Identification of six important amino acid residues of MenA from Bacillus subtilis natto for enzyme activity and formation of menaquinone.

The enzyme 1, 4-dihydroxy-2-naphthoic acid (DHNA) prenyltransferase (MenA) is a critical player in determining the efficiency of the menaquinone (MK) synthesis pathway and is an attractive target for the development of novel chemotherapeutics against pathogenic Gram-positive bacteria. However, there has been no report on structural properties or active region of MenA. To solve this challenge, we predicted the three-dimensiona structure and critical amino acid sites of MenA by bioinformatics analysis. Six amino acid sites were chosen by alligning the amino acid sequence of MenA from Bacillus subtilis natto with 4-hydroxybenzoate octaprenyl transferase (UbiA) from Escherichia coli, Aeropyrum pernix and Archaeoglobus fulgidus. Among them, four Asp sites located in two Asp-rich motifs (D78XXXXXD84 and D208XXXD212) were found to be indispensable amino acid residues in maintaining MenA activity. Site-directed mutagenesis of two other sites (Q67th, N74th) positively affected the catalytic activity of MenA and the MK titer. Q67R resulted in more than a 5-fold increase in specific 2-demethylmenaquinone (DMK) content (YP1/x) compared to wild-type, and the hydrophobic interaction between Cys63 and Arg67 could be the main reason according to the three-dimensional structure analysis. Moreover, a dramatic increase in specific MK content (YP2/x) was realized by co-expressing menG in EcMenA (Q67R). The results obtained could be useful not only in developing novel chemotherapeutics to combat potentially pathogenic Gram-positive bacteria, but also in regulating and optimizating E. coli mutant cultures for the efficient production of MK metabolites.

Mussel-Inspired Catechol-Functionalized Hydrogels and Their Medical Applications.

Mussel adhesive proteins (MAPs) have a unique ability to firmly adhere to different surfaces in aqueous environments via the special amino acid, 3,4-dihydroxyphenylalanine (DOPA). The catechol groups in DOPA are a key group for adhesive proteins, which is highly informative for the biomedical domain. By simulating MAPs, medical products can be developed for tissue adhesion, drug delivery, and wound healing. Hydrogel is a common formulation that is highly adaptable to numerous medical applications. Based on a discussion of the adhesion mechanism of MAPs, this paper reviews the formation and adhesion mechanism of catechol-functionalized hydrogels, types of hydrogels and main factors affecting adhesion, and medical applications of hydrogels, and future the development of catechol-functionalized hydrogels.

Preparation and Properties of Carboxymethyl Chitosan/Alginate/Tranexamic Acid Composite Films.

In this study, the porous composite films of carboxymethyl chitosan/alginate/tranexamic acid were fabricated, with calcium chloride as the crosslinking agent and glycerin as a plasticizer. The composite films were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The properties of the composite films, including water absorption, air permeability, and cumulative release rate, were tested. In addition, their hemostatic performance was evaluated. The results showed that the appearance of the films with good adhesion was smooth and porous. FTIR showed that chemical crosslinking between carboxymethyl chitosan and sodium alginate was successful. The excellent cumulative release of tranexamic acid in the composite films (60⁻80%) gives the films a significant procoagulant effect. This has good prospects for the development of medical hemostasis materials.

Thermal degradation of agar: Mechanism and toxicity of products.

The mechanism of the thermal degradation and the toxicity of the thermal degradation products of agar were studied using TG/DTA, Fourier-transform infrared spectroscopy and pyrolysis gas chromatography/mass spectrometry. It was found that the thermal degradation of agar is a single-step reaction, the thermal degradation temperature (T0, Tp, Tf) increases with increasing gel strength (P) and the influence of P on the thermal degradation rate is modest. The thermal degradation of agar is an exothermic reaction, and the activation energy of the reaction increases with increasing P. In the thermal degradation, agar is first decomposed into 3,6-anhydropyran galactopyranose and galactopyranose, then 3,6-anhydropyran galactopyranose, and finally furyl hydroxymethyl ketone, through loop opening, dehydration and hydrogen transfer. Galactopyranose follows three degradation pathways, and its final degradation products are 3,4-atrosan, d-allose, furfural and 5-(hydroxymethyl)-2-furancarboxaldehyde. Of the degradation products, furyl hydroxymethyl ketone, furfural, and 5-(hydroxymethyl)-2-furancarboxaldehyde show some toxicity to humans.

Bioconversion of farnesol and 1,4-dihydroxy-2-naphthoate to menaquinone by an immobilized whole-cell biocatalyst using engineered Elizabethkingia meningoseptica.

