C-phycocyanin decreases proliferation and migration of melanoma cells: In silico and in vitro evidences.

The incidence and number of deaths caused by melanoma have been increasing in recent years, and the pigment C-phycocyanin (C-PC) appears as a possible alternative to treat this disease. So, the objective of this study was to combine in silico and in vitro analysis to understand the main anti-melanoma pathways exerted by C-PC. We evaluated the ability of C-PC to bind to the main cellular targets related in the progression of melanoma through molecular docking, and the reflection of this bind in the biological effects in the B16F10 cell line through in vitro analysis. Our results showed that C-PC was able to bind BRAF and MEK, which are related to the signal transduction pathway for proliferation and survival. There was also an interaction between C-PC and cyclin-dependent kinase 4 and 6. In vitro analysis demonstrated that C-PC decreased B16F10 cell proliferation, as observed by cell viability and mitotic index assays. C-PC also interacted with matrix metalloproteinase 2 and 9 and N-cadherin, which may have caused the decrease in cell migration observed in vitro. Besides that, C-PC interacts with VEGF, a factor responsible for regulating the proliferation and cellular invasion pathways. Finally, C-PC did not alter the cell viability of the non-tumoral melanocytes. Therefore, C-PC is a strong anti-tumor candidate for the treatment of melanoma, since it acts in different cellular pathways of melanoma, without causing damage to non-tumoral cells.

Purification of xylanases from Aureobasidium pullulans CCT 1261 and its application in the production of xylooligosaccharides.

Xylanolytic enzymes are involved in xylan hydrolysis, the main ones being endo-ß-1,4-xylanases (xylanases). This can be applied in the bioconversion of lignocellulosic materials into value-added products such as xylooligosaccharides (XOS). This study aimed to establish a protocol for the purification of xylanases, as well as to characterize and apply the purified enzyme extract in the production of XOS. The enzyme purification techniques studied were ammonium sulfate ((NH4)2SO4) and ethanol precipitation. Purification of xylanase by fractional precipitation (20-60%) with (NH4)2SO4 was more efficient than with ethanol because the salt precipitation reached a purification factor of 10.27-fold and an enzymatic recovery of 48.6% The purified xylanase exhibited optimum temperature and pH of 50 °C and 4.5, respectively. The Michaelis-Menten constant using beechwood xylan for the enzyme was 74.9 mg/mL. The addition of salts such as CaCl2, ZnCl2, and FeCl3 in the reaction medium increased the xylanase activity. Xylanase showed greater thermal stability (half-life = 169 h) at 45 °C and pH 4.5. Under these conditions and in the presence of Ca2+ (10 mmol/L) the enzyme was even more stable (half-life = 231 h). Total XOS contents (6.7 mg/mL) and the conversion of xylan to XOS (22.3%) between 2 and 24 h were statistically equal. The hydrolysates showed the majority composition of xylobiose, xylotriose, and xylose. The addition of Ca2+ ions did not contribute to an increase in the total XOS content or to a greater conversion of xylan into XOS, but the hydrolysate was richer in xylobiose and had a lower xylose content.

Xylooligosaccharide Production with Low Xylose Release Using Crude Xylanase from Aureobasidium pullulans: Effect of the Enzymatic Hydrolysis Parameters.

Xylooligosaccharides (XOS) are non-digestible and fermentable oligomers that stand out for their efficient production by enzymatic hydrolysis and beneficial effects on human health. This study aimed to investigate the influence of the main reaction parameters of the beechwood xylan hydrolysis using crude xylanase from Aureobasidium pullulans CCT 1261, thus achieving the maximum XOS production. The effects of temperature (40 to 50 °C), reaction time (12 to 48 h), type of agitation, substrate concentration (1 to 6%, w/v), xylanase loading (100 to 300 U/g xylan), and pH (4.0 to 6.0) on the XOS production were fully evaluated. The most suitable conditions for XOS production included orbital shaking of 180 rpm, 40 °C, and 24 h of reaction. High contents of total XOS (10.1 mg/mL) and XOS with degree of polymerization (DP) of 2-3 (9.7 mg/mL), besides to a high percentage of XOS (99.1%), were obtained at 6% (w/v) of beechwood xylan, xylanase loading of 260 U/g xylan, and pH 6.0. The establishment of the best hydrolysis conditions allowed increasing both the content of total XOS 1.5-fold and the percentage of XOS by 9.4%, when compared to the initial production (6.7 mg/mL and 89.7%, respectively). Thus, this study established an efficient enzymatic hydrolysis process that results in a hydrolysate containing XOS with potential prebiotic character (i.e., rich in XOS with DP 2-3) and low xylose amounts.

