Enhanced keratinase production by Bacillus subtilis amr using experimental optimization tools to obtain feather protein lysate for industrial applications.

The poultry industry produces millions of tons of feathers waste that can be transformed into valuable products through bioprocess. The study describes the enhanced keratinase and feather hydrolysate production by Bacillus subtilis AMR. The metabolism of each microorganism is unique, so optimization tools are essential to determine the best fermentation parameters to obtain the best process performance. The evaluation of different propagation media indicated the constitutive production of two keratinases of approximately 80 kDa. The combination of Mn2+, Ca2+, and Mg2+ at 0.5 mM improved the keratinolytic activity and feather degradation 1.5-fold, while Cu2+ inhibited the enzymatic activity completely. Replace yeast extract for sucrose increased the feather hydrolysate production three times. The best feather concentration for hydrolysate production was 1.5% with an inoculum of 108 CFU/mL and incubation at 30 °C. None of the inorganic additional nitrogen sources tested increased hydrolysate production, although (NH4)2SO4 and KNO3 improved enzymatic activity. The optimization process improved keratinolytic activity from 205.4 to 418.7 U/mL, the protein concentration reached 10.1 mg/mL from an initial concentration of 3.9 mg/mL, and the feather degradation improved from 70 to 96%. This study characterized keratinase and feather hydrolysate production conditions offering valuable information for exploring and utilizing AMR keratinolytic strain for feather valorization. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03153-y.

Quantification of schizophyllan directly from the fermented broth by ATR-FTIR and PLS regression.

Non-destructive methods that allow the quantification of bioproducts in a simple and quick manner during fermentation are extremely desirable from a practical point of view. Therefore, a 9 day fermentation experiment with Schizophyllum commune was carried out to investigate the possibility of using ATR-FTIR to quantify the schizophyllan biopolymer (SPG) directly from the culture medium. On each day, aliquots of the fermentation were taken, and the cell-free supernatant was analyzed by ATR-FTIR. The main objective of this step was to evaluate whether FTIR would be able to detect the appearance of specific peaks related to the production of SPG. The results of the PCA analysis showed that there was a reasonable separation of the days through the FTIR spectra. Then PCA-LDA was applied to the same dataset, which confirmed the formation of groups for each day of fermentation, after which, a calibration and test set was developed. Through a matrix generated by an experimental design with 2 factors and 5 levels, 25 samples were created with variations in the concentration of the culture medium and SPG. The ATR-FTIR spectra of this data set were modeled using PLS regression with backward selection of predictors. The results revealed that the amount of SPG produced can be quantified directly in the culture medium with excellent precision with R2CV = 0.951, R2P = 0.970, RMECV = 0.205 g, RMSEP = 0.170 g, RPDcv = 4.53 and RPDp = 5.88. The traditional method to quantify SPG is time consuming, requires several steps and uses solvents. In contrast, the method proposed in this work is a viable, faster, and a simpler alternative, which does not use reagents and does not require extensive pre-treatment of the samples.

New method for rapid identification and quantification of fungal biomass using ergosterol autofluorescence.

This research reports on the development of a method to identify and quantify fungal biomass based on ergosterol autofluorescence using excitation-emission matrix (EEM) measurements. In the first stage of this work, several ergosterol extraction methods were evaluated by APCI-MS, where the ultrasound-assisted procedure showed the best results. Following an experimental design, various quantities of the dried mycelium of the fungus Schizophyllum commune were mixed with the starchy solid residue (BBR) from the babassu (Orbignya sp.) oil industry, and these samples were subjected to several ergosterol extraction methods. The EEM spectral data of the samples were subjected to Principal Component Analysis (PCA), which showed the possibility to qualitatively evaluate the presence of ergosterol in the samples by ergosterol autofluorescence without the addition of any reagent. In order to assess the feasibility of quantifying fungal biomass using ergosterol autofluorescence, the EEM spectral data and known amounts of fungal biomass were modeled using partial least squares (PLS) regression and a procedure of backward selection of predictors (AutoPLS) was applied to select the Excitation-Emission wavelength pairs that provide the lowest prediction error. The results revealed that the amount of fungal biomass in samples containing interfering substances (BBR) can be accurately predicted with R2CV = 0.939, R2P = 0.936, RPDcv = 4.07, RPDp = 4.06, RMSECV = 0.0731 and RMSEP = 0.0797. In order to obtain an easy-to-understand equation that expresses the relationship between fungal biomass and fluorescence intensity, multiple linear regression (MLR) was applied to the VIP variables selected by the AutoPLS method. The MLR model selected only 2 variables and showed a very good performance, with R2CV = 0.862, R2P = 0.809, RPDcv = 2.18, RPDp = 2.35, RMSECV = 0.137 and RMSEP = 0.138. This study demonstrated that ergosterol autofluorescence can be successfully used to quantify fungal biomass even when mixed with agroindustrial residues, in this case BBR.

