CA1179283A - SUCROSE MUTASE, IMMOBILISED SUCROSE MUTASE AND THE USE OF THIS IMMOBILISED SUCROSE MUTASE FOR THE PREPARATION OF ISOMALTULOSE (6-0-.alpha.-D- GLUCOPYRANOSIDO-D-FRUCTOSE) - Google Patents

Abstract

Abstract of the Disclosure An enzyme, which converts sucrose into isomaltulose, has been isolated from Protaminobacter. It can be immobilised by inclusion in hollow fibers, by adsorption onto a solid carrier, by covalent bonding to a carrier or by bonding to a membrane. The immobilised enzyme is used for the production of isomaltulose from sucrose.

Description

11'iJ9283 The invention reLates to a certain new enzyme which converts sucrose into isomaltulose ln a high yield, to this enzyme in i~mobilised form and to the ccntinuous conversion of sucrose into isomaltulose (6-0-a-D-glucopyranosido-D-fructose) with the aid of this immobilised enzyme.
A mixture of 6-o-a-D-glucopyranosido-D-sorbitol and 6-0-a-D-glucopyranosido-D-mannitol, which can be used as a sugar substitute, can be obtained from isomaltulose by hydrogenation in accordance with the method of DE-AS
(German Published Specification) 2,520,173 or German Patent Specification 2,217,628.
It is known that sucrose can be converted into isomaltulose in a good yield by cells of Protaminobacter rubrum (DE-OS (German Published Specification) 1,049,800).
According to German Patent Specification 2,217,628, this reaction proceeds in an optimum manner in 15-40~ ~
strength sucrose solution at 20-37C, with vigorous stirring and aeration.
According to DE-OS (German Published Specification)

2,806,216, the multiplication of the bacteria and the conversion of sucrose into isomaltulose can be carried out simultaneously and continuously.
Instead of Protaminobacter rubrum, it is also possible to use other bacteria, such as Erwinia carotovor~, Serratia marcescens (NCIB 8285), Serratia plymuthica (ATCC 15928) and Leuconostoc mesenteroides (NRRL B-512f) (ATCC 10830a), in such processes.
These fermentation processes have various dis-advantages:

3 1. Some of the sucrose employed is consumed as a source of carbon for multiplication of the cells.
2. The stirring and aeration necessary for cell multiplication requires a high consumption of energy.
3. Some of the product is lost when the cells ~re separated off.

Le A 20 647 .

1~'79'~83

4. According to DE-OS (German Published Specification) 2,806,216, the optimum sucrose concentration is about 25go~ The sugar must therefore first be diluted to this concentration, and must be concentrated again, with a high con-sumption of energy, after the reaction in order to crystallize the product.

5. In addition to the source of carbon provided by sucrose, multipli-cation of the bacteria requires nitrogen-containing substances and salts, the presence of which during crystallization of the product leads to losses.
All these disadvantages could in principle be avoided if it were possible to separate the conversion of sucrose into isomaltulose from the cell multiplication process and to stabilize the conversion over a prolonged period.
It has now been found, surprisingly, that an enzyme which converts highly concentrated solutions of pure sucrose into isomaltulose in a very good yield can be isolated from the cells of isomaltulose-forming bacteria, in particu-lar Protaminobacter rubrum CBS 57 477.
According to the present invention we therefore provide an enzyme which has been isolated from bacteria selected from Protaminobacter, Erwinia and Serratia, especially from Protaminobacter, and which converts sucrose into isomaltulose.
The enzyme according to the present invention, having been isolated, can be immobilized and stabilized by various processes which are known in principle. Preferred immobilized enzyme according to the present invention has been immobilized by inclusion in hollow fibres, by adsorption onto a solid carrier, by covalent bonding to a solid water-insoluble carrier, by covalent bonding to a water-soluble carrier, or by bonding to a membrane.
According to the present invention we further provide a process for the production of isomaltulose which comprises converting sucrose to isomaltulose by the action of an immobilized enzyme according to the present invention the reaction being carried out batchwise or continuously at 30 to 60 C and at a 1~7~Z83 sucrose concentration of 10 to 75% by weight, preferably 40 to 60% by weight.
Isolation of the enzyme was particularly surprising because enzymes having a similar activity, that is to say mutases whieh effect an intramolecular transfer reaction on oligosaccharides, having not hitherto been disclosed. As far as is known, disaccharides are formed in the living cell either by degrada-tion of higher polymers or by energy-dependent synthesis via nucleotide deriva-tives. The enzyme isolated is to be known as "sucrose mutase" (sucrose isomaltulose mutase), and is so referred to hereinafter.
The process according to the invention can be divided into the follow-ing four steps: 1. multiplication of the cells; 2. isolation of the enzyme;
3. immobilization of the enzyme; and 4. conversion of sucrose into isomaltulose with the immobilized enzyme.
To isolate the enzyme, cells of Protaminobacter rubrum (CBS 57 477), in particular, were eultured in a manner whieh is known in prineiple: for the purpose of optimum enzyme produetion, a medium of suerose-eontaining inspissated juice, whieh is diluted to a solids eontent of 5 to 25%l with the addition of 2% of corn-steep liquor and 0.1% of (NH4)2HPO4, is advantageously used.
A yield of 10 to 50 g of sedimented cells/litre of fermentation solu-tion and an enzymatic activity of 10 to 30 U/ml (1 U (unit) _ conversion of 1 ~mol of sucrose/minute at 30 C; for the test, see Example lb) are achieved with this system in a fermentation time of 14 to 16 hours at 30 C.
After eoneentration and breaking down of the cells, the enzyme is isolated by the generally known methods of enzyme purification. The following procedure has proved particularly suitable:

