Glucoamylases (EC 3.2.1.3, alpha-1,4-glucan glucohydrolase) are exo-acting enzymes that catalyze hydrolysis of both alpha-1,4 and alpha-1,6 glycosidic linkages from non-reducing ends of glucose-containing di-, oligo- and poly-saccharides, releasing glucose units one at a time (1960, Pazur and Ando, J. Biol. Chem. 235:297-302). Cleavage occurs at the glycosidic bond connecting the anomeric carbon with oxygen (1962, Fleetwood and Weigel, Nature 196:984). Alpha-1,4, alpha-1,6, and alpha-1,3 bonds are the only linkages hydrolyzed at significant rates by glucoamylase (1957, Barker et al., J. Chem. Soc. 4865-4871).
Glucoamylase is also capable of hydrolyzing the glycosidic bond between the two glucosyl units linked by alpha-1,2 (e.g., Kojibiose) or alpha-1,1 (e.g., trehalose). However, this enzymatic activity occurs at a much lower rate and at more dilute substrate concentrations compared to glucoamylase activity toward disaccharides with alpha-1,4 (maltose) or alpha-1,6 (isomaltose) linkages.
Glucoamylase has been widely used for producing high-glucose syrup from starch. High-glucose syrup is useful as a feedstock for producing various value-added compounds such as fuel alcohol, high-fructose corn syrup, organic acids, amino acids and vitamins. Glucoamylase has been isolated from numerous microorganisms, animals and plants, and among microorganisms, many fungi are good sources of this enzyme. Glucoamylase produced in fungal organisms, such as Aspergillus niger, is commonly used for commercial applications such as high-glucose syrup production.
Transglucosidases (EC.2.4.1.24, 1,4-alpha-glucan 6-alpha-glucosyltransferase) are D-glucosyltransferase enzymes that catalyze both hydrolytic and transfer reactions on incubation with alpha-D-gluco-oligosaccharides (1951, Pazur and French, J. Amer. Chem. Soc. 73:3536). Maltose is the most preferred substrate for transglucosylation reactions with this enzyme. Transfer occurs most frequently to HO-6, producing isomaltose from D-glucose, or panose (6-O-alpha-glucosyl maltose) from maltose. Transglucosidase can also transfer a glucosyl residue to the HO-2 or HO-3 of another D-glucosyl unit to form Kojibiose or Nigerose. This enzyme can further transfer a D-glucosyl unit back to HO-4 to reform maltose.
As a result of transglucosylation reactions with transglucosidase, malto-oligosaccharide residues are converted to isomalto-oligosaccharides (IMO) containing a higher proportion of glucosyl residues linked by alpha-D-1,6 glycosidic linkages from the non-reducing end. IMO sugars are used in many food and beverage formulations in Asia. Brier et al. (U.S. Patent Appl. Publ. No. 2003/0167929) disclosed using transglucosidase to produce IMO from barley wort.
Poulose et al. (U.S. Patent Appl. Publ. No. 2008/0229514) disclosed using transglucosidase to degrade polysaccharides such as xanthan and guar gums. Xanthan gum comprises a cellulosic backbone in which alternate glucoses are 1,3-linked to branches containing mannose and glucuronic acid. The backbone of guar gum comprises beta-1,4-linked mannose residues to which galactose residues are alpha-1,6-linked at every other mannose.
Lantero et al. (U.S. Pat. No. 5,770,437) disclosed using a transglucosidase to degrade sucrose, melezitose and trehalulose. These sugars comprise glucose linked to fructose via 1,2-(sucrose), 1,3-(melezitose), or 1,1-(trehalulose) linkages.
Although various hydrolytic activities of glucoamylase and transglucosidase have been disclosed, these enzymes are generally considered to be alpha-glucosidases, given their ability to hydrolyze alpha-linkages between two glucosyl residues. For example, both glucoamylase and transglucosidase are associated with having maltase activity (hydrolysis of the alpha-1,4 glycosidic link between the two glucosyl residues of maltose), which is a type of alpha-glucosidase activity.
Notwithstanding the foregoing disclosures, surprisingly, it has now been found that alpha-glucosidases such as transglucosidase (EC 2.4.1.24), glucoamylase (EC 3.2.1.3), and other alpha-glucosidases can hydrolyze alpha-1,5 glycosidic linkage of glucosyl-fructose. Alpha-glucosidases are disclosed herein as being useful for degrading disaccharides and oligosaccharides containing glucosyl-alpha-1,5-fructose.