Trehalose phosphorylase

The trehalose phosphorylase of any of the previous claims, wherein the amino acid sequence of the trehalose phosphorylase comprises one or more substitutions, wherein the one or more substitution is/are selected from the group consisting of

• an amino acid substitution at position 712 of SEQ ID NO:
• 1 with the substitution being L712A, L712G, L712I, L712M, L712P or L712V, preferably L712M;
• an amino acid substitution at position 383 of SEQ ID NO:
• 1 with the substitution being P383A, P383G, P383I, P383L, P383M, P383V, P383N, P383C, P383Q, P383S or P383T, preferably P383A, P383G, P383M, P383V, P383N, P383C, P383Q, P383S or P383T, more preferably P383G, P383V, P383C or P383S, or P383T, even more preferably P383V or P383T, and most preferably P383V;
• an amino acid substitution at position 10 of SEQ ID NO:
• 1 with the substitution being V10R, V10H or V10K, preferably V10R;
• an amino acid substitution at position 114 of SEQ ID NO:
• 1 with the substitution being L114A, L114G, L114I, L114M, L114P or L114V, preferably L114I;
• an amino acid substitution at position 118 of SEQ ID NO:
• 1 with the substitution being I118A, I118G, I118L, I118M, I118P or I118V, preferably I118V;
• an amino acid substitution at position 192 of SEQ ID NO:
• 1 with the substitution being S192A, S192G, S192I, S192L, S192M, S192P or S192V, preferably S192V;
• an amino acid substitution at position 197 of SEQ ID NO:
• 1 with the substitution being S197A, S197G, S197I, S197L, S197M, S197P or S197V, preferably S197G;
• an amino acid substitution at position 220 of SEQ ID NO:
• 1 with the substitution being Y220F or Y220W, preferably Y220F;
• an amino acid substitution at position 225 of SEQ ID NO:
• 1 with the substitution being N225A, N225G, N225I, N225L, N225M, N225P or N225V, preferably, N225I, N225L, N225M or N225V, and more preferably N225V;
• an amino acid substitution at position 304 of SEQ ID NO:
• 1 with the substitution being A304G, A304I, A304L, A304M, A304P or A304V, preferably A304I or A304L, and more preferably A304I;
• an amino acid substitution at position 306 of SEQ ID NO:
• 1 with the substitution being D306R, D306H or D306K, preferably D306H;
• an amino acid substitution at position 318 of SEQ ID NO:
• 1 with the substitution being P318R, P318H or P318K, preferably P318H;
• an amino acid substitution at position 323 of SEQ ID NO:
• 1 with the substitution being T323A, T323G, T323I, T323L, T323M, T323P, or T323V, preferably T323I or T323V, and more preferably T323I;
• an amino acid substitution at position 339 of SEQ ID NO:
• 1 with the substitution being L339A, L339G, L339I, L339M, L339P or L339V, preferably L339I;
• an amino acid substitution at position 349 of SEQ ID NO:
• 1 with the substitution being F349W or F349Y, preferably F349Y;
• an amino acid substitution at position 357 of SEQ ID NO:
• 1 with the substitution being G357A, G357I, G357L, G357M, G357P or G357V, preferably G357A;
• an amino acid substitution at position 459 of SEQ ID NO:
• 1 with the substitution being A459N, A459C, A459Q or A459S, A459T, preferably A459S;
• an amino acid substitution at position 476 of SEQ ID NO:
• 1 with the substitution being Q476A, Q476G, Q476I, Q476L, Q476M, Q476P or Q476V, preferably Q476G;
• an amino acid substitution at position 481 of SEQ ID NO:
• 1 with the substitution being E481A, E481G, E481I, E481L, E481M, E481P or E481V, preferably E481I;
• an amino acid substitution at position 484 of SEQ ID NO:
• 1 with the substitution being A484N, A484C, A484Q, A484S or A484T, preferably A484S;
• an amino acid substitution at position 487 of SEQ ID NO:
• 1 with the substitution being Q487A, Q487G, Q487I, Q487L, Q487M, Q487P or Q487V, preferably Q487A, Q487G, Q487L, Q487M or Q487V, more preferably Q487A;
• an amino acid substitution at position 488 of SEQ ID NO:
• 1 with the substitution being K488A, K488G, K488I, K488L, K488M, K488P or K488V, preferably K488A;
• an amino acid substitution at position 506 of SEQ ID NO:
• 1 with the substitution being A506N, A506C, A506Q, A506S or A506T, preferably A506S;
• an amino acid substitution at position A511 of SEQ ID NO:
• 1 with the substitution being A511N, A511C, A511Q, A511S or A511T, preferably A511S;
• an amino acid substitution at position 526 of SEQ ID NO:
• 1 with the substitution being R526D or R526E, preferably R526E;
• an amino acid substitution at position 530 of SEQ ID NO:
• 1 with the substitution being E530A, E530G, E530I, E530L, E530M, E530P, E530V, preferably E530V;
• an amino acid substitution at position 532 of SEQ ID NO:
• 1 with the substitution being G532R, G532H or G532K, preferably G532R;
• an amino acid substitution at position 533 of SEQ ID NO:
• 1 with the substitution being D533A, D533G, D533I, D533L, D533M, D533P or D533V, preferably D533G;
• an amino acid substitution at position 537 of SEQ ID NO:
• 1 with the substitution being D537A, D537G, D537I, D537L, D537M, D537P or D537V, preferably D537M;
• an amino acid substitution at position 550 of SEQ ID NO:
• 1 with the substitution being V550A, V550G, V550I, V550L, V550M or, V550P, preferably V550I or V550P, and more preferably V550I;
• an amino acid substitution at position 556 of SEQ ID NO:
• 1 with the substitution being S556N, S556C, S556Q or S556T, preferably S556T;
• an amino acid substitution at position 564 of SEQ ID NO:
• 1 with the substitution being T564D or T564E, preferably T564E;
• an amino acid substitution at position 590 of SEQ ID NO:
• 1 with the substitution being D590N, D590C, D590Q, D590S, D590T, D590A, D590G, D590I, D590L, D590M, D590P or D590V, preferably D590N, D590G or D590A, and more preferably D590N;
• an amino acid substitution at position 649 of SEQ ID NO:
• 1 with the substitution being A649D or A649E, preferably A649E;
• an amino acid substitution at position 667 of SEQ ID NO:
• 1 with the substitution being R667D, R667E, R667R, R667H or R667K, preferably R667E or R667K, more preferably R667E;
• an amino acid substitution at position 703 of SEQ ID NO:
• 1 with the substitution being A703D or A703E, preferably A703E;
• and
• an amino acid substitution at position 705 of SEQ ID NO:
• 1 with the substitution being K705N, K705C, K705Q, K705S or K705T, preferably K705N.

