Purification and Characterization of 2,6-β- d -Fructan 6-Levanbiohydrolase from Streptomyces exfoliatus F3-2

Abstract

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a ␤-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only ␤-2,6-linkage of levan, but also ␤-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-␤-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).It has been reported that oligosaccharides appear to exhibit various physiological functions according to their structures. As the functions of the oligosaccharides are recognized, the production methods become important. An attempt to synthesize a large number of novel oligosaccharides has been made by using sugar hydrolases or sugar transferases. We have been studying the effective production of novel oligosaccharides (11,(13)(14)(15)(19)(20)(21)(22) from such unused natural fructans as levan and inulin by the microbial polysaccharide-degrading enzymes. Also we confirmed that two of these oligosaccharides, di-Dfructose-1,2Ј:2,3Ј-dianhydride (DFA III) (17) and di-D-fructose-2,6Ј:6,2Ј-dianhydride (DFA IV) (12), had effects on increasing calcium absorption in the small intestine of rats, which could be expected to have practical applications in such fields as treatment with functional food or medicine to prevent osteoporosis.In the course of these studies, Streptomyces exfoliatus F3-2 was isolated as a strain producing a levan-degrading enzyme (LDE) that effectively produces levanbiose from levan (21). Levan is a polymer consisting of ␤-2,6-linked fructose residues and found in monocotyledons as a reserve carbohydrate or synthesized by microbial levansucrase (EC 2.4.1.10) from sucrose (3, 4). In contrast to a plant levan, a microbial levan generally has a lot of branching points at the C-1 position, except for a levan from Serratia levanicum (4, 5). In our previous study (21), we demonstrated that the combination of the LDE from S. exfoliatus F3-2 and levan from S. levanicum was quite efficient for the large-scale preparation of levanbiose, with a maximum production yield of levanbiose of 84% (wt/wt) from 50 mg of levan per ml. The linear nature of the levan molecule from S. levanicum seems to contribute to this high yield.To date, three types of microbial LDEs have been reported:...

Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus

“…A variety of enzymes have been associated with microbial utilization of FOS, namely: fructosidase EC 3.2.1.26 (33,34), inulinase EC 3.2.1.7 (35-37), levanase EC 3.2.1.65 (38), fructofuranosidase EC 3.2.1.26 (39)(40)(41), fructanase EC 3.2.1.80 (7), and levan biohydrolase EC 3.2.1.64 (42,43). Despite the semantic diversity, these enzymes are functionally related and should be considered as members of the same ␤-fructosidase superfamily that incorporates members of both glycosyl family 32 and 68 (44).…”

“…Additionally, microbial fructosidases associated with FOS hydrolysis such as M. laevaniformans LevM (43) and Streptomyces exfoliatus levanbiohydrolase (42) have been reported as extracellular enzymes as well. In contrast, the L. acidophilus NCFM fructosidase does not contain an anchoring signal, thus is likely a cytoplasmic enzyme requiring transport of its substrate(s) through the cell membrane.…”

Functional Analysis of the Fructooligosaccharide Utilization Operon in Lactobacillus paracasei 1195

“…Although FOS transport in bifidobacteria has not been reported, the presence of at least seven gene loci encoding oligosaccharide transport and metabolism in the genome sequence of Bifidobacterium longum (44) suggests that uptake of FOS may also be mediated by specific oligosaccharide transporters. In contrast, extracellular enzymes that hydrolyze FOS have also been reported for nonintestinal bacteria and include a fructan ␤-fructosidase from Lactobacillus pentosus and levan biohydrolases from Streptomyces exfoliatus and Microbacterium laevaniformans (39,41,45).…”

Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by Rhizopus oryzae

“…Soluble sugars were analyzed by thinlayer chromatography using a solvent system of n-butanol:i-propanol:water:acetate (7:5:4:2). Spots were visualized on the plate by spraying with anisaldehyde-sulfuric acid reagent [17].…”