1~'792~3 The ceIls are concentrated 20-fold in a tube centrifuge or a self-desludging separator. To release the enzyme from the inside of the cells, the concentrated cells are broken down, for example by a high-pressure homogeniser ("Manton-Gaulin") under 500 bars.
The resulting solution of the enzyme containing suspended cell debris can be clarified by centrifugation or filtration, if necessary after adjusting the pH to a value which is advantageous for the precipitation of impurities (for example pH 5). This clarified solution of the enzyme can already be used for the immobilisation step. However, for mcst immobilisation processes it is more advantageous first to concentrate the enzyme still further. Precipitation processes, for example using ammonium sulphate or polyethylglycol, and chromatographic processes, for example molecular sieve chromatography (gel chromatography), ion exchange chromatography and adsorption chromatography, are suitable for this.
Chromatography on CM-Sephadex (a Trade Mark for carboxy-methyl-substituted crosslinked dextran) has proved particu-larly effective. A practical embodiment is described in Example lc. A specific activity of the enzyme of 50-250 U/mg of protein is achieved by subjecting the solution to chromatography once. Enzyme which is virtually pure according to electrophoresis and has a specific activity of 450 U/mg is obtained by repeated chromatography on CM-Sephadex .
The purified enzyme changes sucrose into iso-maltulose in a high yield. For example, 86.5% of isomaltulose, 8.6% of l-O-~-D-glucopyranosido-D-fructose, 4.0% of fructose and 0.9% of glucose have been obtained from a 48% strength sucrose solution at 35C. Other characteristic properties of the new enzyme are:
molecular~.~eighJ: about 50,000 (by gel chromatography) Le A 20 647 isoeIectric point: 8.o + 0.3 pH optimum: pH 6.5 + 0.5 stability optimum: pH 5.5 + 0.5 The free enzyme is rapidly inactivated at temp-eratures ~ 40C. The reaction is greatly inhibitedby Cu and Zn ions. Ca, Mn2+, Mg and borate have no effect on the enzymatic activity.
Immobilisation of the enzyme can be carried out by a large number of processes which are known in principle, such as the processes described, for example, in a summary by I. Chibata: Immobilized Enzymes, Halsted Press (a division of I. Wiley & Sons) New York 1978.
The following immobilisation processes, which can all in principle be used for immobilising the sucrose mutase, can be differentiated in a systematic manner:
inclusion process; crosslinking process; bonding to solid carriers by adsorption, covalent bonding or cross-linking; bonding to membranes: bonding to water-soluble carriers.
An example of the inclusion process is the inclusion of the enzyme solution in semi-permeable hollow fibres with a pore size which permits passage of the sugar molecules but retains the enzyme molecules inside the hollow fibres.
A practical embodiment of this process is described in Example 2.
Suitable solid carriers for the adsorption of the enzyme are, for example, ion exchangers. Cation exchangers on a polysaccharide basis, for example CM-cellulose (see Examples 3 and 4) have proved particularly 3 advantageous. The adsorptive bond is so firm that even high sucrose concentrations, for example 60%, do not detach the enzyme from the carrier. It is also possible to make the bond still f-~rmer by crosslinking, for example with glutarodialdehyde (see Example 5).
A particularly firm bond is achieved by covalent .
Le ~ 20 647

