Conversion of guar gum to gel-forming polysaccharides by the action of .alpha.-galactosidase

Water-soluble polysaccharide guar gum, guaran, is altered to convert its properties from those of producing a highly viscous stable dispersion to enable production of a sequence of products that may be separately produced through the selective removal of D-galactopyranosyl side units so as to produce different lengths of molecular segments containing no derivatizing .alpha.-D-galactopyranosyl groups. The structure therefore yields a high viscosity polysaccharide with the ability to form gels of various strengths ranging from very weak gels to strong gels by appropriate selection of the extent to which the .alpha.-D-galactopyranosyl groups are removed.

This invention relates to gel-forming polysaccharides, and particularly to 
a mechanism by which guar gum can be converted to gel-forming 
polysaccharides similar to locust bean gum polysaccharide. 
Guar gum is 78-82% of the endosperm component of guar seed (see "Guar: 
Agronomy, Production, Industrial Use and Nutrition" by Roy L. Whistler and 
Theodore Hymowitz, Purdue University Press, West Lafayette, IN, 1979). 
Guar is Cyanopsis tetragonolobus, of the family Leguminosae. Guar has been 
grown for centuries in India and Pakistan, where it is one of the crops 
used as a food for both humans and animals. In the United States, it is 
grown in north Texas and southern Oklahoma, Guar seeds contain 
approximately 14-17% hull, 35-42% endosperm, and 43-47% germ. They are 
commonly dry-milled to separate out the endosperm which is the industrial 
guar gum of commerce (see "Industrial Gums", Roy L. Whistler, editor, 
Academic Press, New York, 1973). Although guar gum is normally 
commercially used in its commercially produced crude form as the ground 
endosperm containing small amounts of cellulose, protein, and other 
impurities, its principal component and the component giving it industrial 
value is the polysaccharide guaran. Guaran is a galactomannan consisting 
of 34.6% D-galactopyranosyl units and 63.4% D-manopyranosyl units. Guaran 
has been shown to have a structure of 1.fwdarw.4-linked 
.beta.-D-manopyranosyl units, with every second chain sugar unit bearing a 
single .alpha.-D-galactopyranosyl unit linked 1.fwdarw.6. As a 
consequence, guaran readily dissolves in water to form highly viscous 
solutions even at low concentrations of gum. The solutions remain stable 
because molecular segments of guaran cannot bind to each other when they 
collide in solution. This occurs essentially since the manopyranosyl 
chains are separated from each other by the derivatizing 
.alpha.-D-galactopyranosyl side groups. 
Another important industrial gum is locust bean gum. This gum is derived 
from locust bean or carob seeds. These are the seeds from the Ceratonia 
siliqua plant. The plant belongs to the family Leguminosae, sub-family 
Caesalpiniaceae. The ground endosperm of the carob tree seed or the locust 
bean is also a galactomannan widely used in industry. "Industrial Gums", 
supra. Locust bean and carob seed gum have many of the properties of guar 
gum but, in addition, have a tendency to gel and synergistically to 
produce gels in combination with certain other gums such as xanthan gum 
and carrageenan. An examination of the structure of carob seed gum or 
locust bean gum (C. W. Baker and R. L. Whistler, "Distribution of 
D-galactopyranosyl Groups in Guaran and Locust Bean Gum", 45 Carbohydrate 
Research, 237-243 [1975]) demonstrates that the structure of carob seed 
gum or locust bean gum differs from that of guaran in that the 
D-galactopyranosyl groups are not evenly distributed along the chains as 
they are in guaran, but rather are irregularly grouped along the chain, so 
that on the average, 25 D-galactopyranosyl groups are grouped together 
with an intermediate stretch of some 85 adjacent denuded D-manopyranosyl 
units in between. It thus appears that the long chains of 85 or so 
adjacent denuded D-manopyranosyl units associate intermolecularly in 
solution to form junction zones which promote formation of weak gel 
structures. It further appears that the same denuded segments of chain may 
also combine with segments of xanthan gum and carrageenan gum to produce 
cross-linked gel structures. It further appears that by variations in the 
length of the denuded chain segment exposed, variations in the strength of 
the gel could be predicted and achieved. Essentially, a gel having a 
desired strength for a particular application, ranging from a weak gel to 
a strong gel, could be prepared by shortening, or lengthening, 
respectively, the denuded molecular segments (that is, the segments 
containing no D-galactopyranosyl groups). 
According to the invention, guar gum can be modified through the use of 
enzymes, for example, a commercially available .alpha.-D-galactosidase 
enzyme. This .alpha.-D-galactosidase enzyme is useful to remove the 
.alpha.-D-galactopyranosyl side groups. In modification of guar gum, by 
controlling the length of time that the guar gum is exposed to the 
.alpha.-D-galactosidase enzyme, and therefore by controlling the extent of 
the removal of the .alpha.-D-galactopyranosyl side groups, the length of 
denuded mannan chain exposed by the enzyme's activity on the guar gum can 
be controlled. Of course, control of the length of denuded mannan chain 
exposed provides a direct control on the amount of intermolecular 
association that adjacent guaran molecules in solution experience. To 
control this amount of intermolecular association is, of course, to 
control the relative strength or weakness of a gel produced from guar gum 
by the activity of the .alpha.-D-galactosidase enzyme. 
Commercial D-galactosidase enzymes are available from various sources. Of 
course, it is important that the D-galactosidase enzymes used do not 
contain mannosidases which would cleave the principal mannan backbone or 
chain of the guaran. Breaking of the mannan backbone or chain lowers the 
viscosity of the solution to levels unacceptable for certain important 
industrial applications. If commercial D-galactosidase enzymes containing 
no mannosidase enzymes are unavailable, mannosidase enzyme-free 
D-galactosidase enzymes can be produced from germinated guar seeds. The 
resulting galactosidase enzyme can be separated from any mannosidase 
enzymes present by fractional precipitation from solution by varying the 
concentration of ammonium sulfate, or by gel filtration techniques known 
in enzyme chemistry. 
As previously mentioned, the limitation in the use of galactosidase enzyme 
on guaran is the amount of time that the guaran is exposed to the activity 
of the galactosidase enzyme. If the period of exposure is too long, all of 
the .alpha.-D-galactopyranosyl side chains will be stripped away, leaving 
only a mannan backbone chain which will join with other mannan chains to 
form an insoluble precipitate. Such an extended degree of hydrolysis 
produces an essentially useless product. Therefore, according to the 
invention, low concentrations of galactosidase enzyme are used, and they 
are used only for carefully controlled periods of time. Higher 
concentrations of galactosidase enzyme can be used to treat the guaran, 
but the higher concentrations require somewhat more carefully controlled 
reaction times. Reaction time will vary depending upon whether the 
galactosidase enzyme is added to an aqueous solution of guar gum or 
guaran, or whether the enzyme is fixed, or immobilized, by any one of a 
number of methods for immobilizing enzymes known in the enzyme industry. 
If the enzyme is immobilized on a support, the amount of guar gum or 
guaran that is passed over the enzyme support to be exposed to enzyme 
activity can be controlled by the volume of flow. The contact period 
between the guar gum or guaran and the enzyme can thereby be carefully 
controlled. 
The precise conditions of use for a particular galactosidase enzyme depend 
upon the concentration of the galactosidase enzyme and upon the activity 
of the enzyme. Both of these factors depend, in turn, upon whether the 
enzyme is added to the aqueous solution of guar gum or guaran, or whether 
the enzyme is fixed, and, if fixed, the methods of fixation and 
configuration of the bed. Consequently, it is believed best to treat the 
guar gum or guaran with galactosidase enzyme in a batch process, by which 
the enzyme is added directly to a test solution of guaran or guar gum. 
Alternatively, a test solution of guaran or guar gum can be exposed to the 
immobilized enzyme fixed bed. Test runs can be made for different time 
intervals and the degrees of gel formation at the end of each test run can 
be determined. A graph can be generated for a particular concentration of 
guaran or guar gum in solution showing degree of gel formation versus 
time. When several solutions of guar gum or guaran have been treated with 
the enzyme and the tests giving the desired results have been identified, 
further preparations utilizing the thus-determined treatment parameters 
can be made to yield guar gum or guaran gels of the desired strength.

