José Carlos Curvelo Santana

Extraction of lipase from Burkholderia cepacia by PEG/Phosphate ATPS and its biochemical characterization

ABSTRACT This work aimed to study the partitioning of a lipase produced by Burkholderia cepacia in PEG/Phosphate aqueous two phase system (ATPS) and its characterization. Lipase was produced by B. cepacia strains in a fermenter. Enzyme partitioning occurred at pH 6.0 and 8.0, using PEG 1500 and 6000 on two tie lines. Metal ions, pH and temperature effects on enzyme activity were evaluated. Five milliliter of 7.5% olive oil emulsion with 2.5% gum-arabic in 0.1M sodium phosphate buffer at pH 8.0 and 37oC were used for the activity determinations. Results showed that crude stratum from B. cepacia was partitioned by PEG1500/phosphate ATPS at pH 6.0 or 8.0 for, which the partitioning coefficients were 108- and 209-folds. Lipase presented optimal activity conditions at 37 o C and pH 8.0; it showed pH-stability for 4 h of incubation at different pH values at 37 o C. Metal ions such as Mn 2+ , Co 2+ , I - and Ca 2+ sustained enzymatic activities; however, it was inhibited by the presence of Fe

Expanded bed adsorption of bromelain (E.C. 3.4.22.33) from Ananas comosus crude extract

This work focuses on the adsorption of Bromelain in expanded bed conditions, such as the adsorption kinetics parameters. The adsorption kinetics parameters showed that after 40 minutes equilibrium was achieved and maximum adsorption capacity was 6.11 U per resin mL. However, the maximum adsorption capacity was only determined by measuring the adsorption isotherm. Only by the Langmuir model the maximum adsorption capacity, Qm, and dissociation constant, kd, values could be estimated as 9.18 U/mL and 0.591, respectively, at 25 oC and 0.1 mol/L phosphate buffer pH 7.5. A column made of glass with an inner diameter of 1 cm was used for the expanded bed adsorption (EBA). The residence time was reduced 10 fold by increasing the expansion degree 2.5 times; nonetheless, the plate number (N) value was reduced only 2 fold. After adsorption, the bromelain was eluted in packed bed mode, with a downward flow. The purification factor was about 13 fold and the total protein was reduced 4 fold. EBA showed to be feasible for purification of bromelain.

Production and characterization of amylases from Zea mays malt

In this work the α and β-amylase enzymes were obtained from maize (Zea mays) malt and were biochemistry characterized. A germination study to obtain the maize malt with good amylase activity was made. The maize seeds were germinated in laboratory and the enzymatic activity was measured daily. Activity dependence to germination time were fitted to an exponential model (A=A0eµt), which showed that the behaviour of enzymatic activity in the germination process was similar to the growth of the microorganism. Its model could be applied to describe the mechanism of α-amylase production for each maize varieties and others cereals. Maize malt characterization showed that α and β-amylase had optimal pH between 4-6.5, optimal temperature 50 and 90oC, and molecular weight of 67.4 and 47.5kDa, respectively. This work contributed with the advances in biotechnology generating of conditions for application of a new and of low price amylases source.

Partitioning optimization of proteins from Zea mays malt in ATPS PEG 6000/CaCl2

This work aimed to establish the relationship between the compositions and pH of ATPS PEG 6000/CaCl2 and the proteins partition from maize malt and also to simplify the process optimization in ATPS for a statistical model, established by response surface methodology (RSM). Results showed that these were no influence of pH on the phase diagrams and on the composition of tie line length of PEG 6000/CaCl2 ATPS. SRM analyses showed that elevated pH and larger tie line length were the best conditions for recovering of maize malt proteins. The maximum partition coefficient by PEG 6000/CaCl2 ATPS was about 4.2 and was achieved in ATPS in a single purification step. The theoretical maximum partition coefficient was between 4.1-4.3. The process was very suitable for continuous aqueous two-phase purification due to the stability of proteins (e.g. and -amylases) and could increase their content into middle.