- 6 -Iinking. A covalent bond to carriers with hydroxyi groups or amino groups can be achieved, for example, with reagents such as cyanogen bromide, cyanuric chloride or FCP (trifluorochloro-pyrimidine). Examples are bonds to ceIlulose and "Sepharose" obtained with various reagents (see Examples 6 to 8), to polymers with anhydride groups (see Example 9) and to silicate (see Example 10).
For kinetic reasons, it is particularly advan-tageous to use membrane-bonded enzymes (DE-OS (German ~Published Specification) 2,553,649). Practical embodiments of the bonding of sucrose mutase to membranes are described in Examples 11 and 12.
Immobilisation by covalent bonding to water-soluble carriers, for example water-soluble starch, is also advanta-geous. The enzyme is thereby stabilised and, as a result of the high molecular weight of the carrier, can easily be separated from the isomaltulose solution with semi-permeable membranes in a simple manner and can be re-used (see Examples 13 to 17).
The appropriate procedure for converting sucrose into isomaltulose with the enzyme sucrose mutase which has been immobilised by one of the abovementioned methods depends on the particular characteristics of the immobilised enzyme. In all cases, a ~0 to 70% strength solution of sucrose is brought into contact with the immobilised enzyme at 30 to 60C until the desired degree of reaction is achieved In a typical case at 35 C and 48 %
strength sucrose, a product mixture composed of 5 %
of fructose, 2 % of glucose, 9 % of 1-0-~-D-glucopyrano-sido-D-fructose and 84 % if isomaltulose is obtained if the contact time is just sufficient for complete consuption of the sucrose. A substantially longer contact time leads to a lower yield of isomaItulose and higher yields of fructose, glucose and 1-0- .-D-glucopyranosido-D-fructose.
The formation of the by-products is also catalysed, in a virtually unchanged ratio, Le A 20 547 Z.~33 by hi~hly ~urified preparations of sucrose mutase, and is therefore to be regarded as a characteristic property of this enzyme. The product ra~io is influenced by temperature and substrate concentration: lower substra~e concentration results in an increased formation of fructose and glucose whereas higher temperatures fa~tour the formation of 1-0- ~ D-glucopyranosido-D-fructose and other yet unidentified side-products which are observed in HPLC analysis at higher retention times. These compounds, which are observed in a total yield of 0-7 % are probably of trisaccharide nature.
If the free enzyme or the enzyme bonded to a soluble carrier is used for converting sucrose into iso-maltulose, the reaction can be carried out, for example, in a hollow fibre reactor (see Example 2).
Another possibility is a vessel which is closed by a semi-permeable membrane and in which the reaction can be carried out either continuously or batchwise.
Enzyme bonded to a solid carrier can likewise be employed for batchwise operation, for example in stirred kettles, or for continuous operation in column reactors with ascending or descending flow. It must be possib~
to heat such column reactors, which typically have a diameter : packing height ratio of l:l to l:lO.
Enzyme bonded to membranes can be in the form of a flat membrane, a hollow fibre membrane or a filter cartridge. The sucrose solution flows once or several times, until the desired conversion is reached, through the membrane, which is charged with enzyme and is in a suitable support which can be heated.
Some of the isomaltulose formed can be isolated as crystals, by cooling, from the sucrose solutions reacted in one of these ways. A further amount of isomaltulose can be made to crystallise by evaporaJion of the mother liquor, and a total of 90 to 92% of the iso-maltulose formed can thus be isolated.
The production of the enzyme according to the present invention, its immobilisation and its use in converting sucrose into isomaltulose are illustrated by the following Examples.

