Modified glucansucrase and related methods
Inventors
Cote, Gregory L. • Skory, Christopher D.
Assignees
US Department of Agriculture USDA
Publication Number
US-9644190-B2
Publication Date
2017-05-09
Expiration Date
2035-01-27
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Abstract
Disclosed is a genetically modified enzyme belonging to glycosyltransferases type of glucansucrase comprising at least one mutation at position 654 of said enzyme, wherein modified enzyme is capable to producing a glucan polymer.
Core Innovation
Disclosed is a genetically modified enzyme belonging to glycosyltransferases type of glucansucrase comprising at least one mutation at position 654 of said enzyme, wherein the modified enzyme is capable of producing a glucan polymer. The modified enzyme is derived from Leuconostoc mesenteroides and produces glucan polymers with altered properties compared to wild-type glucansucrases, including variations in yields of 1,3-disubstituted and 1,6-disubstituted α-D-glucopyranosyl units, and differences in water solubility.
The problem being solved is the need to establish a range of water-insoluble glucans with varying properties due to the limited applications of naturally occurring water-insoluble glucans. The mechanisms controlling the type of glycosidic linkage formed by glucansucrases remain unclear, and modifying amino acid residues downstream from the transition state stabilizer site, specifically at position 654, is investigated to alter glucan structure and solubility.
The invention specifically targets the amino acid at position 654, which follows the transition state stabilizer, to modify the catalytic properties of glucansucrase enzymes. Substitutions at this position with various amino acids produce modified enzymes that generate glucan polymers with higher yields of specific types of glucosidic linkages or altered solubility. This approach provides further insight into the structure-function relationship of glucansucrases and enables the synthesis of glucans with novel properties.
Claims Coverage
The claims include two independent claims covering modified glucansucrase enzymes with specific amino acid substitutions at position 654 and methods for producing glucan polymers using these modified enzymes. There are two main inventive features described regarding the amino acid substitutions and their effects on glucan polymer production.
Modified glucansucrase with specific amino acid substitutions at position 654
A glucansucrase enzyme having an amino acid sequence at least 96% identical to SEQ ID NO:11, with the threonine at position 654 substituted with one of glutamic acid, lysine, glutamine, cysteine, arginine, asparagine, isoleucine, serine, histidine, glycine, or aspartic acid, wherein the enzyme is capable of producing a glucan polymer.
Modified glucansucrase with threonine 654 replaced by tyrosine
A glucansucrase enzyme with threonine at position 654 replaced by tyrosine, wherein the modified enzyme produces a glucan polymer having a higher yield of 1,6-disubstituted α-D-glucopyranosyl units compared to the wild-type enzyme, and the glucan polymer is more water insoluble.
Methods for producing glucan polymers with modified glucansucrases
Methods comprising expressing the modified glucansucrase enzymes with substitutions at position 654 in recombinant host cells, and using the expressed enzymes to catalyze synthesis of glucan polymers from carbohydrate sources.
The independent claims cover genetically modified glucansucrase enzymes with specific amino acid substitutions at position 654 that alter glucan polymer properties, as well as methods for producing these glucan polymers using recombinant expression of the modified enzymes.
Stated Advantages
Modified glucansucrases produce glucan polymers with higher yields of specific glycosidic linkages compared to wild-type enzymes.
Ability to produce glucan polymers with altered water solubility, including higher or lower solubility than wild-type glucans.
Certain amino acid substitutions activate the enzyme's glucan synthesis rate in the presence of dextran, enhancing catalytic efficiency.
Modified enzymes produce glucans with varying physical appearances and solubility properties, providing useful diversity in glucan materials.
Documented Applications
Production of water-insoluble glucan polymers with specific linkage characteristics for potential material applications.
Recombinant expression of modified glucansucrase enzymes to synthesize glucan polymers with designed structural and solubility properties.
Use of modified glucansucrases in enzymatic synthesis processes to generate glucan polymers from carbohydrate sources.
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