The trehalose phosphorylase of any one of the previous claims, wherein the amino acid sequence of the trehalose phosphorylase comprises

• (a) an amino acid substitution at the three amino acid positions 383, 556, and 590 of SEQ ID NO:
• 1;
• (b) an amino acid substitution at the four amino acid positions 712, 383, 114 and 118 of SEQ ID NO:
• 1;
• and/or 383, 487, 556, and 590 of SEQ ID NO:
• 1;
• and/or 383, 225, 556, and 590 of SEQ ID NO:
• 1;
• (c) an amino acid substitution at the five amino acid positions 712, 383, 114, 118 and 304 of SEQ ID NO:
• 1;
• and/or 712, 383, 114, 118 and 357 of SEQ ID NO:
• 1;
• and/or 383, 225, 304, 556, and 590 of SEQ ID NO:
• 1;
• and/or 383, 225, 487, 556, and 590 of SEQ ID NO:
• 1;
• (d) an amino acid substitution at the six amino acid positions 712, 383, 114, 118, 304 and 357 of SEQ ID NO:
• 1;
• and/or 383, 225, 304, 487, 556, and 590 of SEQ ID NO:
• 1.

The trehalose phosphorylase of any one of the previous claims, wherein the trehalose phosphorylase, has at least one of characteristics (A), (B), (C), (D) and (E), or any combination thereof,

• wherein
• characteristic (A) is thermal stability after incubation at 52° C. for 15 minutes defined by a residual activity of from 30% to 100%;
• characteristic (B) is thermal stability after incubation at 52° C. for 15 minutes which is characterized by
• i) a Tm30-value of at least 52° C., and/or
• ii) a Tm50-value of at least 52° C.;
• characteristic (C) is thermal stability characterized by
• i) a Tm30-value between 52° C. and 90° C. and/or
• ii) a Tm50-value between 52° C. and 90° C.;
• and
• characteristic (D) is thermal stability characterized by
• i) a process stability characterized by a half-life at 45° C. of from 3 hours to 9 days or more;
• or
• ii) a process stability characterized by a half-life at 45° C. of from 24 hours to 9 days or more, or
• iii) a process stability characterized by a half-life at 45° C. of 4 days to 9 days or more;
• and
• characteristic (E) is relative activity expressed as 100/500-ratio of between 0.65 and 1.0, wherein
• the 100/500-ratio is defined as the ratio of
• [trehalose activity at 100 mM glucose and 100 mM alpha-glucose-1 phosphate]/
• [trehalose activity at 500 mM glucose and 100 mM alpha-glucose-1 phosphate].

