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TREHALOSE PHOSPHORYLASE

20190276808 ยท 2019-09-12

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention is related to 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 amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions of SEQ ID NO: 1 712, 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.

    Claims

    1. 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 amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein 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.

    2. The trehalose phosphorylase of claim 1, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions 712, 383, 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, 667, 703 and 705 of SEQ ID NO: 1.

    3. The trehalose phosphorylase of any one of the previous claims, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions 383, 225, 304, 323, 487, 550, 556, 564, 590, and 705 of SEQ ID NO: 1.

    4. The trehalose phosphorylase of any one of the previous claims, wherein the one or more amino acid positions is at amino acid position 383 of SEQ ID NO: 1.

    5. The trehalose phosphorylase of any one of the previous claims, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions L712M, P383G, P383V, P383C, P383S, P383T, V10R, L114I, 118V, S192V, S197G, Y220F, N225I, N225L, N225M, N225V, A304L, A304I, D306H, P318H, T323I, T323V, L339I, F349Y, G357A, A459S, Q476G, E481I, A484S, 487A, Q487G, Q487L, Q487M, Q487V, K488A, A506S, A511S, R526E, E530V, G532R, D533G, D537M, V550I, V550P, S556T, T564E, D590N, D590G, D590A, A649E, R667E, R667K, A703E, and K705N of SEQ ID NO: 1.

    6. The trehalose phosphorylase of any one of the previous claims, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions P383G, P383V, P383C, P383S, P383T, V10R, L114I, S192V, S197G, N225I, N225L, N225M, N225V, A304L, A304I, D306H, P318H, T323I, T323V, L339I, F349Y, G357A, A459S, Q476G, E481I, A484SQ487G, Q487L, Q487M, Q487V, K488A, A506S, A511S, R526E, E530V, G532R, D533G, D537M, V550I, V550P, S556T, T564E, D590G, D590A, A649E, R667E, R667K, A703E, and K705N of SEQ ID NO: 1.

    7. The trehalose phosphorylase of any one of the previous claims, wherein the trehalose phosphorylase comprises an amino acid substitution at two or more amino acid positions, wherein the two or more amino acid positions are selected from the group consisting of 10 and 114, 10 and 712, 114 and 118, 114 and 304, 114 and 357, 114 and 383, 114 and 590, 114 and 712, 118 and 304, 118 and 357, 118 and 383, 118 and 556, 118 and 564, 118 and 590, 118 and 712, 225 and 304, 225 and 383, 225 and 487, 225 and 550, 225 and 556, 225 and 590, 304 and 323, 304 and 357, 304 and 383, 304 and 487, 304 and 556, 304 and 564, 304 and 590, 304 and 712, 323 and 357, 323 and 487, 323 and 556, 323 and 564, 323 and 590, 323 and 649, 349 and 383, 349 and 590, 357 and 383, 357 and 590, 357 and 705, 357 and 712, 383 and 487, 383 and 550, 383 and 556, 383 and 564, 383 and 590, 383 and 649, 383 and 712, 487 and 564, 487 and 590, 487 and 649, 550 and 590, 556 and 564, 556 and 590, 556 and 649, 564 and 590, 564 and 712, 590 and 649, 590 and 712, and 649 and 712.

    8. The trehalose phosphorylase of any one of the previous claims, wherein the trehalose phosphorylase comprises an amino acid substitution at two or more amino acid positions, wherein the two or more amino acid positions are selected from the group consisting of 118 and 383, 118 and 556, 118 and 564, 118 and 590, 225 and 304, 225 and 383, 225 and 487, 225 and 550, 225 and 556, 225 and 590, 304 and 323, 304 and 383, 304 and 487, 304 and 556, 304 and 564, 304 and 590, 323 and 357, 323 and 487, 323 and 556, 323 and 564, 323 and 590, 323 and 649, 349 and 383, 349 and 590, 357 and 383, 357 and 590, 383 and 487, 383 and 550, 383 and 556, 383 and 564, 383 and 590, 383 and 649, 383 and 712, 487 and 564, 487 and 590, 487 and 649, 550 and 590, 556 and 564, 556 and 590, 556 and 649, 564 and 590, 564 and 712, 590 and 649, 590 and 712, 649 and 712, and 705 and 712.

    9. The trehalose phosphorylase of any one of the previous claims, wherein the trehalose phosphorylase comprises an amino acid substitution at two or more amino acid positions, wherein the two or more amino acid positions are selected from the group consisting of 118 and 383, 118 and 590, 225 and 383, 225 and 590, 304 and 383, 304 and 590, 323 and 590, 349 and 383, 349 and 590, 357 and 383, 357 and 590, 383 and 487, 383 and 550, 383 and 556, 383 and 564, 383 and 590, 383 and 649, 383 and 712, 487 and 590, 550 and 590, 556 and 590, 564 and 590, 590 and 649, 590 and 712.

    10. 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.

    11. 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.

    12. The trehalose phosphorylase of any one of the previous claims, wherein the identity of the amino acid sequence of the trehalose phosphorylase with the amino acid sequence of SEQ ID NO: 1 is at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, still more preferably at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, yet more preferably at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, and most preferably at least 96%, or at least 97%, or at least 98%, or at least 99%, at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8%, and in particular at least 99.9%, or 100%.

