Rational engineering S1' substrate binding pocket to enhance substrate specificity and catalytic activity of thermal-stable keratinase for efficient keratin degradation.

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.

Analysis of the Genomic Sequences and Metabolites of Bacillus velezensis YA215.

Discovering more novel antimicrobial compounds has become a keen research problem. In this study, YA215 genome was sequenced by the Illumina HiSeq + PacBio sequencing platform. Genome assembly was performed by Unicycler software and the gene clusters responsible for secondary metabolite biosynthesis were predicted by antiSMASH. The genome comprised 3976514 bp and had a 46.56% G + C content. 3809 coding DNA sequences, 27 rRNAs, 86 tRNAs genes, and 79 sRNA were predicted. Strain YA215 was re-identified as Bacillus velezensis based on ANI and OrthoANI analysis. In the COG database, 23 functional groups from 3090 annotations were predicted. In the GO database, 2654 annotations were predicted. 2486 KEGG annotations linked 41 metabolic pathways. Glycosyl transferases, polysaccharide lyases, auxiliary activities, glycoside hydrolases, carbohydrate esterases, and carbohydrate-binding modules were predicted among the 127 annotations in the CAZy database. AntiSMASH analysis predicted that B. velezensis YA215 boasted 13 gene clusters involved in synthesis of antimicrobial secondary metabolites including surfactin, fengycin, macrolactin H, bacillaene, difficidin, bacillibactin, bacilysin, and plantazolicin. Three of the gene clusters (gene cluster 5, gene cluster 9, and gene cluster 10) have the potential to synthesize unknown compounds. The research underscore the considerable potential of secondary metabolites, identified in the genomic composition of B. velezensis YA215, as versatile antibacterial agents with a broad spectrum of activity against pathogenic bacteria.

In vitro inhibitory effect of five natural sweeteners on α-glucosidase and α-amylase.

A promising and efficacious approach to manage diabetes is inhibiting α-glucosidase and α-amylase activity. Therefore, the inhibitory activities of five natural sweeteners (mogrosides (Mog), stevioside (Ste), glycyrrhizinic acid (GA), crude trilobatin (CT), and crude rubusoside (CR)) against α-glucosidase and α-amylase and their interactions were evaluated in vitro using enzyme kinetics, fluorescence spectroscopy, Fourier infrared spectroscopy, and molecular docking. The inhibitor sequence was CT > GA > Ste, as GA competitively inhibited α-glycosidase activity while CT and Ste exhibited mixed inhibitory effects. Compared to a positive control acarbose, the inhibitory activity of CT was higher. For α-amylase, the mixed inhibitors CT, CR, and Mog and the competitive inhibitor Ste effectively inhibited the enzyme, with the following order: CT > CR > Ste > Mog; nevertheless, the inhibitors were slightly inferior to acarbose. Three-dimensional fluorescence spectra depicted that GA, CT, and CR bound to the hydrophobic cavity of α-glucosidase or α-amylase and changed the polarity of the hydrophobic amino acid-based microenvironment and structure of the polypeptide chain backbone. Infrared spectroscopy revealed that GA, CT, and CR could disrupt the secondary structure of α-glucosidase or α-amylase, which decreased enzyme activity. GA, trilobatin and rubusoside bound to amino acid residues through hydrogen bonds and hydrophobic interactions, changing the conformation of enzyme molecules to decrease the enzymatic activity. Thus, CT, CR and GA exhibit promising inhibitory effects against α-glucosidase and α-amylase.

Effects of electric field and light on resistivity switching of Eu0.7Sr0.3MnO3 thin films.

Based on the excellent piezoelectric properties of 0.7Pb(Mg1/3Nb2/3)O3-0.3PbTiO3 (PMN-PT) single crystals, a hole-doped manganite film/PMN-PT heterostructure has been constructed to achieve electric-field and light co-control of physical properties. Here, we report the resistivity switching behavior of Eu0.7Sr0.3MnO3/PMN-PT(111) multiferroic heterostructures under different in-plane reading currents, temperatures, light stimuli and electric fields, and discuss the underlying coupling mechanisms of resistivity change. The transition from the electric-field induced lattice strain effect to polarization current effect can be controlled effectively by decreasing the in-plane reading current at room temperature. With the decrease of temperature, the interfacial charge effect dominates over the lattice strain effect due to the reduced charge carrier density. In addition, light stimulus can lead to the delocalization of eg carriers, and thus enhance the lattice strain effect and suppress the interfacial charge effect. This work helps to understand essential physics of magnetoelectric coupling and also provides a potential method to realize energy-efficient multi-field control of manganite thin films.

Physicochemical properties and in vitro hypoglycemic activities of hsian-tsao polysaccharide fractions by gradient ethanol precipitation method.

Hsian-tsao polysaccharides fractions (HPs), including HP20, HP40, HP60, and HP80, were fractioned by gradient precipitation of 20 %, 40 %, 60 %, and 80 % (v/v) ethanol, respectively. Their physicochemical properties and in vitro hypoglycemic activities (inhibitory activities on α-amylase and α-glucosidase, glucose adsorption capacity, and glucose diffusion retardation) were determined. The results showed that, with ethanol upward, the average particle size, molecular weight, and apparent viscosity of HPs were decreased while carbohydrate and uronic acid contents, absolute zeta potential, and thermal stability were increased. Each of the HPs contained Rha, Ara, Gal, Xyl, Man, and GalA with different molar ratios, indicative of anionic heteropolysaccharides with uronic acid. HPs, with diverse structures and surface morphologies as proved by FTIR and SEM, whose solutions were pseudoplastic fluids, exhibited elastic behavior of weak gel networks at concentrations of >1 %. Moreover, HPs showed inhibitory activities on α-amylase and α-glucosidase, of which HP80 was the strongest. For α-amylase, HP20 and HP60 behaved as mixed inhibitors, while HP40 and HP80 were non-competitive. For α-glucosidase, HPs acted as mixed inhibitors. Additionally, HPs possessed glucose adsorption capacity and glucose diffusion retardation, with the best for HP20. These results suggested that HPs possessed hypoglycemic activities, which could be developed as functional food or hypoglycemic drugs.

