Heterologous Expression And Catalytic Mechanism Of Cycloinulooligosaccharide Fructanotransferase From Paenibacillus Sp. Lfos16

Cyclofructans(CFs) are a family of cyclic oligosaccharides with a characteristic crown core ether in the central part of the molecule, which are obtained by inulin with cycloinulooligosaccharide fructanotransferase(CFTase). CFTase catalyzes degradation of inulin into cycloinulooligosaccharides by intramolecular transfructosylation, and it also catalyzes intermolecular transfructosylation between β-(2-1)-fructooligosaccharides by disproportionation and coupling reactions.A novel CFTase gene(cft) was cloned from the genomic DNA isolated from Paenibacillus sp. Lfos16, using primers designed according to the conserved amino acid sequences in different fructosidases. TAIL PCR and SEFA PCR was used to amplify the flanking sequences of cft. The ORF of the cft was composed of 4,017 nucleotides and encoded a polypeptide of 1339 amino acids with a calculated Mr of 150 kDa, including a 31-amino-acid signal sequence. The cft was inserted into the pET28a(+) which contained a region coding for the His-tag and expressed in E.coli BL21(DE3). The constructed plasmid was designated as pCFT150. The CFTase activity of recombinant E.coli was 73.80 U/L, which was increased by 1.2 times compared with the wide CFTase activity from Paenibacillus sp. Lfos16. Sequence analysis indicated that cft could be divided into six distinct regions:N-terminal signal peptide, three regions of repeat sequences(R1, R3, and R4) at the N-terminus and C-terminus, endo-inulinase region of homology(ERH) and core region(CR). Each domain function was investigated by comparison of original type CFTase enzyme(CFT150) with deletion mutant proteins of CFTase. The CFT140 and CFT106 mutant had both inulin cyclization and CF hydrolyzing activity as CFT150 did. The results indicated that the C-terminal R4 region of Paenibacillus sp. Lfos16 CFTase was necessary for inulin cyclization and CF hydrolyzing activity. While the ERH and CR were considered to be responsible for disproportionation and inulin hydrolyzing.