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

Abstract

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 (5g), 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 (1.07-1.54mM). A relative thermostability was observed at 55-60°C as indicated by half-lives values (I: 169.06-137.27min; S: 173.29-141.52min) and D-values (I: 561.61-456.00min; S: 575.65-470.11min) which were further confirmed by the high activation energy (123.01 and 143.29kJmol-1). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80g 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.