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, 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.

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.