TREHALOSEPHOSPHORYLASE

Description

Field of the Invention

[0001] The present invention is related to a phosphorylase, a method for reacting a glucosyl monosaccharide and alpha-D-glucose 1-phosphate, a method for reacting a glucosyl monosaccharide and alpha-D-glucose 1-phosphate into trehalose and inorganic phosphate, and the use of the phosphorylase for producing trehalose.Background of the Invention

[0002] Phosphorylases are enzymes which catalyze the addition of a phosphate group from an inorganic phosphate to an acceptor molecule. Phosphorylases which catalyse the reversible phosphorolytic cleavage of trehalose are known in the art and referred to as trehalose phosphorylases. Trehalose phosphorylases can be a distinguished based on the mechanism underlying the reaction catalysed by them.

[0003] A first group of trehalose phosphorylases catalyzes phosphorolytic cleavage of trehalose with net retention of the anomeric configuration using inorganic phosphate as a glucosyl acceptor into glucose and alpha-D-glucose 1-phosphate (aG1P) and are therefore classified as retaining phosphorylases. Trehalose phosphorylases of such first group have been assigned EC number EC 2.4.1.231 by the International Union of Biochemistry and Molecular Biology and have been functionally characterized from various eukaryotic fungi.

[0004] A second group of trehalose phosphorylases are inverting trehalose phosphorylases to which EC number EC 2.4.1.64 has been assigned and which are catalyzing phosphorolytic cleavage of trehalose with inversion of configuration into glucose and beta-D-glucose 1-phosphate. These phosphorylases thus have a reaction mechanism different from EC 2.4.1.231 trehalose phosphorylases. Examples of trehalose phosphorylases and related enzymes are, e.g. the glycosyltransferase family 4 protein from Schizophyllum commune (Uniprot ID No. D8PWQ7), the trehalose phosphorylase from Hypsizygus marmoreus (Uniprot ID No. A0A151VW19), the trehalose phosphorylase from Grifola frondosa (Uniprot ID No. E1ACW7), the glycosyltransferase family 4 protein from Ceriporiopsis subvermispora (Uniprot ID No. M2R7V9) and the glycosyltransferase family 4 protein from Phanerochaete carnosa (Uniprot ID No. K5WZK1). Moreover, WO 95 / 11296 describes enzymes of the group EC .14.1. as well as the stereospecific conversion of oxy acids to amino acids and vice versa.

[0005] Specifically this invention relates to certain phosphorylases of EC number EC 2.4.1.231, that are capable of converting, among other reactions, glucose and alpha-D-glucose-1 phosphate ("aG1P") to trehalose, or of phosphorolytic cleavage of trehalose to glucose and aG1P in the presence of inorganic phosphate.

[0006] The industrial use of trehalose phosphorylases results from the fact that the reaction underlying their biochemical characterization, i.e. catalyzing phosphorolytic cleavage of trehalose, is reversible. Because of this, trehalose phosporylases are particularly useful for catalyzing the conversion of glucose and aGIP to trehalose and inorganic phosphate.

[0007] The reaction that is catalyzed by trehalose phosphorylases is reversible (equilibrium reaction) and may undergo substrate or product inhibition, depending on the specific direction of the reaction. In order to obtain industrially relevant amounts of a desired product, trehalose phosphorylases are required that catalyze the conversion of substrates with high specific activity. In addition, other kinetic factors of the trehalose phosphorylases, such as substrate selectivity and K M may play an important role for product yields. Other relevant aspects may include but are not limited to regioselectivity, inhibition by other factors (e.g. crude extract components, substrate contaminants or side products), and recombinant soluble expression in suitable hosts.

[0008] A major shortcoming of wild type trehalose phosphorylases is the rapid loss of enzyme activity in solution even at moderate temperatures between 25°C and 40°C, which significantly limits their application. For industrial applications, high stability over several days at temperatures above 30°C, or even better above 40°C is desirable. Long reaction times with thermally instable enzymes requires larger amounts of enzyme over process time, often realized by repeated addition of enzyme throughout the process. For example, trehalose phosphorylase from Pleurotus ostreatus shows a half-life of approximately 1.3 h at 25°C and of 3 min at approximately 41°C (Schwarz et al., J Biotechnol 129, 140-150 (2007), Han et al., Protein Expression and Purification 30, 194-202 (2003)). The trehalose phosphorylases from Schizophyllum commune and Grifola frondosa are slightly more stable with half-lives of 4.8 h at 30°C and of 1 h at 37°C, respectively (Schwarz et al., J Biotechnol 129, 140-150 (2007)).

