Method For Producing 2-O-Glyceryl-Alpha-D-Glucopyranoside

Abstract

The present invention relates to a method for producing 2-O-glyceryl-α-D-glucopyranoside (αGG; FIG. 1 ) from a glucosyl donor and a glucosyl acceptor comprising the steps:—providing a sucrose phosphorylase (EC 2.4.1.7), incubating said sucrose phosphorylase with a mixture comprising a glucosyl donor and glycerol as glucosyl acceptor and isolating and/or purifying 2-O-glyceryl-α-D-glucopyranoside.

1 . A method of making 2-O-glyceryl-α-D-glucopyranoside (αGG) from a glucosyl donor and a glucosyl acceptor comprising the steps:
 providing a sucrose phosphorylase (EC 2.4.1.7);   incubating said sucrose phosphorylase with a mixture comprising a glucosyl donor and glycerol as glucosyl acceptor; and   isolating and/or purifying 2-O-glyceryl-α-D-glucopyranoside from said mixture.   
   
   
       2 . The method according to  claim 1 , wherein the glucosyl donor is selected from the group consisting of sucrose and analogues of sucrose comprising a fructosyl moiety modified or substituted by another ketosyl residue, α-D-glucose-1-phosphate, α-D-glucose-1-fluoride, further stable, activated glucosyl donors; α-D-glucose-1-azide, and mixtures thereof. 
   
   
       3 . The method according to  claim 1 , wherein the sucrose phosphorylase is of microbial origin or bacterial origin. 
   
   
       4 . The method according to  claim 3 , wherein the sucrose phosphorylase is obtained from  Agrobacterium vitis  (NCBI P33910),  Bifidobacterium adolescentis  (Q84HQ2),  Bifidobacterium longum  (Q84BY1),  Escherichia coli  (P76041),  Escherichia coli  O6 (Q8FHS2),  Lactobacillus acidophilus  (Q7WWP8, Q7WWQ5),  Lactobacillus delbrueckii  subsp.  lactis  (Q71I99),  Leuconostoc mesenteroides  (Q59495, Q9R5Q3),  Listeria monocytogenes  (Q4ENE7, Q4EQR2, Q4ETN7, Q4EHAO, Q4EJW2, Q4ELY7),  Pseudomonas putrefaciens, Pseudomonas saccharophila  (AAD40317),  Rhodopirellula baltica  (Q7UIS9),  Shewanella baltica  (Q3Q4P1),  Shewanella frigidimarina  (Q3NMD1),  Solibacter usitatus  (Q43TL5),  Streptococcus mutans  (P10249) and/or  Synechococcus  sp. (O68858, Q7U3J7). 
   
   
       5 . The method according to  claim 1 , wherein the sucrose phosphorylase is recombinantly produced as full-length protein or a catalytically active fragment thereof, or a fusion protein. 
   
   
       6 . The method according to  claim 1 , wherein the sucrose phosphorylase is prior the incubation step immobilized on a carrier. 
   
   
       7 . The method according to  claim 6 , wherein said carrier is a solid support, 
   
   
       8 . The method according to  claim 1 , wherein the carrier is a chromatography resin, an-anion exchange chromatography resin, a cation exchange chromatography resin, an affinity chromatography resin or a hydrophobic interaction chromatography resin. 
   
   
       9 . The method according to  claim 1 , wherein the incubation is performed at a pH value of 4 to 10. 
   
   
       10 . The method according to  claim 1 , wherein the incubation is performed for at least 15 min, for at least 60 min, for at least 3 hours, or for at least 5 hours. 
   
   
       11 . The method according to  claim 1 , wherein the incubation is performed at a temperature range of 10 to 50° C., 15 to 40° C. 
   
   
       12 . The method according to  claim 1 , wherein the mixture comprises the glucosyl donor in a concentration of 0.01 to 3 mol/l, of 0.05 to 2 mol/l, or of 0.1 to 1.5 mol/l. 
   
