GLUCOSYLTRANSFERASE AMINO ACID MOTIFS FOR ENZYMATIC PRODUCTION OF LINEAR POLY ALPHA-1,3-GLUCAN
Reactions comprising water, sucrose, and one or more glucosyltransferase enzymes are disclosed herein. Glucosyltransferase enzymes used in these reactions comprise certain motifs allowing production of insoluble poly alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages.
1 . A reaction solution comprising water, sucrose, and a glucosyltransferase enzyme, wherein said glucosyltransferase enzyme comprises a catalytic domain comprising the following three motifs:
(i) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:78,
(ii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:79, and
(iii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:80;
wherein said glucosyltransferase enzyme does not comprise residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:4, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20;
and wherein the glucosyltransferase enzyme produces insoluble poly alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages and a weight average degree of polymerization (DP w ) of at least 100.
2 . The reaction solution of claim 1 , wherein the catalytic domain comprises an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65.
3 . The reaction solution of claim 2 , wherein:
(A) the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:78 aligns with amino acid positions 231-243 of SEQ ID NO:65;
(B) the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:79 aligns with amino acid positions 396-425 of SEQ ID NO:65; and/or
(C) the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:80 aligns with amino acid positions 549-567 of SEQ ID NO:65.
4 . The reaction solution of claim 1 , wherein motif (i) comprises SEQ ID NO:78, motif (ii) comprises SEQ ID NO:79, and motif (iii) comprises SEQ ID NO:80.
5 . The reaction solution of claim 1 , wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages.
6 . The reaction solution of claim 1 , wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having a DP w of at least 400.
7 . A method of producing insoluble poly alpha-1,3-glucan comprising:
(a) contacting at least water, sucrose, and a glucosyltransferase enzyme, wherein said glucosyltransferase enzyme comprises a catalytic domain comprising the following three motifs:
(i) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:78,
(ii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:79, and
(iii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:80,
and wherein said glucosyltransferase enzyme does not comprise residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:4, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20;
whereby insoluble poly alpha-1,3-glucan is produced having at least 95% alpha-1,3 glycosidic linkages and a weight average degree of polymerization (DP w ) of at least 100; and
b) optionally, isolating the poly alpha-1,3-glucan produced in step (a).
8 . The method of claim 7 , wherein the catalytic domain comprises an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65.
9 . The method of claim 8 , wherein:
(1) the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:78 aligns with amino acid positions 231-243 of SEQ ID NO:65;
(2) the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:79 aligns with amino acid positions 396-425 of SEQ ID NO:65; and/or
(3) the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:80 aligns with amino acid positions 549-567 of SEQ ID NO:65.
10 . The method of claim 7 , wherein motif (i) comprises SEQ ID NO:78, motif (ii) comprises SEQ ID NO:79, and motif (iii) comprises SEQ ID NO:80.
11 . The method of claim 7 , wherein insoluble poly alpha-1,3-glucan is produced in step (a) having 100% alpha-1,3 glycosidic linkages.
12 . The method of claim 7 , wherein insoluble poly alpha-1,3-glucan is produced in step (a) having a DP w of at least 400.
13 . A method for identifying a glucosyltransferase enzyme, said method comprising: detecting the presence of at least one motif in a glucosyltransferase catalytic domain, said at least one motif selected from the group consisting of:
(i) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:78,
(ii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:79, and
(iii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:80;
thereby identifying a glucosyltransferase enzyme that produces insoluble poly alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages and a weight average degree of polymerization (DP w ) of at least 100.
14 . The method of claim 13 , wherein the detecting step is performed:
(a) in silico,
(b) with a method comprising a nucleic acid hybridization step,
(c) with a method comprising a protein sequencing step, and/or
(d) with a method comprising a protein binding step.
15 . The method of claim 13 , wherein the detecting step comprises detecting the presence of each of motifs (i), (ii) and (iii) in said catalytic domain.