Optimization strategy for inulinase production by Aspergillus niger URM5741 and its biochemical characterization, kinetic/thermodynamic study, and application on inulin and sucrose hydrolysis.

Inulinases are enzymes of great interest in the food industry, especially due to their application in the synthesis of fructose and fructo-oligosaccharides. Moreover, some inulinases (I) also present invertase activity (S), making them useful for sucrose hydrolysis processes. In the present study, the production of inulinase by Aspergillus niger URM5741 was evaluated and optimized using two statistical approaches. First, the composition of the cultivation medium was determined through a simplex centroid mixture design, followed by the selection of optimal fermentation conditions using the Box-Behnken design. Based on these experimental designs, the maximum activities of inulinase (16.68 U mL-1) and invertase (27.80 U mL-1) were achieved using a mixture of wheat, soy, and oat brans (5 g), along with 2.5% inulin and 40% moisture. The inulinase exhibited optimum temperature and pH of 60 °C and 4.0, respectively, displayed a high affinity for both substrates, as evidenced by very-low Michaelis constant values (1.07-1.54 mM). A relative thermostability was observed at 55-60 °C as indicated by half-lives values (I: 169.06-137.27 min; S: 173.29-141.52 min) and D-values (I: 561.61-456.00 min; S: 575.65-470.11 min) which were further confirmed by the high activation energy (123.01 and 143.29 kJ mol-1). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80 g L-1, respectively. These results indicate that the sequential statistical approach proved to be beneficial to produce inulinase by A. niger URM5741, with the obtained enzyme considered promising for long-term industrial applications.

Production, Kinetic/Thermodynamic Study, and Evaluation of the Influence of Static Magnetic Field on Kinetic Parameters of ß-Fructofuranosidase from Aspergillus tamarii Kita UCP 1279 Produced by Solid-State Fermentation.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

Production, Biochemical Characterization, and Kinetic/Thermodynamic Study of Inulinase from Aspergillus terreus URM4658.

Inulinases are enzymes involved in the hydrolysis of inulin, which can be used in the food industry to produce high-fructose syrups and fructo-oligosaccharides. For this purpose, different Aspergillus strains and substrates were tested for inulinase production by solid-state fermentation, among which Aspergillus terreus URM4658 grown on wheat bran showed the highest activity (15.08 U mL-1). The inulinase produced by this strain exhibited optimum activity at 60 °C and pH 4.0. A detailed kinetic/thermodynamic study was performed on the inulin hydrolysis reaction and enzyme thermal inactivation. Inulinase was shown to have a high affinity for substrate evidenced by very-low Michaelis constant values (0.78-2.02 mM), which together with a low activation energy (19.59 kJ mol-1), indicates good enzyme catalytic potential. Moreover, its long half-life (t1/2 = 519.86 min) and very high D-value (1726.94 min) at 60 °C suggested great thermostability, which was confirmed by the thermodynamic parameters of its thermal denaturation, namely the activation energy of thermal denaturation (E*d = 182.18 kJ mol-1) and Gibbs free energy (106.18 ≤ ΔG*d ≤ 111.56 kJ mol-1). These results indicate that A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications.

Extraction and purification of Aspergillus tamarii ß-fructofuranosidase with transfructosylating activity using aqueous biphasic systems (PEG/phosphate) and magnetic field.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.

Extractive fermentation for process integration of protease production by Aspergillus tamarii Kita UCP1279 and purification by PEG-Citrate Aqueous Two-Phase System.

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 from Aspergillus tamarii Kita UCP1279 by extractive fermentation, obtaining as a response the partition coefficient (K), activity yield (Y) and concentration factor (CF). The enzyme preferably partitioned to the top phase and obtained in the system formed by variables MPEG = 400 g mol-1, CPEG = 20% (w w-1), and CCIT = 20% (w w-1) and pH 6, in this condition were obtained CF = 1.90 and Y = 79.90%. The protease showed stability at a temperature of 60 °C for 180 min, with optimum temperature 40 °C and pH 8.0. For the ions and inhibitors effects, the protease activity increased when exposed to Fe2+, Ca2+ and Zn2 + and inhibited by EDTA, being classified as metalloprotease. The kinetic parameters Km (35.63 mg mL-1) and Vmax (1.205 mg mL-1 min-1) were also estimated. Thus, the protease showed desirable characteristics that enable future industrial applications, especially, for beer industry.