Le A 20 6~7 1~79Z83 Example 1: Preparation of the enzyme la) Fermentation of Protaminobacter rubrum:
An inoculum of the strain Protaminobacter rubrum (CBS 57 477) was cultured in 200 ml of a nutrient solution which consisted of inspissated juice which had been diluted to a solids content of 25% and to which 0.5 g/l of (NH4)2HP04 had oeen added and which had been adjusted to pH 7.2, in a 1 litre flask at 30C for 16 hours on a shaking machine at 290 rpm. The culture from 5 to 10 such flasks (each of 1 to 2 litres) was used for inoculating a 300 litre fermenter. The medium consisted of 200 litres of inspissated juice (about 94% of sucrose in the solids content) which had been diluted with water to a solids content of 25% and to which 0.5 g/l of (NH4)2HP04 and 2% of corn-steep liquor had been added.
The ~ermentation was carried out at 30C, without monitoring the pH, with an aeration of 100 l/minute and at a stirring speed of 180 rpm, and had ended after 16 to 18 hours.
lb) Determination of the enzyme activity An incubation batch of 5 ml of 10% strength sucrose solution in 0.01 M phosphate buffer of pH 7.0 was used to determine the enzyme activity. ~.fter adding 1 to 10 U of the enzyme preparation to be tested (cell suspensicn, enzyme solution or immobilised enzyme), the batch was incubated at 30C for 60 minutes, whilst shaking. The reaction was then ended by heating the batch to 100C for one minute. The reaction in a control sample was stopped directly after addition of the enzyme preparation by 3 heating to 100C.
The conversion could be determined by 3 methods:
1. Determination of the unreacted sucrose and of the products by HPLC (high pressure liquid chrom~tography).
2. Enzymatic determination of the unreacted sucrose, for example in accordance with the method of H.U. Berg-Le A 2-0 647 11'79~83 g meyer and E. Bernt in H.U. Bergmeyer, Methoden der enzymatischen Analyse (Methods of Enzymatic Analysis), 2nd edition, volume II, pages 1,143-1,146, Verlag Chemie 1970.
3. By determination of the resulting reducing sugars with dinitrosalicylic acid, since sucrose does not have a reducing action whilst the products of the reaction do. The reagent consisted of an aqueous solution of lO g of dinitrosalicylic acid, 16 g of sodium hydroxide and 300 g of K/Na tartrate in 1 litre.
To carry out the reaction, 0.5 ml of this reagent was added to 0.4 ml of the incubation batch, the mixture was heated to 100C for 5 minutes, cooled by means of water and diluted with 2.5 ml of water and the extinction was measured at 540 nm against a reagent 0 value in a photo-meter. Evaluation was carried out by means of a standard curve, which had been obtained with pure iso-maltulose. The reducing sugars formed were obtained as isomaltulose equivalents in umols/ml of the incubation batch, and the enzymatic activity was calculated there-from by the formula:

U/ml or g =~UmOlS/ml x 5 60 x amount of enzyme sample (ml or g) lc) Isolation of the enzyme 28 litres of bacterial sludge with an activity of 77.5 U/ml (94% yield) were obtained from 200 litres of fermentation solution as described in Example la, with an activity of 11.5 U/ml, by centrifugation in a tube centrifuge (Sharples). The bacterial sludge was diluted to 50 litres with desalinated water and the pH value was 3 kept at 5.5 to 6.0 by adding dilute sodium hydroxide solution. The diluted sludge was broken down in a high-pressure homogeniser ("Manton-Gaulin") under 500 bars at a flow of 20 litres/hour.
.
Le A 20 647 3L1792~3 The pH value was then adjusted to 5.0 with acetic acid and the enzyme solution was freed from cell debris and precipitated impurities by centrifugation in a tube centrifuge. The still turbid centrifugate was further clarified by filtration, for example over a Seitz AS
filter. 44 litres of enzyme solution with an activity of 43 U/ml (87% yield) were obtained. The solution contained 19.1 mg of protein/ml (Lowry method). The specific activity of the enzyme was 2.15 U/mg.
756 ~.1 (32,500 U) of the clarified solution were adjusted to pH 6.o with dilute sodium hydroxide solution and diluted to 3,250 ml with desalinated water in order to establish a conductivity of 4.5 mS. This solution was discharged onto a 11 x 30 cm (2.9 litres) column con-taining CM-Sephadex C-50, equilibrated with potassium phosphate buffer of pH 6.o, with a conductivity of 4.5 mS. The column was then rinsed with 3 litres of the same buffer in order to remove impurities. The column was then eluted with a linear gradient of 5 litres of the rinsing buffer and 5 litres of the same buffer to which 0.5 M NaCl had been added. 25 ml fractions were coll-ected. The sucrose mutase activity appeared in fractions 150 to 200. The combined active fractions contained 27,122 U (83.5%) in 1,230 ml, with a specific activity of 98 U/mg. The enzyme solution was concen-trated to 475 ml by ultrafiltration over a membrane with an exclusion limit of 20,000 daltons ("Ami~o~ UM-20 E).
The concentrate was~desalinated over a polyacrylamide gel ("Biorad Bioge~" P4) column with water as the eluting agent. Yield: 21,535 U (79%) with a specific activity of 107 U/mg.
Example 2 Inclusion of the enzyme in hollow fibres A bundle of semi-permeable hollow fibres (type HlPlO, Messrs. Amicon~ was filled with 23 ml of a solution of sucrose mutase, containing 1,905 U with a specific Le A 20 647 ~ ~rad~ rk~

