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Author: search.filters.author.Naidoo, Kameshnee

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    Purification and characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant
    (University of Zagreb, 2015) Naidoo, Kameshnee; Kumar, Ajit; Sharma, Vikas; Permaul, Kugen; Singh, Suren
    An 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 activ-ity 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 residu-al 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 sub-strate 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 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 syr-ups.
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    Enhanced production of inulinase from Xanthomonas campestris pv. phaseoli
    (2010) Naidoo, Kameshnee; Singh, Suren; Permaul, Kugen
    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.