Molecular Characterization of Cycloinulooligosaccharide Fructanotransferase from Bacillus macerans

Abstract

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of ␤-(231)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated M r of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their K m values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (k cat /K m ) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.

Purification and Properties of Cycloinulooligosaccharide Fructanotransferase from Paenibacillus polymyxa MG-CF6

“…The N terminal sequence was fully consistent with the amino acid sequence of 253 to 266 from Bacillus macerans CFC1 19) and Paenibacillus polymyxa MGL21 10) enzymes and also possessed about 78.5% similarity to the amino acid sequence (235 248) of CFTase from Bacillus circulans MCI 2554.…”

“…9) With respect to the enzymes from P. polymyxa MGL21, the 148 kDa CFTase is reported to be inhibited by Cu 2+ ion 10) whereas no inhibition was observed either in the 108 kDa CFTase 11) or the enzyme in this study. The most remarkable difference between this enzyme and the enzymes from B. circulans OKUMZ31B, 2) B. circulans MCI 2554 7) and B. macerans CFC1 9) was the production ratio of cycloinulohexaose (CF 6) and cycloinuloheptaose (CF7) as shown in Table 3, indicating that the enzyme from P. polymyxa MG CF6 is capable of producing larger amounts of CF7 than the other enzymes. In this connection, crude enzyme prepared from culture broth of P. polymyxa MGL21 was found to produce CF6 and CF7 in a ratio of 2.7:1, 10) which was similar to that (2.5:1) in our purified enzyme.…”

“…The molecular mass of the purified enzyme is estimated to be 121,942 Da, accordingly. The deduced amino acid sequence of the CFTase from P. polymyxa MG CF6 showed 83, 95 and 98% similarity to that of Bacillus circulans MCI 2554, 18) Paenibacillus (Bacillus) macerans CFC1, 19) and Paenibacillus (Bacillus) polymyxa MGL21, 10) respectively, as illustrated in Fig. 3.…”

“…Primers used were designed on the basis of the N terminal amino acid sequence of the CFTase and the nucleotide sequences which are highly conserved regions of three CFTase gene previously reported. 10,18,19) Furthermore, to determine the upstream and the downstream sides of the core sequence inverse PCR was done. These nucleotides of resultant PCR products were sequenced with an Applied Biosystems 373A DNA sequencer and a DNA sequencing kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit).…”

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

“…FG02067.1 is an interesting member of the GH32 gene family as it shows very limited sequence identity to the known enzymes. It is predicted to be an intracellular enzyme and shows the highest identity (e-value 2610 209 ) to a cycloinulooligosaccharide fructanotransferase from Bacillus macerans (Kim & Choi, 2001). M. grisea contains five GH32 enzymes, four of which cluster in a separate branch different from the other fungal proteins (Fig.…”