[0009] Various strategies were applied in the art to address such shortcomings. For example, addition of trehalose, glycerol and polyethylene glycol (PEG) was shown to increase trehalose phosphorylases stability (Klimacek et al., Biotechnology Techniques 13: 243-248, 1999, Eis et al., Biochem J 341, 385-393 (1999), Schwarz et al. J Biotechnol 129, 140-150 (2007)). The best stabilization was achieved by adding 20 % PEG 4000, which resulted in half-lives of trehalose phosphorylase from Schizophyllum commune at 30°C, 40°C and 50 °C of 4.5 days, 2.2 hours and 6 min, respectively (Klimacek et al., Biotechnology Techniques 13: 243-248, 1999, Eis et al., Biochem J 341, 385-393 (1999)). While the addition of PEG 4000 improved the stability of the enzyme, such improvement is still insufficient for applications at or above 40°C and process times of several hours to several days. The presence of PEG 4000 may furthermore interfere with industrial scale cost structure and down-stream processing requirements for the products obtained by the enzymatic reaction.

[0010] Half-life of trehalose phosphorylases from Schizophyllum commune could also be improved by immobilization to 22 days, 3.3 days and 2 hours at 30°C, 40°C and 50 °C, respectively (Klimacek et al., Biotechnology Techniques 13: 243-248, 1999). Immobilization, however, results in higher enzyme production costs due to expensive carrier and manufacturing costs, and is efficient in terms of industrial applicability only if the enzyme can be recovered and reused for multiple cycles. Additionally, immobilization limits process and down-stream processing options for products of the enzymatic reaction.

[0011] It is therefore not surprising that synthesis reactions employing trehalose phosphorylases are conducted at temperatures ranging from 25°C to 35°C (Schwarz et al., J of Biotechnology 129 140-150 (2007), Saito et al.,Appl Microbiol Biotechnol 50:193-198 (1998), Saito et al. Appl Microbiol Biotechnol 64: 4340-4345 (1998)).

[0012] A further approach for improving performance of enzymes and their suitability for use in industrial processes is enzyme engineering. This technique involves developing variants of a starting enzyme with improved properties (for review, see, for example, S. Lutz, U.T. Bomscheuer, Protein Engineering Handbook, Wiley VCH, Weinheim, 2009). Among others, phosphorylases were improved by enzyme engineering. For example, US 2013-0029384 discloses variants of a sucrose phosphorylase belonging to glycosyl hydrolase family 13 having improved thermal stability. Variants of trehalose phosphorylase of EC number EC 2.4.1.231 have so far been limited to variants for elucidation of the reaction mechanism of said trehalose phosphorylase. Based on such variants, Goedl et al. (Biochem J 397; 491-500; 2006) discovered that substitutions at amino acid positions D379, H403, R507 and K512 of trehalose phosphorylase from Shizophyllum commune led to a reduction in activity. The variants having one of the following single mutations D379N, H403A, R507A and K512A showed reduced activity for trehalose phosphorolysis. Goedl et al. (FEBS J 275; 903-913, 2008) more specifically found that mutations R507A and K512A of trehalose phosphorylase from Shizophyllum commune had an impact on catalytic efficiency of trehalose phosphorylase of the wild type (kcat / K M ).

[0013] As the wild type trehalose phosphorylases available from the prior art are not satisfactory in every respect, and attempts to efficiently improve the industrial applicability of trehalose phosphorylases, as described in the art were not successful, there is a need for trehalose phosphorylases which are advantageous compared to wild type trehalose phosphorylases, in particular with respect to process stability at high temperatures for the industrial production of trehalose.

[0014] Accordingly, the problem underlying the present invention is the provision of a trehalose phosphorylase which is suitable for use in industrial production of trehalose.

