Method of stabilizing .alpha.-galactosidase

The activity of mycelial bound .alpha.-galactosidase is stabilized by treating .alpha.-galactosidase containing mycelia with about 5 to about 25 percent by weight glutaraldehyde based upon the dry weight of the mycelia. The glutaraldehyde treated mycelia may be used in the hydrolysis of oligosaccharides containing .alpha.-galactoside linkage without incurring substantial .alpha.-galactosidase activity loss during hydrolysis.

BACKGROUND AND SUMMARY 
This invention relates to the hydrolysis of oligosaccharides containing 
.alpha.-galactoside linkage in the presence of .alpha.-galactosidase and 
more particularly to a method for stabilizing .alpha.-galactosidase enzyme 
activity containing mycelia for subsequent use in the hydrolysis of 
oligosaccharides containing .alpha.-galactoside linkage. 
The hydrolysis of oligosaccharides containing .alpha.-galactoside linkage, 
such as raffinose, stachyose, melibiose, galactobiose, verbascose, and the 
like, in the presence of .alpha.-galactosidase is an important commercial 
practice in some industries. For example, in the production of crystalline 
sugar from sugar beets, the naturally occurring trisaccharide raffinose is 
known to be present in sugar beet diffusion juice in varying quantities. 
Since raffinose forms insoluble calcium saccharates, it is precipitated 
together with sucrose in the conventional Steffen process, and tends to 
build up in Steffen molasses as sucrose is precipitated from the molasses 
solution. As with other impurities, the presence of raffinose in sucrose 
containing liquids detrimentally effects sucrose crystalization, such as 
by depressing the sucrose crystallization velocity and by resulting in the 
formation of cubic, flat or needle-like crystals at varying raffinose 
concentrations. In the past, it has been a common commercial practice to 
discard, or use merely as a by-product, molasses having a raffinose 
buildup of over about 5% by weight on dry substance without attempting to 
recover additional sucrose from the molasses. Such practices have resulted 
in the loss of substantial quantities of potentially recoverable sucrose. 
To overcome the foregoing problems, it has previously been suggested to 
treat raffinose containing beet molasses with .alpha.-galactosidase to 
hydrolize the raffinose into D-galactose and sucrose, thereby permitting 
the recovery of additional sucrose from the molasses solution. For 
example, U.S. Pat. No. 3,647,625 of Suzuki et al relates to such a process 
wherein .alpha.-galactosidase is formed in mycelia of Mortierella vinacea 
var. raffinose-utilizer (ATCC No. 20034) to obtain mycelia substantially 
free of invertase activity. The foregoing .alpha.-galactosidase containing 
mycelia is commercially provided in pellet form to allow for continuous 
enzymatic treatment in a sucrose production process. Although this process 
has been successful in reducing the raffinose content of beet molasses and 
thereby increasing recoverable sucrose yields, it has been found that 
enzyme losses are relatively high due to actual physical loss of the 
mycelia, loss of .alpha.-galactosidase from the mycelia and/or 
inactivation of the .alpha.-galactosidase. Losses of each type 
substantially effect the realized enzyme activity level in a continuous 
raffinose hydrolysis process and the economic desirability of conducting 
such a process. 
In addition, it has previously been known that glucose isomerase activity 
within whole Streptomyces olivaceus (NRRL 3583) bacterial cells can be 
stabilized by treating the whole bacterial cells with glutaraldehyde. See, 
for example, U.S. Pat. No. 3,779,869 of Zienty, which relates to such a 
process. However, there is no disclosure or suggestion in the Zienty 
patent that glutaraldehyde treatment might be effective for stabilization 
of anything other than glucose isomerase activity in whole bacterial 
cells. 
In accordance with the present invention, it has been found that the 
.alpha.-galactosidase activity level in the hydrolysis of oligosaccharides 
containing .alpha.-galactoside linkage can be stabilized by treating 
mycelial bound .alpha.-galactosidase with about 5 to about 25 percent by 
weight glutaraldehyde based upon the dry weight of the mycelia. The 
treatment method is preferably performed by suspending 
.alpha.-galactosidase activity containing mycelia in an aqueous medium, 
adding an active site protection agent to the aqueous medium, adding the 
glutaraldehyde to the aqueous medium, maintaining the pH of the aqueous 
medium within the range of about 6.5 to about 8.5, mixing the aqueous 
medium for a period of at least about 0.25 hours, separating the mycelia 
from the aqueous medium and then washing the mycelia to remove excess 
glutaraldehyde. The treated .alpha.-galactosidase activity containing 
mycelia may then be used in a conventional manner in the hydrolysis of 
oligosaccharides containing .alpha.-galactoside linkage. 
DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS 
The method of the present invention is useful for the treatment of mycelia 
containing .alpha.-galactosidase activity. Mycelia presently particularly 
preferred for use in the practice of the invention is mycelia of the mold 
Montierella vinacea var. raffinose-utilizer, or other 
.alpha.-galactosidase mycelia, which has been cultured in a manner so as 
to minimize invertase activity. An illustrative example of such mycelia is 
that available from the Agency of Industrial Science and Technology and 
the Hokkaido Sugar Company, Tokyo, Japan, under the tradename Melibia-D, 
an .alpha.-galactosidase containing mycelia of the mold Montierella 
vinacea var. raffinose-utilizer which is provided in dry pellet form. 
In order to ensure uniform stabilization of the .alpha.-galactosidase 
enzyme, the mycelia is initially suspended in an aqueous medium having 
sufficient water content to permit uniform dispersion of the mycelia 
throughout the medium upon subsequent agitation. It has been found that 
about 5 liters of aqueous medium per kg. of mycelia is sufficient to 
adequately disperse the mycelia for further treatment. 
In order to protect the .alpha.-galactosidase activity from potential 
detrimental effects of glutaraldehyde treatment, it is a presently 
preferred practice to add a sufficient amount of an active site protection 
agent to the medium to substantially decrease any such detrimental 
effects. Suitable active site protection agents include agents which will 
enter the active site of the .alpha.-galactosidase enzyme and prevent 
glutaraldehyde bridging across the active site thereby decreasing 
availability of the active site to raffinose or other oligosaccharide 
acceptance. Illustrative examples of suitable active site protection 
agents include raffinose, sucrose, melibiose, galactose, glucose and 
fructose. The presently particularly preferred active site protection 
agent is glucose. The active site protection agent is preferably added in 
a greater than stoichiometric amount relative to available enzyme active 
sites to ensure a high level of protection. When the active site 
protection agent is glucose, it has been found that about 1 to about 10, 
preferably about 2 to about 5, and more preferably about 2 to about 3 
percent by weight glucose, based upon the dry weight of the mycelia, is 
effective for this purpose. 
The mycelia-containing aqueous medium is then treated by adding about 5 to 
about 25, preferably about 7.5 to about 15, and more preferably about 9 to 
about 12 percent by weight glutaraldehyde, based upon the dry weight of 
the mycelia, to the dispersion. The pH of the dispersion is adjusted to 
about 6.5 to about 8.5, preferably about 7.5 to about 8.5, with a suitable 
pH adjusting agent, such as NaOH, and is then mixed for a sufficient time 
to obtain maximum glutaraldehyde stabilization, usually at least about 
0.25 and more preferably at least about 1.0 hours. To obtain optimum 
results, the glutaraldehyde may be continuously added to the aqueous 
medium over the treatment period with frequency NaOH additions to maintain 
the desired pH levels. 
During the mixing period, it is particularly critical to limit, and 
preferably to minimize, inclusion of atmospheric oxygen in the aqueous 
medium. It has been found that inclusion of relatively high levels of 
atmospheric oxygen results in an immediate decline or suppression of 
.alpha.-galactosidase activity. Mixing of the aqueous medium is therefore 
carried out in a manner so as to limit, and preferably to minimize, oxygen 
inclusion in the medium. For example, mixing may be accomplished by 
stirring the aqueous medium in a closed container having a layer of inert 
gas overlaying the aqueous medium, by gently agitating the aqueous medium 
or by other suitable means. 
After mixing for sufficient time to obtain the desired degree of 
glutaraldehyde stabilization of the enzyme, the mycelia are separated from 
the dispersion, such as by settling and decantation, by filtration, by 
centrifugation, or by other suitable means. The mycelia are then washed to 
remove excess residual glutaraldehyde. Washing of the treated mycelia may 
be accomplished by resuspending the mycelia in an aqueous medium, such as 
tap water, distilled water or deionized water, followed by separation of 
the mycelia from the medium in a conventional manner. Preferably, the 
mycelia are washed at least twice to insure adequate excess glutaraldehyde 
removal prior to subsequent use of the mycelia. The washed, stabilized 
.alpha.-galactosidase containing mycelia are then ready to be used in a 
conventional process for the hydrolysis of oligosaccharides containing 
.alpha.-galactoside linkage.

The foregoing principles may be better understood in association with the 
following illustrative examples. As used in these examples, an activity 
unit is defined as the amount of enzyme required to liberate 1.mu.g of 
glucose per hour at 50.degree. C. from melibose at 30 Brix and a pH of 
5.2. 
