Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV.
ABSTRACT To clone and overexpress a novel levan fructotransferase gene lftA from Arthrobacter ureafaciens K2032.
The lftA gene, encoding a levan fructotransferase (LFTase) of 521 amino acids (aa) residues, was cloned from the genomic DNA of A. ureafaciens K2032, and overexpressed in Escherichia coli. The recombinant LFTase overexpressed in E. coli was then used to produce a difructose dianhydride (DFA IV) from levan. DFA IV crystals with 97% purity could be obtained from the reaction mixture in 83.7% yield by using a natural crystallization method.
The lftA gene cloned from A. ureafaciens K2032 encode a novel levan fructotransferase which produces difructose dianhydride (DFA IV) from levan.
Levan fructotransferase is a useful enzyme with great promise in the production of DFA IV and various fructosides.
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ABSTRACT: Di-D-fructose-2,6':6,2'-dianhydride (DFA-IV) is a disaccharide consisting of two fructose residues that are prepared from levan by levan fructotransferase. Levan is a homopolysaccharide composed of D-fructofuranosyl residues joined by -(2,6) and -(2,1) linkages. We compared the immunomodulatory effects of levan with DFA-IV. Tumoricidal activity, phagocytosis and nitric oxide (NO) production were examined in levan- and DFA-IV-treated RAW264.7 cells. The NO production, tumoricidal and phagocytic activities were significantly increased in both treated cells. The results indicate that levan has significantly greater effects on tumoricidal activity than DFA-IV at low concentrations (1 ) and its effect on NO production shows a similar pattern. These results suggest that tumoricidal activity induced by both samples is mediated by NO production.Journal of Food Science and Nutrition. 01/2008; 13(1):1-6.
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ABSTRACT: Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.Journal of Biological Chemistry 07/2012; 287(37):31233-41. · 4.65 Impact Factor
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ABSTRACT: We developed di-d-fructofranosyl-2,6′:2′,6-anhydride (DFA IV) production system with single culture of Bacillus subtilis directly from sucrose. This system can avoid the purification procedure of levan which organic solvent was used for precipitation. The levan fructotransferase (LFTase) gene was cloned from Arthrobacter nicotinovorans GS-9 (AHU1840, FERM P-15285) and expressed in levan producing B. subtilis 168. LFTase activity was detected in the culture supernatant of the transformant with maximal activity of 0.062 U/ml after 15.5 h post induction. Then sucrose was added as substrate and incubated. About 78 h after addition of sucrose, 20.5 g/l of DFA IV was produced from 139.3 g/l of sucrose consumed. The yield of DFA IV from sucrose was 14.7 wt.%.Enzyme and Microbial Technology. 01/2007;