Novel trehalose synthase from Picrophilus torridus and methods of use thereof
AssigneeAcademia Sinica
InventorsJei-Fu Shaw, Guan-Chiun Lee, Yi-Shan Chen
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Abstract
The present invention relates to the isolation and functional identification of a novel acid and heat resistant trehalose synthase enzyme cloned from Picrophilus torridus. More particularly, the present invention discloses the DNA sequence for the Picrophilus torridus trehalose synthase gene, PTTS, which when expressed in a heterologous host such as Escherichia coli, provides enzymatic activity that catalyzes the direct interconversion of maltose and trehalose through intramolecular transglycosylation. Additionally, the present invention teaches methods of use of PTTS for production of trehalose as well as for production of various useful compounds comprising trehalose.
Claims72
An isolated and purified nucleic acid sequence encoding a functional Picrophilus torridus trehalose synthase protein such that the protein has a catalytic activity of catalyzing the intramolecular rearrangement of the α1,4-linkage of maltose to the α1,1-linkage of trehalose, or vice versa.
An isolated and purified protein molecule having functional Picrophilus torridus trehalose synthase activity such that the protein has a catalytic activity of catalyzing the intramolecular rearrangement of the α1,4-linkage of maltose to the α1,1-linkage of trehalose, or vice versa.
A method for producing a mutein of Picrophilus torridus trehalose synthase with at least one altered property selected from the group consisting of regiospecificity and stereospecificity comprising the steps of:
- (a) threading the wild-type Picrophilus torridus trehalose synthase amino acid sequence onto the three-dimensional structure of a mammalian analogue enzyme;
- (b) docking at least one maltose substrate into the predicted active site pocket;
- (c) mapping amino acid residues within a defined distance of the modeled substrate molecules and rationalizing the amino acid residues with predicted chemical transformations catalyzed by the Picrophilus torridus trehalose synthase to identify amino acid residues capable of steric, ionic, electronic and hydrophobic interactions with the substrate molecules;
- (d) selecting at least one altered amino acid residue for its potential for substrate binding and positioning of the substrate molecule relative to the heme-catalytic center;
- and
- (e) introducing the at least one altered amino acid amino acid residue by site-directed mutagenesis to produce the mutein.
Dependencies are inferred from the claim text.