Synthesis of glucan comprising beta-1,3 glycosidic linkages with phosphorylase enzymes

Abstract

Reaction compositions are disclosed herein comprising at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and a beta-1,3-glucan phosphorylase enzyme. These reactions can synthesize oligosaccharides and polysaccharides with beta-1,3 glycosidic linkages. Further disclosed are methods of isolating beta-1,3-glucan.

1 - 18 . (canceled) 
     
     
         19 . A reaction composition comprising at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and a beta-1,3-glucan phosphorylase enzyme comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:2, 4, 8, 10, or 12, wherein said amino acid sequence has beta-1,3-glucan phosphorylase activity,
 wherein the acceptor molecule (i) comprises one or more glucose monomeric units, (ii) does not consist of glucose, and (iii) has one or more glycosidic linkages,   wherein the beta-1,3-glucan phosphorylase enzyme synthesizes a beta-1,3-glucan in the reaction composition and the reaction composition is cell-free.   
     
     
         20 . The reaction composition of  claim 19 , wherein the beta-1,3-glucan has at least about 90% beta-1,3 glycosidic linkages. 
     
     
         21 . The reaction composition of  claim 20 , wherein the beta-1,3-glucan has at least about 99% beta-1,3 glycosidic linkages. 
     
     
         22 . The reaction composition of  claim 19 , wherein the degree of polymerization (DP) of the beta-1,3-glucan is at least 3. 
     
     
         23 . The reaction composition of  claim 22 , wherein the DP of the beta-1,3-glucan is at least about 15. 
     
     
         24 . The reaction composition of  claim 19 , wherein the acceptor molecule comprises a monosaccharide, disaccharide, or oligosaccharide. 
     
     
         25 . The reaction composition of  claim 24 , wherein the acceptor molecule comprises laminaribiose. 
     
     
         26 . The reaction composition of  claim 24 , wherein the acceptor molecule comprises cellobiose or p-nitrophenyl beta-D-glucopyranoside. 
     
     
         27 . The reaction composition of  claim 19 , wherein the acceptor molecule comprises a polysaccharide. 
     
     
         28 . The reaction composition of  claim 27 , wherein the polysaccharide comprises beta-glucan. 
     
     
         29 . The reaction composition of  claim 28 , wherein the beta-glucan comprises laminarin. 
     
     
         30 . The reaction composition of  claim 19 , wherein the amino acid sequence is at least 97% identical to SEQ ID NO:2, 4, 8, 10, or 12. 
     
     
         31 . A method for producing beta-1,3-glucan, said method comprising:
 contacting at least water, alpha-glucose-1-phosphate (alpha-G1P), an acceptor molecule, and a beta-1,3-glucan phosphorylase enzyme comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:2, 4, 8, 10, or 12, wherein said amino acid sequence has beta-1,3-glucan phosphorylase activity, wherein the acceptor molecule (i) comprises one or more glucose monomeric units, (ii) does not consist of glucose, and (iii) has one or more glycosidic linkages,   wherein a beta-1,3-glucan is produced, and   wherein the contacting step is performed under cell-free conditions.   
     
     
         32 . The method of  claim 31 , wherein said alpha-G1P is provided in the contacting step by providing a second reaction, wherein the products of the second reaction comprise alpha-G1P. 
     
     
         33 . The method of  claim 32 , wherein the second reaction is provided in the same vessel in which the contacting step is performed, and wherein the second reaction is performed before and/or continuously with the contacting step. 
     
     
         34 . The method of  claim 32 , wherein the second reaction produces alpha-G1P by contacting (i) water, (ii) inorganic phosphate, (iii) a glucose-comprising disaccharide, oligosaccharide, or polysaccharide, and (iv) a phosphorylase that phosphorolyzes the disaccharide, oligosaccharide, or polysaccharide. 
     
     
         35 . The method of  claim 31 , wherein said acceptor molecule is provided in the contacting step by providing a third reaction, wherein the products of the third reaction comprise said acceptor molecule. 
     
     
         36 . The method of  claim 35 , wherein said acceptor molecule provided by the third reaction is laminaribiose. 
     
     
         37 . The method of  claim 31 , further comprising isolating the beta-1,3-glucan produced in the contacting step. 
     
     
         38 . The method of  claim 31 , wherein the amino acid sequence is at least 97% identical to SEQ ID NO:2, 4, 8, 10, or 12.