    13. The trehalose phosphorylase of any one of the previous claims, wherein the amino acid sequence of the trehalose phosphorylase comprises or consists of any one of amino acid sequences of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 87, 88, 89, 91, 93, 94, 95, 97, 98, 101, 103, 104, 105, 107, 109, 110, 111, 113, 114, 115, 117, 119, 121, 123, 125, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 146, 147, 149, 150, 151, 152, 154, 155, 156, 157, 158, 159, or 190, preferably wherein the amino acid sequence of the trehalose phosphorylase comprises or consists of the amino acid sequence of SEQ ID NO: 14.

    14. The trehalose phosphorylase of any one of the previous claims, wherein the trehalose phosphorylase is capable of catalyzing conversion of glucose and alpha-D-glucose-1 phosphate to trehalose and inorganic phosphate and/or conversion of trehalose and inorganic phosphate to glucose and alpha-D-glucose-1 phosphate.

    15. 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].

    16. 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].

    17. The trehalose phosphorylase of claim 16, wherein the trehalose phosphorylase is a trehalose phosphorylase as defined in any one of claims 1 to 15.

    18. A trehalose phosphorylase comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 85% identical to and/or at least 86% homologous to an amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 160, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions 108, 112, 221, 300, 319, 345, 379, 483, 544, 550, 558, 584, 643, and 707 of SEQ ID NO:81 or SEQ ID NO: 160.

    19. A trehalose phosphorylase of claim 18, comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 85% identical to and/or at least 86% homologous to an amino acid sequence of SEQ ID NO: 160, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions of L108, V112, N221, A300, T319, F345, P379, V483, V544, S550, Q558, N584, A643, and L707 of SEQ ID NO: 160.

    20. A trehalose phosphorylase of claim 18, comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 85% identical to and/or at least 86% homologous to an amino acid sequence of SEQ ID NO: 81, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions of L108, V112, N221, A300, T319, F345, P379, 1483, V544, S550, Q558, N584, A643, and L707 of SEQ ID NO:81.

    21. A trehalose phosphorylase of any one of claims 18 to 20, wherein the trehalose phosphorylase comprises an amino acid substitution at two or more amino acid positions, wherein the pair of two amino acid positions is selected from the group consisting of 108 and 112, 108 and 221, 108 and 300, 108 and 319, 108 and 345, 108 and 379, 108 and 483, 108 and 544, 108 and 550, 108 and 558, 108 and 584, 108 and 643, 108 and 707, 112 and 221, 112 and 300, 112 and 319, 112 and 345, 112 and 379, 112 and 483, 112 and 544, 112 and 550, 112 and 558, 112 and 584, 112 and 643, 112 and 707, 221 and 300, 221 and 319, 221 and 345, 221 and 379, 221 and 483, 221 and 544, 221 and 550, 221 and 558, 221 and 584, 221 and 643, 221 and 707, 300 and 319, 300 and 345, 300 and 379, 300 and 483, 300 and 544, 300 and 550, 300 and 558, 300 and 584, 300 and 643, 300 and 707, 319 and 345, 319 and 379, 319 and 483, 319 and 544, 319 and 550, 319 and 558, 319 and 584, 319 and 643, 319 and 707, 345 and 379, 345 and 483, 345 and 544, 345 and 550, 345 and 558, 345 and 584, 345 and 643, 345 and 707, 379 and 483, 379 and 544, 379 and 550, 379 and 558, 379 and 584, 379 and 643, 379 and 707, 483 and 544, 483 and 550, 483 and 558, 483 and 584, 483 and 643, 483 and 707, 544 and 550, 544 and 558, 544 and 584, 544 and 643, 544 and 707, 550 and 558, 550 and 584, 550 and 643, 550 and 707, 558 and 584, 558 and 643, 558 and 707, 584 and 643, 584 and 707, and 643 and 707.

    22. The trehalose phosphorylase of any one of claims 18 to 21, wherein the trehalose phosphorylase comprises an amino acid substitution at two or more amino acid wherein the pair of two amino acid positions is selected from the group consisting of (i) L108 and T319, L108 and P379, L108 and V483, L108 and S550, L108 and Q558, N221 and T319, N221 and P379, N221 and V483, N221 and V544, N221 and S550, N221 and Q558, T319 and P379, T319 and V483, T319 and V544, T319 and S550, T319 and Q558, P379 and V483, P379 and V544, P379 and S550, P379 and Q558, V483 and V544, V483 and S550, V483 and Q558, and S550 and Q558 of SEQ ID NO:160. (ii) L108 and T319, L108 and P379, L108 and I483, L108 and S550, L108 and Q558, N221 and T319, N221 and P379, N221 and I483, N221 and V544, N221 and S550, N221 and Q558, T319 and P379, T319 and I483, T319 and V544, T319 and S550, T319 and Q558, P379 and I483, P379 and V544, P379 and S550, P379 and Q558, 1483 and V544, 1483 and S550, 1483 and Q558, and S550 and Q558 of SEQ ID NO:81.