activity of 48.7 U/mg. 10% strength sucrose solution was passed through the inside of the hollow fibres.
The following product mixture was found, at 25C, by HPLC
analysis in the solution flowing out: at 60ml/hour:
4% of fructose, 3% of glucose, 1% of sucrose, 86% of iso-maltulose and 6 % of l-o-cc-D-glucopyranosido-D-fructose; and at 100 ml/hour: 4% of fructose, 4% of glucose, 7% of sucrose, 76% of isomaltulose and 9% of 1-c-a-D-glucopyranosido-D-fructose.
These results were virtually unchanged even after continuous operation for 5 days.
Example 3 Adsorptive bonding to CM-cellulose 50 g of moist CM-cellulose~equilibrated to pH
6.o (type CM 52, Messrs. Whatman) were added to 121 ml of a solution of sucrose mutase containing 5,810 U with a specific activity of 49 U/mg. The conductivity of the enzyme solution was 4.2 mS. The suspension was stirred at room temperature for 2 hours and the enzyme adsorbed onto the carrier was filtered off. Yield: 52.0 U/g =
2,965 U (51%).
The filtrate contained 759 U (13%).
Example 4 Adsorptive bonding to CM-cellulose In the same manner, 222 ml of a solution of sucrose mutase containing 4,770 U (77 U/mg) were reacted with 25 g of CM-cellulose~(type CM 11, Messrs. Whatman) at pH 5.5-Yield: 26.6 with an activity of 50.9 U/g - 1,354 U
28%.
The filtrate contains 730 U (15%).
Example 5 Adsorption with subsequent crosslinking 10 g of the carrier-adsorbed enzyme from Example 3 were stirred with a solutlon of 50 ml of 0.2% strength glutarodialdehyde for 1 hour and then filtered off.
Yield: 10.4 g with an activity of 49.2 U/g (98%) Exampl-e 6 Coupling of sucrose mutase to Sepharose 4B CL
by means of cyanogen bromide Le ~: 20 647 d~ra ~

