Purification and Characterization of an Endoinulinase from Xanthomonas campestris pv. phaseoli KM 24 Mutant

Abstract

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.

Directed evolution of an endoinulinase from Talaromyces purpureogenus toward efficient production of inulooligosaccharides

“…For example, A. ficuum endoinulinase expressed and purified from E. coli exhibited a specific activity of 75.22 U/mg, the Bacilus smithii T7 endoinulinase had a reported specific activity of 833 U/mg and the Xanthomonas campestris pv. phaseoli KM 24 mutant endoinulinase showed a specific activity of 119 U/mg . Recently, expression of the A. fumigatus Cl1 endoinulinase in E. coli using high cell‐density fermentation, yielded an enzyme with specific activity similar to that of our evolved variants (1590 U/mg) …”

“…phaseoli KM 24 mutant endoinulinase showed a specific activity of 119 U/mg. 35 Recently, expression of the A. fumigatus Cl1 endoinulinase in E. coli using high celldensity fermentation, yielded an enzyme with specific activity similar to that of our evolved variants (1590 U/mg). 36 Analysis of IOS distribution resulting from inulin hydrolysis using the evolved variants TLC and HPLC were used to characterize the distribution of products resulting from inulin hydrolysis by the purified reTP and its evolved variants.…”

Efficient Precipitation Methods of Inulinase from Endophytic Bacteria Bacillus aquimaris Isolated from Jerusalem Artichoke

“…The inulinase of X. campestris pv. phaseoli KM24 was precipitated at 20-100% of ammonium sulfate for 16 h [19]. The requirement of salt concentration for precipitation is dependent on the properties and concentration of proteins present in the starting solution.…”

“…inulinase of Bacillus cereus MU-31 was precipitated at 80% [17], inulinase of Pseudomonas putida was 40-80% [18] and inulinase of Xanthomonas campestris pv. phaseoli KM24 was 20-100% [19]. The 3 phase protein portioning of inulinase was reported in Aspergillus niger using 30% of ammonium sulfate with 1.0 : 0.5 v/v ratio of t-butanol, giving 88% recovery and 10.2 fold of purification [20].…”

A Novel Inulin-Mediated Ethanol Precipitation Method for Separating Endo-Inulinase From Inulinases for Inulooligosaccharides Production From Inulin

“…Therefore, several approaches have been carried out in recent years to promote endo-inulinase functionality. One reported approach involved chromatographic isolation of endo-inulinase from natural inulinases produced by varying microorganisms ( Park et al, 1999 ; Cho and Yun, 2002 ; Jin et al, 2005 ; Naidoo et al, 2015 ). Unfortunately, the use of column chromatography is an expensive and complex approach which places significant cost burden upon an IOS process ( Golunski et al, 2011 ).…”