A microplate assay for extracellular hydrolase detection.

This article presents a new qualitative method to detect enzyme activity replacing the conventional Agar-Petri dishes. This new method is a simple rapid and low-cost technique that uses 24-well microplates. The detection of hydrolases producing microorganisms in bioprospecting studies by qualitative methods is time consuming, costly and requires a large quantity of strains or enzymatic extracts. Tests with different substrate concentrations (0 to 20 g/L) in agar solution for the enzymatic hydrolysis analysis were performed to determine the best substrate concentrations in 24-well microplates. Other quantitative and analytical methods, such as enzymatic assays and thin layer chromatography, were performed to validate this new method and to compare the relationship between enzymatic activity and substrate degradation. Statistically relevant results were observed for amylase, endoglucanase and polygalacturonase enzymes, even when there was a low substrate concentration in agar, where the halo diameter was high. The results also indicated that the concentrations for efficient enzyme index measurements were 4 g/L carboxymethylcellulose for endoglucanase detection and 8 g/L for amylase and polygalacturonase assays. The results were presented according to the traditional methods for detection of enzymatic activity. This new method can be used as a general test for the detection of important industrial hydrolases. It is a faster and less costly alternative for screening microbial enzyme producing microorganisms and is useful for studying the production of microbial enzymes under different growing conditions.

Inhibitory effect of linalool-rich essential oil from Lippia alba on the peptidase and keratinase activities of dermatophytes.

Abstract Lippia alba (Miller) N.E. Brown is an aromatic plant known locally as "Erva-cidreira-do-campo" that has great importance in Brazilian folk medicine. The aim of our study was to evaluate the antidermatophytic potential of linalool-rich essential oil (EO) from L. alba and analyze the ability of this EO to inhibit peptidase and keratinase activities, which are important virulence factors in dermatophytes. The minimum inhibitory concentrations (MICs) of L. alba EO were 39, 156 and 312 µg/mL against Trichophyton rubrum, Epidermophyton floccosum and Microsporum gypseum, respectively. To evaluate the influence of L. alba EO on the proteolytic and keratinolytic activities of these dermatophytes, specific inhibitory assays were performed. The results indicated that linalool-rich EO from L. alba inhibited the activity of proteases and keratinases secreted from dermatophytes, and this inhibition could be a possible mechanism of action against dermatophytes. Due to the effective antidermatophytic activity of L. alba EO, further experiments should be performed to explore the potential of this linalool-rich EO as an alternative antifungal therapy.

Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste.

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml(-1)). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.

Keratinase Production by Three Bacillus spp. Using Feather Meal and Whole Feather as Substrate in a Submerged Fermentation.

Three Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B. licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B. subtilis 1273, with 412 U/mL and 463 U/ml. The three strains were able to degrade feather meal (62-75%) and feather (40-95%) producing 3.9-4.4 mg/ml of soluble protein in feather meal medium and 1.9-3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest enzymatic activity.

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml(-1)). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15-140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40-50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50-70°C and pH 7.0-11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.

Increased levels of the CD40:CD40 ligand dyad in the cerebrospinal fluid of rats with vitamin B12(cobalamin)-deficient central neuropathy.

