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The present study evaluated the influence of the variables polyethylene glycol (PEG) molar mass, pH, PEG concentration and sodium citrate concentration in the integrated production of the protease ...

β-fructofuranosidases (EC3.2.1.26) are members of the GH32 family of glycoside hydrolases, which include more than 390 enzymes of vegetable and microbial origins, used in several biotechnological applications. Thus, this research aimed to produce a β-fructofuranosidase obtained by Aspergillus tamarii through solid state fermentation, and to purify by Aqueous Two-Phase System (ATPS). Summary results presented the optimal parameters to produce the β-fructofuranosidase used wheat bran as a substrate at 30 °C for 48 h, and purification process using ATPS with polyethylene glycol and sodium citrate (PEG/sodium citrate), where the β-fructofuranosidase preferably partitioned to the salt-rich phase, the best run (24% of PEG 400, 20% sodium citrate, pH 8) which presented a higher purification factor 6.42 with 12.39 U/mL activity and 352% yield. Optimum parameter was pH 5.15 and temperature of 55 °C, respectively. The purified enzyme showed excellent thermal stability and exhibited a half-life of 60 min at 65 °C. Kinetics results for enzyme showed for Sucrose substrate the enzyme showed Km of 42.9 ± 2.21 mM and Vmax of 180.2 ± 2.8 μM min−1 mg−1 of protein. Although Vmax was the highest for 1-Kestose (219.4 ± 2.7 μM min−1 mg−1 of protein) the preferred substrate of Aspergillus tamarii β-fructofuranosidase (β-FFase) was Nystose (Km of 3.8 ± 0.15 mM). SDS-PAGE revealed a single band of protein at ~66 kDa. Finally, this study demonstrated the potential of ATPS to purify a β-fructofuranosidase with application in biotechnological field aiming to functional foods.

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 23 full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000 g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000 g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDSPAGE and fibrin zymography showed that the purified protease has a molecular mass of 97 kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37° C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150 min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification., The authors acknowledge the financial support of the Brazilian Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE), and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq). The authors also thank the project approved in the REN-NORFUN network (MCT/CNPq/MMA/MEC/CAPES/FNDCT, Acao Transversal/FAPs, No.47/2010, Sistema Nacional de Pesquisa em Biodiversidade - SISBIOTA/Brazil).

This work explores the possibility of using a polyethylene glycol (PEG)/sodium citrate aqueous two-phase system (ATPS) as a first step in a process for the purification of lectin from Canavalia grandiflora seed. Purification of lectins is a limiting step for its uses in the area of biotechnology. Extraction by an aqueous two-phase system is a powerful technique for separation, concentration, and purification of biomolecules and pharmaceutical products. Four factors (PEG's molar mass, PEG's concentration, pH, citrate concentration) affecting the lectin ConGF partitioning were studied. A two-level factorial design (24) was carried out. Lectin ConGF preferentially partitioned to the top (polyethylene glycol) phase. The statistical analysis showed that the citrate concentration chloride significantly affects the KL (partitioning coefficient for protein) value for lectin partitioning. An ATPS composed of 20% (w/w) PEG 400 and 20% (w/w) citrate, at pH 6, allowed the recovery of lectin ConGF with an 8.67 partition coefficient and 104% yield. Consequently, the system proved to be efficient and can be used as a first step in lectin purification from crude extract of ConGF seeds.

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (MPEG) and concentration (CPEG), the citrate concentration (CC) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing CC, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing CC. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using MPEG = 10,000 g/mol, CPEG = 22% (w/w) and CC = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131%) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth.

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.