1~7~2~3 3 g of moist Sepharose 4B CL were activated with 50 mg of cyanogen bromide at pH 11.0 for 30 minutes, the pH value of the suspension was adjusted to 7.0 with 1 N HC1 and the suspension was stirred with 25 ml of sucrose mutase solution (containing an activity of 6,350.7 units and 30.62 mg of protein, measured by the Lowry method) at pH 7.0 for 2 hours. The enzyme carrier was filtered off and washed with distilled water.
Yield of enzyme carrier: 2.7 g with an activity of 50.0 units/g = 135 units = 2.1% of the activity employed.
Filtrate: 120 ml with an activity of 6,486 units.
~ Covalent bonding of sucrose mutase to Sepharose 4B CL after activation with cyanuric chloride 90 g of moist Sepharose 4B CL were activated with 1.5 g of cyanuric chloride in a mixture of 100 ml of distilled water and 100 ml of dioxane at pH 6 to 7 for 30 minutes. The activated Sepharose 4B was filtered off, washed with dioxane and distilled water and finally suspended in 100 ml of distilled water. 59 ml of sucrose mutase solution with an activity of 6,661 units and containing 32.1 mg of protein, measured by the Lowry method, were added and the mixture was stirred at room temperature and at pH 7.0 for 4 hours. The enzyme carrier was filtered off and washed on the filter with distilled water.
Yield of enzyme carrier: 93.2 g with an activity 3 of 18.54 units/g = 1,728 units - 27.2% of the activity employed.
Filtrate: 280 ml with an activity of 85.7 units = 1.35% of the activity employed.
~xample 8 Covalent bonding of sucrose mutase to cellu-lose after activation with cyanuric chloride Le 4 20 647 ~179Z~3 ~ 50 g of cellulose ("Avicel" from Messrs. Merck, IL~ br Darmstadt) were activated with 5 g of cyanuric chloride as described in Example 7. 25 ml of sucrose mutase solution with an activity of 6,350.7 units and containing 5 30.62 mg of protein, measured by the Lowry method, were added to the activated carrier and the mixture was worlced up as described in Example 7.
Yield of enzyme carrier: 119.7 g with an acti-vity of 15.5 units/g = 1,850.7 units - 29.1% of the 10 activity ~mployed.
Filtrate: 275 ml with an activity of 39.7 units = 0.63% of the activity employed Example 9 Covalent bonding of sucrose mutase to anhy-dride resin 10 g of anhydride resin (copolymer of 80% by weight of tetraethylene glycol dimethacrylate, 10% by weight of maleic acid and 10% by weight of maleic anhydride, particle size: 0.5-0.8 mm) were suspended in acetone and rinsed on a frit several times with acetone.
20 The washed resin was suspended in 20 ml of distilled water and the suspension was stirred with 66 ml of sucrose mutase solution (2,003.1 units and 19.8 mg of protein, measured by the Lowry method) at pH 6.2 overnight.
The enzyme carrier was filtered off and washed on the 25 frit with distilled water, 10% strength NaCl solution and distilled water again.
Yield of enzyme carrier: 14.48 g with an acti-vity of 18.9 units/g = 273.5 units - 13 . 6% of the activity employed.
3 Fiitrate: 315 ml with an activity of 85.1 units = 4.27% of the activity employed.
Example 10 Covalent bonding of sucrose mutase to silicate Qbout 100 ml of 50% strength sulphuric acid were added to 500 ml of sodium silicate solution (available, 35 for example, from Messrs Merck, Darmstadt), whilst stir-.. ..
Le .4 20 647 ~r~de~ k ~lt79Z~3 ring, and the precipitate was filtered off, pressed dry and dried at 160 to 180C in a circulating air drying cabinet. The resulting silicate was comminuted in a mortar.
50 g of silicate were boiled under reflux with 1.33 litres of a 10% strength solution of ~-aminopropyl-triethoxysilane in benzene for 8 hours in order to intro-duce amino groups, and, after cooling, the silicate was washed three times with in each case 330 ml of benzene and 330 ml of acetone. The silicate carrying amino groups was washed with about 1 litre of 0. 05 M phosphate buffer of pH 7.0 in portions.
Yield: 39.0 g of amino-silicate 19.5 g of amino-silicate were suspended in 200 ml 15 of distilled water/dioxane (1:1) and activated with 1.95 g of cyanuric chloride at pH 5 to 7 for 60 minutes. The activated resin was washed with dioxane and distilled water. lO0 ml of sucrose mutase solution with an activity of 15,030 units were added, whilst stirring, and 20 the suspension was stirred at room temperature for 4 hours. The enzyme carrier was filtered off and washed on the filter.
Yield of enzyme carrier: 25.1 g with an activity of 58.7 units/g = 1,473 units - 9.8% of the acti~ity 25 employed.
Filtrate: no activity Example 11 Isomerisation of the sucrose into isomaltulose in an enzyme-membrane reactor 30 a) A filter cartridge with a ~jilicate polymer mem-brane, for example an "Amerace H30~G cartridge with a mem-brane surface of 0.25 m , was inserted into an appropriate filter housing and was prewashed with distilled water at room temperature by means of a pump. By circulatory 35 pumping of a dilute sucrose mutase solution (25 U/ml) at Le A 20 647 .
~ ~rade ~1ark +4C, the enzyme W25 immobilised by adsorption in the pores of a membrane cartridge pretreated in this manner.
The remainder of the enzyme solution was washed out with distilled water at +4C and the enzyme-membrane reactor was kept under moist conditions.
b) To isomerise the sucrose into isomaltulose, a filtered 10% strength sucrose solution was pumped con-tinuously (as shown schematically in Figure 1) from the container (S) by means of an infinitely adjustable pump (p), via a heat exchanger (W) and through a flow meter (D), through the enzyme-membrane reactor (EMR) into the iso-maltulose container (I) at 34C. The composition of the isomaltulose solution was determined by HPLC.
According to Table 1 which follows, the degree of conversion depended on the flow rate.
Table 1 Composition of the isomaltulose solution as a function of the flow rate (0. 25 m2 membrane surface; 10% strength sucrose solution) 20 Flow rate inIsomaltuloseResidual sucrose litres/hour % %
0.68 72.6 0.0 1.9 70.3 9.4 4.2 65.3 16.1 8.2 31.~1 59.1 Example 12 Isomerisation of sucrose into isomaltulose in an enzyme-membrane reactor.
The process was carried out as described in Example lla) and b) but using a 20% strength sucrose solution in-stead of the 10% strength sucrose solution of Example 11 b).
The content of the resulting isomaltulose solution was determined by HPLC. As shown in Table 2, the degree 30 of conversion depended on the flow rate. At a flow rate of 0.58 lit,re per,hour and ar. isomaltulose content of 74.1%

