Abstract:
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.
Description:
Submitted in complete fulfillment for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2010.