Enzymatic synthesis optimization of isoamyl butyrate from fusel oil

Many food, cosmetic and pharmaceutical industries have increased their interest in short-chain esters due to their flavor properties. From the industrial standpoint, enzyme reactions are the most economical strategy to reach green products with neither toxicity nor damage to human health. Isoamyl butyrate (pear flavor) was synthesized by isoamyl alcohol (a byproduct of alcohol production) and butyric acid with the use of the immobilized lipase Lipozyme TL IM and hexane as solvents. Reaction variables (temperature, butyric acid concentration, isoamyl alcohol:butyric acid molar ratio and enzyme concentration) were investigated in ester conversion (%), concentration (mol L-1) and productivity (mmol ester g-1 mixture . h), by applying a sequential strategy of the Fractional Factorial Design (FFD) and the Central Composite Rotatable Design (CCRD). High isoamyl butyrate conversion of 95.8% was achieved at 24 hours. At 3 hours, the highest isoamyl butyrate concentration (1.64 mol L-1) and productivity (0.19 mmol ester g-1 mixture . h) were obtained under different reaction conditions. Due to high specificity and selectivity of lipases, process parameters of this study and their interaction with the Lipozyme TL IM are fundamental to understand and optimize the system so as to achieve maximum yield to scale up. Results show that fusel oil may be recycled by the green chemistry process proposed by this study.

Xylooligosaccharides production from wheat middlings bioprocessed with Bacillus subtilis.

Prebiotic compounds are substrates selectively metabolized by beneficial gut microbiota causing a health-promoting effect. Despite some prebiotic carbohydrates have been largely studied, xylooligosaccharides (XOS) are important prebiotics derived from arabinoxylans, which are polysaccharides found in cereals. This study aimed to investigate the production of xylanolytic enzymes and XOS during bioprocessing of wheat middlings, a product derived from wheat flour production, using a probiotic Bacillus subtilis. The composition of XOS and the enzymatic and prebiotic activities of resulting B. subtilis cultures were evaluated. The activity of xylanolytic enzymes continuously enhanced during the 72 h bacterial growth, where ß-xylosidase presented the highest value (70.31 U/mL). XOS profile and concentration varied considerably between control and bioprocessed samples and among these at different times. Maximum prebiotic activity score was found for the 24 h and 72 h bioprocessed samples (1.73 and 1.61, respectively) using the commercial probiotic Lactobacillus acidophilus LA-5. Wheat middlings showed to be a promising substrate for production of prebiotics like XOS and B. subtilis FTC01 appears to be a good source of xylanolytic enzymes.

Cell pretreatment with ethylenediaminetetraacetic acid for selective extraction of C-phycocyanin with food grade purity.

C-phycocyanin (C-PC) is a natural blue dye, and depending on its purity, which is measured by the ratio between the absorbance of the chromophore (A620 ) and the absorbance of the proteins (A280 ), it can be used in food (purity > 0.7), cosmetics (purity > 1.5), and therapeutic treatments (purity > 4.0). Several physical, chemical, and enzymatic methods of extraction are reported, however, few are able to extract C-PC with purity above 0.7. An innovative method of C-PC extraction with food grade purity from wet Spirulina platensis biomass is proposed. The cells were pretreated with ethylenediaminetetraacetic acid and subsequent C-PC extraction was performed with tris-(hydroxymethyl) aminomethane-SO4 buffer. C-PC was released after 12 h of cell pretreatment. Six variables of the extraction process were evaluated. The extraction temperature significantly influenced C-PC extraction yield and purity. In the best condition of cell pretreatment and extraction, C-PC with purity of 1.0 and extraction yield of 129.0 mg/g could be obtained to be used as a food dye without any purification process. Lastly, an ultrafiltration process was integrated and C-PC was concentrated 8.8-fold, resulting in purity of 1.6 and recovery of 93.4%.