Le A 20 647 ~9 Z~3 (= 148.2 g/litre), a capacity of about 8.3 kg of isomaltulose per m of membrane area per day was calculated for the enzyme-membrane reactor.
Table 2 Composition of the isomaltulose solution as a function of the flow rate (0.25 m of membrane surface, 20%
strength sucrose solution) Flow rate in Isomaltulose Residual sucrose litres/hour % %
.
o.58 74.1 0.0 1.1 72.8 2.8 2.3 69.1 14.7 3.9 41.2 48.7 0 Example 13 Covalent bonding of sucrose mutase to starch after activation with cyanogen bromide 2 g of starch according to Zulkowsky (obtained from Messrs. Merck, Darmstadt) were dissolved in 50 ml of distilled water and activated with 0.2 g of cyanogen bromide at pH 11.0 for 30 minutes, and the pH was then adjusted to

7.0 with 2 N HCl. The starch was coupled with 20 ml of sucrose mutase solution with an activity of 10,000 units at room temperature and at pH 6.5 to 7.0 for 2 hours.
The soluble enzyme carrier was separated from the sucrose mutase via an "Amicon" XM 100 membrane and was washed several times with a total of 500 ml of 0.1 M phosphate buffer of pH 7Ø
150 ml of starch-sucrose mutase with an activity of 7,020 units = 70.2% of the activity employed resulted.
Example 14 Covalent bonding of sucrose mutase to Dextran 60 after activation with cyanogen bromide 2.5 g of Dextran 60 (Molecular weight 60,000 -9l~ Messrs. Serva, Heidelberg) were activated with .. .... . . . . . . . . .
Le A 20 647 1179Zfl3 cyanogen bromide as described in Example 13 and coupled with 102 ml of sucrose mutase solution (12,500 units and 250 mg of protein, measured by the Lowry method), and the mixture was worked up as described in Example 13.
104 ml of Dextran 60-sucrose mutase with an activity of 8,580 units - 68.6% of the activity employed resulted.
Example 15 Covalent bonding of sucrose mutase to Manucol LD after activation with cyanogen bromide ~L
2.5 g of "Manucol" LD (soluble alginate obtained from Messrs. Alginate Industries GmbH, Hamburg) were coupled with sucrose mutase as described in Example 13, and the mixture was worked up.
192 ml of"Manucol"sucrose mutase with an activity of 11,712 units - 93.7% of the activity employed resulted.
After dropwise addition into 1% strength CaC12 solution, "Manucol-sucrose mutase could be precipitated as an insoluble enzyme carrier. 0 Example 16 Covalent bonding of sucrose mutase to carrageenan after activation with cyanogen bromide 2.5 g of carrageenan (soluble polysaccharide sulphuric acid ester from the marine alga Chandrus crispus, obtained from Messrs. Serva, Heidelberg) were coupled with sucrose mutase as described in Example 13, and the mix-ture was worked up.
210 ml of carrageenan-sucrose mutase with an activ-ity of 8,421 units - 67.4% of the activity employed 3 resulted.
Example 17 Covalent bonding of sucrose mutase to a soluble anhyd~ide polymer 0.5 g of "Gantrez' AN 179 (soluble copolymer of ma eic anhydride and methyl vinyl ether, obtained from Messrs.. Serva, Heidelbe~g)..we~e. dissolved. in 50 ml.of r e A 20 647 ~ ~Qde ~

