Antimycobacterial compound of chitosan and ethambutol: ultrastructural biological evaluation in vitro against Mycobacterium tuberculosis.

Chitosan (CS) is a promising biopolymer and has been tested as a complement to the action and compensation of toxicity presented by anti-tuberculosis drugs. The present work studied the adjuvant effect of CS with the drug ethambutol (EMB) as a compound (CS-EMB), to explore its antimicrobial and cytotoxic activity, using transmission electron microscopy (TEM), to examine ultracellular changes that represent possible antimycobacterial action of CS on Mycobacterium tuberculosis (Mtb). Antimycobacterial activities were tested against reference strains Mtb ATCC® H37Rv and multidrug resistant (MDR). In vitro cytotoxicity tests were performed on Raw 264.7. For the studied compounds, morphological, ultrastructural, and physical-chemical analyses were performed. Drug-polymer interactions that occur through the H bridges were confirmed by physical-chemical analyses. The CS-EMB compound is stable at pHs of 6.5-7.5, allowing its release at physiological pH. The antibacterial activity (minimum inhibitory concentration) of the CS-EMB compound was 50% greater than that of the EMB in the H37Rv and MDR strains and the ultrastructural changes in the bacilli observed by TEM proved that the CS-EMB compound has a bactericidal action, allowing it to break down the Mtb cell wall. The cytotoxicity of CS-EMB was higher than that of isolated EMB, IC50 279, and 176 µg/mL, respectively. It is concluded that CS-EMB forms a promising composite against strains Mtb H37Rv and multidrug resistant (MDR-TB).Key points• Our study will be the first to observe ultrastructurally the effects of the CS-EMB compound on Mtb cells.• CS-EMB antimicrobial activity in a multidrug-resistant clinical strain.• The CS-EMB compound has promising potential for the development of a new drug to fight tuberculosis.

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate.

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-D: -glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50 degrees C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K(HILL) and Vmax of CDA were 288+/-34 nmol/l and 0.08+/-0.01 U mg protein(-1) min(-1), respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50 degrees C.

Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization.

Chitosan from a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung (Northeast-Brazil), was used as a film support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weight(-1); 15.2% of dry mycelia weight) among the nine strains screened, which presented 89% D-glucosamine. A chitosan film was used for lipase (EC 3.1.1.3) immobilization using glutaraldehyde as a bifunctional agent. The immobilized lipase retained 47% (12.6 micromol s(-1) m(-2)) of its initial catalytic activity after four cycles of reaction. This result is comparable (same order of magnitude) to that of the enzyme immobilized on film made from commercially available crustacean chitosan.