No 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.

SummaryAn extracellular endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 mutant was purifi ed to homogeneity by gel fi ltration chromatography and showed a specifi c activity of 119 U/mg. The optimum pH and temperature of the purifi ed 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 K m and v max values of the inulinase when using inulin as a substrate were 1.15 mg/mL and 0.15 μM/min, respectively, whereas the k cat value was found to be 0.145 min -1 . The calculated catalytic effi ciency of the enzyme was found to be 0.126 (mg·min)/mL. The purifi ed inulinase can be used in the production of high fructose syrups.

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase on various carbon sources. The highest inulinase production of 9.24 ± 0.03 IU ml¯¹by X. campestris pv. phaseoli was attained using an optimized medium comprising of 3% sucrose and 2.5% tryptone. Inulinase production in X. campestris pv. phaseoli was further enhanced through ethylmethanesulfonate mutagenesis. The resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated enhanced inulinase production of 22.09 ± 0.03 IU ml¯¹after 24 h, which was 2.4 – fold higher than that of the wild type. Inulinase production by this mutant was scaled up in a 5 L fermenter yielding a final activity of 21.87 ± 0.03 IU ml¯¹with an inulinase/invertase (I/S) ratio of 2.6 after 18 h. Maximum volumetric (21 865 IU 1¯¹ h¯¹) and specific (119 025 IU g¯¹ h¯¹) productivities of inulinase were attained in a fermenter after 18h growth. Inulin hydrolysis by the crude inulinase and subsequent detection of mono- and oligosaccharides indicated the presence of an endoinulinase. The extracellular endoinulinase from the mutant KM 24 was purified to homogeneity by gel filtration chromatography and had a specific activity of 174.74U/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 over 60% activity for 30 min, but activity rapidly declined at temperatures above 60°C. The pure inulinase enzyme was also found to be stable between pH 6-9. The Lineweaver-Burk plots showed that the apparent Km and Vmax values of the inulinase for inulin were 1.15 mg/ml and 15µM/min, respectively. The Kcat value was found to be 0.145 min¯¹ with an enzyme catalytic efficiency of 0.126 mg¯¹.ml.min¯¹.This mutant demonstrated good potential for large scale production of inulinase and fructooligosaccharides.

No abstract