[0015] A further problem underlying the present invention is the provision of a trehalose phosphorylase which is thermally stable and shows a residual activity ranging from 30% to 90 % or from 55 % to 100 % after incubation at 52°C for 15 minutes without the necessity of immobilizing the enzyme or adding any stabilizing agent (e.g., PEG or glycerol) apart from sucrose.

[0016] Another problem underlying the present invention is the provision of a trehalose phosphorylase which is thermally stable and shows a half-life at 45 °C from 3 hours to 9 days or more without the necessity of immobilizing the enzyme or adding any stabilizing agent (e.g., PEG or glycerol) apart from sucrose.

[0017] Another problem underlying the present invention is the provision of a trehalose phosphorylase which has a 100 / 500-ratio ranging between 0.65 and 1.0.

[0018] A still further problem is the provision of means which allow converting glucose and alpha-D-glucose 1-phosphate into trehalose and inorganic phosphate.Summary of the Invention

[0019] These and other problems are solved by the attached independent claims. Preferred embodiments may be taken from the attached dependent claims.

[0020] More specifically, these and other problems are solved in a first aspect, which is also the first embodiment of the first aspect, by a trehalose phosphorylase, comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical 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, wherein when the amino acid substitution comprises a substitution at amino acid position 649, then said amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, preferably 649E; and wherein when the amino acid substitution comprises a substitution at amino acid position 10, then said amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and, or V10H , preferably V10R, 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]; or wherein the trehalose phosphorylase is characterized by a thermal stability after incubation at 52.5°C for 15 minutes defined by a residual activity of 15% to 100%, or wherein the trehalose phosphorylase compared to the trehalose phosphorylase of SEQ ID NO:1, is characterized by an increase in thermal stability, whereby the increase in thermal stability is an increase of the Tm30-value of at least 2 °C up to 40.5°C, or an increase of the Tm50-value of at least 2 °C up to 42.5°C, or an improved process stability, characterized by an increased half-life at 45 °C of at least 3-fold up to 216-fold or more, or at least 24-fold up to 216-fold or more. Preferably, the one or more amino acid positions is / are individually and independently 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. Even more preferably, the one or more amino acid positions is / are individually and independently 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. As surprisingly found in the context of the present invention, the mutation of one or more of these positions resulted in improved characteristics in comparison to the wild type sequence of SEQ ID NO: 1, for example in an increased thermal stability of the enzyme leading to a residual activity after 15 min incubation at 52°C of at least 22 % in comparison to 19 % of trehalose phosphorylase encoded by the wild type amino acid sequence of SEQ ID NO: 1.

[0021] In another embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions of L712, P383, V10, L114, I118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO: 1.

[0022] In another embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions 712, 383, 10, 114, 118, 220, 225, 304, 318, 323, 349, 357, 481, 487, 488, 506, 511, 550, 556, 564, 590, 649, 703 and 705 of SEQ ID NO: 1.

[0023] In another embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, Y220, N225, A304, P318, T323, F349, G357, E481, Q487, K488, A506, A511, V550, S556, T564, D590, A649, A703 and K705 of SEQ ID NO: 1.

[0024] In another embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

[0025] In another embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions L712, P383, L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1.

[0026] In another preferred embodiment of the first aspect, 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.

[0027] In another preferred embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions 383, 225, 304, 487, 556, 590 and 705 of SEQ ID NO: 1.

[0028] In another preferred embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions 383, 225, 304, 487, 556, and 590 of SEQ ID NO: 1.

[0029] In another preferred embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions 383, 556, and 590 of SEQ ID NO: 1.

[0030] In another preferred embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions 383 and 590 of SEQ ID NO: 1.

[0031] In another preferred embodiment of the first aspect, the one or more amino acid positions isthe amino acid position 383 of SEQ ID NO: 1.

[0032] In another preferred embodiment of the first aspect, the one or more amino acid positions is / are selected from the group consisting of amino acid positions P383, N225, A304, T323, Q487, V550, S556, T564, D590 and N705 of SEQ ID NO: 1.

