Trehalose phosphorylase

A method for preparing trehalose comprising reacting glucose and alpha-D-glucose-1 phosphate at a temperature of at least 40° C. in the presence of a trehalose phosphorylase, wherein the trehalose phosphorylase

• (i) retains at least 30% of its activity after incubation for 15 minutes at 52° C. in a buffer containing 1 M sucrose compared to its activity without thermal treatment;
• and/or
• (ii) retains at least 50% of its activity after incubation for 15 minutes at 52° C. in a buffer containing 1 M sucrose compared to its activity without thermal treatment;
• and/or
• (iii) has a ratio of activity at 100 mM glucose to activity at 500 mM glucose of at least 0.65, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical to and/or at least 80% homologous to an amino acid sequence of SEQ ID NO:
• 1, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein
• (a) the one or more amino acid positions is/are selected from the group consisting of amino acid positions 712, 383, 10, 114, 118, 192, 197, 220, 225, 304, 306, 318, 323, 339, 349, 357, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 590, 649, 667, 703 and 705 of SEQ ID NO:
• 1,
• (b) when the amino acid substitution comprises a substitution at amino acid position 649, then the amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, and
• (c) when the amino acid substitution comprises a substitution at amino acid position 10, then the amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and V10H.

A method for increasing thermal stability of a trehalose phosphorylase, wherein the method comprises:

• aligning an amino acid sequence of a first trehalose phosphorylase with an amino acid sequence of a second trehalose phosphorylase,
• identifying one or more amino acid positions of the amino acid sequence of the second trehalose phosphorylase which correspond to one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase, wherein substitution of an amino acid residue at the one or more amino acid position of the amino acid sequence of the first trehalose phosphorylase increases thermal stability of the first trehalose phosphorylase,
• substituting an amino acid residue at the one or more amino acid positions of the second trehalose phosphorylase corresponding to the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase, wherein substitution of an amino acid residue at the one or more amino acid position of the amino acid sequence of the first trehalose phosphorylase increases thermal stability of the first trehalose phosphorylase,
• wherein the first trehalose phosphorylase is a trehalose phosphorylase comprising an amino acid sequence according to SEQ ID NO:
• 1,
• wherein the substitution of an amino acid residue in the second trehalose phosphorylase is performed at one or more of the amino acids positions corresponding to amino acid positions 712, 383, 10, 114, 118, 192, 197, 220, 225, 304, 306, 318, 323, 339, 349, 357, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 590, 649, 667, 703 and 705 of SEQ ID NO:
• 1, wherein
• (a) when the amino acid substitution comprises a substitution at amino acid position 649, then the amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, and
• (b) when the amino acid substitution comprises a substitution at amino acid position 10, then the amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and V10H.