An overview of Bacillus proteases: from production to application

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.     

Modification of a Reaction Vessel for the Determination of Total Mercury in Hair by Cold Vapor Atomic Absorption Spectrometry

A classical cold vapor atomic absorption spectrometric (CVAAS) method for the determination of total mercury in blood has been previously adapted for application to samples of human hair. The reaction vessel specified in the original adaptation was, however, large and difficult to use with small hair samples. In the present study, the reaction vessel has been modified and reduced in size and the protocols have been optimized in order to provide an analytical method that is more efficient, less time-consuming, and gives lower blank values than the original adaptation. The optimized method was validated by multiple, independent, replicated assays of certified reference hair samples, and the mean recovery obtained (98.7%) indicated an efficient performance of the digestion and detection processes. The method was applied to the assay of 144 hair samples derived from populations that had or had not been exposed to mercury from environmental sources. The results from all of the samples analyzed were consistent with those published previously for similar samples.     

Functional approach based on morphology as a model of phytoplankton variability in a subtropical floodplain lake: a long-term study

Long-term phytoplankton dynamics in a floodplain lake, between periods of limnophase and potamophase was studied using the morphology-based functional classification (MBFG). The work was carried out to test the hypothesis that the temporal distribution of MBFGs is influenced by the hydrosedimentological regime of the Paraná River, and that these differences can be registered by analyzing the dominant MBFGs in the two periods. Samples were taken in an isolated floodplain lake on the Upper Paraná River floodplain, Brazil, from 2000 to 2012, and water level, water temperature, conductivity, pH, dissolved-oxygen, euphotic zone, maximum depth, and nutrients were used to explain the distribution of MBFGs. 478 taxa were identified and distributed in seven MBFGs (I–VII). MBFG V (flagellates algae) and VI (diatoms) showed 100% frequency of occurrence. MBFG II was associated exclusively with the limnophase; MBFG IV and VII were associated with limnophase periods with higher pH and dissolved-oxygen content; and MBFG I, III, V, and VI were associated with limnophase and potamophase, mainly associated with transparency, nutrients, and conductivity. The MBFG approach represented the trends of each group in terms of its occurrence and biovolume, according to the hydrosedimentological regime, providing broad-scale information on changes in the phytoplankton.     

Spectroscopic Investigation of Local Mechanical Impedance of Living Cells

We studied nanoscale mechanical properties of PC12 living cells with a Force Feedback Microscope using two experimental approaches. The first one consists in measuring the local mechanical impedance of the cell membrane while simultaneously mapping the cell morphology at constant force. As the interaction force is increased, we observe the appearance of the sub-membrane cytoskeleton. We compare our findings with the outcome of other techniques. The second experimental approach consists in a spectroscopic investigation of the cell while varying the tip indentation into the membrane and consequently the applied force. At variance with conventional dynamic Atomic Force Microscopy techniques, here it is not mandatory to work at the first oscillation eigenmode of the cantilever: the excitation frequency of the tip can be chosen arbitrary leading then to new spectroscopic AFM techniques. We found in this way that the mechanical response of the PC12 cell membrane is found to be frequency dependent in the 1 kHz - 10 kHz range. In particular, we observe that the damping coefficient consistently decreases when the excitation frequency is increased.     

UVA tanning is involved in the increased incidence of skin cancers in fair-skinned young women

Melanomas are the most prevalent cancers in 25–29 yr old females and compose roughly 12% of cancers in 20–40 yr old women; under the age of 40, women have a higher incidence of melanomas than do men. Within the past few decades, the alarming trend to use commercial sunlamps for cosmetic pigmentation is of particular concern, especially since 71% of those patrons are women with 50% of patrons under the age of 29. A major problem may be the use of UVA-rich sunlamps which produce a visible tan but afford little to no protection from subsequent UV exposure. We hypothesize that the additional exposure of adolescents to unnaturally large amounts of UVA from artificial UV sources is implicated in the increasing incidence of malignant melanomas disproportionately in young women.     

Enantioselective benzylic microbial hydroxylation of indan and tetralin

A screening with 15 strains of bacteria and fungi targeted at the production of specific hydroxylated benzylic derivatives of indan 1 and tetralin 2 was carried out. Mortierella isabellina, Mortierella ramanniana and Beauveria bassiana were shown to mediate the respective conversions to 1-indanol (3) and 1-tetralol (4), the most satisfactory results being obtained with M. isabellina, which gave 78% conversion of 1 to (1R)-3 (64% yield, 86% ee) after a 2-day-incubation, and 52% conversion of 2 to (1R)-4 (38% yield, 92% ee) in a 4-day-incubation. Over-oxidation of alcohols 3 and 4 during the reactions resulted on the formation of 1-indanone and 2-tetralone, respectively.     

Trypanosoma cruzi: description of a highly purified surface antigen defined by human antibodies

A glycoprotein of 25,000 daltons (G25) purified from T. cruzi extracts is recognized by serum antibodies of Chagas' disease patients. These human antibodies were isolated by affinity chromatography and were used to demonstrate that G25 antigenic determinants are i) represented at the parasite surface, and ii) are expressed in all developmental stages of the parasite's life cycle, as well as in several T. cruzi strains. This antigen-antibody system may be useful for the diagnosis of Chagas' disease because antibodies to radiolabeled G25 are found in the serum of 96.5% of 173 chagasic patients from different endemic areas, but are not found in the serum from other individuals. Taken collectively, the data suggest that antibodies to G25 define highly conserved determinants of the species T. cruzi. Moreover, its remarkable immunogenicity to infected humans offers an opportunity to investigate the role of specific immunologic responses in the pathogenicity of Chagas' disease.