Simultaneous production of proteases and antioxidant compounds from agro-industrial by-products.

The use of processes for simultaneous production of bioproducts as enzymes and bioactive compounds is an interesting alternative to reduce environmental impacts. Thus, the aim of this study was to produce simultaneously, using the biorefinery concept, both proteases and bioactive compounds with antioxidant activity from Bacillus sp. P45 cultivation by using different by-products. The integrated process developed in this study enabled to obtain enzymes with proteolytic and keratinolytic properties in a process with alternate substrates from agro-industrial by-products (feather meal, residual feather meal and biomass), thus, creating an interesting alternative to managing them. The residual biomass provided the highest protease activity (1306.6U/mL) and the reused feather meal reached the highest keratinolytic activity (89U/mL), both at 32h of cultivation. Moreover, hydrolysates produced in cultivation using feather meal and residual biomass had high antioxidant activity, they have great potential as natural antioxidants.

Co-production of carbonic anhydrase and phycobiliproteins by Spirulina sp. and Synechococcus nidulans.

The aim of this work was to study the co-production of the carbonic anhydrase, C-phycocyanin and allophycocyanin during cyanobacteria growth. Spirulina sp. LEB 18 demonstrated a high potential for simultaneously obtaining the three products, achieving a carbonic anhydrase (CA) productivity of 0.97U/L/d and the highest C-phycocyanin (PC, 5.9µg/mL/d) and allophycocyanin (APC, 4.3µg/mL/d) productivities. In the extraction study, high extraction yields were obtained from Spirulina using an ultrasonic homogenizer (CA: 25.5U/g; PC: 90mg/g; APC: 70mg/g). From the same biomass, it was possible to obtain three biomolecules that present high industrial value.

Production of Nanofibers Containing the Bioactive Compound C-Phycocyanin.

C-phycocyanin (C-PC) is a water-soluble phycobiliprotein present in light-harvesting antenna system of cyanobacteria. The nanostructures have not been widely evaluated, precluding improvements in stability and application of the C-PC. Electrospun nanofibers have an extremely high specific surface area due to their small diameter, they can be produced from a wide variety of polymers, and they are successfully evaluated to increase the efficacy of antitumor drugs. The incorporation of C-PC into nanofibers would allow investigations of potential uses in alternative cancer treatments and tissue engineering scaffolds. In this paper, C-phycocyanin were incorporated into the polymer polyethylene oxide (PEO) in various concentrations for nanofiber production via an electrospinning process. Nanofibers structures were analyzed using digital optical microscopy and scanning electron microscopy (SEM). Thermogravimetric analysis was performed on the pure starting compounds and the produced nanofibers. At a concentration of 2% (w/w) of PEO, nanofibers were not produced, and concentrations of 4% (w/w) of PEO failed to produce nanofibers of good quality. Solutions with 6% (w/w) PEO, 6% (w/w) PEO plus 1% (w/w) NaCI, and 8% (w/w) PEO promote the formation of bluish, homogeneous and bead-free nanofibers with average diameters varying between 542.1 and 759.9 nm, as evaluated by optical microscopy. SEM analysis showed that nanofibers produced from polymer solutions containing 6% (w/w) PEO, 1% (w/w) NaCl and 3% (w/w) C-PC have an average diameter of 295 nm. Thermogravimetric analysis detected an increase in thermal resistance with the incorporation of C-phycocyanin into nanofibers.

Interference of salts used on aqueous two-phase systems on the quantification of total proteins.