EXAMPLE I 
350 g. of dry, pelletized Melibia-D, an .alpha.-galactosidase activity 
containing mycelium obtained from the Hokkaido Sugar Company, Tokyo, 
Japan, is added to 2 liters (1.) of a 0.015 M aqueous solution of 
melibiose, and the resulting mixture is mixed for two hours at a 
temperature of 5.degree. C. to protect the enzyme active sites of the 
.alpha.-galactosidase. The mixture is then divided into four equal 
samples. To each of the four samples is added the percentage of reagent 
grade glutaraldehyde shown in Table I, based upon the dry weight of the 
mycelia in the sample, to form four separate reaction mixtures. The pH of 
the reaction mixtures is maintained at 8.5 by the addition of NaOH, and 
the reaction mixtures are allowed to react under constant gentle agitation 
at 23.degree. C. for one and one-half hours. The reaction mixtures are 
allowed to settle, and excess liquid is decanted from the settled mycelia. 
The mycelia are washed twice in 2 l. of deionized water, with supernatant 
wash water being decanted from the mycelia of each sample. Each sample is 
then added to 500 ml. of a 30 RDS molasses solution having a pH of 5.2 to 
form hydrolysis mixtures. 50 g. of sea sand is added to each hydrolysis 
mixture, and the hydrolysis mixtures are placed in a shaker bath at 
50.degree. C. After a two hour hydrolysis reaction period, no significant 
difference is apparent between the enzyme activities of the various 
hydrolysis mixtures. After three weeks, the .alpha.-galactosidase 
activities of the hydrolysis mixtures are as shown in TABLE I: 
TABLE I 
______________________________________ 
Activity 
(Units/g .times. 10.sup.3) 
Hydrolysis 
% % of Initial 
Mixture Glutaraldehyde 
0 Weeks 3 weeks 
Control 
______________________________________ 
1 0 839 86 10 
2 0.1 842 108 13 
3 1.0 853 104 12 
4 10.0 831 378 45 
______________________________________ 
EXAMPLE II 
The procedure of Example I is repeated with the substitution of glucose for 
melibiose, at the glutaraldehyde treatment levels shown in Table II, and 
for a hydrolysis reaction time of two weeks. The .alpha.-galactosidase 
activities of the hydrolysis mixtures are as shown in TABLE II: 
TABLE II 
______________________________________ 
Activity 
Hydrol- 
% % of Initial 
ysis Glut- Units/g .times. 10.sup.3 
Control 
Mix- aralde- 0 1 2 Activity Remaining 
ture hyde Weeks Week Weeks 1 Week 2 Weeks 
______________________________________ 
1 0 1004 176 68 18 7 
2 5 1339 428 316 43 32 
3 10 1138 605 385 60 39 
4 15 868 493 437 49 44 
______________________________________ 
EXAMPLE III 
The procedure of Example II is repeated without the addition of melibiose, 
glucose or any other active site protection agent at 0% and 10% 
glutaraldehyde treatment levels. The .alpha.-galactosidase activity levels 
of the hydrolysis mixtures are shown in TABLE III: 
TABLE III 
______________________________________ 
Activity 
Hydrol- 
% % of Initial 
ysis Glut- Units/g .times. 10.sup.3 
Control 
Mix- aralde- 0 1 2 Activity Remaining 
ture hyde Weeks Week Weeks 1 Week 2 Weeks 
______________________________________ 
1 0 1004 176 57 18 6 
2 10 875 587 619 58 62 
______________________________________ 
The data of Tables I, II and III illustrate that the .alpha.-galactosidase 
enzyme activity level of the mycelia is significantly stabilized by 
glutaraldehyde treatment, particularly at the 5%-15% glutaraldehyde 
treament levels, and that pre-treatment of the mycelia with an active site 
protection agent is effective in reducing initial enzyme activity losses 
incurred in the subsequent glutaraldehyde treament. 
EXAMPLE IV 
The procedure of Example II is followed with treatment in the 
glutaraldehyde reaction mixture for 0.25, 0.5, 1.0 and 2.0 hours. Similar 
results are obtained in the 0.5, 1.0 and 2.0 hour treatments, with a 
lesser degree of stabilization being obtained in the 0.25 hour treatment. 
EXAMPLE V 
The procedure of Example II is repeated while maintaining the pH of the 
glutaraldehyde reaction mixture at 6.5. Similar results are obtained. 
While the invention has been described in association with certain 
presently preferred embodiments, certain modifications will be apparent to 
those skilled in the art. Such modifications are intended to be within the 
scope of the appended claims except as precluded by the prior art.