    23. The trehalose phosphorylase of any one of claims 18 to 22, wherein the one or more substitution is/are selected from the group consisting of an amino acid substitution at position L108 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being L108A, L108G, L108I, L108M, L108P or L108V, preferably L108I; an amino acid substitution at position V112 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being V112A, V112G, V112L, V112M, V112P or V112I, preferably V112I; an amino acid substitution at position N221 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being N221A, N221G, N221I, N221L, N221M, N221P or N221V, preferably N221I, N221L, N221M or N221V, and more preferably N221V; an amino acid substitution at position A300 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being A300G, A300I, A300L, A300M, A300P or A300V, preferably A300I or A300L, and more preferably A300I; an amino acid substitution at position T319 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being T319A, T319G, T319I, T319L, T319M, T319P, or T319V, preferably T319I or T319V, and more preferably T319I; an amino acid substitution at position P379 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being P379A, P379G, P3791, P379L, P379M, P379V, P379N, P379C, P379Q, P379S or P379T, preferably P379A, P379G, P379M, P379V, P379N, P379C, P379Q, P379S or P379T, more preferably P379G, P379V, P379C or P379S, or P379T, even more preferably P379V or P379T, and most preferably P379V; an amino acid substitution at position I483 of SEQ ID NO: 81 with the substitution being I483A, I483G, I483I, I483L, I483M, I483P or I483V, preferably I483A, I483G, I483L, I483M or I483V, more preferably I483A; an amino acid substitution at position Q483 of SEQ ID NO: 160 with the substitution being Q483A, Q483G, Q483I, Q483L, Q483M, Q483P or Q483V, preferably Q483A, Q483G, Q483L, Q483M or Q483V, more preferably Q483A; an amino acid substitution at position V544 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being V544A, V544G, V544I, V544L, V544M or V544P, preferably V544I or V544P, and more preferably V544I; an amino acid substitution at position S550 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being S550N, S550C, S550Q or S550T, preferably S550T; an amino acid substitution at position Q558 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being Q558D or Q558E, preferably Q558E; an amino acid substitution at position D558 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being D558N, D558C, D558Q, D558S, D558T, D558A, D558G, D558I, D558L, D558M, D558P or D558V, preferably D558N, D558G or D558A, and more preferably D558N; an amino acid substitution at position A643 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being A643D or A643E, preferably A643E; an amino acid substitution at position L707 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being L707A, L707G, L707I, L707M, L707P or L707V, preferably L707M.

    24. The trehalose phosphorylase of any one of claims 18 to 23, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid sequence according to any one of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, preferably according to any one of SEQ ID NO: 163, 165, 166, 168, 171, 173, 176, 177, 178, 179, 180, 184, 185, 186, 187, 188, 189, more preferably according to any one of SEQ ID NO: 176, 178, 180, 185, 188.

    25. 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].

    26. The trehalose phosphorylase of claim 25, wherein the trehalose phosphorylase is a trehalose phosphorylase as defined in any one of claims 18 to 24.

    27. 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.

    28. The trehalose phosphorylase variant of claim 27, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 77% homologous to the amino acid sequence of SEQ NO: 1, wherein the polypeptide comprises an amino acid substitution at two or more of amino acid positions selected from the group consisting of amino acid position 383, 114, 225, 304, 323, 349, 357, 550, 556, 564 and 649 wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    29. The trehalose phosphorylase variant of claim 28, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 77% homologous to the amino acid sequence of SEQ NO: 1, wherein the polypeptide comprises an amino acid substitution at two or more of amino acid positions selected from the group consisting of amino acid position 383V, 383T, 114I, 225I, 225L, 225M, 225V, 304I, 304L, 323I, 323V, 349Y, 357A, 550I, 550P, 556T, 564E and 649E wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    30. The trehalose phosphorylase variant of any one of claims 28 to 29, wherein the variant comprises at least one further amino acid substitution at amino acid positions selected from amino acid position 712, 118, 487 and 590, preferably selected from amino acid position 712M, 118V, 487A, 487G, 487L, 487M, 487V, and 590N, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    31. The trehalose phosphorylase variant of claim 27, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 68% homologous to the amino acid sequence of SEQ NO: 1, wherein the polypeptide comprises an amino acid substitution at two or more of amino acid positions selected from the group consisting of amino acid position 383, 114, 225, 304, 323, 349, 357, 550, 556, and 564 wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    32. The trehalose phosphorylase variant of claim 31, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 68% homologous to the amino acid sequence of SEQ NO: 1, wherein the polypeptide comprises an amino acid substitution at two or more of amino acid positions selected from the group consisting of amino acid position 383V, 383T, 114I, 225I, 225L, 225M, 225V, 304I, 304L, 323I, 323V, 349Y, 357A, 550I, 550P, 556T, and 564E wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    33. The trehalose phosphorylase variant of any one of claims 31 to 32, wherein the variant comprises at least one further amino acid substitution at amino acid positions selected from amino acid position 712, 118, 487, 590, and 649, preferably selected from amino acid position 712M, 118V, 487A, 487G, 487L, 487M, 487V, 590N, and 649E, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    34. The trehalose phosphorylase variant of claim 27, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 63% homologous to the amino acid sequence of SEQ NO: 1, wherein the polypeptide comprises an amino acid substitution at two or more of amino acid positions selected from the group consisting of amino acid position 383, 114, 225, 304, 323, 349, 357, 556, and 564 wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    35. The trehalose phosphorylase variant of claim 34, wherein the variant comprises an amino acid sequence, wherein the amino acid sequence is at least 63% homologous to the amino acid sequence of SEQ NO: 1, wherein the polypeptide comprises an amino acid substitution at two or more of amino acid positions selected from the group consisting of amino acid position 383V, 383T, 114I, 225I, 225L, 225M, 225V, 304I, 304L, 323I, 323V, 349Y, 357A, 556T, and 564E wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    36. The trehalose phosphorylase variant of any one of claims 34 to 35, wherein the variant comprises at least one further amino acid substitution at amino acid positions selected from amino acid position 712, 118, 487, 550, 590, and 649, preferably selected from amino acid position 712M, 118V, 487A, 487G, 487L, 487M, 487V, 550I, 550P, 590N, and 649E, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    37. A thermally stable trehalose phosphorylase variant, wherein the variant retains at least 30% of its initial activity after incubation for 15 minutes at 52? C. in a buffer containing 1 M sucrose, and the initial activity is determined after incubation for 15 minutes at room temperature and/or wherein the variant retains at least 50% of its initial activity after incubation for 15 minutes at 52? C. in a buffer containing 1 M sucrose, and the initial activity is determined after incubation for 15 minutes at room temperature.