In this study the interference of potassium phosphate, sodium citrate, sodium chloride and sodium nitrate salts on protein quantification by Bradford's method was assessed. Potassium phosphate and sodium citrate salts are commonly used in aqueous two-phase systems for enzyme purification. Results showed that the presence of potassium phosphate and sodium citrate salts increase the absorbance of the samples, when compared with the samples without any salt. The increase in absorptivity of the solution induces errors on protein quantification, which are propagated to the calculations of specific enzyme activity and consequently on purification factor. The presence of sodium chloride and sodium nitrate practically did not affect the absorbance of inulinase, probably the metals present in the enzyme extract did not interact with the added salts.

Raw Glycerol and Parboiled Rice Effluent for Carotenoid Production: Effect of the Composition of Culture Mediumand Initial pH.

Search for naturally grown food has stimulated the biotechnological production of carotenoids. Therefore, the use of the yeast Xanthophyllomonas dendrorhous has been researched due to its abilities to assimilate different sources as substrates and to produce high amounts of carotenoids. Furthermore, alternative sources have been used as the culture medium to reduce costs and environmental impact. A potent carotenoid is astaxanthin in view of its health-promoting and antioxidative properties. It consists of different geometrical isomers with trans and cis configuration. In X. dendrorhous this carotenoid is mostly found in the trans form, but cis isomers can also be found. Carotenoid production was investigated in culture medium containing by-products such as raw glycerol (from biodiesel) and parboiled rice effluent. The effects of the culture medium components on biomass concentration and specific and volumetric productions of carotenoids were verified by the Plackett-Burman design. Cultivations were carried out with yeast Xanthophyllomonas dendrorhous NRRL Y-17268 at 25 °C and 150 rpm for 168 h. In this study, maximum production of carotenoids was obtained under the following conditions (in g/L): raw glycerol 10, glucose 10, yeast extract 10, malt extract 10 and peptone 1 at pH=6. Resulting specific and volumetric productions of carotenoids were 326.8 and 4.1 µg/g, respectively.

Purification of C-phycocyanin from Spirulina platensis in aqueous two-phase systems using an experimental design

C-phycocyanin from Spirulina platensis was purified in aqueous two-phase systems (ATPS) of polyethylene glycol (PEG)/potassium phosphate, varying the molar mass of the PEG. Results using a full factorial design showed that an increase in the concentration of salt and decrease in the concentration of PEG caused an increment in the purification factor for all the ATPS studied. Optimization of the conditions of the purification was studied using a central composite rotatable design for each molar mass of PEG. The ATPS composed of 7% (w/w) PEG 1500 or 4% (w/w) PEG 8000 (g/gmol) and 23 or 22.5% (w/w) of phosphate resulted a purification factor of 1.6-fold for C-phycocyanin, with total and 57% recovery, respectively. Process conditions were optimized for the purification factor for the system with PEG 1500. The ATPS with 4% (w/w) PEG 4000 or 4% (w/w) PEG 6000 and 21% (w/w) phosphate resulted purification factors of 2.1 and 2.2-fold, recovering 100% and 73.5%, respectively of C-phycocyanin in the top phase.

Modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin.

This work is focused on the experimental evaluation and mathematical modeling of ion exchange expanded-bed chromatography for the purification of C-phycocyanin from crude fermentative broth containing Spirulina platensis cells. Experiments were carried out in different expansion degree to evaluate the process performance. The experimental breakthrough curves were used to estimate the mass transfer and kinetics parameters of the proposed model, using the Particle Swarm Optimization algorithm (PSO). The proposed model satisfactorily fitted the experimental data. The results from the model application pointed out that the increase in the initial bed height does not influence the process efficiency, however enables the operation of expanded-bed column at high volumetric flow rates, improving the productivity. It was also shown that the use of mathematical modeling was a good and promising tool for the optimization of chromatographic processes.

Effect of compressed fluids treatment on ß-galactosidase activity and stability.

