WO1987005936A1 - Process for the manufacture of oligosaccharides - Google Patents

Process for the manufacture of oligosaccharides Download PDF

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Publication number
WO1987005936A1
WO1987005936A1 PCT/SE1987/000170 SE8700170W WO8705936A1 WO 1987005936 A1 WO1987005936 A1 WO 1987005936A1 SE 8700170 W SE8700170 W SE 8700170W WO 8705936 A1 WO8705936 A1 WO 8705936A1
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WIPO (PCT)
Prior art keywords
reaction
process according
carrying
gal
gnac
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Application number
PCT/SE1987/000170
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French (fr)
Inventor
Hanna Maria Elisabet Johansson
Lars Olof Hedbys
Per-Olof Larsson
Klaus Mosbach
Alf Gunnar Ingemar Gunnarsson
Sigfrid Edvin Svensson
Original Assignee
Johansson Hanna Maria E
Lars Olof Hedbys
Larsson Per Olof
Klaus Mosbach
Alf Gunnar Ingemar Gunnarsson
Sigfrid Edvin Svensson
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Application filed by Johansson Hanna Maria E, Lars Olof Hedbys, Larsson Per Olof, Klaus Mosbach, Alf Gunnar Ingemar Gunnarsson, Sigfrid Edvin Svensson filed Critical Johansson Hanna Maria E
Publication of WO1987005936A1 publication Critical patent/WO1987005936A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the present invention relates to a process for synthesizing enzymatically oligosaccharides starting from lower building entities, which for example may be constituted by monomeric sugars.
  • glycos idases are enzymes that normally hydrolyze carbohydrates, for example di-, oligo- and polysaccharides, to smaller entities, for example mono-saccharides.
  • the reaction is based on an equilibrium which in the normal case is shifted far in direction to the smaller constituents. Since the reaction involves an equilibrium in the course of hydrolysis the reaction 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 substances, for example peptides. It has also been described in connection with carbohydrate synthesis and a number of publications have been made where the principal also has been used for preparative synthesis. However, a characterizing and common feature of these known preparative syntheses is that the yields of the desired 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 saccharides than the starting constituents, unexpected high yields are obtained.
  • the invention has also surprisingly shown, that the glycosidases used are substantially 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 extremely 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 temperature is the fact that the reaction proceeds at a substantially higher rate.
  • the present invention has for its purpose to provide a process for enzymatic synthesis of oligosaccharides starting from lower building stones down to monomers, and the invention is characterized thereby that reverse hydrolysis is carried out in the presence of a glycosidas by carrying out the reaction in a solution containing substrate in a high concentration, i.e. a solution of a correspondingly low water content.
  • 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.
  • reaction it is suitable to perform the reaction in a solution containing at least about 50% by weight of substrate and particularly at least about 70% by weight of substrate.
  • the reaction is suitably carried out starting from monomeric building stones, oligosaccharides 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 formation of oligosaccharides containing 2-5 monomeric entities.
  • the invention can be applied to all known sugars while using the corresponding glycosidase it is, however, preferred to use in the reaction building stones selected from the following sugars: Ribose, arabin ⁇ se, 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 rhamnos. It is particulary preferred to use in the reaction one or several of the following sugars:
  • Glucose mannose, galactose, N-acetylglucose amine, N-acetylgalactose 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 combinations of sugars are selected from: Man-GNAc, Gal-GNAc, Gal-GalNAc, NeuNAc-Gal, NsuNAc-GNAc, NeuNAc-GalNAc, fucos-Gal and fucos-GNAc.
  • the invention may also, of course, be applied for the manufacture of oligomers composed of one and the same monomeric building entity.
  • monomeric building entities it is preferred to direct the process on to the preparation of dimers built up of one and the same type of monomer or corresponding to the above-mentioned partisular 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 reaction 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 temperature.
  • 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 product; c) substantially improved rate of reaction; d) improved stability of the enzyme and thereby extended life.
  • substrate solution containing this 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 temperature.
  • the reaction can be carried cut at about 75°C, the reaction rate being about thirty times higher than at room temperature.
  • the high substrate contents obviously results in stabilization of the enzyme so that no heat inactivation takes place.
  • Such enzymes are of course used as result in the desired product and as correspond to the carbohydrate type used.
  • a suitable monosidase for example ⁇ -mannosidase.
  • Mannose Yield determination at different mannose concentrations.
  • 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), respectively.
  • concentration of mannose became 20, 30, 55 and 85% (w/w), respectively.
  • 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 mannose could be dissolved at room temperature.
  • the samples were incubated 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.
  • 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 corresponding glycosidase as an enzyme.