    38. The thermally stable trehalose phosphorylase variant of claim 37, wherein the trehalose phosphorylase is a trehalose phosphorylase as defined in any one of claims 1 to 36.

    39. A thermally stable variant of trehalose phosphorylase from Schizophyllum commune or Grifola frondosa, wherein the variant has a residual activity of at least 30% after incubation at 52? C. for 15 minutes, in a buffer containing 1 M sucrose, preferably wherein the variant has a residual activity of at least 50% of its initial activity after incubation for 15 minutes at 52? C., in a buffer containing 1 M sucrose, and the initial activity is determined after incubation for 15 minutes at room temperature.

    40. The thermally stable variant of trehalose phosphorylase from Schizophyllum commune of claim 39, wherein the trehalose phosphorylase is a trehalose phosphorylase as defined in any one of claims 1 to 17 and 37 to 38.

    41. The thermally stable variant of trehalose phosphorylase from Grifola frondosa, wherein the trehalose phosphorylase is a trehalose phosphorylase as defined in any one of claims 18 to 26 and 37 to 38.

    42. A thermally stable variant of a trehalose phosphorylase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, or SEQ ID NO: 160, or SEQ ID NO: 81, wherein the variant has a residual activity of at least 30% after incubation at 52? C. for 15 minutes, in a buffer containing 1 M sucrose, preferably wherein the variant has a residual activity of at least 50% of its initial activity after incubation for 15 minutes at 52? C., in a buffer containing 1 M sucrose, and the initial activity is determined after incubation for 15 minutes at room temperature.

    43. The thermally stable variant of a trehalose phosphorylase of claim 42, wherein the trehalose phosphorylase is a trehalose phosphorylase as defined in any one of claims 1 to 41, preferably as in any of of claims 1 to 26.

    44. A method for reacting a glucosyl monosaccharide and alpha-D-glucose-1 phosphate, wherein the method comprises reacting the glucosyl monosaccharide and alpha-D-glucose-1 phosphate with a trehalose phosphorylase as defined in any one of claims 1 to 43.

    45. 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.

    46. The method according to claim 44, wherein the trehalose phosphorylase is any of the trehalose phosphorylase variant of claims 1 to 43.

    47. 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.

    48. The method for increasing thermal stability of a trehalose phosphorylase, wherein the substitution of an amino acid residue in the second trehalose phosphorylase is done in one or more of the amino acids positions corresponding to 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, preferably corresponding to 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

    49. 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.

    50. The method for increasing the relative activity expressed as 100/500-ratio of a trehalose phosphorylase, wherein the substitution of an amino acid residue in the second trehalose phosphorylase is done in one or more of the amino acids positions corresponding to 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, preferably corresponding to 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

    51. Use of a trehalose phosphorylase according to any one of claims 1 to 43 for producing trehalose, preferably for producing trehalose according to any one of claims 44 to 46.

    Description

    [0711] FIG. 1 is a diagram showing residual activity in % as a function of temperature for wild type trehalose phosphorylase of SEQ ID NO: 1 in the presence and in the absence of 1M sucrose added a stabilizing agent; and

    [0712] FIG. 2 is a diagram showing residual activity in % as a function of time for wild type trehalose phosphorylase of SEQ ID NO: 1 and various trehalose phosphorylases of the invention; and

    [0713] FIG. 3 shows an alignment of the wild type TPs from Grifola frondosa (UniProtKB/Swiss-Prot: Accession No: 075003.1 and Genebank Accession No: ADM15725.1), Pleurotus ostreatus (Genebank Accession No: KDQ33172.1), Lentinus sajor-caju (Synonym: Pleurotus sajor-caju, Genebank Accession No: Q9UV63.1). The alignment was done using Clustal omega (Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R Nucleic acids research 2010 July, 38 Suppl: W695-9).

