Immobilization of microogranisms on weakly basic anion exchange substance for producing isomaltulose

Isomaltulose-forming microorganisms are immobilized on a carrier that is a weakly basic anion exchange substance in the form of a substantially non-compressible porous particulate solid material, and are used for isomerization of sucrose to isomaltulose. A preferred carrier contains microfibers or microparticles of diethylaminoethyl cellulose adherently bound by agglomeration with polystyrene. The isomerization may be a continuous conversion in one or more columns packed with the carrier. Isomaltulose may be hydrogenated to form isomalt for use in sweetening. Microorganisms can be immobilized on the carrier by feeding microorganisms to a column containing the carrier. After microorganism immobilization, the carrier may be treated with a crosslinking and/or flocculating compound. Regeneration of the carrier is carried out by removing microorganisms, washing and reloading with fresh microorganisms.

FIELD OF THE INVENTION 
The present invention relates to a process for the isomerization of sucrose 
into isomaltulose with the aid of viable immobilized isomaltulose-forming 
micro-organisms. The invention also relates to a carrier material 
comprising an anion exchange material and having immobilized thereon 
viable isomaltulose-forming micro-organism cells. The invention further 
relates to the production of isomalt from sucrose via isomaltulose. 
BACKGROUND OF THE INVENTION 
Isomaltulose (or palatinose) is a reducing disaccharide having the 
systematic name of 6-O-.alpha.-D-glucopyranosyl-D-fructofuranose. 
Isomaltulose has been proposed for use as a sweetener in the food industry 
and it is a raw material for the production of isomalt (palatinit) by 
hydrogenation. Isomalt is a substantially equimolar mixture of 
.alpha.-D-glucopyranosyl-(1,6)-sorbitol and 
.alpha.-D-glucopyranosyl-(1,6)-mannitol. Isomalt is a non-cariogenic 
special sweetener described as having a gentle sweetening flavour. 
Various processes for the isomerization of sucrose to isomaltulose have 
been reported. The isomerization is believed to be performed by 
.alpha.-glucosyl transferase (saccharose mutase) enzyme which has been 
found to exist in micro-organisms such as Protaminobacter rubrum, 
Serratica plymuthica, Erwinia rhapontici, etc. The known isomerization 
techniques include isomerization with viable or dead micro-organism cells 
or with the enzyme in extracted form. Various techniques for the 
immobilization of the enzyme have also been reported. 
Thus, for instance EP-B1-0 028 900 suggests immobilization of the enzyme of 
various isomerizing micro-organisms, preferably E. rhapontici. The 
preferred immobilization technique comprises entrapping dead microbe cells 
within calcium alginate pellets. Immobilization in a thick aqueous slurry 
of diethylaminoethyl cellulose (DEAE-cellulose) is also disclosed but is 
reported to provide poorer results. 
U.S. Pat. No. 4,386,158 discloses improving the physical strength of 
calcium alginate gels used to immobilize .alpha.-glucosyl transferase by 
treatment with polyethyleneimine and glutaraldehyde. 
U.S. Pat. No. 4,640,894 describes the production of isomaltulose by using a 
reactor including immobilized dead cells of P. rubrum. Various 
immobilization techniques, such as entrapment in gels and flocculation 
with flocculants are disclosed. 
U.S. Pat. No. 4,373,313 and EP-B-0 077 971 disclose the immobilization of 
viable cells of P. rubrum by flocculation with tannin and a long chain 
polyamine, reaction with an adduct of epihalohydrin/polyamine copolymer 
and glutaraldehyde, and drying. 
EP-B-0 049 801 discloses the immobilization of a sucrose converting enzyme 
isolated from P. rubrum on various carrier materials such as hollow fibers 
or cation exchange resins. 
EP-B-0 200 069 discloses the selective immobilization of extracted 
saccharose mutase enzyme on an anionisable carrier material, especially a 
sulphonic acid cation-exchange matrix. 
EP-A-0 160 253 discloses immobilization of P. rubrum cells through 
entrapment in a polymer system during polymerization to provide 
biocatalysts for the conversion of sucrose to isomaltulose. 
The prior art processes for the conversion of sucrose to isomaltulose are 
not entirely satisfactory. The carrier material is generally produced 
on-site. The immobilization processes are complicated and care must be 
exercised not to harm the sensitive enzyme. Entrapping in alginate or 
carrageenan gels requires several separate process steps to be performed 
and a final cross-linking with glutaraldehyde is often necessary. The 
prior art flocculation with a flocculating agent in solution requires 
several subsequent production steps involving the enzyme, including 
drying, extrusion and granulation. Inclusion of the enzyme in a polymer 
during polymerization may provide a physically strong product but this 
process is also complicated and may affect the activity of the sensitive 
enzyme. 
