C, higher than at 25 °C in the case of the conjugated enzyme.

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.

A new kind of bionanohybrids, synthesized by using 63 kDa‐lipase from Candida rugosa (CRL) in the presence of polyethyleneimine (PEI) by in situ formation of Pd (0) nanoparticles (PdNPs) in aqueous media at room temperature were obtained. Addition of Triton X‐100 as additive was also used. XRD confirmed the formation of crystalline Pd (0) as metal species. TEM analyses revealed PdNPs ranging from 6 to 8 nm, whereas using Triton X‐100 the size range of the nanoparticles was 9 to 24 nm (mainly being 13 nm). PdNPs of biohybrid created only with Triton X‐100 ranged from 8 to 21 nm. The different heterogeneous nanobiohybrids were applied as catalyst in the oxidation of benzyl alcohol under mild conditions. In all cases, they were selective to the synthesis of alcohol to benzaldehyde without traces of carboxylic acid. More than >99 % conversion was obtained in THF at 50 °C under air as oxidant. The addition of hydrogen peroxide improved the conversion initially but the reaction was stopped at around 50–68 % conversion after 24 h in the best of cases. These new nanocatalysts synthesized using PEI showed a very high recyclability, maintaining >95 % activity after 5 cycles of use. Also successful results were obtained in the oxidation of other aromatic primary and secondary alcohols. The new PEI‐nanobiohybrids conserved the enzymatic activity and showed excellent results in metallic‐enzymatic domino reaction.

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.

A novel superparamagnetic ultrathin FeCO₃ nanorods–enzyme bionanohybrid heterogeneous catalyst has been developed.

This review is focused on the recent advances in the design of iron nanostructures and their catalytic applications.

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.