Alkaline alpha-galactosidase

Details Alkaline alpha-galactosidase Alkaline alpha-galactosidase

2001-04-25

2003-08-19

Prouty, Rececca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S004000, C435S014000, C435S183000, C435S200000, C435S091530, C435S096000, C435S262000

Reexamination Certificate

active

06607901

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to enzymes for hydrolysis of sugars and particularly to an alkaline alpha-galactosidase which hydrolyzes a broad spectrum of galactosyl-saccharides such as melibiose, raffinose and stachyose and guar gum, at neutral to alkaline pH conditions.
BACKGROUND OF THE INVENTION
The enzyme alpha-galactosidase (E.C. 3.2.1.22; alpha-D-galactoside galactohydrolase) catalyzes the hydrolysis of the terminal linked alpha-galactose moiety from galactose-containing oligosaccharides. These include, for example, the naturally occurring disaccharide melibiose (6-O-alpha-D-galactopyranosyl-D-glucose), the trisaccharide raffinose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside) and the tetrasaccharide stachyose (O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside). Alpha-galactosidases have potential use in various applications, and some examples are described by Margolles-Clark et al. (“Three alpha-galactosidase genes of
Trichoderma reesi
cloned by expression in yeast”, Eur. J. Biochemistry, 240:104-111, 1996). They may hydrolyze alpha-galactose residues from polymeric galactomannans, such as in guar gum; modification of guar gum galactomannan with alpha-galactosidase has been used to improve the gelling properties of the polysaccharide (Bulpin, P. V., et al., “Development of a biotechnological process for the modification of galactomannan polymers with plant alpha-galactosidase”, Carbohydrate Polymers 12:155-168, 1990). Alpha-galactosidase can hydrolyze raffinose from beet sugar syrup, which can be used to facilitate the sugar crystallization from molasses, since the raffinose presents an obstacle to the normal crystallization of beet sugar (Suzuki et al., “Studies on the decomposition of raffinose by alpha-galactosidase of mold” Agr. Biol. Chem., 33:501-513, 1969). Additionally, alpha-galactosidase can be used to hydrolyze stachyose and raffinose in soybean milk, thereby reducing or eliminating the undesirable digestive side effects which are associated with soybean milk (Thananunkal et al., “Degradation of raffinose and stachyose in soybean milk by alpha-galactosidase from
Mortierella vinacea
” Jour. Food Science, 41:173-175, 1976). The enzyme can also remove the terminal alpha-galactose residue from other glycans, such as the erythrocyte surface antigen conferring blood group B specificity. This has potential medical use in transfusion therapy by converting blood group type B to universal donor type O (Harpaz et al. “Studies on B-anticenic sites of human erythrocytes by use of coffee bean alpha-galactosidase”, Archives of Biochemistry and Biophysics, 170:676-683, 1975, and by Zhu et al. “Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes”, Archives of Biochemistry and Biophysics, 327:324-329, 1996).
Plant alpha-galactosidases from numerous sources have been studied and multiple forms of the enzyme have been described, such as in Keller F. and Pharr D. M., “Metabolism of Carbohydrates in Sinks and Sources: Galactosyl-Sucrose Oligosaccharides”, In: Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 7, pp. 168-171, 1996, Marcel Dekker, Publ., N.Y. These can be classified into two broad groups, acid or alkaline, according to the pH at which they show optimal activity. Practically all studies of alpha-galactosidases have dealt with the acidic forms of the enzyme and these play important roles in seed development and germination. Alpha-galactosidases with optimal activity at alkaline pH are uncommon in eucaryotic organisms.
Alpha-galactosidases which show preferred activity to the disaccharide melibiose are often referred to as melibiases. These may have optimal activity at alkaline pH but are relatively specific to melibiose, with only little activity and low affinity to the trisaccharide raffinose. In addition, they characteristically function as a multimeric protein. For example, the bacterial alpha-galactosidase that has been described from