A further disadvantage associated with the prior art methods of including 
viable or dead micro-organism cells in a carrier during the production of 
the carrier matrix itself lies in the fact that when the activity has been 
lost, the whole carrier must be discarded. 
SUMMARY OF THE INVENTION 
An object of the present invention is to overcome disadvantages of the 
prior art techniques for immobilization of isomaltulose-forming microbes 
and to provide a technically feasible process for production of 
isomaltulose and isomalt from sucrose. 
An object of the present invention is also to provide a process for 
converting sucrose to isomaltulose by immobilized viable micro-organisms 
without exposing the microbes to such reactions as are necessary for the 
production of a solid carrier body. 
A further object of the invention is to provide a process, wherein 
immobilization of viable cells of an isomaltulose-forming micro-organism 
onto a carrier material is technically uncomplicated. 
A further object of the invention is to provide a continuous process for 
converting sucrose to isomaltulose in a packed column, wherein the 
immobilized cells can be easily refreshed or re-activated during 
production. 
An object of the invention is also to provide a process for producing 
isomaltulose in a packed column including a carrier having good physical 
strength and causing only a low pressure drop at flow of a sucrose 
solution through said columnn. 
A specific object of the invention is to provide a process using a carrier 
system with isomaltulose-forming viable micro-organism cells, wherein the 
carrier can be regenerasted after use. 
DETAILED DESCRIPTION OF THE INVENTION 
It has surprisingly been found that a technically uncomplicated process for 
converting sucrose to isomaltulose by immobilized viable micro-organism 
cells can be provided by using as a carrier a solid porous material having 
weakly basic anion exchange properties. 
The present invention is defined in the appended claims. 
Accordingly, the invention relates to a process for the isomerization of 
sucrose into isomaltulose with the aid of viable isomaltulose-forming 
micro-organism cells immobilized on an anion exchange material. In said 
process a sucrose containing solution is contacted with said 
isomaltulose-forming micro-organism cells which have been immobilized on a 
porous particulate carrier material comprising a weakly basic anion 
exchange material in the form of an inert and substantially 
non-compressible solid matrix, whereafter the isomerization product is 
recovered from the solution. 
The process of the invention may be performed either batch-wise or as a 
continuous process. A continuous process is preferably performed in a 
column packed with the carrier. 
The viable micro-organism cells are preferably immobilized onto the surface 
of the porous carrier after the formation of the carrier matrix. The 
carrier may be either continuously or intermittently refreshed, for 
instance by feeding nutrients and/or growth medium through the column. 
According to a specifically preferred embodiment the carrier material can 
be regenerated after use by removing all microbial cells, washing and 
reloading with fresh viable cells. 
The present invention also relates to a process for the production of 
isomalt from sucrose, said process including the steps of contacting a 
sucrose containing solution with viable iso-maltulose-forming 
micro-organism cells immobilized on a porous particulate carrier material 
comprising a weakly basic anion exchange material in the form of an inert 
and substantially non-compressible solid matrix; hydrogenating said 
isomaltulose to isomalt; and recovering said isomalt. 
The present invention also provides a carrier for use in a process for 
producing isomaltulose and/or isomalt from sucrose, said carrier 
comprising a porous particulate material comprising a weakly basic anion 
exchange material in the form of an inert and substantially 
non-compressible solid matrix having viable isomaltulose-forming 
micro-organism cells immobilized onto the surface thereof. 
The term "isomaltulose-forming micro-organisms" as used in the present 
specification and in the claims is intended to mean any micro-organism 
which is capable of converting sucrose to isomaltulose and which can be 
immobilized onto the surface of a weakly basic anion exchange carrier 
material. Examples of such micro-organisms include, but are not limited to 
those mentioned in the above mentioned prior art patent specifications. A 
specially preferred micro-organism is Protaminobacter rubrum (CBS 574.77) 
since it is very effective and can safely be used for the production of 
food ingredients. Good results have also been obtained with, for instance, 
Serratia ptymurhica and Erwinia rhapontici, but these microbes have 
pathogenic properties and are therefore less safe to use. Other suitable 
isomaltulose-forming micro-organisms may also be isolated from sugar beets 
and similar sources or may be derived from such micro-organisms by various 
techniques, as is evident to those skilled in the art. 
