Fructan Depolymerization

Depolymerization of grass fructans has been documented in many species to be both hydrolytic and exolytic. To date, no endolytic fructan depolymerizing enzyme has been documented from grasses. Two different enzymes have been documented to catalyze fructan depolymerization. These are fructan exohydrolase (FEH, E.C. and FFT. The action by FFT from wheat to transfer a fructosyl moiety from 1-kestose and from P-2,1-fructans of DP 4 to sucrose was discussed in section 2.2.1. The action of FFT would not result in net degradation of fructan, but in the shortening of one fructan polymer while elongating another. At this time, it appears that FEHs are the predominant or sole enzymes responsible for the net degradation of fructans in grasses.

Two of the concerns expressed by Pollock and Cairns (10) about the literature on fructan synthesis apply to the literature on fructan degradation as well. These are, that the purity of the enzymes being studied needs to be rigorously established and that the substrates of these enzymes should be precisely identified and be physiologically relevant.

3.1. Fructan exohydrolase (FEH)

Yamamoto and Mino (28) studied a fructan exohydrolase isolated from stem bases of Dactylis glomerata. Dactylis fructans are primarily P-2,6-linked fructans based on 6-kestose that have limited branch points (5). After four steps of purification, the enzyme was examined by discontinuous electrophoresis followed by amido black staining for protein. Only one band was visualized and it corresponded precisely with distribution of the enzyme activity eluted from gel sections. Although these methods of electrophoresis and of protein staining are not the most stringent for determining purity, there was only one protein detected and it was an active FEH. This protein had a native molecular weight of 57 kD as determined by size exclusion chromatography. It was maximally active at pH 5.5. It released the terminal fructose from substrate. The preferred substrate was the fructan purified from Dactylis and the Km for this fructan, with a mean DP of 60, was 91 mM. The FEH hydrolyzed a P-2,6 and P-2,1 mixed linkage fructan isolated from rhizomes of Agropyron repens at 14% the rate of the fructan from Dactylis and hydrolyzed inulin at 9% the rate of hydrolysis of the Dactylis fructan. This FEH did not hydrolyze sucrose.

A fructan exohydrolase has also been purified from stems of Avena sativa (29) which store mainly P-2,6-linked fructans of DP 3 to 14 elaborated from neokestose (2,30). To purify a [3-2,6-hydrolyzing enzyme from oat, the authors used neokestin, sucrose and chicory inulin as substrates for screening eluate from chromatography columns. The neokestin, a mixture of neokestose-based polymers with DP 7-14 isolated from oat, was used to monitor hydrolysis of (3-2,6-linkages. The authors also extracted, purified and characterized the degree of polymerization and the linkage patterns of five fructans from oat for use in defining the reactions catalyzed by the FEH purified from oat. The enzyme was purified by salt precipitation and three types of chromatography. Invertase activity was separated from the FEH activity by methyl-hydrophobic interaction chromatography, but the ability to hydrolyze chicory inulin was not separated from the FEH activity. Examination of the purified enzyme by SDS-PAGE and staining with silver nitrate for protein revealed a single protein of 43 kD, indicating that this FEH was purified to homogeneity. This FEH exhibited maximal activity at pH 4.5 to 5.0. The purified enzyme hydrolyzed the terminal p-2,6-linkage of 6G,6-kestotetraose, the preferred substrate, 3.5 times more rapidly than it hydrolyzed the terminal p-2,6 linkage of 6G-kestotriose and approximately 10 times faster than it hydrolyzed the terminal [3-2,1 linkage of chicory inulin. Sucrose and 1-kestose were not substrates of the FEH purified from oat. Hydrolysis of 6G,6-kestotetraose showed Michaelis-Menten kinetics. The Km for 6G,6-kestotetraose was 5.58% (w/v). The FEH from oat is very much like the FEH from Dactylis as both preferentially hydrolyzed p-2,6-fructosyl linkages, slowly hydrolyzed P-2,l-fructosyl linkages and could not hydrolyze sucrose.

