PROCESS FOR THE MANUFACTURE OF OLIGOSACCHARIDES

Abstract: A process for enzymatic synthesis of oligosaccharides starting from lower building stones down to monomers, characterized by performing reverse hydrolysis in the presence of a glycosidase by carrying out the reaction in a solution having a high substrate content, i.e. a low water content.

Description: process for the manufacture of oliαosaccharides The present invention relates to a process for synthesizing enzy atically oligosaσcharidβs starting from lower building enti¬ ties, which for example may be constituted by monomeric sugars. It is known that glycoβidaβes are enzymes that normally hy- drolyze carbohydrates, for example di-, oligo- and polyβacchari- des, to smaller entities, for example mono-saccharides . The reac- tion is based on an equilibrium which in the normal case is shif¬ ted far in direction to the smaller constituents. Since the reac¬ tion involves an equilibrium in the course of hydrolysis the reac¬ tion can, of course, in the usual manner proceed in the opposite direction provided the conditions for this reversed hydrolysis are favourable for such shifting of the equilibrium. The present invention relates to such type of carbohydrate synthesis and will in the present disclosure be referred to as reversed hydrolysis since the course of catalysis is reverse in relation to the normal one. The principal of reverse as such is previously known and has been utilized practically in the synthesis of other types of sub¬ stances, for example peptideβ. It has also been described in con¬ nection with carbohydrate synthesis and a number of publications have been made where the principal also has been used for prepara- tive synthesis. However, a characterizing and common feature of these known preparative syntheses is that the yields of the desi¬ red product are low. The invention is based on the surprising discovery that in the presence of high sugar concentrations, i.e. high substrate contents, such substantial shift of the equilibrium in direction to the formation of higher saccharideβ than the starting consti¬ tuents, unexpected high yields are obtained. The invention has also surprisingly shown, that the glycosidaseβ used are substan¬ tially stabilized in view of the high substrate concentration. This enzyme stabilization is so pronounced that the reaction can be carried out at substantially increased temperature. This fact in turn means that the reverse reaction can be performed at ex¬ tremely high substrate concentration since the sugar used can be dissolved to a very high concentration. A further advantage of carrying out the reaction at a substantially increased temperatur is the fact that the reaction proceeds at a substantially higher rate. Thus, the present invention has for its purpose to provide process for enzymatic synthesis of oligosaccharides starting from lower building stones down to monomers, and the invention is cha¬ racterized thereby that reverse hydrolysis is carried out in the presence of a glycosidas by carrying out the reaction in a solu- tion containing substrate in a high concentration, i.e. a solutio of a correspondingly low water content. In a preferred embodiment of the process according to the invention the reaction is performed at an increased temperature, for example at at least about 30°C, and the temperature may be as high as up to temperatures lying close to the boiling point of water, for example about 75°C. It is suitable to perform the reaction in a solution contai¬ ning at least about 50% by weight of substrate and particularly at least about 70% by weight of substrate. Although the invention is not limited hereto the reaction is suitably carried out starting from monomeric building stones, oli¬ gosaccharides containing up to about 15 monomers being prepared. It is particularly preferred to carry out in accordance with the techniques of the invention the enzymatic synthesis with the for- mation of oligosaccharides containing 2-5 monomeric entities. Although the invention can be applied to all known sugars while using the corresponding glycosidase it is, however, prefer¬ red to use in the reaction building stones selected from the fol¬ lowing sugars: Riboβe, arabinσse, xylose, glucose, mannose (Man), galactoβe (Gal) , N-acetylglucσse amine <GNAc), N-acetylgalactose amine <GalNAc), N-acetylmannose amine (ManNAc), N-acetylneuraminic acid (NeuNAc), glucoronic acid, galacturonic acid, mannuronic acid, iduronic acid, fucos and rhamnos . It is particulary preferred to use in the reaction one or several of the following sugars: Glucose, mannose, galactose, N-acetylglucose amine, N-ace¬ tylgalactose amine, N-acetylneuraminic acid and fucos. The invention is applicable also in processes where in one and the same reaction there are included two or more of the above- -mentioned types of sugars, and particularly preferred combina¬ tions of sugars are selected from: Man-GNAc, Gal-GNAc, Gal-GalNAc, NeuNAc-Gal, NβuNAc-GNAc, NeuNAc-GalNAc, fucos-Gal and fucos-GNAc . The invention may also, of course, be applied for the manu¬ facture of oligomers composed of one and the same monomeric buil¬ ding entity. When using monomeric building entities it is preferred to direct the process on to the preparation of dimerβ built up of one and the same type of monomer or corresponding to the above-mentio¬ ned partiβular combinations. The reaction may also of course be carried out in such a manner as to obtain a mixture of oligomers, wherein the separate types of oligomers may then be separated in a suitable manner. The technique according to the present invention results in substantial advantages mainly originating from the utilization