The anion exchange material useful as a carrier in the present invention 
should have an anion exchange capacity which renders it suitable for 
binding the viable microbial cells by adsorption. Weakly basic anion 
exchangers are preferred. 
Weakly basic anion exchangers are materials having primary and/or secondary 
/or tertiary amino groups. They dissociate and have exchange capability in 
acidic solutions. The materials having tertiary amino groups have rather 
basic properties and they are also called medium basic anion exchangers. 
The carrier should be inert in the sense that it should not affect the 
conversion of sucrose to isomaltulose. The carrier should, however, 
preferably promote the binding of the microbe cells to the surface 
thereof. The term "porous" as used of the carrier according to the 
invention is intended to mean that the solid carrier comprises a multitude 
of hollows and pores providing a surface area which is very large compared 
to, for instance, the surface area of a sphere having approximately the 
same radius. 
The term "substantially non-compressible" as used in the specification and 
claims is intended to mean that the solid carrier does not deform to any 
appreciable extent at the pressure prevailing during the conversion 
process. This is a clear distinction from the deformable alginate gel 
pellets used in the prior art. 
The carrier material of the invention should have a solid porous matrix 
capable of resisting deformation and allowing flow of the sucrose solution 
through the column without causing a large pressure drop. 
Carrier materials which may be used according to the present invention are 
weakly basic anion exchangers in the form of solid granules or particles. 
Examples of commercially available carriers of this type are those used in 
the working examples of the present specification. The carrier material 
particles are preferably of a generally spherical form so as to provide 
less resistance to flow.The carrier material should also preferably be of 
a macro-porous nature for providing a large surface area. 
The preferred carrier material of the present invention comprises a weakly 
basic anion exchange material in the form of diethylaminoethyl modified 
cellulose (DEAE cellulose) which includes microfibers or microparticles 
agglomerated with polystyrene, as described in U.S. Pat. No. 4,355,117, 
the disclosure of which is incorporated herein by reference. 
The advantage of using a weakly basic anion exchanger in the form of a 
porous carrier material with a large surface area, is that the microbes 
will be immobilized on the surface of the carrier and not entrapped within 
the carrier material, as is the case with alginate gels and in situ formed 
polymers. The sucrose solution need not penetrate into the carrier itself 
and, thus, the conversion rate will be enhanced compared to systems 
wherein the microbes are entrapped within the carrier material. 
The viable microbial cells are generally immobilized onto the carrier 
material on the conversion site. However, the carrier material may also be 
loaded with the microbial cells prior to use and the loaded carrier may be 
stored or transported for use at another location. One aspect of the 
present invention, in fact, concerns the porous solid carrier material 
including viable isomaltulose-forming micro-organism cells. 
A third aspect of the invention concerns the production of isomalt by 
hydrogenation of the isomaltulose produced from sucrose by the conversion 
process using the immobilized viable micro-organism cells. 
After the reaction in batch or column, the isomaltulose may be purified and 
recovered from the reaction solution. The isomaltulose may, for instance, 
be purified by ion-exchanging to remove impurities and by-products and 
crystallized by cooling crystallization. Isomalt can be produced from the 
isomaltulose by hydrogenation. The hydrogenation may, for instance, be 
performed as a catalytic hydrogenation in solution (30-50% w/w) using 
Raney nickel as a catalyst. The temperature is generally set at about 100 
to 130.degree. C., the hydrogen pressure at about 40 to 100 kg/cm.sup.2, 
the process duration being from about 3 to 10 hours. 
After completion of the hydrogenation the catalyst is separated from the 
reaction solution. The solution is filtered and, if desired, the resulting 
isomalt may be purified again by ion exchange. The isomalt may be used in 
liquid form or it may be recovered from the solution in ways which are 
well known to those skilled in the art. Alternatively, the isomaltulose 
may be hydrogenated into isomalt without a preceding crystallization and 
purification. 
The process according to the invention for converting sucrose to 
isomaltulose may be performed as a batch-wise process in a reaction tank 
including the immobilized viable microbial cells on the particulate 
carrier. After a sufficient reaction time for conversion of up to 80% or 
more of the sucrose, the solid carrier may be removed from the solution by 
filtration. After the reaction the solution contains isomaltulose as the 
main reaction product, some trehalulose, fructose and glucose, as well as 
any unreacted sucrose. 
In the preferred method the process is performed as a continuous process in 
a column packed with the carrier. There may be several columns which may 
be connected in series and/or in parallel, for instance for allowing one 
column to undergo regeneration while the other ones are in operation. The 
sucrose solution may also be recirculated through one or several columns 
to increase the conversion. 
