Identification and characterization of a novel inulin binding module (IBM) from the CFTase of Bacillus macerans CFC1.
A novel inulin-binding module (IBM), which was identified from the N-terminal region of the cycloinulinooligosaccharide fructanotransferase (CFTase) in Bacillus macerans CFC1, was characterized using the discrete entity of IBM produced by the recombinant Escherichia coli strains. Deletion analyses located the inulin binding activity in the N-terminal region between 241 and 389 amino acid residues, which was removed from the mature enzyme by processing when secreted from the B. macerans CFC1 cells. IBM bound specifically to polyfructans such as inulin and levan but it did not interact with any of the glycan polymers tested in this study including cellulose, xylan, and starch. Binding studies on the IBM revealed that the equilibrium dissociation constant K(d) and the maximum amount of protein bound [(PC)(max)] were 4.7 microM and 22 microM g(-1), respectively. Together, these results indicate that the IBM of CFTase has a relatively high and specific affinity for inulin. Adsorption of the IBM to inulin was highest at pH 7.0 and lowered slowly with decreasing pH down to 3.0. At pH 7.0, the binding activity was enhanced about twofold by the presence of 1 M MgCl(2). Chemical modification experiments with the aromatic amino acid-specific modifiers implied that tryptophan and tyrosine residues in the IBM are likely to participate in the interaction with the inulin molecules.
Lee
Ji-Hyun
JH
Graduate School of Life Science and Biotechnology, Korea University, Seoul 136-701, Republic of South Korea.
Kim
Kyung-Nam
KN
Choi
Yong-Jin
YJ
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005630:Fructans / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D005630:Fructans; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014990:Xylans; D015636:Magnesium Chloride; D012965:Sodium Chloride; D002482:Cellulose; D013213:Starch; D007444:Inulin; C072599:levan; D006602:Hexosyltransferases; C062589:cycloinulo-oligosaccharide fructanotransferase