[0033] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid sequence according to any one of SEQ ID NO: 2, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, preferably according to any one of SEQ ID NO: 2, 84, 85, 87, 88, 89, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, more preferably according to any one of SEQ ID NO: 84, 85, 87, 88, 89, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, equally preferably according to any one of SEQ ID NO: 87, 89, 91, 97, 98, 99, 100, 101, 102, 103, 104, 109, even more preferably according to any one of SEQ ID NO: 87, 91, 97, 98, 99, 100, 101, 102, 103, 104, 109, and most preferably according to any one of SEQ ID NO: 87, 97, 98, 99, 100, 101, 102, 103, 104.

[0034] In another embodiment of the first aspect the one or more amino acid positions is two or more amino acid positions.

[0035] In another embodiment of the first aspect, the two or more amino acid positions comprises a pair of two amino acid positions, wherein the pair of two amino acid positions is selected from the group consisting of 10 and 114, 10 and 118, 10 and 220, 10 and 225, 10 and 304, 10 and 323, 10 and 349, 10 and 357, 10 and 383, 10 and 487, 10 and 506, 10 and 550, 10 and 556, 10 and 564, 10 and 590, 10 and 649, 10 and 705, 10 and 712, 114 and 118, 114 and 220, 114 and 225, 114 and 304, 114 and 323, 114 and 349, 114 and 357, 114 and 383, 114 and 487, 114 and 506, 114 and 550, 114 and 556, 114 and 564, 114 and 590, 114 and 649, 114 and 705, 114 and 712, 118 and 220, 118 and 225, 118 and 304, 118 and 323, 118 and 349, 118 and 357, 118 and 383, 118 and 487, 118 and 506, 118 and 550, 118 and 556, 118 and 564, 118 and 590, 118 and 649, L118 and 705, 118 and 712, 220 and 225, 220 and 304, 220 and 323, 220 and 349, 220 and 357, 220 and 383, 220 and 487, 220 and 506, 220 and 550, 220 and 556, 220 and 564, 220 and 590, 220 and 649, 220 and 705, 220 and 712, 225 and 304, 225 and 323, 225and 349, 225 and 357, 225 and 383, 225 and 487, 225 and 506, 225 and 550, 225 and 556, 225 and 564, 225 and 590, 225 and 649, 225 and 705, 225 and 712, 304 and 323, 304 and 349, 304 and 357, 304 and 383, 304 and 487, 304 and 506, 304 and 550, 304 and 556, 304 and 564, 304 and 590, 304 and 649, 304 and 705, 304 and 712, 323 and 349, 323 and 357, 323 and 383, 323 and 487, 323 and 506, 323 and 550, 323 and 556, 323 and 564, 323 and 590, 323 and 649, 323 and 705, 323 and 712, 349 and 357, 349 and 383, 349 and 487, 349 and 506, 349 and 550, 349 and 556, 349 and 564, 349 and 590, 349 and 649, 349 and 705, 349 and 712, 357 and 383, 357 and 487, 357 and 506, 357 and 550, 357 and 556, 357 and 564, 357 and 590, 357 and 649, 357 and 705, 357 and 712, 383 and 487, 383 and 506, 383 and 550, 383 and 556, 383 and 564, 383 and 590, 383 and 649, 383 and 705, 383 and 712, 487 and 506, 487 and 550, 487 and 556, 487 and 564, 487 and 590, 487 and 649, 487 and 705, 487 and 712, 506 and 550, 506 