The carrier includes viable glucosyltransferase enzyme containing 
micro-organism cells (isomaltulose-forming micro-organism cells) 
immobilized onto the surface thereof. The immobilization of the microbes 
onto the carrier surface may be performed either through "on column 
loading" by feeding a cell suspension through a column packed with 
carrier; or "in shake flask loading" by introducing carrier to a 
cultivation medium during propagation in a shake flask or the like 
container. 
In both methods it is preferable to further enhance the loading in column 
by feeding fresh nutrient medium through the column. 
The "on column loading" allows the column to be packed with the new carrier 
material before any immobilization takes place. Loading with the microbes 
is easy and no additional reaction steps are necessary after the 
immobilization has taken place. Thus, the microbes are not exposed to any 
stringent treatment conditions. 
If desired, the immobilization of the microbial cells onto the carrier may 
be enchanced by adding crosslinking agents such as glutaraldehyde to the 
solution. It is also possible to add flocculating agents such as those 
known in the prior art. Flocculation has been found to enchance the 
conversion and prolong the half life of the immobilized system. However, 
since the flocculation in the present invention is performed with the 
microbial cells immobilized on a solid carrier matrix, no drying step is 
required. The flocculated and crosslinked carrier is ready to use as such. 
The reaction is preferably carried out in a packed column, at an optimal 
temperature of 25 to 35.degree. C., preferably about 30.degree. C. 
Temperatures of 35 to 45.degree. C. have proven partly inactivating. 
Temperatures above 55.degree. C. totally inactivated a P. rubrum column in 
24 hours. 
A solution containing sucrose in purified form or as molasses is made to 
flow through the packed column, preferably from bottom to top to 
facilitate the removal of CO.sub.2, which will rise with the flow together 
with any aeration air to the top of the column. 
The sucrose concentration of the solution should preferably be kept at 
about 20 to 40%, more preferably at 25 to 35%. Sucrose concentrations 
above 55% have been found to significantly reduce the conversion in a 
short space of time. The pH of the sucrose solution should preferably be 
kept at about 4 to 8, more preferably at 5.5 to 7. 
Although the flow rate of the sucrose solution can be very slow to provide 
a very high conversion of sucrose to isomaltulose, a commercial process 
will preferably be operated at less than maximum conversion to save 
reactor volume. A conversion of up to about 80% is suitable in most 
applications. The isomerization may be continued until the isomaltulose 
yield drops. It may be preferable or necessary to refresh the microbial 
activity from time to time by feeding nutrients into the column. A 
constant low nutrient feed may also be used. 
According to the preferred embodiment of the process according to the 
present invention the isomerization is performed as a continuous process 
in a packed column reactor containing immobilized microbial cells bonded 
to the surface of a substantially incompressible carrier having weakly 
basic anion exchange properties. The carrier is preferably composed of a 
continuous porous bed, or alternatively of dimpled or reticulated porous 
granules. The matrix or granules may be composed of individual 
microparticles or microfibres. This carrier structure provides a very 
large surface area for the immobilization of microbial cells. 
The particulate or matrix character of the preferred carrier is produced by 
loosely binding, felting, weaving, gluing or agglomerating together the 
individual microparticles which comprise microfibres, microgranules, 
microspheres, microbeads, etc. The binding is accomplished by establishing 
chemical, adherent or mechanical links at some of the contact points 
between the individual microparticles. Chemical binding is accomplished by 
causing a chemical cross-linking reaction at these points. Adherent 
binding is accomplished by agglomerating or gluing the microparticles 
together through the use of an additional ingredient such as a 
thermoplastic resin. Mechanical binding is accomplished by entangling or 
knotting the fibres at the contact point or by joining the particles by 
meshing their surfaces together. In the latter form, the matrix will 
comprise a continuous structure throughout the reactor, much like cotton 
fluff of filter paper packed into a tube. Also in that case, in their 
final form, the particles will be discrete and individual. 
The microparticles are composed of a weakly basic anion exchange substance 
that can be formed into the desired, rough-surfaced microparticles. These 
substances include native or regenerated cellulose or rayon that is 
derivatized to provide anion exchange character; synthetic anion exchange 
resins such as phenolformaldehyde resins, acrylic resins and polystyrene 
resins, as well as agarose or dextrin based anion exchange resins. The 
preferred carrier is a porous, particulate anion exchange substance 
derived from cellulose or rayon that has been chemically modified to 
provide anion exchange character. Especially preferred embodiments include 
microfibres or microparticles of diethylaminoethyl substituted cellulose, 
adherently bound by agglomeration with polystyrene. 