This study aimed at assessing the influence of different pressurized fluids treatment on the enzymatic activity and stability of a lyophilized ß-galactosidase. The effects of system pressure, exposure time and depressurization rate, using propane, n-butane, carbon dioxide and liquefied petroleum gas on the enzymatic activity were evaluated. The ß-galactosidase activity changed significantly depending on the experimental conditions investigated, allowing the selection of the proper compressed fluid for advantageous application of this biocatalyst in enzymatic reactions. The residual activity ranged from 32.1 to 93.8 % after treatment. The storage stability of the enzyme after high-pressure pre-treatment was also monitored, and results showed that the biocatalyst activity presents strong dependence of the fluid used in the pretreatment. The activity gradually decreases over the time for the enzyme treated with LGP and propane, while the enzyme treated with n-butane maintained 96 % of its initial activity until 120 days. For CO(2), there was a reduction of around 40 % in the initial activity 90 days of storage. The enzyme treated with n-butane also showed a better thermostability in terms of enzymatic half-life.

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Purification of carbonic anhydrase from bovine erythrocytes and its application in the enzymic capture of carbon dioxide.

This work presents a study of industrially applicable techniques to obtain a biologically supported carbon dioxide capture system, based on the extraction of carbonic anhydrase from bovine blood. Carbonic anhydrase is a metalloenzyme which catalyzes the reversible hydration of carbon dioxide. The objective of this study was to establish conditions to obtain carbonic anhydrase from bovine erythrocytes and apply it in the capture of carbon dioxide. To achieve this, two different purification techniques were evaluated: one by extraction with the organic solvents chloroform and ethanol, where different solvent proportions were studied; and the other by ammonium sulfate precipitation, testing percent saturations between 10% and 80%. Carbon dioxide was enzymatically captured by its precipitation as calcium carbonate with the enzyme obtained by both techniques. The enzyme extracted by ethanol and chloroform showed an activity of 2623 U mL(-1), recovery of 98% and purification factor of 104-fold. That precipitated by ammonium sulfate showed an activity of 2162 U mL(-1), recovery of 66% and purification factor of 1.4-fold using 60% ammonium sulfate saturation. The results obtained in the carbon dioxide capture experiments showed that the carbonic anhydrase extracted in this study not only enhanced the hydration of CO(2), but also promoted the formation of CaCO(3).

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis.

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v (5-1) fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Maximization of beta-galactosidase production: a simultaneous investigation of agitation and aeration effects.

In this work, the agitation and aeration effects in the maximization of the beta-galactosidase production from Kluyveromyces marxianus CCT 7082 were investigated simultaneously, in relation to the volumetric enzyme activity and the productivity, as well as the analysis of the lactose consumption and production of glucose, and galactose of this process. Agitation and aeration effects were studied in a 2 L batch stirred reactor. A central composite design (2(2) trials plus three central points) was carried out. Agitation speed varied from 200 to 500 rpm and aeration rate from 0.5 to 1.5 vvm. It has been shown in this study that the volumetric enzyme production was strongly influenced by mixing conditions, while aeration was shown to be less significant. Linear models for activity and productivity due to agitation and aeration were obtained. The favorable condition was 500 rpm and 1.5 vvm, which lead to the best production of 17 U mL(-1) for enzymatic activity, 1.2 U mL(-1) h(-1) for productivity in 14 h of process, a cellular concentration of 11 mg mL(-1), and a 167.2 h(-1) volumetric oxygen transfer coefficient.

Strategy for a protein purification design using C-phycocyanin extract.

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.

Mathematical modeling and simulation of inulinase adsorption in expanded bed column.

A mathematical model for an expanded bed column was developed to predict breakthrough curves for inulinase adsorption on Streamline SP ion-exchange adsorbent, using a crude fermentative broth with cells as the feedstock. The kinetics and mass transfer parameters were estimated using the PSO (particle swarm optimization) heuristic algorithm. The parameters were estimated for each expansion degree (ED) using three breakthrough curves at initial inulinase concentrations of 65.6UmL(-1). In sequence, the model parameters for an ED of 2.5 were validated using the breakthrough curve at an initial concentration of 114.4UmL(-1). The applicability of the validated model in process optimization was investigated, using the model as a process simulator and experimental design methodology to optimize the column and process efficiencies. The results demonstrated the usefulness of this methodology for expanded bed adsorption processes.