and 556, 506 and 564, 506 and 590, 506 and 649, 506 and 705, 506 and 712, 550 and 556, 550 and 564, 550 and 590, 550 and 649, 550 and 705, 550 and 712, 556 and 564, 556 and 590, 556 and 649, 556 and 705, 556 and 712, 564 and 590, 564 and 649, 564 and 705, 564 and 712, 590 and 649, 590 and 705, 590 and 712, 649 and 712, 649 and 705, and 705 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of V10 and L114, V10 and I118, V10 and Y220, V10 and N225, V10 and A304, V10 and T323, V10 and F349, V10 and G357, V10 and P383, V10 and Q487, V10 and A506, V10 and V550, V10 and S556, V10 and T564, V10 and D590, V10 and A649, V10 and K705, V10 and L712, L114 and I118, L114 and Y220, L114 and N225, L114 and A304, L114 and T323, V10 and F349, L114 and G357, L114 and P383, L114 and Q487, L114 and A506, L114 and V550, L114 and S556, L114 and T564, L114 and D590, L114 and A649, L114 and K705, L114 and L712, I118 and Y220, I118 and N225, I118 and A304, I118 and T323, I118 and F349, I118 and G357, I118 and P383, I118 and Q487, I118 and A506, I118 and V550, I118 and S556, I118 and T564, I118 and D590, 118 and A649, L118 and K705, I118 and L712, Y220 and N225, Y220 and A304, Y220 and T323, Y220 and F349, Y220 and G357, Y220 and P383, Y220 and Q487, Y220 and A506, Y220 and V550, Y220 and S556, Y220 and T564, Y220 and D590, Y220 and A649, Y220 and K705, Y220 and L712, N225 and A304, N225 and T323, N225 and F349, N225 and G357, N225 and P383, N225 and Q487, N225 and A506, N225 and V550, N225 and S556, N225 and T564, N225 and D590, N225 and A649, N225 and K705, N225 and L712, A304 and T323, A304 and F349, A304 and G357, A304 and P383, A304 and Q487, A304 and A506, A304 and V550, A304 and S556, A304 and T564, A304 and D590, A304 and A649, A304 and K705, A304 and L712, T323 and F349, T323 and G357, T323 and P383, T323 and Q487, T323 and A506, T323 and V550, T323 and S556, T323 and T564, T323 and D590, T323 and A649, T323 and K705, T323 and L712, F349 and G357, F349 and P383, F349 and Q487, F349 and A506, F349 and V550, F349 and S556, F349 and T564, F349 and D590, F349 and A649, F349 and K705, F349 and L712, G357 and P383, G357 and Q487, G357 and A506, G357 and V550, G357 and S556, G357 and T564, G357 and D590, G357 and A649, G357 and K705, G357 and L712, P383 and Q487, P383 and A506, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and K705, P383 and L712, Q487 and A506, Q487 and V550, Q487 and S556, Q487 and T564, Q487 and D590, Q487 and A649, Q487 and K705, Q487 and L712, A506 and V550, A506 and S556, A506 and T564, A506 and D590, A506 and A649, A506 and K705, A506 and L712, V550 and S556, V550 and T564, V550 and D590, V550 and A649, V550 and K705, V550 and L712, S556 and T564, S556 and D590, S556 and A649, S556 and K705, S556 and L712, T564 and D590, T564 and A649, T564 and K705, T564 and L712, D590 and A649, D590 and K705, D590 and L712, A649 and L712, A649 and K705, and K705 and L712.