It is believed that the electric forces established between the positively 
charged resin and the negatively charged microbial cells are primarily 
responsible for the binding of the microbe cells to the surfaces of the 
resin. This binding substantially reduces the leaching of the microbes, 
while still permitting intimate contact between the microbes and the 
sucrose solution. 
Weakly basic anion exchangers of the above presented kind have proven 
especially advantageous in providing a stable and active carrier material 
for the conversion of sucrose to isomaltulose. These carriers are solid 
and non-deformable, which provides a long operation life and low 
resistance to flow. They have a large surface area, which allows a large 
microbial population per volume unit to be provided for the conversion. 
A special advantage of the carrier system of the present invention is that 
when conversion drops after several refreshment cycles, the carrier may be 
regenerated, reloaded and taken into new use. This is especially so with 
the preferred carrier of the present invention, i.e. a DEAE-modified 
cellulose, comprising a solid polystyrene matrix. In case crosslinking 
with glutaraldehyde is performed, regeneration may, however, be 
complicated.

The invention will now be illustrated in detail with the following 
non-limiting examples. 
EXAMPLE 1 
Production of a carrier (DEAE) for immobilization Granular derivatized 
cellulose was manufactured according to U.S. Pat. No. 4,355,117 as 
follows. 
25 parts of fibrous cellulose was mixed with 25 parts of titanium dioxide 
and the mixture was compounded with 50 parts of food grade high-impact 
polystyrene using a twin-screw extruded. The extrudate was cooled in 
water, and sieved to a particle size of 0.35-0.85 mm. 
The sieved granular agglomerated cellulose particles were derivatized to 
form DEAE cellulose as described in the above mentioned U.S. Patent. 
20 g of the granular DEAE-cellulose produced was hydrated by soaking in 
distilled water for 5 hours. The hydrated DEAE-cellulose carrier was 
sanitized by soaking in ethanol (75%) for one hour and rinsed with sterile 
water before being transferred to a sterile glass column (60 cm high, 1,5 
cm diameter). 
Production of a Cell Suspension of Protaminobacter rubrum (CBS 574.77) 
Cells from a culture of Protaminobacter rubrum strain (CBS 574.77) were 
diluted with 10 ml of saline. 0.1 ml aliquots of the resultant suspension 
were used to inoculate 300 ml of growth medium in 1000 ml sterilized shake 
flasks. The medium was as follows: sucrose 40 g/l, peptone 10 g/l, yeast 
extract 5 g/l, meat extract 3 g/l, Na.sub.2 HPO.sub.4 2 g/l, pH 7. The 
inoculated flasks 3.times.300 ml were shaken 230 rpm at 30.degree. C. for 
20 hours to reach concentration over 5.times.10.sup.9 cells/ml. 
Immobilization of the Bacteria on the DEAE-Cellulose Carrier 
750 ml of the cell suspension were pumped through the carrier bed in column 
at a flow rate of 35 ml/h (=0.5 BV/h) at 25.degree. C. from the top to the 
bottom. The amount of immobilized microbes in the column was enhanced by 
feeding the column with 750 ml of fresh growth medium after feeding the 
cell suspensions. The carrier in the column was now ready for the 
production stage. 
Production of Isomaltulose in Column 
A sucrose solution of 25% (pH 7.5) was fed at 30.degree. C. continuously to 
the bottom of the column of immobilized P. rubrum on DEAE-cellulose 
carrier and product solution was removed from the top. This system allowed 
the CO.sub.2 to be released and removed from the top of the column. 
Different conversions were achieved depending on the flow rate or sucrose 
concentration (see table below). 
TABLE 1 
______________________________________ 
Sucrose concentration 25% 
Flow rate (BV/h)* 
0.6 0.3 0.15 0.08 
______________________________________ 
isomaltulose 12 22 35 61 
trehalulose 1 2 2.5 4.5 
fructose 1 2 2 2.5 
glucose 1 1.5 1.5 2 
sucrose 85 73 60 30 
______________________________________ 
Flow rate 0.08 BV/h 
Sucrose concentration 
25% 35% 55% 
______________________________________ 
isomaltulose 61 43 12 
trehalulose 4.5 2 1 
fructose 3 3 1 
glucose 2 2.5 0.6 
sucrose 30 50 86 
______________________________________ 
(BV = Bed Volume) 
The column of immobilized P. rubrum on DEAE-cellulose carrier was 
maintained in operation for two weeks. The conversion diminished as time 
went on. The immobilized cells were refreshed after two weeks by feeding 
the column with a fresh sterile cultivation medium (10 BV). After this 
refreshing cycle the flow rate of sucrose solution (25%, 30.degree. C.) 
was adjusted to 0.08 BV/h which converted sucrose to the following product 
(analyzed by HPLC; ion exchange resin in Pb.sup.++ -form): isomaltulose 
79%, trehalulose 0.9%, fructose 0.4%, glucose 0.6, sucrose 18.5%. 