Kinetic/thermodynamic study of immobilized ß-fructofuranosidase from Aspergillus tamarii URM4634 in chitosan beads and application on invert sugar production in packed bed reactor.

ß-fructofuranosidase (FFase) from Aspergillus tamarii URM4634 was immobilized covalently in chitosan beads. It was characterized biochemically, studied in terms of kinetic and thermodynamic parameters, and applied on conversion of sucrose for invert sugar production in a packed bed reactor (PBR). The optimum reactional conditions were determined and obtained at pH 5.0 and 60 °C. FFase was thermostable at 50-55°C. At 50°C, the enzyme shows longer half-life (t1/2) (594.13 min) and a higher D-value (1,973.64 min). This indicates that immobilized FFase was stable at temperature commonly used in invert sugar production. The following thermodynamic parameters were obtained: activation energy (E*d = 301.57 kJ mol-1), enthalpy (298.76 ≤ ΔH*d ≤ 298.89 kJ mol-1), entropy (579.88 ≤ ΔS*d ≤ 589.27 J K-1 mol-1) and Gibbs free energy (100.29 ≤ ΔG*d ≤ 108.47 kJ mol-1). The high E*d, ΔH*d and ΔG*d values confirmed FFase thermostability. The high and positive values for ΔS*d indicate an increase in disorder due opening of the enzyme structure. The sucrose hydrolysis in PBR showed a maximum invert sugar yield (96.0%) at 15 min of operation. The hydrolysis process remained efficient up to 100 min (70.22%). The results obtained in the present study provide a good indication that immobilized FFase on chitosan beads in PBR is efficient to invert sugar production for food industry.

Production, extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems.

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (MPEG), concentrations of PEG (CPEG) and citrate (CCIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°m (-10.89 kJ mol-1), ΔHm (-5.0 kJ mol-1) and partition ΔSm (19.74 J mol-1 K-1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.

Fructo-oligosaccharides production by an Aspergillus aculeatus commercial enzyme preparation with fructosyltransferase activity covalently immobilized on Fe3O4-chitosan-magnetic nanoparticles.

Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4-chitosan- magnetic nanoparticles. Immobilization carried out according to a 23-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g L-1, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase with transfructosylating activity.

This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.

Thermodynamic and kinetic studies on pectinase extracted from Aspergillus aculeatus: Free and immobilized enzyme entrapped in alginate beads.

The kinetics and thermodynamics of Aspergillus aculeatus pectinase, either free or immobilized in alginate beads, were investigated. Pectinase immobilization ensured an enzyme immobilization yield of 59.71%. The irreversible denaturation of pectinase in both preparations was evaluated at temperatures ranging from 30 to 60 °C. When temperature was raised, the first-order thermal denaturation constant increased from 0.0011 to 0.0231 min-1 for the free enzyme and from 0.0017 to 0.0700 min-1 for the immobilized one, respectively. The results of residual activity tests enabled us to estimate, for denaturation of both free and immobilized pectinase, the activation energy (E⁎d = 85.1 and 101.6 kJ·mol-1), enthalpy (82.59 ≤ ΔH⁎d ≤ 82.34 kJ·mol-1 and 99.11 ≤ ΔH⁎d ≤ 98.86 kJ·mol-1), entropy (-63.26 ≤ ΔS⁎d ≤ -63.85 J·mol-1·K-1 and -5.50 ≤ ΔS⁎d ≤ -5.23 J·mol-1·K-1) and Gibbs free energy (101.8 ≤ ΔG⁎d ≤ 104.7 kJ·mol-1 and 100.6 ≤ ΔG⁎d ≤ 102.0 kJ·mol-1). The integral activity of a continuous system using the free and immobilized enzyme was also predicted, whose results indicated a satisfactory enzyme long-term thermostability in both preparations at temperatures commonly used to clarify juice. These results suggest that both free and immobilized pectinase from A. aculeatus may be profitably exploited in future food industrial applications, with special concern to the immobilized enzyme because of its reusability.