Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

Abstract

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 x 10(10) cells mL(-1) and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds(-1) h(-1). The maximum activity was obtained at pH 5.0 and 55.0 degrees C. Within the investigated range, the enzyme extract was more thermostable at 50.0 degrees C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol(-1). The extract showed highest stability from pH 4.5 to 4.8. Apparent K(m) and V(max) are 7.1 mM and 17.79 M min(-1), respectively.

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.…”

“…Using 50wt% of butyl acetate, free inulinase showed the highest catalytic power, since the activity was higher than at 25 and 70 wt% for both pH and temperature. Mazutti et al (2007) reported an optimal temperature and pH of 55.0°C and 5.0, respectively, for free inulinase obtained by solidstate fermentation using the same strain in sodium acetate buffer.…”

“…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.…”