The isomaltulose solution received from the column of immobilized P. rubrum 
was evaporated at a temperature of 70-85.degree. C. to the concentration 
of 70% and crystallized by linear cooling from 65.degree. C. to 25.degree. 
C. Isomaltulose crystals were recovered by centrifugation with washing and 
dried at 50.degree. C. The centrifugation yield was 70% (isomaltulose from 
isomaltulose) and the isomaltulose content of the crystals was 95 to 100%. 
Isomaltulose solution (50% w/w) was hydrogenated to isomalt using Raney 
nickel as a catalyst (pH 9, temperature 100.degree. C., hydrogen pressure 
40 kg/cm.sup.2, 3 hours). After completion of the hydrogenation the Raney 
nickel was separated from the reaction solution. The solution was 
concentrated by evaporation and isomalt was crystallized from the solution 
in a conventional way. 
After one month in use the DEAE-cellulose carrier was regenerated in column 
by washing out immobilized P. rubrum cells with 1 M NaOH (60.degree. C.) 
until the original color of the carrier was achieved, washing with water, 
buffering to pH 5 and washing with sterile water. After that the carrier 
was ready for reloading and for a new production period. 
EXAMPLE 2 
Production of Cell Suspension of Erwinia rhapontici (ATCC 29284) 
Cells from a culture of Erwinia rhapontici (ATCC 29284) were diluted with 
10 ml of saline, 0.1 ml of aliquots of the resultant suspension were used 
to inoculate 300 ml of growth medium in 1000 ml sterilized shake flasks. 
The medium was as follows: sucrose 40 g/l, peptone 10 g/l, meat extract 6 
g/l, KH.sub.2 PO.sub.4 0.01 M, pH 7. The inoculated flasks 3.times.300 ml 
were shaken 230 rpm at 30.degree. C. for 24 hours to reach a concentration 
over 2.times.10.sup.9 cells/ml. 
Production of Cell Suspension of Serretia plymuthica (ATCC 15928) 
Cells from a culture of Serretia plymuthica (ATCC 15928) were diluted with 
10 ml of saline. 0.1 ml of aliquots of the resultant suspension were used 
to inoculate 300 ml of growth medium in 1000 ml sterilized shake flasks. 
The medium was as follows: sucrose 40 g/l, peptone 10 g/l, meat extract 6 
g/l, KH.sub.2 PO.sub.4 0.01 M, pH 7. The inoculated flasks 3.times.300 
were shaken 230 rpm at 30.degree. C. for 24 hours to reach a concentration 
over 2.times.10.sup.9 cells/ml. 
Production of Isomaltulose in Column by Immobilized Serretia plymuthica 
(ATCC 15928) and Erwinia rhapontici (ATCC 29284) 
Immobilization of S.plymuthica and E.rhapontici on a DEAE carrier, spezyme 
GDC (producer Cultor Ltd) was carried out as described in Example 1. A 25% 
sucrose (pH 7.5) solution was pumped at 30.degree. C. continuously through 
the immobilized cell column. The flow rate was adjusted to maximize 
isomaltulose concentration in the outflow. Sucrose was converted to the 
following products (analyzed by HPLC; ion exchange resin in Pb.sup.++ 
-form); 
______________________________________ 
S. plymuthica 
E. rhapontici 
flow rate 0.12 BV/h 0.02 BV/h 
______________________________________ 
isomaltulose 80% 79% 
trehalulose 7.5% 15% 
fructose 5.5% 0.5% 
glucose 3% 0.5% 
sucrose -- --% 
______________________________________ 
Both columns with their specific immobilized microbial cells were 
maintained in operation for a couple of weeks. The production diminished 
as time went on. The microbes were refreshed from time to time by feeding 
the column with a fresh sterile cultivation medium (10 Bed Volume, BV). 
Regeneration 
After the several refreshing cycles (one month's use) the Spezyme GDC 
carrier was regenerated in column by washing out immobilized microbial 
cells with 1M NaOH (60.degree. C.) until the original color of the carrier 
was achieved, washing with water and buffering to pH 5 and washing with 
sterile water. After that the carrier was ready for reloading and for a 
new production period. 
