Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

The screening for hydrolases-producing, halotolerant, and spore-forming gram-positive bacteria from the root, rhizosphere, and non-rhizosphere soil of Blutaparon portulacoides, a plant found in the Restinga de Jurubatiba located at the northern region of Rio de Janeiro State, Brazil, resulted in the isolation of 22 strains. These strains were identified as Halobacillus blutaparonensis (n = 2), Oceanobacillus picturae (n = 5), and Oceanobacillus iheyensis (n = 15), and all showed the ability to produce different extracellular enzymes. A total of 20 isolates (90.9 %) showed activity for protease, 5 (22.7 %) for phytase, 3 (13.6 %) for cellulase, and 2 (9.1 %) for amylase. Some bacterial strains were capable of producing three (13.6 %) or two (9.1 %) distinct hydrolytic enzymes. However, no bacterial strain with ability to produce esterase and DNase was observed. The isolate designated M9, belonging to the species H. blutaparonensis, was the best producer of protease and also yielded amylase and phytase. This strain was chosen for further studies regarding its protease activity. The M9 strain produced similar amounts of protease when grown either without or with different NaCl concentrations (from 0.5 to 10 %). A simple inspection of the cell-free culture supernatant by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of three major alkaline proteases of 40, 50, and 70 kDa, which were fully inhibited by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) (two classical serine protease inhibitors). The secreted proteases were detected in a wide range of temperature (from 4 to 45 °C) and their hydrolytic activities were stimulated by NaCl (up to 10 %). The serine proteases produced by the M9 strain cleaved gelatin, casein, albumin, and hemoglobin, however, in different extensions. Collectively, these results suggest the potential use of the M9 strain in biotechnological and/or industrial processes.

ABSTRACT. It has been reported in the literature that the Malpighian tubules of Neuroptera in the third instar undergo drastic histological changes, when they stop functioning in osmoregulation and start to secrete silk fibers for a cocoon. Therefore, to increase our knowledge about these cellular alterations that occur in the larvae of Neuroptera, we analyzed the cells that constitute the Malpighian tubules of each larval instar of the species Myrmeleon uniformis, with emphasis on nucleolar activity. Malpighian tubules, after being removed, were fixed on a slide using liquid nitrogen and stained by silver impregnation. In Nucleolar activity in antlions addition, total protein of the tubules was quantified. By analyzing the cells in the first instar larval stage, we observed only two silver-stained nucleolar regions. In cells of second instar larvae, there was an increase in the number of stained regions, and in the third instar, the number of nucleolar regions was very large. Agarose gel electrophoresis indicated that third instar larvae had high synthetic activity, where the total amount of proteins was larger in third instar stage than in the other larval stages. Furthermore, the most abundant proteins displayed molecular weights of about 32-43 kDa and were probably precursors of silk fibers. Thus, the results obtained showed that nucleolar alterations occur in the cells of the Malpighian tubules of larval instars of M. uniformis and this is directly related to the production of silk fibers used by the pupa to ensure the completion of metamorphosis.

No abstract

Myrmeleon unifomis Malpighian tubules cells were analyzed under two aspects: chromatin structure and total protein quantification in the three larval phases (L 1 , L 2 and L 3 ) and in adults. This insect Malpighian tubules present polyploid tissue resultant from endoreduplication, which probably begins in the first larval phases. The Malpighian tubule proximal region in L 2 , L 3 and adult cells has polyploid nuclei, although the polyploid degree does not cause change in its morphology; nevertheless in the L 3 larvae distal region, polyploidization changes the cell morphology from a round to a multilobulate shape. Chromatin analysis in these nuclei by cytochemical techniques showed that the whole nucleus is filled out with a granular chromatin. In multilobulate nuclei during L 3 the chromatin is formed by a network of chromatin fibers. The nucleolar markings made in the several development phases of Myrmeleon sp showed the gradual increase that occurs during the larval phases and their reduction in the adult phase. This transcriptional activity was confirmed by total protein analyses, which were quantified by SDS-PAGE. The 39.8-kDa band deserves prominence, because it can be seen from L 2 phase, increasing its intensity during L 3 and decreasing in the adult phase, indicating that this protein is related to production of the pupation cocoon.