The levels of the soluble (s) CD40:sCD40 ligand (L) dyad, which belongs to the tumor necrosis factor (TNF)-alpha:TNF-alpha-receptor superfamily, are significantly increased in the cerebrospinal fluid (CSF), but not the serum of cobalamin (Cbl)-deficient (Cbl-D) rats. They were normalized or significantly reduced after treatment with Cbl, transforming growth factor-beta1 or S-adenosyl-L-methionine, and the normal myelin ultrastructure of the spinal cord was concomitantly restored. The concomitance of the two beneficial effects of these treatments strongly suggests that the increases in CSF sCD40:sCD40L levels may participate in the pathogenesis of purely myelinolytic Cbl-D central neuropathy in the rat. In keeping with this, an anti-CD40 treatment prevented myelin lesions.

Molecular alterations of cells resistant to platinum drugs: role of PKCalpha.

Development of resistance to platinum compounds may involve not only overexpression of defence mechanisms but also alterations in cellular response to the drug-induced genotoxic stress. To investigate the cellular bases of response to platinum compounds, we examined the profile of gene expression of ovarian carcinoma cells exhibiting sensitivity (A2780) or resistance (A2780/BBR3464) to platinum compounds. Using display PCR, we found that acquisition of resistance to the multinuclear platinum complex BBR3464 was associated with modulation of several transcripts, including up-regulation of the major substrate of protein kinase C (PKC), the myristoylated alanine-rich C kinase substrate (MARCKS). This feature was associated with PKCalpha down-regulation. To explore the role of PKCalpha in cellular sensitivity to platinum compounds, resistant cells were transfected with a PKCalpha-containing vector. PKCalpha-overexpressing resistant cells exhibited a decrease in sensitivity to cisplatin, whereas no significant change in sensitivity to BBR3464 was observed. A number of approaches designed to modulate the function or expression of PKCalpha support that the isoenzyme may play a role in determining resistance only to cisplatin but not to BBR3464, which is known to activate a different pathway of cell response. In conclusion, in spite of PKCalpha down-regulation in our model, its regulatory function was not apparently implicated in the development of resistance to platinum compounds and the present results do not support a general role of PKCalpha as a determinant of the resistance status.

Effect of glucose stress conditions in BL6T murine melanoma cells.

Elevated expression of stress proteins can be a characteristic of human cancer and may be involved in the development of resistance to some types of chemotherapeutic agent. In this paper, the effect of physiological stress conditions, such as glucose deprivation, was investigated in overexpressing nPKCdelta murine melanoma BL6 (BL6T) cells. Glucose stress conditions decreased the proliferative capacity, increasing the percentage of BL6T cells in the G0/G1 phase of the cell cycle. Furthermore, under such conditions, nPKCdelta, whose subcellular localization is cell cycle dependent, showed a cytoplasmic and perinuclear localization by immunohistochemistry, this being typical for cells in G0/G1 phase. Moreover, these cells expressed GRP-78, a known stress protein. On the other hand, glucose depletion enhanced intracellular melanin as well as tyrosinase activity and expression. In summary, these data demonstrate that stress conditions can modify the biological characteristics of BL6T cells, and therefore can select a quiescent cellular population.

Inorganic mercury changes the fate of murine CNS stem cells.

Stem cells isolated from the central nervous system of both embryonic and adult mice can generate neurons and glia through the activation of different patterns of differentiation in dependence of exposure to appropriate epigenetic signals. On the other hand, environmental conditions might affect the proliferation, migration, and differentiation of these cells. We report here, for the first time, that inorganic mercury affects the proliferative and differentiative capacity of adult neuronal stem cells (ANSCs). Actually, inorganic mercury increases apoptosis in ASNC. Furthermore, in stem cell-derived astrocytes, high levels of the 70 kDa heat shock protein (HSP-70) occur, while the levels of GTP-beta-tubulin activity dramatically decrease. Interestingly, when induced to differentiate, inorganic mercury modifies morphological proprieties of astrocytes, while the neuron population is reduced. These results demonstrate that inorganic mercury produces toxicity in the ANSC-derived neuronal population and affects the biological properties of the glial-derived population.