EXAMPLE 3 
Testing of Anion and Cation Exchange Resins 
Protaminobacter rubrum (CBS 574.77) was cultivated in the same conditions 
as in Example 1. After 5 hours cultivation 3 grams of each tested ion 
exchange material was added to the 75 ml of the cultivation medium. 
Cultivation in shake flasks was continued up to 13 hours to load the ion 
exchange materials with cells of P. rubrum. The ion exchange materials 
were separated from the cultivation medium by filtration and washed with 
water. One of two Spezyme GDC carrier samples was exposed to 
glutaraldehyde treatment for cross-linking. 3 g of Spezyme GDC with 
immobilized P. rubrum was mixed with 15 ml of 0.3% glutaraldehyde. After 
0.5 hour's slow agitation the Spezyme GDC was washed with 150 ml of 
distilled water. The activity of immobilized P. rubrum cells on each ion 
exchange material, including the glutaraldehyde treated Spezyme GDC, was 
determined as glucosyltransferase activity. The results of the tests are 
shown in Table 2. The properties of the carrier materials are shown in 
Table 3. 
TABLE 2 
______________________________________ 
The activity of P. rubrum cells immobilized on various carriers 
Glucosyl- 
transferase 
Ion exchanger 
Solid matrix 
Trade name activity U/g 
______________________________________ 
Weakly basic anion exchangers: 
DEAE-cellulose 
polystyrene 
Spezyme GDC.sup.1 
1.0 
DEAE-cellulose 
polystyrene 
Spezyme GDC + 
0.8 
glutaraldehyde 
tertiary amine 
phenolform- 
Duolite A 568.sup.2 
1.3 
aldehyde 
tertiary amine 
styrene-di- 
Amberlite 0.6 
vinyl-benzene 
IRA 93.sup.2 
tertiary amine 
polystyrene 
Macronet MN-100.sup.3 
0.3 
Weakly acidic cation exchangers: 
carboxylic polyacrylic 
Duolite C 464.sup.2 
0 
Strongly basic anion exchangers: 
quaternary polystyrene 
Macronet MN 400.sup.3 
0 
ammonium 
quaternary styrene-di- 
Amberlite 0 
ammonium vinyl-benzene 
IRA 900.sup.2 
Strongly acidic cation exchangers: 
sulphonic polystyrene 
Residion EXC.sup.4 
0 
sulphonic styrene-di- 
Amberlite 0 
vinylbenzene 
IRC 200.sup.2 
______________________________________ 
The ion exchangers were purchased from 
.sup.1 Cultor Ltd. 
.sup.2 Rohm et Haas Ltd. 
.sup.3 Purolite Ltd. 
.sup.4 Mitsubishi Chemicals Co 
TABLE 3 
______________________________________ 
Properties of the carrier materials 
Capacity 
Surface Pores 
meq/ml m.sup.2 /g 
.ANG. 
______________________________________ 
Weakly basic anion exchangers: 
Spezyme GDC-carrier 
0.2-0.25 
Duolite A 568 1.2 1 (ml/g) 150-250 
Amberlite IRA 93 0.85 25 300-1000 
Macronet MN-100 0.15 800-1000 850-950 
Weakly acidic cation exchanger: 
Duolite C 464 2.7 
Strongly basic anion exchangers: 
Macronet MN 400 0.3 800-1000 850-950 
Amberlite IRA 900 
1.0 18 100-1000 
Strongly acidic cation exchangers: 
Residion EXC 1.1 200 
Amberlite IRC 200 
1.75 45 100-500 
______________________________________ 
Determination of the Glucosyltransferase Activity 
Incubation test: 5 ml of 10% sucrose solution in 0.05 M KH.sub.2 PO.sub.4 
buffer pH 7 was mixed with 1 g of the immobilisate in 5 ml of water in 
test tube. The mixture was incubated at 30.degree. C. for 60 minutes 
shaking mildly. The enzyme reaction was stopped by heating the test tubes 
at 100.degree. C. in a water bath for 2 minutes. A control sample was 
prepared likewise but the enzyme reaction was stopped directly after the 
addition of the enzyme sample. Isomaltulose formed in the reaction was 
determined as a reduced sugar (DNS-method). U/g=(.mu.Mol/ml.times.5 
ml)/(60 min..times.amount of sample, g). 