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 oligosaccharides
The present invention relates to a process for synthesizing enzymatically oligosaccharides starting from lower building entities, which for example may be constituted by monomeric sugars.
It is known that glycos idases are enzymes that normally hydrolyze carbohydrates, for example di-, oligo- and polysaccharides, to smaller entities, for example mono-saccharides. The reaction is based on an equilibrium which in the normal case is shifted far in direction to the smaller constituents. Since the reaction involves an equilibrium in the course of hydrolysis the reaction 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 substances, for example peptides. It has also been described in connection with carbohydrate synthesis and a number of publications have been made where the principal also has been used for preparative synthesis. However, a characterizing and common feature of these known preparative syntheses is that the yields of the desired 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 saccharides than the starting constituents, unexpected high yields are obtained. The invention has also surprisingly shown, that the glycosidases used are substantially 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 extremely 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 temperature is the fact that the reaction proceeds at a substantially higher rate. Thus, the present invention has for its purpose to provide a process for enzymatic synthesis of oligosaccharides starting from lower building stones down to monomers, and the invention is characterized thereby that reverse hydrolysis is carried out in the presence of a glycosidas by carrying out the reaction in a solution containing substrate in a high concentration, i.e. a solution 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 containing 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, oligosaccharides 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 formation 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, preferred to use in the reaction building stones selected from the following sugars: Ribose, arabinσse, 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 rhamnos. It is particulary preferred to use in the reaction one or several of the following sugars:
Glucose, mannose, galactose, N-acetylglucose amine, N-acetylgalactose 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 combinations of sugars are selected from: Man-GNAc, Gal-GNAc, Gal-GalNAc, NeuNAc-Gal, NsuNAc-GNAc, NeuNAc-GalNAc, fucos-Gal and fucos-GNAc.
The invention may also, of course, be applied for the manufacture of oligomers composed of one and the same monomeric building entity. When using monomeric building entities it is preferred to direct the process on to the preparation of dimers built up of one and the same type of monomer or corresponding to the above-mentioned partisular 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 reaction 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 temperature. 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 product; c) substantially improved rate of reaction; d) improved stability of the enzyme and thereby extended life.
As an example of application of the techniique according to the invention there may be mentioned the manufacture of oligosaccharides starting from monomeric entities of mannose. In heating to about 75°C mannose can be solved to a very high concentration, more than 85% by weight, and such highly concentrated mannose 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, mannosidase, accepted this high substrate concentration without being negatively affected.
Since substrate solution containing this 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 temperature. Thus, the reaction can be carried cut at about 75°C, the reaction rate being about thirty times higher than at room temperature. The high substrate contents obviously results in stabilization of the enzyme so that no heat inactivation 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 for example oligosaccharides of mannose there may thus be used a suitable monosidase, for example α-mannosidase.
The invention will now be illustrated further by non-limiting 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 -galactosidase 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 allowed 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 enzyme 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 according to GC was 10% disaccharide (w/w).
EXAMPLE 2
Reversal with 8-galactosidase from E.coli starting from galactose and N-acetylgalactose amine. 9 mg galactose were dissolved in 21 mg 20mM phosphate buffer, 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 phosphate-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 α- 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 concentrations. 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), respectively. 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 mannose could be dissolved at room temperature. The samples were incubated 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 disaccharides 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 corresponding glycosidase as an enzyme.

Claims

CLAI MS
1. 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.
2. A process according to claim 1, characterized by carrying out the reaction at an increased temperature.
3. A process according to claim 2, characterized by carrying out the reaction at at least about 30°C.
4. A process according to any preceding claim, characterized 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 carrying out the reaction in a solution containing at least about 70% by weight of substrate.
6. A process according to any preceding claim, characterized by carrying out the reaction starting from monomeric building stones.
7. A process according to any preceding claim, characterized 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, characterized by carrying out the reaction starting from building stones selected from: Ribose, arabinose, 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 rhamnos.
10. A process according to claim 9, characterized by carrying out the reaction starting from building stones selected from: Glucose, mannose, galactose, N-acetylglucose amine, N-acetylgalactose amine, N-acetylneuraminic acid and fucos.
11. A process according to claim 9, characterized by carrying 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.
PCT/SE1987/000170 1986-04-02 1987-04-01 Process for the manufacture of oligosaccharides WO1987005936A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE8601482A SE452776B (en) 1986-04-02 1986-04-02 PROCEDURE FOR THE PREPARATION OF Oligosaccharides
SE8601482-6 1986-04-02

