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The synergistic effectiveness of chitosan with zinc oxide nanomicelles (CZNPs) on broad spectrum of multidrug resistance (MDR) was previously evidenced in our labs, requiring elucidation of the therapeutic index (TI) for safe in vivo use. This in vitro assessment estimated the effective dose (ED 50) of micellar CZNPs for eradication of the MDR Enterococcus faecium 1449 model and the corresponding cytotoxic dose (LD 50) against rat small intestinal epithelial cells as functions of TI. In order to visually determine the mechanistic effects of micellar CZNPs on bacterial biofilm size reduction, LIVE/DEAD viability assay was used in conjunction with advanced fluorescence imaging and 3D confocal microscopy. Biofilm quantification was performed through the measure of the fluorescence intensity, using the Biotek Synergy Neo2 for calculating the ED 50. To generate the LD 50 , the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was implemented. Quantification results revealed, at the same concentration (200 µg/mL), micellar CZNPs had average biofilm reduction of approximately 50.22% at 24 h (ED 50 = 199.13 µg/mL, LD 50 = 240.20 µg/mL, TI = 1.2062), compared to chitosan (15.66%) and ZnO (13.94%) alone. Conclusively, the ED 50 of micellar CZNPs on MDR bacterial biofilms (199.13 µg/mL) as a function of TI reveals a promising nanotherapeutic agent in comparison to either Chitosan or ZnO alone. [ABSTRACT FROM AUTHOR]

• Microwave assisted extraction of fungal chitosan has been found to be efficient. • Optimization of the microwave power and duration of incubation was performed. • Higher chitosan yield of 13.43% w/w dry biomass. • Chitosan of 94% degree of deacetylation. • The energy consumption bill significantly lower, 1.1 vs 50 cents. Microwave-assisted extraction (MAE) of chitosan from dried fungal biomass of Rhizopus oryzae NRRL1526, obtained by culturing on potato dextrose broth (PDB), was performed and the optimal conditions required were identified using statistical analysis for the first time in this study. This microwave-assisted extraction (MAE) was compared against the conventional autoclave assisted method of chitosan extraction. The full factorial experimental design was used to investigate the impact of operating parameters of MAE, microwave power (100 W–500 W), and duration (10 min–30 min), on alkaline insoluble material (AIM) yield, chitosan yield, and degree of deacetylation (DDA). The effect of operating conditions was then evaluated using full factorial data analysis and optimum condition for MAE of chitosan was identified using response surface methodology to be 300 W and 22 min. This optimum condition identified was then further evaluated and the chitosan obtained characterized. Higher chitosan yield of 13.43 ± 0.3% (w/w) of fungal biomass was obtained when compared to that obtained, 6.67% ± 0.3% (w/w) of dry biomass, for the conventional extraction process. MAE yielded chitosan of higher degree of deacetylation, 94.6 ± 0.9% against 90.6 ± 0.5% (conventional heating), but the molecular weight was observed to be similar to that obtained by using conventional autoclave heating. MAE of chitosan was observed to yield a higher quantity of chitosan when compared to conventional extraction process and obtained chitosan exhibited a higher degree of deacetylation as well as molecular weight. The lower energy consumption of 0.11 kW h for MAE (5 kW h for conventional process) and the concomitant reduction in the energy bill to 1.1 cents from 50 cents, in addition to the above results, show that microwave irradiation is a more efficient and environment-friendly means to obtain chitosan from fungal biomass. [ABSTRACT FROM AUTHOR]

Abstract Laccase is one of the widely used enzymes for biotechnological processes. Immobilization of enzymes is a universally accepted approach to increase their reusability and stability. In this study, laccase enzyme from Trametes versicolor was encapsulated for the first time in a chitosan-nanobiochar matrix. The chitosan-tripolyphosphate gel formation technique was employed to produce homogeneous biocatalyst nanoparticles, with 35% effective binding efficiency and 3.5 Units/g apparent activity under the best configuration. The reusability of the encapsulated laccase was demonstrated towards the oxidation of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) for several consecutive cycles, exhibiting 30% of the initial activity after 5 cycles. The encapsulated laccase showed a moderate increase in enzyme stability against pH and temperature variation compared to the free enzyme. Moreover, the storage stability of laccase at both 4 °C and 25 °C was increased after immobilization. Only 2% of laccase was leaked during a 5-day period from biocatalyst. Laccase in its free form showed no antibacterial activity against Gram positive and Gram-negative model microorganisms, while encapsulated laccase showed antibacterial activity towards Gram-positive ones. Thus, the encapsulation of the laccase is an efficient method to keep the enzyme active and stable for different applications. Highlights • The biocatalyst showed 35% of binding efficiency and 3.5 Units/g apparent activity. • The encapsulated laccase showed higher stability compare to free enzyme. • Encapsulated laccase onto chitosan-nanobiochar exhibited antimicrobial activity. [ABSTRACT FROM AUTHOR]