RESUMO
The present study evaluates the protective properties of boric acid (BA) against the toxic effects induced by ochratoxin A (OTA) in human embryonic kidney cells (HEK293). The focus is on various parameters such as cytotoxicity, genotoxicity, oxidative stress, and apoptosis. OTA is a known mycotoxin that has harmful effects on the liver, kidneys, brain, and nervous system. BA, on the other hand, a boron-based compound, is known for its potential as a vital micronutrient with important cellular functions. The results show that BA administration not only increases cell viability but also mitigates the cytotoxic effects of OTA. This is evidenced by a reduction in the release of lactate dehydrogenase (LDH), indicating less damage to cell membranes. In addition, BA shows efficacy in reducing genotoxic effects, as the frequency of micronucleus (MN) and chromosomal aberrations (CA) decreases significantly, suggesting a protective role against DNA damage. In addition, the study shows that treatment with BA leads to a decrease in oxidative stress markers, highlighting its potential as a therapeutic intervention against the deleterious effects of OTA. These results emphasize the need for further research into the protective mechanisms of boron, particularly BA, in combating cell damage caused by OTA.
RESUMO
Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 ß-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å2 ß-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others.