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JP (1) JPS63502900A (en)
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WO (1) WO1987005936A1 (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0266177A2 (en) * 1986-10-27 1988-05-04 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharides
WO1989009275A1 (en) * 1988-03-24 1989-10-05 Nilsson Kurt G I A method for synthesis of oligosaccharides
US4978751A (en) * 1988-04-14 1990-12-18 Roussel-Ulcaf Low-calorie sweeteners of fructosyl oligosaccharides and the food or dietetic products or drinks containing them
WO1991002806A1 (en) * 1989-08-18 1991-03-07 Procur Ab Process for producing an oligosaccharide compound by using glycosidases from a mollusc
WO1991013164A1 (en) * 1990-03-02 1991-09-05 Procur Ab Biochemical process to produce oligosaccharides
WO1993003168A1 (en) * 1991-08-06 1993-02-18 Kurt Nilsson Enzymatic method for synthesis of carbohydrates
WO1997021822A2 (en) * 1995-12-12 1997-06-19 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
US5716812A (en) * 1995-12-12 1998-02-10 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides, and the products formed thereby
US6284494B1 (en) 1995-12-12 2001-09-04 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
EP1211320A2 (en) * 1995-12-12 2002-06-05 University Of British Columbia Methods and compositions for synthesis of oligosacccharides using mutant glycosidase enzymes
US6972172B2 (en) 1999-02-17 2005-12-06 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US8231914B2 (en) 2003-08-26 2012-07-31 Mannatech Incorporated Antioxidant compositions and methods thereto
US9050313B2 (en) 2008-02-29 2015-06-09 Valeant Canada Lp Activation of innate and adaptive immune responses by a ginseng extract

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Nisizarra, the Carbohydrates, Chemistry and Biochemistry Second Edition, 1970, II A, Academic Press, p 242-268 (see p 244) *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0266177A2 (en) * 1986-10-27 1988-05-04 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharides
EP0266177A3 (en) * 1986-10-27 1989-01-25 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharides
US4957860A (en) * 1986-10-27 1990-09-18 Kabushiki Kaisha Yakult Honsha Method for producing oligosaccharide
WO1989009275A1 (en) * 1988-03-24 1989-10-05 Nilsson Kurt G I A method for synthesis of oligosaccharides
US5246840A (en) * 1988-03-24 1993-09-21 Procur Ab Method for synthesis of oligosaccharides
US4978751A (en) * 1988-04-14 1990-12-18 Roussel-Ulcaf Low-calorie sweeteners of fructosyl oligosaccharides and the food or dietetic products or drinks containing them
WO1991002806A1 (en) * 1989-08-18 1991-03-07 Procur Ab Process for producing an oligosaccharide compound by using glycosidases from a mollusc
WO1991013164A1 (en) * 1990-03-02 1991-09-05 Procur Ab Biochemical process to produce oligosaccharides
WO1993003168A1 (en) * 1991-08-06 1993-02-18 Kurt Nilsson Enzymatic method for synthesis of carbohydrates
US5532147A (en) * 1991-08-06 1996-07-02 Nilsson; Kurt Enzymatic method for synthesis of carbohydrates
WO1997021822A2 (en) * 1995-12-12 1997-06-19 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
WO1997021822A3 (en) * 1995-12-12 1997-08-28 Univ British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
US5716812A (en) * 1995-12-12 1998-02-10 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides, and the products formed thereby
US6284494B1 (en) 1995-12-12 2001-09-04 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
EP1211320A2 (en) * 1995-12-12 2002-06-05 University Of British Columbia Methods and compositions for synthesis of oligosacccharides using mutant glycosidase enzymes
EP1211320A3 (en) * 1995-12-12 2005-03-16 University Of British Columbia Methods and compositions for synthesis of oligosacccharides using mutant glycosidase enzymes
US7078194B2 (en) 1995-12-12 2006-07-18 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides using mutant glycosidase enzymes
US6972172B2 (en) 1999-02-17 2005-12-06 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US8231914B2 (en) 2003-08-26 2012-07-31 Mannatech Incorporated Antioxidant compositions and methods thereto
US9050313B2 (en) 2008-02-29 2015-06-09 Valeant Canada Lp Activation of innate and adaptive immune responses by a ginseng extract

Also Published As

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SE8601482L (en) 1987-10-03
EP0410963A1 (en) 1991-02-06
JPS63502900A (en) 1988-10-27
SE452776B (en) 1987-12-14
SE8601482D0 (en) 1986-04-02

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