A trehalose phosphorylase comprising an amino acid sequence, 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 trehalose phosphorylase has at least one of characteristics (A), (B), (C), (D) and (E), or any combination thereof, wherein
• characteristic (A) is thermal stability after incubation at 52° C. for 15 minutes defined by a residual activity of from 30% to 100%;
• characteristic (B) is thermal stability after incubation at 52° C. for 15 minutes which is characterized by
• i) a Tm30-value of at least 52° C., and/or
• ii) a Tm50-value of at least 52° C.;
• characteristic (C) is thermal stability characterized by
• i) a Tm30-value between 52° C. and 90° C. and/or
• ii) a Tm50-value between 52° C. and 90° C.;
• characteristic (D) is thermal stability characterized by
• i) a process stability characterized by a half-life at 45° C. of from 3 hours to 9 days or more;
• or
• ii) a process stability characterized by a half-life at 45° C. of from 24 hours to 9 days or more, or
• iii) a process stability characterized by a half-life at 45° C. of 4 days to 9 days or more;
• and
• characteristic (E) is relative activity expressed as 100/500-ratio of between 0.65 and 1.0, wherein
• the 100/500-ratio is defined as the ratio of
• [trehalose activity at 100 mM glucose and 100 mM alpha-glucose-1 phosphate]/
• [trehalose activity at 500 mM glucose and 100 mM alpha-glucose-1 phosphate].

A trehalose phosphorylase comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical to and/or at least 85% homologous to an amino acid sequence of SEQ ID NO:

• 160 or SEQ ID NO:
• 81, preferably SEQ ID NO:
• 160, wherein the trehalose phosphorylase, has at least one of characteristics (A), (B), (C), (D) and (E), or any combination thereof, wherein
• characteristic (A) is thermal stability after incubation at 52° C. for 15 minutes defined by a residual activity of from 30% to 100%;
• characteristic (B) is thermal stability after incubation at 52° C. for 15 minutes which is characterized by
• i) a Tm30-value of at least 52° C., and/or
• ii) a Tm50-value of at least 52° C.;
• characteristic (C) is thermal stability characterized by
• i) a Tm30-value between 52° C. and 90° C. and/or
• ii) a Tm50-value between 52° C. and 90° C.;
• characteristic (D) is thermal stability characterized by
• i) a process stability characterized by a half-life at 45° C. of from 3 hours to 9 days or more;
• or
• ii) a process stability characterized by a half-life at 45° C. of from 24 hours to 9 days or more, or
• iii) a process stability characterized by a half-life at 45° C. of 4 days to 9 days or more;
• and
• characteristic (E) is relative activity expressed as 100/500-ratio of between 0.65 and 1.0, wherein
• the 100/500-ratio is defined as the ratio of
• [trehalose activity at 100 mM glucose and 100 mM alpha-glucose-1 phosphate]/
• [trehalose activity at 500 mM glucose and 100 mM alpha-glucose-1 phosphate].

A trehalose phosphorylase variant, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 20% homologous to the amino acid sequence of SEQ NO:

• 1, wherein the variant comprises two or more, three or more, four or more, five or more, six or more, preferably seven or more, of the following amino acid positions selected from the group consisting of
• 712A, 712G, 712I, 712M, 712P or 712V, preferably 712M, and
• 383A, 383G, 383I, 383L, 383M, 383V, 383N, 383C, 383Q, 383S or 383T, preferably 383A, 383G, 383M, 383V, 383N, 383C, 383Q, 383S or 383T, more preferably 383G, 383V, 383C, 383S or 383T, even more preferably 383V or 383T, and most preferably 383V, and
• 114A, 114G, 114I, 114M, 114P or 114V, preferably 114I, and
• 118 is 118A, 118G, 118I, 118L, 118M, 118P or 118V, preferably 118V, and
• 225A, 225G, 225I, 225L, 225M, 225P or 225V, preferably 225I, 225L, 225M or 225V, and more preferably 225V, and
• 304G, 304I, 304L, 304M, 304P or 304V, preferably 304I or 304L, and more preferably 304I, and
• 323A, 323G, 323I, 323L, 323M, 323P, or 323V, preferably 323I or 323V, and more preferably 323I,
• 349W or 349Y, preferably 349Y, and
• 357A, 357I, 357L, 357M, 357P or 357V, preferably 357A, and
• 487A, 487G, 487I, 487L, 487M, 487P or 487V, preferably 487A, 487M, 487G, 487L or 487V, more preferably 487A, and
• 550A, 550G, 550I, 550L, 550M or 550P, preferably 550 I or 550P, and more preferably 550I, and
• 556N, 556C, 556Q or S556T, preferably 556T, and
• 564D or 564E, preferably 564E, and
• 590N, 590C, 590Q, 590S, 590T, 590A, 590G, 590I, 590L, 590M, 590P or 590V, preferably
• 590N, 590G or 590A, more preferably 590N, and
• 649D or 649E, preferably 649E
• with the numbering referring to an aligning position in SEQ ID NO:
• 1.

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.

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.

A method for increasing the relative activity expressed as 100/500-ratio 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 the relative activity expressed as 100/500-ratio 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 the relative activity expressed as 100/500-ratio 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.