Deletion of the natural inhibitory protein Inh1 in Ustilago maydis has no effect on the dimeric state of the F1FO-ATP synthase but increases the ATPase activity and reduces the stability.

Transduction of electrochemical proton gradient into ATP synthesis is performed by F1FO-ATP synthase. The reverse reaction is prevented by the regulatory subunit Inh1. Knockout of the inh1 gene in the basidiomycete Ustilago maydis was generated in order to study the function of this protein in the mitochondrial metabolism and cristae architecture. Deletion of inh1 gen did not affect cell growth, glucose consumption, and biomass production. Ultrastructure and fluorescence analyzes showed that size, cristae shape, network, and distribution of mitochondria was similar to wild strain. Membrane potential, ATP synthesis, and oxygen consumption in wild type and mutant strains had similar values. Kinetic analysis of ATPase activity of complex V in permeabilized mitochondria showed similar values of Vmax and KM for both strains, and no effect of pH was observed. Interestingly, the dimeric state of complex V occurs in the mutant strain, indicating that this subunit is not essential for dimerization. ATPase activity of the isolated monomeric and dimeric forms of complex V indicated Vmax values 4-times higher for the mutant strain than for the WT strain, suggesting that the absence of Inh1 subunit increased ATPase activity, and supporting a regulatory role for this protein; however, no effect of pH was observed. ATPase activity of WT oligomers was stimulated several times by dodecyl-maltoside (DDM), probably by removal of ADP from F1 sector, while DDM induced an inactive form of the mutant oligomers.

Production of chitosan-oligosaccharides by the chitin-hydrolytic system of Trichoderma harzianum and their antimicrobial and anticancer effects.

Chitosan-oligosaccharides (COS) are low-molecular weight chitosan derivatives with interesting clinical applications. The optimization of both COS production and purification is an important step in the design of an efficient production system and for the exploration of new COS applications. Trichoderma harzianum is an innocuous biocontrol agent that represents a novel biotechnological tool due to the production of extracellular enzymes, including those that produce a COS mixture. In this work, we propose different systems for the production of COS using the T. harzianum chitinolitic system. A complete qualitative and quantitative analysis of a partially purified COS mixture were performed. Also, an evaluation of the anticancer and antimicrobial effects of the COS mixture was carried out. Three chitosan variants (colloidal, solid and solution) and two fungus stages (spores and mycelia) were tested for COS production. The best system consisted of the interaction of the solid chitosan and the fungal spores, producing a COS mixture containing species from the monomer to the hexamer, in a concentration range of 7-238 mg/mL, according to chromatographic analysis. The proposed purification method isolated the monomer and the dimer from the COS mixture. Moreover, the COS mixture has an inhibitory effect on the growth of bacteria and changes the morphology of yeasts. As anticancer compounds, COS inhibited the growth of cervical cancer cells at concentration of 4 mg/mL and significantly reduced the survival rate of the cells. In conclusion, T. harzianum proved to be an efficient system for COS mixture production.