Bacillus stearothermophilus
(Talbot, G. and Sygusch, J., “Purification and characterization of thermostable b-mannanase and alpha-galactosidase from
Bacillus stearothermophilus
”, Applied and Environmental Microbiology, 56:3503-3510, 1990) has over a 15-fold higher activity with melibiose, as compared to raffinose and functions as a trimer. The alpha-galactosidase described from
Escherichia coli
K12 similarly has only about 4% of the activity with raffinose as compared to melibiose, with Km values of 60 mM and 3.2 mM, respectively, in addition to functioning as a tetrameric protein (Schmid and Schmitt, “Raffinose metabolism in
Escherichia coli
K12: purification and properties of a new alpha-galactosidase specified by a transmissible plasmid”, Eur. J. Biochemistry, 67:95-104, 1976). Similarly, the enzyme from
Pseudomonas fluorescens
H-601 (Hashimoto, H. et al., “Purification and some properties of alpha-galactosidase from
Pseudomonas fluorescens
H-601”, Agric. Biol. Chem., 55:2831-2838, 1991) has relative Km values for raffinose and melibiose of 17 and 3.2 mM, respectively, and functions as a tetramer.
There are obvious advantages to the use of a monomer protein with the desired enzyme activity, as compared to multimeric proteins. This has clearly been shown, for example, with the alpha-galactosidases from mung bean seeds (del Campillo, E., et al., “Molecular properties of the enzymic phytohemagglutinin of mung bean”, J. Biol. Chem. 256:7177-7180, 1981) in which the retrameric form of the enzyme disassociated into the monomeric form during storage, and this was accompanied by loss of activity.
The galactosyl-sucrose sugars, stachyose and raffinose, together with sucrose, are the primary translocated sugars in the phloem of cucurbits, which includes muskmelons, pumpkins and cucumber. The very low concentrations of raffinose and stachyose in fruit tissues of muskmelon suggest that galactosyl-sucrose unloaded from phloem is rapidly metabolized, with the initial hydrolysis by alpha-galactosidase, as described in “Cucurbits”, Schaffer, A. A., Madore, M. and Phan, D. M., In : Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioning in Plants and Crops: Source-Sink Relationships, ch. 31, pp. 729-758, 1996, Marcel Decker Publ., N.Y.
P.-R. Gaudreault and J. A. Webb have described in several publications, (such as “Alkaline alpha-galactosidase in leaves of
Cucurbita pepo
”, Plant Sci. Lett. 24, 281-288, 1982, “Partial purification and properties of an alkaline alpha-galactosidase from mature leaves of
Cucurbita pepo
”, Plant Physiol., 71, 662-668, 1983, and “Alkaline alpha-galactosidase activity and galactose metabolism in the family Cucurbitaceae”, Plant Science, 45, 71-75, 1986), a novel alpha-galactosidase purified from young leaves of
Cucurbita pepo
, that has an optimal activity at alkaline conditions (pH 7.5). In addition to the alkaline alpha-galactosidase, they also reported three acid forms of the enzyme, and distinct substrate preferences were found for the acid and alkaline forms. Raffinose was found to be the preferred substrate of the acidic forms. The alkaline form had high affinity (Km=4.5 mM) and high activity (1.58 &mgr;mol galactose formed per min per mg protein) only with stachyose. It had low affinity for (Km=36.4 mM) and low activity (0.14 &mgr;mol galactose formed per min. per mg protein) toward the trisaccharide raffinose and hydrolyzed melibiose very slowly and therefore affinity and activity on that sugar was not calculated. Thus, this previously reported alkaline alpha-galactosidase can be described as having activity at alkaline pH but with only a narrow spectrum of substrates.
A further characteristic of the alkaline alpha-galactosidase from young leaves of
Cucurbita pepo
is that alpha-D-galactose, the product of the enzymatic reaction, is a strong inhibitor of the enzyme's activity (Ga

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