    [0714] The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

    EXAMPLES

    Example 1: General Methods

    [0715] Cloning of the wild type TP: The trehalose phosphorylase gene from S. commune was codon-optimized for expression in E. coli and synthesized by Eurofins MWG Operon. The gene was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmids were used for transformation of E. coli BL21(DE3) cells.

    [0716] Molecular biology methods: Mutants of the TP enzymes were created by standard site-directed mutagenesis technologies as known in the state of the art.

    [0717] Expression of recombinant TPs: Recombinant TPs were routinely expressed by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone, 25 mM Na2HPO4*12H2O, 25 mM KH2PO4, 50 mM NH4Cl2, Na2SO4, 5 g/L glycerol, 0.5 g/L glucose*1H2O, 2 mM MgSO4, 50 ?g/mL kanamycin) with a fresh overnight culture. Cultures were grown at 37? C. up to an optical density at 600 nm of 0.6-0.8. Cultures were induced with 0.1 mM IPTG final concentration. Expression was at 24-25? C. overnight.

    [0718] Preparation of TP enzyme preparations: Preparation of cell free extract was done using procedures well known as described elsewhere. Cells were harvested by centrifugation and suspended in a buffer containing 50 mM potassium phosphate-buffer pH 7, 2 mM MgCl2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. 1 M sucrose was at times added as a stabilizing agent. Cell disruption was achieved by sonication or repeated freeze/thaw cycles. Cell free extract containing soluble enzyme was separated from the debris by centrifugation.

    [0719] Activity measurements: Activity of trehalose phosphorylase can be determined in both the direction of trehalose cleavage (phosphorolytic activity) and synthesis (synthetic activity) as described in Assay I and Assay II:

    [0720] Assay I: Phosphorolytic activity was routinely assayed at 30? C. using a continuous coupled assay in which the aG1P produced from trehalose is converted to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphate and NADP is converted to 6-phospho-gluconate and NADPH by glucose 6-phosphate dehydrogenase. The detection is based on measuring the absorbance of NADPH at 340 nm. The assay solution contained: 75 mM potassium phosphate buffer pH 7, 2.5 mM NADP, 10 ?M glucose 1,6-bisphosphate, 10 mM MgCl2, 225 mM trehalose, 3 U/mL phosphoglucomutase and 3.4 U/mL glucose 6-phosphate dehydrogenase.

    [0721] Assay II: Synthetic activity was routinely assayed at 40? C. using the following conditions: 50 mM sodium MES buffer pH 7, 100 mM aG1P and 100 or 500 mM glucose concentrations as given. Reaction progress was determined discontinuously by measuring liberated phosphate with an assay based on the complex formation with molybdate under acidic conditions. The molybdate complex is reduced by ferrous sulfate and yields a blue color, which is analyzed photometrically at 750 nm. For the analysis 250 ?L of sample are mixed with 250 ?L 0.5 M HCl and 500 ?L molybdate-reagent (73.2 g/L Fe(II)SO4*7H2O and 10 g/L ammonium molybdate*4H2O in 3.5% sulfuric acid). After incubation at RT for 15-30 min, absorbance is measured at 750 nm. The amount of inorganic phosphate in the sample is quantified using external standards.

    Example 2: Effect of Sucrose on Thermal Stability

    [0722] Expression of recombinant TPs: The wild-type enzyme SEQ ID NO: 1 was expressed in shaking flasks by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone, 25 mM Na2HPO4*12H2O, 25 mM KH2PO4, 50 mM NH4Cl2, Na2SO4, 5 g/L glycerol, 0.5 g/L glucose*1H2O, 2 mM MgSO4, 50 ?g/mL kanamycin) with a fresh overnight culture. Cultures were induced in the logarithmic phase with 0.1 mM IPTG and expressed overnight at 24-25? C.

    [0723] Preparation of TP enzyme preparations: For the preparation of cell extract without sucrose cells were harvested by centrifugation and suspended in a buffer containing 50 mM potassium phosphate-buffer pH 7, 2 mM MgCl2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. Cells were disrupted by sonication. Cell free extract containing soluble enzyme was separated from the debris by centrifugation. For the preparation of cell extract with sucrose as a stabilizing agent, cells were harvested by centrifugation and suspended in a buffer containing 100 mM potassium phosphate-buffer pH 7, 2 mM MgCl2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. Cells were disrupted by sonication. Cell free extract containing soluble enzyme was separated from the debris by centrifugation and diluted 1:2 with 2 M sucrose solution.

    [0724] Determination of denaturation profile: 50 ?L aliquots of enzyme preparations with and without 1 M sucrose were incubated for 15 min at temperatures ranging from 36 to 53.7? C. Denatured protein was separated by centrifugation. The activity of the resulting supernatants as well as cell extract without a heat inactivation step was determined using Assay I. FIG. 1 is a denaturing profile of SEQ ID NO: 1 with and without 1 M sucrose as a stabilizing agent showing the obtained residual activities compared to the enzyme preparations without heat inactivation. The addition of 1 M sucrose results in an increase of Tm50 from approx. 40? C. to 47.5? C. 1 M sucrose was therefore chosen as a stabilizing agent for TP.