   
       13 . The method according to  claim 1 , wherein the mixture comprises glycerol in a concentration of 0.01 to 10 mol/l, of 0.05 to 5 mol/l, of 0.1 to 3 mol/l, or of 0.1 to 1.5 mol/l. 
   
   
       14 . The method according to  claim 1 , wherein the ratio of glycerol to glucosyl donor in the mixture ranges from 0.1:1 to 10:1, from 0.5:1 to 5:1, or from 1:1 to 3:1. 
   
   
       15 . The method according to a  claim 1 , wherein α-D-glucosylglycerol is isolated by elution chromatography on activated charcoal in a yield of 55%, 70%, 85% or greater than 85%. 
   
   
       16 . The method according to  claim 1  to  15 , wherein the sucrose phosphorylase is obtained from  Leuconostoc mesenteroides  (Q59495, Q9R5Q3) and immobilised on an acrylic polymer, wherein the acrylic polymer comprises a copolymer of methacrylamide, N,N′-methylen-bis(acrylamide) and a polymer carrying oxirane groups, wherein the immobilized sucrose phosphorylase is incubated with sucrose as glucosyl donor. 
   
   
       17 . A method of producing a mixture containing α-D-glucosylglycerol and fructose comprising the steps:
 providing a sucrose phosphorylase (EC 2.4.1.7);   incubating said sucrose phosphorylase with a mixture comprising sucrose and glycerol as glucosyl acceptor; and   isolating and/or purifying the α-D-glucosylglycerol and fructose mixture.   
   
   
       18 . A product made by the process of  claim 17 , wherein the product comprises a α-D-glucosylglycerol. 
   
   
       19 . The product of  claim 18 , wherein the fructose is in an equimolar amount to the α-D-glucosylglycerol. 
   
   
       20 . A cosmetic preparation comprising α-D-glucosylglycerol and an acceptable carrier. 
   
   
       21 . A pharmaceutical preparation comprising α-D-glucosylglycerol and a pharmaceutically acceptable carrier. 
   
   
       22 . A food supplement comprising α-D-glucosylglycerol made by the steps of  claim 18 . 
   
   
       23 . A method of sweetening a sample comprising the steps of:
 adding a composition comprising a α-D-glucosylglycerol or product comprising α-D-glucosylglycerol to a sample.   
   
   
       24 . A method of stabilizing of biomolecules comprising the steps of:
 administering a composition comprising an α-D-glucosylglycerol to a sample to stabilize one or more a biomolecules, selected from proteins, protein fragments and lipid-derived structures during processing and storage.   
   
   
       25 . The method of  claim 24 , wherein the sample comprises one or more living microorganisms during processing and storage. 
   
   
       26 . The method of  claim 24 , wherein the proteins are dried or lyophilized. 
   
   
       27 . The method of  claim 24 , wherein the α-D-glucosylglycerol or product comprising α-D-glucosylglycerol assists protein refolding from inclusion bodies and preventing protein misfolding and aggregation in vivo. 
   
   
       28 . The method of  claim 24 , wherein the α-D-glucosylglycerol or product comprising α-D-glucosylglycerol improves cell viability during recombinant protein production. 
   
   
       29 . The method of  claim 24 , wherein the α-D-glucosylglycerol or product comprising α-D-glucosylglycerol improves the stability of enzymes under reaction conditions. 
   
   
       30 . The method of  claim 24 , wherein the sample comprises  Escherichia coli.    
   
   
       31 . The method according to  claim 7 , wherein said carrier comprises an acrylic polymer, a methacrylamide, a N,N′-methylen-bis(acrylamide) or a monomer comprising one or more oxirane groups. 
   
   
       32 . The method according to  claim 1 , wherein the incubation is performed at a pH value of 5 to 9. 
   
   
       33 . The method according to  claim 1  wherein the incubation is performed at a pH value of 6 to 8. 
   
   
       34 . The method according to  claim 1  wherein the incubation is performed at a pH value of about 7. 
   
   
       35 . The method according to  claim 1 , wherein the incubation is performed at a temperature range of about 30° C.