Effects of arginine and leucine substitutions on anti-endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α-amylase.

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice α-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI-1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50 ) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 µm, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 µm), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti-inflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved anti-endotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Preparation and gel properties of low molecular weight curdlan by hydrolysis of curdlan with commercial α-amylase.

Low molecular weight curdlan (LMWC) was prepared by hydrolysis of curdlan with commercial α-amylase. The hydrolysis reaction was conducted using 31.94 mg α-amylase per 500 mL reaction mixture, which contained 5 g curdlan. The hydrolysis was performed at pH 5.98 and 55.92 °C for 10 min. The molecular weight and structure of LMWC were characterized by high-performance liquid chromatography and Fourier transform infrared spectroscopy, respectively. Generally, LMWC showed lower gel strength than high molecular weight curdlan (HMWC). Unlike HMWC, LMWC could form into a gel at 50 °C. By contrast, HMWC could form into a gel at pH 11, but LMWC gel failed to form at this pH level. The strength of LMWC and HMWC gels increased with increasing temperature and decreased with increasing pH level.

Development of enteric submicron particles formulation of α-amylase for oral delivery.

Enteric submicron particles (SPs) formulations of α-amylase were prepared by w/o/w emulsion solvent evaporation using hydroxypropyl methylcellulose phthalate (HPMCP) and Eudragit L 100, to avoid gastric inactivation of α-amylase. Smaller internal and external aqueous phase volume provided maximum encapsulation efficiency (71.92-73.40%), least particle size (546.4-595.4 nm) and 23-26% loss of enzyme activity. Release studies in 0.1 N HCl confirmed the gastro-resistance of formulations. The anionic SPs aggregated in 0.1 N HCl (i.e. gastric pH 1.2), due to protonation of carboxylic groups of enteric polymer. The aggregates being < 500 µm size would not impede gastric emptying. However, at pH >5.0 (duodenal pH), SPs showed de-aggregation due to restoration of surface charge. HPMCP and Eudragit L 100 SPs facilitated almost complete release of α-amylase within 30 min at pH 6.0 and 6.8, respectively, following Higuchi kinetics. PXRD and DSC indicated amorphous character and scanning electron microscope showed spherical shape of SPs. In simulated gastro-intestinal pH condition, HPMCP and Eudragit L 100 SPs showed good digestion of cooked rice and could serve as potential carrier for oral enzyme delivery. Stability studies indicated the formulations as quite stable to ensure 2 years shelf life at room temperature.

A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions.

We developed a new protein crystallization method has been developed using a simplified counter-diffusion method for optimizing crystallization condition. It is composed of only a single capillary, the gel in the silicon tube and the screw-top test tube, which are readily available in the laboratory. The one capillary can continuously scan a wide range of crystallization conditions (combination of the concentrations of the precipitant and the protein) unless crystallization occurs, which means that it corresponds to many drops in the vapor-diffusion method. The amount of the precipitant and the protein solutions can be much less than in conventional methods. In this study, lysozyme and alpha-amylase were used as model proteins for demonstrating the efficiency of this method. In addition, one-dimensional (1-D) simulations of the crystal growth were performed based on the 1-D diffusion model. The optimized conditions can be applied to the initial crystallization conditions for both other counter-diffusion methods with the Granada Crystallization Box (GCB) and for the vapor-diffusion method after some modification.

Caracterizacao e sintese dos inibidores de 'alfa'-amilase do feijao (Phaseolus vulgaris) / Characterization and synthesis of 'alfa'-amylase inhibitors of beans (Phaseolus vulgaris)

Inibidores de alfa amilase de feijao (Phaseolus vulgaris) foram caracterizados. O inibidor da variedade jalo apresentou peso molecular de 50kDa (por filtracao em gel), ponto isoeletrico de 4,75 e 9,6 porcento de carboidratos. O inibidor da variedade argentino apresentou peso molecular de 48kDa, ponto isoeletrico de 4,90 e 7,6 porcento de carboidratos. Ambos inibidores apresentaram pH otimo de interacao com alfa amilase pancreatica de porco de 5,0 e, a pH 6,9, a complexacao com a mesma enzima se deu na proporcao de 1:1. Esses resultados, mais a composicao de aminoacidos, indicaram que as caracteristicas desses inibidores sao semelhantes as de outros ja purificados de diferentes variedades de feijao