    Example 3: Residual Activity of TP Variants after Incubation at 52? C. for 15 Min

    [0725] Expression of recombinant TPs: Recombinant TPs were expressed in deep-well plates by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone, 25 mM Na2HPO4*12H2O, 25 mM KH2PO4, 50 mM NH4Cl2, Na2SO4, 5 g/L glycerol, 0.5 g/L glucose*1H2O, 2 mM MgSO4, 50 ?g/mL kanamycin) with a fresh overnight culture. Cultures were grown at 37? C. up to an optical density at 600 nm of 0.6-0.8. Cultures were induced with 0.1 mM IPTG final concentration. Expression was at 24-25? C. overnight.

    [0726] Preparation of TP enzyme preparations: Cells were harvested by centrifugation and suspended in 100 mM potassium phosphate-buffer pH 7, 2 mM MgCl2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. Cells were disrupted by repeated freeze/thaw cycles. Cell free extract containing soluble enzyme was separated from the debris by centrifugation. The cell free extract was diluted 1:2 with 2 M sucrose solution.

    [0727] Heat-inactivation and activity measurement: A 50 ?L aliquot of each TP was incubated at 52? C. for 15 min. Denatured protein was separated by centrifugation. The activity of the supernatant was determined using Assay II with 500 mM glucose. Another aliquot of each TP was assayed directly for activity without heat-inactivation using Assay II with 500 mM glucose. The resulting residual activities are listed in Table 6. All variants showed a higher residual activity than the wild-type enzyme which means they possess an improved thermal stability compared to the wild-type.

    TABLE-US-00006 TABLE 6 Residual activity of TP variants after incubation at 52? C. for 15 min residual activity in % after 15 min incubation at 52? C. SEQ ID [%] SEQ ID NO: 1 19 SEQ ID NO: 2 30 SEQ ID NO: 3 64 SEQ ID NO: 4 30 SEQ ID NO: 5 39 SEQ ID NO: 6 54 SEQ ID NO: 7 55 SEQ ID NO: 8 42 SEQ ID NO: 9 63 SEQ ID NO: 10 68 SEQ ID NO: 11 39 SEQ ID NO: 12 55 SEQ ID NO: 13 61 SEQ ID NO: 14 30 SEQ ID NO: 15 75 SEQ ID NO: 16 43 SEQ ID NO: 17 50 SEQ ID NO: 18 41 SEQ ID NO: 19 48 SEQ ID NO: 20 75 SEQ ID NO: 21 52 SEQ ID NO: 22 37 SEQ ID NO: 23 36 SEQ ID NO: 24 47 SEQ ID NO: 25 41 SEQ ID NO: 26 45 SEQ ID NO: 27 42 SEQ ID NO: 28 31 SEQ ID NO: 29 55 SEQ ID NO: 30 38 SEQ ID NO: 31 33 SEQ ID NO: 32 39 SEQ ID NO: 33 31 SEQ ID NO: 34 53 SEQ ID NO: 35 51 SEQ ID NO: 36 34 SEQ ID NO: 37 37 SEQ ID NO: 38 39 SEQ ID NO: 39 35 SEQ ID NO: 40 46 SEQ ID NO: 41 51 SEQ ID NO: 42 43 SEQ ID NO: 43 42 SEQ ID NO: 44 55 SEQ ID NO: 45 32 SEQ ID NO: 46 43 SEQ ID NO: 47 41 SEQ ID NO: 48 35 SEQ ID NO: 49 53 SEQ ID NO: 50 96 SEQ ID NO: 51 106 SEQ ID NO: 52 99 SEQ ID NO: 53 105 SEQ ID NO: 54 105 SEQ ID NO: 55 77 SEQ ID NO: 56 106 SEQ ID NO: 57 84 SEQ ID NO: 58 88 SEQ ID NO: 59 86 SEQ ID NO: 60 113 SEQ ID NO: 61 117 SEQ ID NO: 62 101 SEQ ID NO: 63 79 SEQ ID NO: 64 101 SEQ ID NO: 65 99 SEQ ID NO: 66 105 SEQ ID NO: 67 99 SEQ ID NO: 68 92 SEQ ID NO: 69 104 SEQ ID NO: 70 76 SEQ ID NO: 71 97 SEQ ID NO: 72 90 SEQ ID NO: 73 105 SEQ ID NO: 74 108 SEQ ID NO: 75 107 SEQ ID NO: 76 97 SEQ ID NO: 78 109 SEQ ID NO: 79 97 SEQ ID NO: 84 29 SEQ ID NO: 85 23 SEQ ID NO: 86 28 SEQ ID NO: 87 38 SEQ ID NO: 88 38 SEQ ID NO: 89 42 SEQ ID NO: 90 28 SEQ ID NO: 91 40 SEQ ID NO: 92 25 SEQ ID NO: 93 35 SEQ ID NO: 94 31 SEQ ID NO: 95 35 SEQ ID NO: 96 29 SEQ ID NO: 97 38 SEQ ID NO: 98 64 SEQ ID NO: 99 22 SEQ ID NO: 100 22 SEQ ID NO: 101 54 SEQ ID NO: 102 26 SEQ ID NO: 103 41 SEQ ID NO: 104 78 SEQ ID NO: 105 33 SEQ ID NO: 106 29 SEQ ID NO: 107 30 SEQ ID NO: 108 26 SEQ ID NO: 109 33 SEQ ID NO: 110 56 SEQ ID NO: 111 43 SEQ ID NO: 112 25 SEQ ID NO: 113 63 SEQ ID NO: 114 32 SEQ ID NO: 115 72 SEQ ID NO: 116 24 SEQ ID NO: 117 37 SEQ ID NO: 118 25 SEQ ID NO: 119 32 SEQ ID NO: 120 29 SEQ ID NO: 121 72 SEQ ID NO: 122 27 SEQ ID NO: 123 32 SEQ ID NO: 124 27 SEQ ID NO: 125 55 SEQ ID NO: 126 25 SEQ ID NO: 127 23 SEQ ID NO: 128 30 SEQ ID NO: 129 23 SEQ ID NO: 130 51 SEQ ID NO: 131 31 SEQ ID NO: 132 64 SEQ ID NO: 133 67 SEQ ID NO: 134 61 SEQ ID NO: 135 59 SEQ ID NO: 136 63 SEQ ID NO: 137 56 SEQ ID NO: 138 70 SEQ ID NO: 139 62 SEQ ID NO: 140 69 SEQ ID NO: 141 62 SEQ ID NO: 142 26 SEQ ID NO: 143 28 SEQ ID NO: 144 23 SEQ ID NO: 145 29 SEQ ID NO: 146 34 SEQ ID NO: 147 55 SEQ ID NO: 148 26 SEQ ID NO: 149 43 SEQ ID NO: 150 34 SEQ ID NO: 151 40 SEQ ID NO: 152 56 SEQ ID NO: 153 26 SEQ ID NO: 154 81 SEQ ID NO: 155 68 SEQ ID NO: 156 94 SEQ ID NO: 157 69 SEQ ID NO: 158 74 SEQ ID NO: 159 87 SEQ ID NO: 190 100 SEQ ID 100/500 ratio SEQ ID NO: 1 0.6 SEQ ID NO: 5 0.7 SEQ ID NO: 6 0.8 SEQ ID NO: 8 0.7 SEQ ID NO: 22 0.7 SEQ ID NO: 24 0.8 SEQ ID NO: 26 0.7 SEQ ID NO: 27 0.9 SEQ ID NO: 29 0.7 SEQ ID NO: 34 0.7 SEQ ID NO: 36 0.7 SEQ ID NO: 37 0.9 SEQ ID NO: 38 0.8 SEQ ID NO: 41 1.0 SEQ ID NO: 42 0.8 SEQ ID NO: 52 0.8 SEQ ID NO: 53 0.9 SEQ ID NO: 56 0.7 SEQ ID NO: 57 0.9 SEQ ID NO: 60 1.0 SEQ ID NO: 61 1.0 SEQ ID NO: 62 0.8 SEQ ID NO: 63 0.9 SEQ ID NO: 64 0.8 SEQ ID NO: 65 0.8 SEQ ID NO: 66 0.9 SEQ ID NO: 68 0.7 SEQ ID NO: 69 0.7 SEQ ID NO: 75 0.9 SEQ ID NO: 76 0.7 SEQ ID NO: 190 0.9

