Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

Abstract

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett-Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 +/- 65 U/ml.

Comparative studies of the stability of free and immobilized inulinase from Kluyveromyces marxianus NRRL Y-7571 in aqueous-organic solutions

“…These values are in agreement with previous work carried out by Mazutti et al (2007) and Treichel et al (2009), who evaluated the free enzyme in 0.1M sodium acetate buffer at pH 4.8. Mazutti et al (2007) found 17.2 U.mL -1 for v max and 7.1 mM for k m , while Treichel et al (2009) reported 10.7 U.mL -1 and 13.0 mM for v max and k m , respectively.…”

“…Since Ed values represents the energy necessary to start the inactivation reaction and this value was lower for the immobilized enzyme than that for the free enzyme, one can conclude that the immobilized enzyme was less stable than the free enzyme when using this immobilization method (Santos et al, 2007). These results are similar to those reported by Mazutti et al (2007) (343.9 kJ.mol -1 ) and lower than those of Treichel et al (2009) (527 kJ.mol -1 ) for free inulinase.…”

“…These values are quite different from those reported in the literature for the free enzyme in aqueous systems. Mazutti et al (2007) reported half-lives of 2224.0 and 322.0 minutes at 50°C and 55°C, respectively, and Treichel et al (2009) found half-lives of 4158.0 and 594.0 min at 50°C and 52.5°C, respectively, for the free inulinase from K. marxianus NRRL Y-7571 in sodium acetate buffer. These results suggest that the use of an organic solvent in the reaction medium decreases the free enzyme stability, as previously reported (Klibanov, 2001).…”

“…Mazutti et al (2007) reported that the free inulinase in sodium acetate buffer retained 67% of its initial activity after 3300.0 minutes of reaction at pH values of 4.5 and 4.8 and Treichel et al (2009) found a half-life of 4920.0 min at pH 4.4. The temperature used in all these experiments was 50°C.…”

<b>Agave syrup as a substrate for inulinase production by <i>Kluyveromyces marxianus</i> NRRL Y-7571

“…Although high levels of inulinase production have been reported for Aspergillus niger (Poorna & Kulkarne, 1995;Singh & Gill, 2006;Mutanda, Wilheimi, & Whiteley, 2008;Dinarvand et al, 2012), yeasts of the genus Kluyveromyces have also attracted great interest. Besides being considered as GRAS (Generally Recognized as Safe), those yeasts have been described as some of the best producers of inulinase in several fermentative processes (submerged and solid fermentation) with a wide range of substrates (Selvakumar & Pandey, 1999;Silva-Santisteban & Maugeri, 2005;Xiong, Jinhua, & Dongsheng, 2007;Treichel, Mazutti, Maugeri, & Rodrigues, 2009;Neagu & Bahrim, 2011).…”