Menaquinone (MK) has important applications in the pharmaceutical and food industries. To increase the production rate (QP) of MK-4, we developed a straightforward biotransformation method for MK-4 synthesis directly from its precursors 1,4-dihydroxy-2-naphthoate (DHNA) and farnesol using whole cells of genetically engineered Elizabethkingia meningoseptica. Results showed that MK-4 can be produced directly from farnesol and DHNA using both free and immobilized FM-D198 cells. MK-4 yield peaked at 29.85 ± 0.36 mg/L in the organic phase and 24.08 ± 0.33 mg/g DCW after 12 h of bioconversion using free cells in a two-phase conversion system. MK-4 yield reached 26.34 ± 1.35 mg/L and 17.44 ± 1.05 mg/g DCW after 8 h using immobilized cells. Although this yield was lower than that using free cells, immobilized cells can be re-used for MK-4 production via repeated-batch culture. After ten batch cultures, efficient MK-4 production was maintained at a yield of more than 20 mg/L. After optimizing the catalysis system, the MK-4 yield reached 26.91 ± 1.27 mg/L using the immobilized cells and had molar conversion rates of 58.56 and 76.90% for DHNA and farnesol, respectively.

Development of drug-loaded chitosan-vanillin nanoparticles and its cytotoxicity against HT-29 cells.

Chitosan as a natural polysaccharide derived from chitin of arthropods like shrimp and crab, attracts much interest due to its inherent properties, especially for application in biomedical materials. Presently, biodegradable and biocompatible chitosan nanoparticles are attractive for drug delivery. However, some physicochemical characteristics of chitosan nanoparticles still need to be further improved in practice. In this work, chitosan nanoparticles were produced by crosslinking chitosan with 3-methoxy-4-hydroxybenzaldehyde (vanillin) through a Schiff reaction. Chitosan nanoparticles were 200-250 nm in diameter with smooth surface and were negatively charged with a zeta potential of - 17.4 mV in neutral solution. Efficient drug loading and drug encapsulation were achieved using 5-fluorouracil as a model of hydrophilic drug. Drug release from the nanoparticles was constant and controllable. The in vitro cytotoxicity against HT-29 cells and cellular uptake of the chitosan nanoparticles were evaluated by methyl thiazolyl tetrazolium method, confocal laser scanning microscope and flow cytometer, respectively. The results indicate that the chitosan nanoparticles crosslinked with vanillin are a promising vehicle for the delivery of anticancer drugs.

Synthesis and characterization of chitosan quaternary ammonium salt and its application as drug carrier for ribavirin.

N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) is hydro-soluble chitosan (CS) derivative, which can be obtained by the reaction between epoxypropyl trimethyl ammonium chloride (ETA) and CS. The preparation parameters for the synthesis of HTCC were optimized by orthogonal experimental design. ETA was successfully grafted into the free amino group of CS. Grafting of ETA with CS had great effect on the crystal structure of HTCC, which was confirmed by the XRD results. HTCC displayed higher capability to form nanoparticles by crosslinking with negatively charged sodium tripolyphosphate (TPP). Ribavrin- (RIV-) loaded HTCC nanoparticles were positively charged and were spherical in shape with average particle size of 200 nm. More efficient drug encapsulation efficiency and loading capacity were obtained for HTCC in comparison with CS, however, HTCC nanoparticles displayed faster release rate due to its hydro-soluble properties. The results suggest that HTCC is a promising CS derivative for the encapsulation of hydrophilic drugs in obtaining sustained release of drugs.

[Inhibitory effect of PNS on drug metabolism enzyme CYP3A in rat livers and its kinetic analysis].

OBJECTIVE: To study the inhibitory effect of total saponins of the root and rhizome of Panax notoginseng (PNS) on drug metabolism enzyme CYP3A in rat livers and its kinetic analysis. METHOD: Microsome enzyme was prepared by differential velocity centrifugation. Michaelis constant (Km) and maximum velocity (Vmax) of CYP3A, 50% inhibitory concentration of PNS on CYP3A, and the inhibition type and the inhibition constant of CYP3A (Ki, Kis) of PNS on CYP3A were calculated by Lineweaver-Burk and the low of semi-effect-probit. RESULT: Total saponins of the root and rhizome of panax notoginseng inhibited CYP3A activity, with IC50 of 689.54 mg x L(-1). Compared with the substrate aminopyrine, CYP3A showed Km of 0.036 mmol x L(-1) and Vmax of 21.01 micromol min(-1) x g(-1). Total saponins of the root and rhizome of panax notoginseng showed a mixed inhibition on CYP3A, with the inhibition constants of 247.79 mg x L(-1) (Ki) and 321.79 mg x L(-1) (Kis). CONCLUSION: Total saponins of the root and rhizome of panax notoginseng have a significant effect on CYP3A activity in rat livers.