A fructan exohydrolase has been purified from Lolium perenne and characterized for its catalytic properties using a variety of purified fructans (31). The FEH preparation exhibited a single band (69 kD) when analyzed by SDS-PAGE followed by silver nitrate staining and was 65 kD by size exclusion chromatography under native conditions indicating the protein was pure and was a monomer. Isoelectric focusing of this FEH preparation revealed a major band with a pi of 4.7 and faint bands on either side of the main band. These very minor bands are not necessarily unexpected of a purified glycoprotein as they often exhibit microheterogeneity when analyzed by isoelectrofocusing. The conclusion that this FEH is a glycoprotein was based upon its selective binding to a Con A-lectin affinity chromatography column. Examination of the substrate specificity of this FEH revealed that it preferentially hydrolyzed the p-2,6-fructosyl linkages in 6,6-kestotetraose, 6-kestotriose, and land 6-kestotetraose. This purified FEH, like those from Dactylis glomerata and Avena sativa, also hydrolyzed p-2,l-fructosyl linkages such as those found in 1-kestotriose and in 1,1-kestotetraose, but did so at about 20-25% of the rates of 6,6-kestotetraose hydrolysis. The purified FEH from Lolium, in contrast to those from Dactylis and Avena, did hydrolyze sucrose, albeit at a low level (3% of the rate of 6,6-kestotetraose hydrolysis). Kinetic parameters of substrate hydrolysis were not possible to be determined because the enzyme was not saturable with concentrations of 6,6-kestotetraose, the preferred substrate, as high as 25 mM. The optimal pH for hydrolysis of 6,6-kestotetraose was shown to be between 5.1 and 5.6. Product inhibition of FEH activity was examined using fructose concentrations as high as 50 mM, but no effect was observed. Sucrose was shown to be a strong inhibitor with concentrations as low as 0.5 mM resulting in 15% inhibition. FEH activity was 70% inhibited by 10 mM sucrose.

A fructan exohydrolase from Hordeum uulgare has also been purified and characterized (32). This enzyme was first isolated by screening for activity with chicory inulin as the substrate (33). This FEH was reported to hydrolyze a native, but uncharacterized, fructan isolated from barley. It was unable to hydrolyze levan, a highly branched chain fructan with a (3-2,6-linked backbone and (3-2,1 linkages in the side chains, from Aerobacter levanicum. The original report of this FEH (33) did not include characterization of the hydrolytic properties with well defined substrates and it was stated that two bands were detected on silver stained gels from SDS-PAGE. This FEH has since been purified to homogeneity and reexamined using the five purified and well defined fructans isolated from oat as reported by Henson and Livingston (29). The purity of the FEH isolated from barley was determined by silver staining gels from IEF and SDS-PAGE. A single band was identified in both electrophoresis systems. Under denaturing conditions, the enzyme had a molecular mass of 33 kD while under native conditions the enzyme mass was 62.5 kD (33), indicating that the enzyme is a homodimer. Other plant fructan hydrolases are monomeric (28, 29, 31, 34), although several microbial fructan degrading enzymes are polymeric (35, 36). The purified FEH from barley was shown to hydrolyze (3-2,1-linkages in 6G,l-kestotetraose, 1 and 6G-kestotetraose, 1,1-kestotetraose, and 1-kestotriose with relative rates of 100:96:85:88. This enzyme slowly hydrolyzed the (3-2,6-linkages in 6G-kestotriose and in 6G,6-kestotetraose and sucrose with relative rates of 5:4:3 compared to 6G,l-kestotetraose hydrolysis rates arbitrarily set at 100. The substrate attack pattern, determined by identifying products from hydrolysis of purified fructan tetrasaccharides, was of the multichain type as has been reported for other plant fructan exohydrolases (28,29,34). Sucrose was a mixed-type inhibitor of inulin hydrolysis.

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