distilled water~ 12,250 units of sucrose mutase were added and the solution was stirred at pH 6.2 and at room temperature overnight. Non-bonded enzyme was separated off by ultrafiltration ov~r an "Amiconti XM 100 membrane.
310 ml of "Gantrez" AN 179-sucrose mutase with ar.
activity of 3,040 units - 24.3% of the activity employed resulted.
Example 18 Adsorptive bonding of sucrose mutase to titanium dioxide with subsequent cross-linking by me~ns of glutarodialdehyde 10 g of "Enzacry~fi titanium dioxide (obtained f.om Koch-Light Laboratories Ltd., Colnbrook, England) were suspended in 10 ml of distilled water and the suspension was stirred with 4 ml of sucrose mutase solution at pH 6.5. The enzyme carrier was filtered off and washed with distilled water.
Yield of enzyme carrier: 12.88 g of titanium dioxide-sucrose mutase with an activity of 22.3 units/g = 286.3 units.
Filtrate: 75 ml with an activity of 448.5 units The titanium dioxide-sucrose mutase carrier could be crosslinked by treatment with 0.1% strength glutaro-dialdehyde solution for 30 minutes.
Example 19 Conversion of sucrose into isomaltulose with adsorbed sucrose mutase a) A 10% strength sucrose solution was allowed to flow through 21.3 g of the carrier-bonded enzyme from Example 3 in a 1.6 x 14 cm column at 30C. At a flow rate of 60 ml/hour, the following composition of the 3 runnings from the column was found by HPLC analysis:
80.7% of isomaltulose, 0% of sucrose, 7.4% of l-O-a-D-glucopyranosido-D-fructose, 6.9% of fructose and 5% of glucose.
b) 60% strength sucrose solution was converted into isomaltulose in the same column at a flow rate of 20 ml/
. . . . . . . . ..
Le A 20 647 ~ r~e l~k hour. HPLC analysis of the runnings: 85.6% of iso-maltulose, 0~ of sucrose, 10.6~ of l-0--D-glucopyrano-sido-D-fructose, 3.1% of fructose and 0.7% of glucose.
The column reactor was operated over a period of 540 hours with a loss in activity of less than 30%.
c) The reaction was carried out in the same manner with 60% strength sucrose solution at 45C and at a flow rate of 30 ml/hour. Under these conditions, the con-version fell to 50% in 430 hours.
Example 20 Conversion of sucrose into isomaltulose with covalently bonded sucrose mutase A 2 x 17 cm column was filled with 50 ml of amoist preparation of carrier-bonded sucrose mutase according to Example 8, and 48% strength sucrose solution was allowed to flow through the column at 11 m]/hour.
The reaction was carried out first at 35C for 500 hours and then at 45C for a further 330 hours. The column runnings typically had the composition:
80% of isomaltulose, 6% of sucrose, 8% of l-0-a-D-glucopyranosido-D-fructose, 4% of fructose and 2% of glucose. The loss in activity of the reactor was less than 20% throughout the entire period of the experiment.

Le A 20 647

Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An enzyme which has been isolated from bacteria selected from Protaminobacter, Erwinia and Serratia and which converts sucrose into iso-maltulose.

2. An enzyme according to claim 1, which has been isolated from Protaminobacter.

3. An enzyme according to claim 1 or 2 which has been isolated and immo-bilized.

4. An enzyme according to claim 1 or 2, which has been immobilized by inclusion in hollow fibres.

5. An enzyme according to claim 1 or 2, which has been immobilized by adsorption onto a solid carrier.

6. An enzyme according to claim 1 or 2, which has been immobilized by covalent bonding to a solid water-insoluble carrier.

7. An enzyme according to claim 1 or 2, which has been immobilized by covalent bonding to a water-soluble carrier.

8. An enzyme according to claim 1 or 2, which has been immobilized by bonding to a membrane.

9. An enzyme according to claim 1 which has been isolated from Protaminobacter rubrum.

10. An enzyme according to claim 1 which has been isolated from Erwinia carotovora.

11. An enzyme according to claim 1 which has been isolated from Serratia marescens.

12. An enzyme according to claim 1 which has been isolated from Serratia plymuthica.

13. A process for the production of isomaltulose which comprises convert-ing sucrose to isomaltulose by the action of an immobilized enzyme according to claim 1 or 2 is used, the reaction being carried out batchwise or continuously at 30 to 60 C and at a sucrose concentration of 10 to 75%.

14. A process for the production of isomaltulose which comprises convert-ing sucrose to isomaltulose by the action of an immobilized enzyme according to claim 1 or 2 is used, the reaction being carried out batchwise or continuously at 30 to 60 C and at a sucrose concentration of 40 to 60%.

15. A process for the production of isomaltulose which comprises convert-ing sucrose to isomaltulose by the action of an immobilized enzyme isolated from Protaminobacter, the reaction being carried out batchwise or continuously at 30 to 60 C and at a sucrose concentration of 10 to 75%.

16. A process for the production of isomaltulose which comprises convert-ing sucrose to isomaltulose by the action of an immobilized enzyme isolated from Protaminobacter, the reaction being carried out batchwise or continuously at 30 to 60°C and at a sucrose concentration of 40 to 60%.