    Example 4: 100/500-Ratio of TP Variants

    [0728] Cell extract of TP-variants were prepared as described in Example 2. The activity was determined using Assay II with 500 mM glucose and Assay II with, 100 mM glucose, respectively and the 100/500-ratio of each variant calculated. The resulting 100/500-ratios are listed in Table 7. As can be seen, some variants showed, in addition to an improved thermal stability, also a higher 100/500-ratio compared to the wild-type. This is an indication for an improved Km-value for glucose.

    [0729] Table 7: 100/500 ratio of TP variants

    Example 5: Denaturation Profiles of TP Variants

    [0730] 14 TP variants, which had shown high improvements in thermal stability in Example 3, were selected for the determination of their Tm30- and Tm50-values. Denaturation profiles were determined in 50 mM potassium phosphate buffer pH 7 containing 1 M sucrose as described in Example 2. The following Tm30- and Tm-50-values were extrapolated from the denaturation profiles:

    TABLE-US-00007 TABLE 8 Tm30- and Tm50-values of TP-variants in 50 mM potassium phosphate buffer pH 7 containing 1M sucrose SEQ ID Tm30 value Tm50 value SEQ ID NO: 1 49.5 47.5 SEQ ID NO: 44 53.5 52 SEQ ID NO: 50 54.5 53.5 SEQ ID NO: 54 55.5 54.5 SEQ ID NO: 57 55.5 54.5 SEQ ID NO: 58 56 54.5 SEQ ID NO: 62 57.5 56 SEQ ID NO: 64 57.5 56 SEQ ID NO: 65 57.5 56 SEQ ID NO: 71 57 56 SEQ ID NO: 72 58 56.5 SEQ ID NO: 73 58.5 57.5 SEQ ID NO: 74 57.5 56 SEQ ID NO: 78 58 56 SEQ ID NO: 79 57.5 56

    Example 6: S/P-Ratio

    [0731] The wild-type enzyme and 23 TP-variants were selected for the determination of the S/P-ratio. Cell extracts were prepared as described in Example 2. The activity was determined in the direction of trehalose phosphorolysis (Assay I) and trehalose synthesis (Assay II, 500 mM glucose). The ratio between synthesis and phosphorolysis activity (S/P-ratio) was 0.3 for the wild-type enzyme, which means that the enzyme shows higher reaction rates in the direction of trehalose cleavage. The tested TP variants all showed S/P-ratios above 0.3. SEQ ID NO: 42 and SEQ ID NO: 53 showed the highest improvements with an over 3-fold higher S/P-ratio compared to the wild-type enzyme.