of high substrate concentrations, i.e. low water contents in the rβ- action solution used. The improved enzyme stability obtained in using the high substrate concentrations in accordance with the technique of this invention in turn results in a situation whereby the reaction can be carried out at a substantially increased tem¬ perature. The added result of these features of the invention give the following advantages: a) substantially increased product yields; b) substantially increased concentration of the desired pro¬ duct; c) substantially improved rate of reaction; d) improved stability of the enzyme and thereby extended life. As an example of application of the techn ique according to the invention there may be mentioned the manufacture of oligo¬ saccharides starting from monomeric entities of mannose. In heat- ing to about 75°C mannose can be solved to a very high concentra¬ tion, more than 85% by weight, and such highly concentrated man¬ nose solution can be maintained at room temperature for some hours before sugar crystallization occurs. In connection with the crea- tion of the invention the surprising discovery was now made that the enzyme used, mannosidaβe, accepted this high substrate concen¬ tration without being negatively affected. Since substrate solution containing th 3 high concentration of sugar is easily subjected to crystallization of the sugar it is suitable to increase the temperature, and in this connection it is surprisingly found that the enzyme is not affected by a high tem¬ perature. Thus, the reaction can be carried cut at about 75°C, the reaction rate being about thirty times higher than at room tempe- rature. The high substrate contents obviously results in stabili¬ zation of the enzyme so that no heat inactiv»tion takes place. With regard to the enzymes used such enzymes are of course used as result in the desired product and as correspond to the carbohydrate type used. When manufacturing f_r example oligo- saccharideβ of mannose there may thus be used a suitable monosi- dase, for example α-mannosidaβe . The invention will now be illustrated further by non-limi¬ ting examples. These examples illustrate the manufacture of some different types of oligosaccharides while using the technique of the invention. The percentages given in the examples relate to weight if not stated otherwise. EXAMPLE 1 Reversal of 8 -αalactosidase from E.coli in 36% galactose. 0.8 g galactose and 1,2 g H2O were heated on a water bath until all galactose had dissolved, and the solution was then allo¬ wed to cool down to room temperature. To 1.5 ml of the galactose solution there were added 20/ul 1M phosphate buffer, pH 7.2 and 0.15 ml enzyme solution containing 185 enzy ε units. The mixture was incubated at room temperature and the products were detected using HPLC. After equilibrium had been reached the reaction was interrupted by heating on a boiling water bath for 10 minutes, and final determination of the yield was done using GC. The yield ac¬ cording to GC was 10% disaccharide (w/w) . EXAMPLE 2 Reversal with 8 -αalactosidase from E.coli starting from galactose and N-acetylgalactose amine. 9 mg galactose were dissolved in 21 mg 20mM phosphate buf¬ fer, pH 7,5, by heating on a boiling water bath. After cooling to room temperature 24 mg GalNAc were added under stirring. Freeze dried pure β -galactosidase (450 enzyme units (U) ) were added to 200 U/ml. After 2 days the reaction was interrupted by heating on a boiling water bath for 5 minutes. Analyses using GC-MS showed that 5% galactose (w/w) based on the total sugar amount had been bound as disaccharide . Of this disaccharide 2-3% were present as Gal- GalNAc. Only the 1.6-isomer could be found. EXAMPLE 3 Reversal on a large scale in 83% mannose. 5 g mannose were dissolved in 0.883 g buffer (20 mM phos- phate-citrate, 50 mM KCl, 1 mM ZnCl and 0.02% azide) by heating on a boiling water bath in a test tube having a tight threaded lid, and the solution was then allowed to cool somewhat. 0.3 ml Ct- -mannosidase from jack bean in buffer (130 enzyme units/ml) were added and the mixture was incubated at 75°C. The activity of the reaction mixture after 1 day was 6 U/ml and was unchanged while the reaction proceeded. Products were detected using HPLC. After 3 days the reaction was interrupted by heating on a boiling water bath, and final analysis and determination of yield were carried out using GC-MS. The yield of di-, tri-, tetra- and higher oligosaccharides was 30, 25, 10 and 5%, respectively. EXAMPLE 4 Reversal in mannose: Yield determination at different mannose con¬ centrations . Mannose, buffer (se Example 3 above) and enzyme solution (about 160 enzyme units/ml) were weighed and mixed so that the concentration of mannose became 20, 30, 55 and 85% (w/w), respec¬ tively. In the case of 85% mannose heating on a boiling water bath of mannose and buffer was required before the enzyme was added in order to dissolve the mannose, whereas in the other cases the man¬ nose could be dissolved at room temperature. The samples were in¬ cubated at 50°C and the formation of disaccharides was followed using HPLC. When the amount of disaccharide had ceased to increase in the respective samples the reaction was interrupted by heating on a boiling water bath for 12 minutes. Final determination of the yield was made using GC . The yield of disaccharideβ in 20, 30, 55 and 85% mannose was 22, 25, 31 and 39%, respectively, based on the total sugar quantity. It should be observed that the present invention in no way is limited to the types of sugars given in the above examples but that it can be applied to all known types of sugars using the cor¬ responding glycosidase as an enzyme.