In an illustrative preparation technique, guar gum is treated with a 
commercially available D-galactosidase enzyme, and a 1% by weight solution 
of the treated guaran or guar gum in water is permitted to stand for 
thirty minutes. At the end of thirty minutes, a viscosity measurement is 
conducted. A reduction, and specifically a large reduction, in the 
viscosity of the solution indicates the presence of mannosidase enzymes in 
the starting enzyme treatment. The mannosidase enzymes have cleaved, or 
broken, the mannan backbone or main chain of the guaran and rendered the 
treated solution useless. On the other hand, gel formation in the 1% 
solution after standing for thirty minutes will be indicated by an 
increase in the viscosity of the 1% solution. The promotion of gel-forming 
characteristics in the 1% solution, and the relative "strength" of the 
resultant gel, can be made by any of a number of methods for measuring gel 
strength known in the industry. 
Another method for measuring the success of the treatment with 
D-galactosidase enzyme is the change in viscosity of the treated guaran or 
guar gum when a standard concentration of the treated guaran or guar gum 
is mixed with a standard concentration of a synergistically active gum 
such as xanthan gum of carrageenan. The synergistic increase in viscosity 
of a mixture of these two normally water-soluble polysaccharides will be 
indicative of the degree of removal of D-galactopyranosyl side groups by 
D-galactosidase enzyme. 
The invention may further be understood by reference to the drawings and 
the accompanying descriptions of the drawings. 
FIG. 1 illustrates diagrammatically guaran molecules 8, each consisting of 
a mannan main chain 10 with spaced galactopyranosyl side groups 12. 
FIG. 2 illustrates the guaran molecules 8 after treatment with 
galactosidase enzyme. Each includes a mannan main chain 10, fewer 
galactopyranosyl side units 12 and vacant, or denuded, sections 14 for 
junction zone formation. 
FIG. 3 illustrates the treated guaran molecules 8 of FIG. 2 associated 
intermolecularly to produce a gel. Again, each treated guaran molecule 
includes the mannan main chain 10, galactopyranosyl side units 12, and 
junction zones 14. The junction zones 14 of the treated guaran molecules 
lie in contact, or closely adjacent, one another in a manner forming the 
gel. 
FIG. 4 illustrates guaran molecules 20 treated excessively with 
galactosidase enzyme. The inordinately long junction zones 24 in these 
molecules 20 are to be noted. As previously mentioned, in this excessively 
treated guaran, essentially only the mannan main chain 26 remains. The 
essentially completely denuded mannan main chains 26 with very few, or no, 
remaining galactopyranosyl side units 22 associate intermolecularly to 
form insoluble precipitates which have little value. 
FIG. 5 illustrates the result of treatment of guaran including mannan 
backbones or main chains 30 with enzyme mixtures including both a 
D-galactosidase enzyme and a mannosidase enzyme. It is to be noted that, 
although not all of the galactopyranosyl units 32 have been removed, the 
mannan backbone chains 30 have been attacked by the mannosidase in the 
treating enzyme mixture, cleaving them and resulting in a low viscosity 
solution of little utility. This drawing illustrates the importance of 
isolation of the galactosidase enzymes, or at the very least, separation 
of the undesirable mannosidase enzymes from the treating enzyme mixture.