Batch and continuous extraction of bromelain enzyme by reversed micelles

The main aim of this study was to optimize the conditions for bromelain extraction by reversed micelles from pineapple juice (Ananas comosus). The purification was carried out in batch extraction and a micro-column with pulsed caps for continuous extraction. The cationic micellar solution was made of BDBAC as a surfactant, isooctane as a solvent and hexanol as a co-solvent. For the batch process, a purification factor of 3 times at the best values of surfactant agent, co-solvent and salt concentrations, pH of the back and forward extractions were, 100 mM, 10% v/v, 1 M, 3.5 and 8, respectively. For the continuous operation, independent variables optimal point was determined: ratio between light phase flow rate and total flow rate equal to 0.67 and 1 second for the time interval between the pulses. This optimal point led to a productivity of 1.29 mL/min and a purification factor of 4.96.

Optimization of drying process of Zea Mays malt to use as alternative source of amylolytics enzymes

This work aimed to study the drying process optimization of maize (Zea Mays) malt for obtaining maize malt, without affecting enzymatic activity of a e b-amylases from maize malt. Results showed that dryer operation must occur in zone at 54°C and 5.18-6 h process time. The maize malt obtained had good enzymatic properties.

The effect of pH on bromelain partition from Ananas comosus by PEG4000/phosphate ATPS

In this work, the thermodynamic equilibrium and applying of PEG4000/Phosphate ATPS on the purification of bromelain extracted from pineapple was studied. A rigorous study of the equilibrium curves and tie-line length from PEG4000/phosphate ATPS were done for the pH 6-11 at 25oC. Results showed that there was augment in the PEG and salt contents with the high pH value from PEG4000/Phosphate ATPS and two-phase formation needed only increasing the PEG content. Two tie-line length at pH 11 from PEG4000/Phosphate ATPS were optimal condition for bromelain purification, one on composition of 14% PEG and 13% salt and other at 12.6% PEG and 12.2% salt, while a 25-62 folds of enzyme was found. SDS-PAGE electrophoreses had one band only, which showed that bromelain was purified. Optimum conditions of bromelain use were found at pH 7 and between 30-40oC.

Response surface methodology to evaluation the recovery of amylases by hollow fiber membrane

This work aimed to study the pH and the transmembrane pressure effects during the recovery of a and b amylases enzymes from corn malt (Zea mays) by hollow fiber membrane. The optimal condition was obtained for a statistical model, established by response surface methodology (RSM). The response surface analysis showed that the best operation condition for amylolitics enzymes recovery by hollow fiber membrane was 0.05 bar and pH 5.00, while the enzymes were purified about of 26 times.

Development of colors with sustainability: a comparative study between dyeing of cotton with reactive and vat dyestuffs

This study compared reactive dyestuffs and vat dyestuffs in the dyeing of cotton. We investigated the possibility of reuse of the effluent generated, the ecological costs, and the colorfastness to water, rubbing, daylight, and perspiration. The experiments with vat dyestuffs showed slight advantage in terms of ecological costs, generating less molecules of carbon dioxide than the experiments conducted with reactive dyestuffs, lower consumption of energy, and greater possibility for reuse of treated effluent. In addition, vat dyestuffs exhibited decolorization efficiency above 99% and rates of total organic carbon removal over 90% in all cases, in addition to higher values in colorfastness properties.

Purificação de amilases de malte de Zea mays

Neste trabalho realizou-se a purificacao das enzimas α e β-amilases de malte de milho combinando os processos de adsorcao em leito expandido com o fracionamento por etanol. Alimentaram-se 5 mL de uma solucao a 2% de malte de milho em tampao fosfato 0,05 M a pH 5 em uma coluna contendo as resinas de troca ionica Amberlite IRA 410, em leito fixo de 4 cm fluidizado para 8 cm. A eluicao foi promovida com NaCl 0,25M com 14 mL/minuto de vazao descendente e o material eluido foi fracionado com alcool etilico nas concentracoes de 40-70%, 50-80% e 60-90% (v/v) a 20 e 30 oC. A recuperacao da atividade e o fator de purificacao foram de 95% e 23 vezes, respectivamente, na primeira etapa. A melhor condicao de fracionamento das enzimas foi apresentada a 50-80% de alcool e 20 oC, e o fator de purificacao ficou acima de 100 vezes.