Claims: CLAI MS 1. A process for enzymatic synthesis of oligosaccharides starting from lower building stones down to monomers, characteri- zed by performing reverse hydrolysis in the presence of a glycosi¬ dase by carrying out the reaction in a solution having a high sub¬ strate content, i.e. a low water content. 2. A process according to claim 1, characterized by carry¬ ing out the reaction at an increased temperature. 3. A process according to claim 2, characterized by carry¬ ing out the reaction at at least about 30°C. 4. A process according to any preceding claim, characteri¬ zed by carrying out the reaction in a solution containing at least about 50% by weight of substrate. 5. A process according to claim 4, characterized by carry¬ ing out the reaction in a solution containing at least about 70% by weight of substrate. 6. A process according to any preceding claim, characteri¬ zed by carrying out the reaction starting from monomeric building stones. 7. A process according to any preceding claim, characteri¬ zed by carrying out the reaction to synthesize oligosaccharides containing up to about 15 monomeric entities. 8. A process according to claim 7, characterized by carrying out the reaction to synthesize oligosaccharides containing 2-5 monomeric entities. 9. A process according to any preceding claim, characteri¬ zed by carrying out the reaction starting from building stones selected from: Ribose, arabinoβe, xylose, glucose, mannose (Man), galactose (Gal), N-acetylglucose amine (GNAc), N-acetylgalactose amine (GalNAc), N-acetylmannose amine (ManNAc), N-acetylneuraminic acid (NeuNAc), glucoronic acid, galacturonic acid, mannuronic acid, iduronic acid, fucos and rhamnoβ . 10. A process according to claim 9, characterized by carry¬ ing out the reaction starting from building stones selected from: Glucose, mannose, galactose, N-acetylglucose amine, N-ace¬ tylgalactose amine, N-acetylneuraminic acid and fucos. 11. A process according to claim 9, characterized by carry¬ ing out the reaction starting from building atone combinations selected from: Man-GNAc, Gal-GNAc, Gal-GalNAc, NeuNAc-Gal, NeuNAc-GNAc, NeuNAc-GalNAc, fucos-Gal and fucos-GNAc. 12. A process according to claim 10 for the manufacture of oligomers built up from one and the same monomeric building stone, 13. A process according to claim 11 for the manufacture of dimers corresponding to said combinations. 14. A process according to claim 10 for the manufacture of oligosaccharides starting from mannose as a monomeric building stone. 15. Oligomers manufactured by the process according to any preceding claim.

Inventors: GUNNARSSON ALF GUNNAR INGEMAR, HEDBYS LARS OLOF, JOHANSSON HANNA MARIA ELISABET, LARSSON PER-OLOF, MOSBACH KLAUS, SVENSSON SIGFRID EDVIN, GUNNARSSON, ALF, GUNNAR, INGEMAR, HEDBYS, LARS, OLOF, JOHANSSON, HANNA, MARIA, ELISABET, LARSSON, PER-OLOF, MOSBACH, KLAUS, SVENSSON, SIGFRID, EDVIN
Applicants: GUNNARSSON ALF GUNNAR INGEMAR, HEDBYS LARS OLOF, JOHANSSON HANNA MARIA E, LARSSON PER OLOF, MOSBACH KLAUS, SVENSSON SIGFRID EDVIN, GUNNARSSON, ALF, GUNNAR, INGEMAR, HEDBYS, LARS, OLOF, JOHANSSON, HANNA, MARIA, ELISABET, LARSSON, PER-OLOF, MOSBACH, KLAUS, SVENSSON, SIGFRID, EDVIN
Application year: 1987
Main classification: C12P19
IPC codes: C07H1, C12P19