Hyperactivation of lipases by immobilization on superhydrophobic graphene quantum dots inorganic hybrid nanoflower.

Various nanoflowers are synthesized for enzyme immobilization. In order to increase the activity of nanoflowers, in this study, 3D flower-like structured organic-inorganic hybrid nanoflowers (hNFs) with various lipases Rhizomucor miehei lipase (RML), Candida antarctica lipase B (CALB), Humicola insolens lipase (HIL), Thermomyces lanuginosus lipase (TLL), Eversa® Transform 2.0 (ET) a genetically modified enzyme derived of TLL and graphene quantum dots (GQDs) were prepared and characterized.Lipase hNFs [lipase-(Cu/Co)3(PO4)2] and lipase@GQDs hNFs [lipase@GQDs-(Cu/Co)3(PO4)2] were straightforwardly prepared through mixing with metal ion (Cu2+or Co2+) aqueous solutions with or without GQDs. The ET@GQDs-(Cu)3(PO4)2 hNFs demonstrated 687 % higher activity than ET-(Cu)3(PO4)2 hNFs and 650 % higher activity than the free ET. Similar results were also observed with other lipase hybrid nanoflowers. For example, TLL@GQDs-(Cu)3(PO4)2 hNFs exhibited a 557 % higher activity than TLL-(Cu)3(PO4)2 hNFs and a 463 % higher activity than free TLL. Additionally, TLL@GQDs-(Co)3(PO4)2 hNFs showed a 141 % higher activity than TLL-(Co)3(PO4)2 hNFs and a 304 % higher activity than free TLL. Upon examining pH and thermal stability, it was revealed that lipase@GQDs hNFs exhibited higher activity compared to free lipase and other hNFs without GQDs. The effect of metal ions, enzyme concentrations and amount of GQDs on the morphology and enzyme activity of the lipase-hNFs was examined.

Peptiligase, an enzyme for efficient chemo-enzymatic synthesis of aviptadil.

Increasing attention to peptides as prospective therapeutics has created a renaissance in searching for new alternatives to the current peptide synthetic approaches as well as their modification. In this context, it is necessary to develop different approaches for peptide ligation. Using enzymes as a novel strategy and powerful tool for the peptide and protein ligation has recently received a lot of attention. We here designed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the synthesis of aviptadil a 28-mer therapeutic peptide with potential therapeutic effects in various medical contexts specially in the treatment of acute respiratory distress syndrome (ARDS) by coupling two peptide segments with four different peptiligase variants in aqueous environments. Our study reveals that peptiligase variants are capable of ligation reaction in 15 min. The overall time of ligation is shorter than those peptides with similar lengths and hinderance to aviptadil which reported for conventional synthesis by full solid-phase peptide synthesis. Yields ranged from 54 % to 76 %.

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Immobilization of Bacillus amyloliquefaciens protease "Neutrase" as hybrid enzyme inorganic nanoflower particles: A new biocatalyst for aldol-type and multicomponent reactions.

Organic-inorganic hybrid nanoflowers (hNFs) with commercial protease "Neutrase" is proposed and characterized as efficient and green biocatalysts for promiscuous catalysis in aldol-type and multicomponent reactions. Neutrase hNFs [Neutrase-(Cu/Ca/Co/Mn)3(PO4)2] are straightforwardly prepared through mixing metal ion (Cu2+, Ca2+, Co2+ or Mn2+) aqueous solutions with Neutrase in phosphate buffer (pH 7.4, 10 mM) resulting in precipitation (3 days). The hNFs were characterized by various techniques including scanning electron microscopy (SEM), energy dispersive X-ray (EDX), element mapping, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). In SEM images, the metal-Neutrase complexes revealed flower-like or granular structures after hybridization. The effect of metal ions and enzyme concentrations on the morphology and enzyme activity of the Neutrase-hNFs was examined. The synthesized Neutrase-Mn hNFs showed superior activity and stability compared to free Neutrase. Traditional organic CC coupling reactions such as aldol condensation, decarboxylative aldol, Knoevenagel, Hantzsch-type reactions and synthesis of 4H-pyran derivatives were used to test the generality and scope of Neutrase promiscuity, while optimizing conditions for the Neutrase-Mn hNF biocatalyst. Briefly, Neutrase-Mn3(PO4)2 hNFs showed excellent enzyme activity, stability and reusability, qualifying as effective reusable catalysts for coupling reactions under mild conditions.

Antiulcer properties of Glycyrrhiza glabra L. extract on experimental models of gastric ulcer in mice.

Glycyrrhiza glabra L. is used in folk medicine for treatment of stomach disorders including peptic ulcers. The hydroalcoholic extract of Glycyrrhiza glabra L. (HEGG) was evaluated for antiulcerogenic activity and acute toxicity profile in mice. Various doses of HEGG (50-200 mg/kg) were administered orally to animals of different groups. Omeprazole and cimetidine at doses of 30 and 100 mg/kg were used as positive controls, respectively. Stomach was opened along the greater curvature then ulceration index was determined examining the inner lining of stomach. Oral administration of the extract at 1600 mg/kg did not produce toxic symptoms and mortality in mice. 2950 mg/kg was determined as the oral LD50. The HEGG (50-200 mg/kg) showed a significant reduction in ulcer index in HCl/Ethanol-induced ulcer. G. glabra extract (50-150 mg/kg) showed antiulcer activity against indomethacin-induced gastric lesions dose dependently. The extract effectively inhibited formation of gastric lesions induced by ethanol. The extract (200 mg/kg) was more potent than omeprazole (30 mg/kg). HEGG reduced the ulcer index in hypothermic stress induced gastric ulcers in mice and the antiulcer effect was comparable to that of cimetidine. The results indicated that G. glabra hydroalcoholic extract exerted an antiulcergenic effect that could be associated with increase in gastric mucosal defensive factors.