Interactions involved in heat-induced gelatin gel containing hsian-tsao gum, and mechanisms to improve its properties.

BACKGROUND: Hsian-tsao gum (HG) has unique gel-promoting and nutritional properties; however, its use in processed foods is limited to starchy foods, partially due to a lack of knowledge related to its interaction with proteins. This study elucidated the interaction mechanism of heat-induced gelatin (G) (50 g kg-1 ) gel fortified with HG (0 ~ 20 g kg-1 ) using rheology, Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), large deformation tests, low-field nuclear magnetic resonance (LF-NMR), and confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). RESULTS: Heating promoted synergistic interactions between G and more HG molecules with enhanced apparent viscosity and higher storage modulus G' than loss modulus G″, thus shortening the gel time (tg ) of G-HG sols into gels. Fourier-transform infrared spectroscopy and DSC also confirmed the chemical interactions that occurred, facilitating the formation of ß-sheet structures of G. The microstructure of G gradually formed separate, coarse strands, and aggregated as HG was added, as observed by CLSM and SEM. This accelerated the gel formation rate and changed the textural properties. Although HG caused a disruptive decrease in the helix structure of G, it was possible to compensate for this by accelerating synergistic interactions, including depletion attractions and Maillard reactions, by heating. CONCLUSION: Hsian-tsao gum interacted synergistically with G as a result of heating and this accelerated the gel formation rate and improved the gel properties. Novel complex gels could be designed by blending HG to improve the gel properties of G in the heat processing of the food gel formulation. © 2022 Society of Chemical Industry.

Isolation and purification of antibacterial lipopeptides from Bacillus velezensis YA215 isolated from sea mangroves.

The increasing burden and health risks of antimicrobial resistance (AMR) pose a great threat to society overall. Lipopeptides exhibit great potential as novel and safe alternatives to traditional antibiotics. In this study, the strain YA215, which was isolated from the mangrove area in Beibu Gulf, Guangxi, China, was identified as Bacillus velezensis. Then, YA215 lipopeptide extracts (YA215LE) from B. velezensis was found to exhibit a wide spectrum of antibacterial and antifungal activities. Additionally, YA215LE was identified and found to contain three groups of lipopeptides (surfactin, iturin, and fengycin). Furthermore, one separation fraction (BVYA1) with significant antibacterial activity was obtained. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of BVYA1 showed three molecular ion peaks ([M + H]+: m/z 980.62; 994.66; 1008.66) corresponding to conventional surfactin homologs. By MS/MS analysis, BVYA1 was identified as sufactin with the precise amino acid sequence Glu-Leu/Ile-Leu-Val-Asp-Leu-Leu/Ile and hydroxyl fatty acids with 11-13 carbons. [M + H]+ at m/z 980.62 was detected for the first time in B. velezensis, which demonstrates that the strain corresponds to a new surfactin variant. In particular, BVYA1 showed antibacterial activity with the minimum inhibitory concentration (MIC) values of 7.5-15 µg/ml. Finally, the preliminary mechanism of inhibiting E. coli treated with BVYA1 showed that BVYA1 effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. In conclusion, this study suggests that the YA215LE from B. velezensis YA215 might be a potential candidate for a bactericide.

Glycosaminoglycan from Ostrea rivularis attenuates hyperlipidemia and regulates gut microbiota in high-cholesterol diet-fed zebrafish.

Hyperlipidemia an immense group of acquired or genetic metabolic disorders that is characterized by an excess of lipids in the bloodstream. Altogether, they have a high prevalence worldwide and constitute a major threat to human health. Glycosaminoglycans (GAG) are natural biomolecules that have hypolipidemic activity. The purpose of this study was to investigate the potential hypolipidemic effect of glycosaminoglycans extracted from Ostrea rivularis (OGAG) on hyperlipidemic zebrafish, as well as the possible underlying mechanism of such effect. Dietary supplementation with OGAG during 4 weeks significantly reduced the serum and hepatic lipid levels and the hepatosomatic index in hyperlipidemic zebrafish. In addition, histopathological showed that OGAG supplementation decreases the volume and number of lipid droplets in hepatocytes. Transcriptome and real-time quantitative polymerase chain reaction analysis revealed that the gene expression levels of PPARγ, SCD, HMGRA, ACAT2, HMGCS, and HMGCR were significantly downregulated by OGAG treatment in hepatocytes, whereas those of CD36, FABP2, FABP6, ABCG5, and CYP7A1 were significantly upregulated. This suggests that the hypolipidemic effect of OGAG relies on increasing the ketogenic metabolism of fatty acids, inhibiting cholesterol synthesis, and enhancing the transformation of cholesterol to bile acid. Furthermore, OGAG treatment improved gut microbiota imbalance by reducing the Firmicutes-to-Bacteroidetes ratio, increasing the relative abundance of beneficial bacteria (Bacteroidetes, Verrucomicrobia, Acidobacteria, and Sphingomonas), and reducing the relative abundance of harmful bacteria (Proteobacteria, Cohaesibacter, Vibrio, and Terrisporobacter). These findings highlight the potential benefit of implementing OGAG as a dietary supplement to prevent and treat hyperlipidemia.