Modified enzymes for producing increased isomelezitose
Inventors
Cote, Gregory L. • Skory, Christopher D.
Assignees
US Department of Agriculture USDA
Publication Number
US-10745675-B2
Publication Date
2020-08-18
Expiration Date
2038-04-30
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Abstract
Provided herein are compositions and methods for the synthesis of the trisaccharide, isomelezitose, using genetically modified glucansucrase enzymes from representative microorganisms, including lactic acid bacteria such as Leuconostoc mesenteroides. Various modified enzymes are detailed, increasing isomelezitose yields and provide the foundation for large-scale production of isomelezitose for food, industrial and biomedical applications.
Core Innovation
Certain lactic acid bacteria produce glucansucrase enzymes that synthesize α-D-glucans via glucosyl transfer from sucrose. This invention provides compositions and methods for synthesizing the trisaccharide isomelezitose at high yields using genetically modified variants of these glucansucrases. The modified enzymes enable large-scale production of isomelezitose for applications in food, industrial processes, and biomedicine.
Isomelezitose, a rare non-reducing trisaccharide composed of glucose and fructose, has previously been produced at low yields (8-11%) by various α-glucosidase enzymes and whole-cell systems. Efforts to genetically modify α-glucosidase or use sucrose isomerase enzymes to increase isomelezitose production have led to modest improvements but yields remained low (around 7-22%) with byproduct formation and degradation issues.
The present disclosure addresses the problem of low isomelezitose production by bacterial enzymes by examining amino acid substitutions of a key conserved leucine residue (L441) in the domain B motif of the wild-type DsrI glucansucrase enzyme from Leuconostoc mesenteroides. Modifications at this locus in various glucansucrases shift product formation, dramatically increasing isomelezitose yields compared to unmodified enzymes. These genetically modified enzymes can be produced recombinantly, purified, and used to convert carbohydrate sources, particularly sucrose, into isomelezitose efficiently.
Claims Coverage
The claims define multiple inventive features encompassing modified glucansucrase enzymes and methods for producing isomelezitose.
Modified glucansucrase enzymes with substituted leucine residues in domain B motif
Glucansucrase enzymes having an amino acid sequence at least 99% identical to specific SEQ ID NOs, wherein the conserved leucine residue at defined positions (e.g., L441 in SEQ ID NO:5) in the domain B motif is substituted with an amino acid other than leucine.
Proline as preferred substituting amino acid
The amino acid substituting the conserved leucine residue in the modified glucansucrase can be proline, enhancing isomelezitose production.
Variants of L. mesenteroides DsrI enzyme
Modified enzymes at positions L441 (full-length) or L400 (mature) with substitution by selected amino acids such as arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, lysine, proline, serine, threonine, or valine.
Variants of L. mesenteroides DsrS enzyme
Modified enzymes with leucine at position 459 (full-length) or 417 (mature) substituted preferably with proline for increased isomelezitose synthesis.
Variants of L. citreum Asr enzyme
Modified enzymes substituting the leucine residue at position 544 (full-length) or 505 (mature) with amino acids like glutamic acid, proline, or serine to improve isomelezitose yield.
Variants of S. sobrinus GtfI enzyme
Modified enzymes substituting leucine at position 350 (full-length) or 312 (mature) with arginine, glutamic acid, proline, or serine for enhanced isomelezitose production.
Variants of L. pseudomesenteroides GtfG enzyme
Modified enzymes with leucine at position 417 (full-length) or 380 (mature) substituted with amino acids, particularly proline, to increase isomelezitose synthesis.
Method of producing isomelezitose
A process comprising contacting any of the above modified enzymes with a carbohydrate solution and allowing conversion to isomelezitose.
Expression and purification steps
Optionally expressing the modified enzymes in recombinant host cells and purifying them prior to the carbohydrate conversion.
Use of sucrose as carbohydrate source
The carbohydrate source used in the method preferably contains sucrose, optionally at about 1.0 M concentration in aqueous solution.
The claims cover genetically modified glucansucrase enzymes with specific leucine substitutions in the domain B motif from various microorganisms that substantially increase isomelezitose production, and methods using these enzymes to produce isomelezitose from carbohydrate sources, particularly sucrose.
Stated Advantages
Genetically modified enzymes produce isomelezitose in high yields, significantly surpassing yields of unmodified enzymes and prior art methods.
The modified enzymes allow for large-scale production of isomelezitose, enabling feasible commercial applications.
Isomelezitose produced is noncariogenic, low calorie, and suitable for diabetic foods and potential biomedical uses.
Documented Applications
Large-scale production of isomelezitose for food applications including diabetic-friendly sweeteners.
Industrial synthesis of isomelezitose as a rare sugar component with specialized properties.
Biomedical applications such as use in vaccines, drugs, and nutritional supplements involving isomelezitose.
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