    TABLE-US-00008 TABLE 9 S/P-ratio SEQ ID S/P-ratio SEQ ID NO: 1 0.3 SEQ ID NO: 14 0.5 SEQ ID NO: 24 0.4 SEQ ID NO: 27 0.5 SEQ ID NO: 29 0.7 SEQ ID NO: 34 0.6 SEQ ID NO: 41 0.5 SEQ ID NO: 42 0.9 SEQ ID NO: 43 0.9 SEQ ID NO: 44 0.5 SEQ ID NO: 46 0.8 SEQ ID NO: 50 0.6 SEQ ID NO: 54 0.5 SEQ ID NO: 57 0.4 SEQ ID NO: 58 0.5 SEQ ID NO: 62 0.5 SEQ ID NO: 64 0.5 SEQ ID NO: 65 0.5 SEQ ID NO: 71 0.5 SEQ ID NO: 72 0.5 SEQ ID NO: 73 0.6 SEQ ID NO: 74 0.5 SEQ ID NO: 78 0.5 SEQ ID NO: 79 0.5

    Example 7: Process Stability at 45? C.

    [0732] Process stability of the wild-type enzyme and the TP variants SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 78 was determined at 45? C. in 50 mM potassium phosphate buffer pH 7 containing 1M sucrose. Cell extracts were prepared as described in Example 2. Samples were incubated at 45? C. for 16 days. Samples were taken over time and the activity was measured using Assay I. The results are shown in FIG. 2. SEQ ID NO: 1 showed a rapid activity loss within the first 3 hours and a half-life of approx. 1 hour. As expected from the Tm50-values, the new variants showed greatly improved process stability. SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 78 showed the highest improvements with half-lives of approx. 8.8 days. This constitutes an over 200-fold improvement compared to the wild-type enzyme.

    Example 8: Alternative TP Enzymes

    [0733] A possibility to identify alternative wild-type enzymes which possess trehalose phosphorylase activity is to compare known trehalose phosphorylases to sequences deposited in sequence databases, such as GenBank. In order to identify alternative TP enzymes, SEQ ID NO:1 was blasted against the non-redundant database of GenBank (NCBI). Alternative trehalose phosphorylases may be chosen from database sequences which either possess high sequence similarity to SEQ ID: 1, such as the putative trehalose phosphorylase from Hypholoma sublateritium FD-334 SS-4 (GenBank accession: KJA27491.1) or functionally characterized trehalose phosphorylases such as the enzymes from Lentinus sajor-caju (Genbank accession: Q9UV63.1), Grifola frondosa (Genbank accession: 075003.1 or ADM15725.1) or Pleurotus ostreatus (Genbank accession: KDQ33172.1). The sequences of these four enzymes were aligned to SEQ ID NO: 1 in FIG. 3.

    [0734] Variants of alternative wild-type trehalose phosphorylases are created using the methods described in Example 1. The variants contain one or more mutations at the positions corresponding to L114, I118, G357, P383, N225, A304, T323, S556, T564, A649 and L712 in SEQ ID NO:1. Variants are tested for improved thermal stability as described in Example 3. The heat inactivation step is carried out at the temperature at which the corresponding wild-type retains approximately 20% residual activity after incubation for 15 min. It is expected, that the new variants will show similar improvements to the variants in Example 3. Further mutations corresponding to positions L114, I118, G357, P383, N225, A304, T323, F349, Q487, V550, S556, T564, A649 and L712 in SEQ ID NO:1 may be added. It is expected, that the addition of one or more of these mutations will lead to a further improvement in thermal stability.

    TABLE-US-00009 TABLE 10 Residual activity after incubation at 52.5? C. for 15 min residual activity in % after 15 min incubation at SEQ ID 52.5? C. [%] SEQ ID NO: 160 9 SEQ ID NO: 161 14 SEQ ID NO: 162 22 SEQ ID NO: 163 33 SEQ ID NO: 164 26 SEQ ID NO: 165 37 SEQ ID NO: 166 37 SEQ ID NO: 167 20 SEQ ID NO: 168 34 SEQ ID NO: 169 12 SEQ ID NO: 170 15 SEQ ID NO: 171 37 SEQ ID NO: 172 29 SEQ ID NO: 173 42 SEQ ID NO: 174 12 SEQ ID NO: 175 25 SEQ ID NO: 176 57 SEQ ID NO: 177 42 SEQ ID NO: 178 56 SEQ ID NO: 179 38 SEQ ID NO: 180 74 SEQ ID NO: 181 16 SEQ ID NO: 182 28 SEQ ID NO: 183 21 SEQ ID NO: 184 43 SEQ ID NO: 185 58 SEQ ID NO: 186 42 SEQ ID NO: 187 49 SEQ ID NO: 188 70 SEQ ID NO: 189 41