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The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

BACKGROUND: Nisin is a commercially available bacteriocin produced by Lactococcus lactis ATCC 11454 and used as a natural agent in the biopreservation of food. In the current investigation, milk whey, a byproduct from dairy industries was used as a fermentation substrate for the production of nisin. Lactococcus lactis ATCC 11454 was developed in a rotary shaker (30 • C/36 h/100 rpm) using two different media with milk whey (i) without filtration, pH 6.8, adjusted with NaOH 2 mol L −1 and without pH adjustment, both autoclaved at 121 • C for 30 min, and (ii) filtrated (1.20 µm and 0.22 µm membrane filter). These cultures were transferred five times using 5 mL aliquots of broth culture for every new volume of the respective media. RESULTS:The results showed that culture media composed of milk whey without filtration supplied L. lactis its adaptation needs better than filtrated milk whey. Nisin titers, in milk whey without filtration (pH adjusted), was 11120.13 mg L −1 in the second transfer, and up to 1628-fold higher than the filtrated milk whey, 6.83 mg.L −1 obtained in the first t transfer.CONCLUSIONS: Biological processing of milk byproducts (milk whey) can be considered a profitable alternative, generating high-value bioproducts and contributing to decreasing river disposals by dairy industries.

Nisin is a natural additive for conservation of food, pharmaceutical, and dental products and can be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of gram-positive and gram-negative bacteria. This study was performed to optimize large-scale nisin production in skimmed milk and subproducts aiming at low-costs process and stimulating its utilization. Lactococcus lactis American Type Culture Collection (ATCC) 11454 was developed in a rotary shaker (30 degrees C/36 h/100 rpm) in diluted skimmed milk and nisin activity, growth parameters, and media components were also studied. Nisin activity in growth media was expressed in arbitrary units (AU/mL) and converted to standard nisin concentration (Nisaplin, 25 mg of pure nisin is 1.0x10(6) AU/mL). Nisin activity in skimmed milk 2.27 g(total solids) was up to threefold higher than transfers in skimmed milk 4.54 g(total solids) and was up to 85-fold higher than transfers in skimmed milk 1.14 g(total solids). L. lactis was assayed in a New Brunswick fermentor with 1.5 L of diluted skimmed milk (2.27 g(total solids)) and airflow of 1.5 mL/min (30 degrees C/36/200 rpm), without pH control. In this condition nisin activity was observed after 4 h (45.07 AU/mL) and in the end of 36 h process (3312.07 AU/mL). This work shows the utilization of a low-cost growth medium (diluted skimmed milk) to nisin production with wide applications. Furthermore, milk subproducts (milk whey) can be exploited in nisin production, because in Brazil 50% of milk whey is disposed with no treatment in rivers and because of high organic matter concentrations it is considered an important pollutant. In this particular case an optimized production of an antimicrobial would be lined up with industrial disposal recycling.

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Serogroup B outer membrane vesicles (OMV) with iron regulated proteins (IRP) from Neisseria meningitidis constitute the antigen for the vaccine against the disease caused by this bacterium. Aiming to enhance final OMV concentration, seven batch experiments were carried out under four different conditions: (i) with original Catlin medium; (ii) with original Catlin medium and lactate and amino acids pulse at the 6th cultivation hour; (iii) with Catlin medium with double initial concentrations of lactate and amino acids and (iv) Catlin medium without glycerol and with double initial concentrations of lactate and amino acids. The cultivation experiments were carried out in a 7-L bioreactor under the following conditions: 36°C, 0.5atm, overlay air 1L/min, agitation: 250-850 rpm, and O(2) control at 10%, 20 h. After lactate and amino acids exhaustion, cell growth reached stationary phase and a significant release increase of OMV was observed. According to the Luedeking & Piret model, OMV liberation is non-growth associated. Glycerol was not consumed during cultivation. The maximum OMV concentration value attained was 162 mg/L with correspondent productivity of 8.1mg/(Lh) employing Catlin medium with double initial concentrations of lactate and amino acids. The obtained OMV satisfied constitution and protein pattern criteria and were suitable for vaccine production.

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Instrumental neutron activation analysis was used to measure concentrations of elements in hair samples from a group of patients of a medical clinic and from a control group. Elements A1, As, Br, Ca, Cd, C1, Co, Cu, Fe, Hg, K, Mg, Mn, Na, Sb, Sc, Se, V and Zn were analyzed and comparisons were made between the results obtained for these two groups of individuals. Normal ranges for elemental hair by commercial laboratories are also presented, for comparison, with those results obtained for the control group of individuals living in Sgo Paulo, Brazil.