EXAMPLE 4 
Production of a Cell Suspension of Protaminobacter rubrum (CBS 574.77) 
Cells from a culture of Protaminobacter rubrum strain (CBS 574.77) were 
diluted with 10 ml of saline. 0.1 ml aliquots of the resultant suspension 
were used to inoculate 300 ml of growth medium in 1000 ml sterilized shake 
flasks. The medium was as follows: sucrose 40 g/l, peptone 10 g/l, yeast 
extract 5 g/l, meat extract 3 g/l, Na.sub.2 HPO.sub.4 2 g/l, pH 7. The 
inoculated flasks 3.times.300 ml were shaken 230 rpm at 30.degree. C. for 
20 hours to reach a concentration over 5.times.10.sup.9 cells/ml. The cell 
suspension was concentrated by centrifugation (5000 rpm, 20 min) to the 
volume of 1/8 of the original. 
Immobilization of the Bacteria on a DEAE-Cellulose Carrier using Tannic 
Acid (TA), Polyethyleneimine (PEI) and Glutaraldehyde (GA) 
A mixture of the above P. rubrum cell suspension and a Spezyme GDC carrier 
material (producer Cultor Ltd) was exposed to the following treatments: 
1. 40 ml of the concentrated cell suspension and 20 ml of wet Spezyme GDC 
carrier were agitated with a magnetic stirrer at room temperature for 30 
minutes. 
2. 40 ml of the concentrated cell suspension and 20 ml of wet Spezyme GDC 
carrier were agitated with a magnetic stirrer at room temperature for 30 
minutes. Thereafter 0.6 ml of a 10% glutaraldehyde (GA) solution was added 
to crosslink cells onto the Spezyme GDC carrier and agitation was 
continued for further 30 minutes. 
3. 40 ml of the concentrated cell suspension and 20 ml of wet Spezyme GDC 
carrier were agitated with a magnetic stirrer at room temperature for 30 
minutes. Thereafter 0.4 ml of polyethyleneiminie (PEI) (25%) was added to 
the same mixture to flocculate cells and mixing was continued for 10 
minutes. The reaction mixture was crosslinked by adding 0.6 ml of 
glutaraldehyde (10%) to the mixture and agitation was continued for 
further 30 minutes. Granular type of flocculants appeared in the solution. 
4. 40 ml of the concentrated cell suspension and 20 ml of wet Spezyme GDC 
carrier were agitated with a magnetic stirrer at room temperature for 30 
minutes. Thereafter 1 ml of tannic acid (TA) (4%) was added to flocculate 
the cell suspension and the mixture was agitated for 30 minutes. 
Flocculation was further enhanced by adding 1 ml of PEI (25%) and by 
allowing the reaction to proceed for 10 minutes under agitation. 
Crosslinking of the flocs and the Spezyme GDC carrier was carried out by 
adding 0.6 ml of glutaraldehyde (10%) to the mixture and the agitation was 
continued for further 30 minutes. The solution comprised a mixture of 
Spezyme GDC granules containing adsorbed cells and agglomerated cells and 
separate granular flocs of cell mass. 
Each one of the above four reaction mixtures 1 to 4 was transformed to its 
respective column (diameter 2 cm, volume 70 ml). Additional reaction 
solution was filtered off in the column through the Spezyme GDC and the 
granular flocs containing P. rubrum cells. Loading the column from the top 
with 32% sucrose solution in 0.05 M phosphate buffer (pH 7) at room 
temperature was started immediately at a feed rate of 4.4 ml/h (=0,25 
BV/h). The conversion ability of each column was tested running the 
columns continuously for 20 days. The results are shown in Table 4 below. 
TABLE 4 
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A summary of the column trials 
2 3 4 
Trial 1 Spezyme + Spezyme + Spezyme + TA + 
Spezyme GA GA + PEI PEI + GA 
______________________________________ 
isomaltulose 
16 14 53 73 
yield % on 
d.s. (1 st 
day) 
isomaltulose 
production 
g/l (column)/ 
after 1 day 
14 12 44 54 
after 20 days 
4.5 6 27 38 
half life, h 
250 500 800 850 
(at 22- 
24.degree. C.) 
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The monitoring period of the column trials was 20 days and no changes in 
the resistance of flow was observed. The Spezyme GDC carrier had a 
combined effect as an immobilization carrier. It offered a good base for 
adsorbtion of the cells in solution and served as a flowing aid for the 
separate flocs. 
The present invention has been described herein by way of some specific 
examples. These examples are, however, only of an illustrative nature and 
it is obvious to those skilled in the art that the invention may be varied 
in a number of ways without deviating from the scope of the appended 
claims.