ABSTRACT
An extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purified to homogeneity by gel filtration chromatography and showed a specific activity of 119 U/mg. The optimum pH and temperature of the purified enzyme were found to be 6.0 and 50 °C, respectively. The enzyme was stable up to 60 °C, retaining 60% of residual activity for 30 min, but inactivated rapidly above 60 °C. The enzyme was found to be stable at pH=6-9 when it retained 100% of its residual activity. The Lineweaver-Burk plot showed that the apparent Km and vmax values of the inulinase when using inulin as a substrate were 1.15 mg/mL and 0.15 µM/min, respectively, whereas the kcat value was found to be 0.145 min-1. The calculated catalytic efficiency of the enzyme was found to be 0.126 (mg·min)/mL. The purified inulinase can be used in the production of high fructose syrups.
ABSTRACT
Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 +/- 0.03 U mL(-1)) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 +/- 0.03 U mL(-1) after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L(-1) h(-1)) and specific (119,025 U g(-1) h(-1)) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L(-1) h(-1) and specific productivity of 72 g g(-1) h(-1) FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.