[0036] In another embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 10 and 114, 10 and 220, 10 and 383, 10 and 506, 10 and 705, 10 and 712, 114 and 118, 114 and 220, 114 and 225, 114 and 304, 114 and 225, 114 and 349, 114 and 357, 114 and 383, 114 and 487, 114 and 506, 114 and 550, 114 and 556, 114 and 564, 114 and 590, 114 and 649, 114 and 705, 114 and 712, 118 and 225, 118 and 304, 118 and 323, 118 and 349, 118 and 357, 118 and 383, 118 and 487, 118 and 550, 118 and 556, 118 and 564, 118 and 590, 118 and 649, 118 and 712, 220 and 383, 220 and 506, 220 and 705, 220 and 712, 225 and 304, 225 and 323, 225 and 349, 225 and 357, 225 and 383, 225 and 487, 225 and 550, 225 and 556, 225 and 564, 225 and 590, 225 and 649, 225 and 712, 304 and 323, 304 and 349, 304 and 357, 304 and 383, 304 and 487, 304 and 550, 304 and 556, 304 and 564, 304 and 590, 304 and 649, 304 and 712, 323 and 349, 323 and 357, 323 and 383, 323 and 487, 323 and 550, 323 and 556, 323 and 564, 323 and 590, 323 and 649, 323 and 712, 349 and 357, 349 and 383, 349 and 487, 349 and 550, 349 and 556, 349 and 564, 349 and 590, 349 and 649, 349 and 712, 357 and 383, 357 and 487, 357 and 550, 357 and 556, 357 and 564, 357 and 590, 357 and 649, 357 and 705, 357 and 712, 383 and 487, 383 and 506, 383 and 550, 383 and 556, 383 and 564, 383 and 590, 383 and 649, 383 and 705, 383 and 712, 487 and 550, 487 and 556, 487 and 564, 487 and 590, 487 and 649, 487 and 712, 506 and 705, 506 and 712, 550 and 556, 550 and 564, 550 and 590, 550 and 649, 550 and 712, 556 and 564, 556 and 590, 556 and 649, 556 and 712, 564 and 590, 564 and 649, 564 and 712, 590 and 649, 590 and 712, 649 and 712, 705 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of V10 and L114, V10 and Y220, V10 and P383, V10 and A506, V10 and K705, V10 and L712, L114 and I118, L114 and Y220, L114 and N225, L114 and A304, L114 and T323, L114 and F349, L114 and G357, L114 and P383, L114 and Q487, L114 and A506, L114 and V550, L114 and S556, L114 and T564, L114 and D590, L114 and A649, L114 and K705, L114 and L712, I118 and N225, I118 and A304, I118 and T323, I118 and F349, I118 and G357, I118 and P383, I118 and Q487, I118 and V550, I118 and S556, I118 and T564, I118 and D590, 118 and A649, I118 and L712, Y220 and P383, Y220 and A506, Y220 and K705, Y220 and L712, N225 and A304, N225 and T323, N225 and F349, N225 and G357, N225 and P383, N225 and Q487, N225 and V550, N225 and S556, N225 and T564, N225 and D590, N225 and A649, N225 and L712, A304 and T323, A304 and F349, A304 and G357, A304 and P383, A304 and Q487, A304 and V550, A304 and S556, A304 and T564, A304 and D590, A304 and A649, A304 and L712, T323 and F349, T323 and G357, T323 and P383, T323 and Q487, T323 and V550, T323 and S556, T323 and T564, T323 and D590, T323 and A649, T323 and L712, F349 and G357, F349 and P383, F349 and Q487, F349 and V550, F349 and S556, F349 and T564, F349 and D590, F349 and A649, F349 and L712, G357 and P383, G357 and Q487, G357 and V550, G357 and S556, G357 and T564, G357 and D590, G357 and A649, G357 and K705, G357 and L712, P383 and Q487, P383 and A506, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and K705, P383 and L712, Q487 and V550, Q487 and S556, Q487 and T564, Q487 and D590, Q487 and A649, Q487 and L712, A506 and K705, A506 and L712, V550 and S556, V550 and T564, V550 and D590, V550 and A649, V550 and L712, S556 and T564, S556 and D590, S556 and A649, S556 and L712, T564 and D590, T564 and A649, T564 and L712, D590 and A649, D590 and L712, A649 and L712, K705 and L712.

[0037] In another embodiment of the first aspect, the pair of two amino acid positions is 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, and preferably the pair of two amino acid positions is selected from the group consisting of V10 and L114, V10 and L712, L114 and I118, L114 and A304, L114 and G357, L114 and P383, L114 and D590, L114 and L712, I118 and A304, I118 and G357, I118 and P383, I118 and S556, I118 and T564, I118 and D590, I118 and L712, N225 and A304, N225 and P383, N225 and Q487, N225 and V550, N225 and S556, N225 and D590, A304 and T323, A304 and G357, A304 and P383, A304 and Q487, A304 and S556, A304 and T564, A304 and D590, A304 and L712, T323 and G357, T323 and Q487, T323 and S556, T323 and T564, T323 and D590, T323 and A649, F349 and P383, F349 and D590, G357 and P383, G357 and D590, G357 and K705, G357 and L712, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and L712, Q487 and T564, Q487 and D590, Q487 and A649, V550 and D590, S556 and T564, S556 and D590, S556 and A649, T564 and D590, T564 and L712, D590 and A649, D590 and L712, and A649 and L712.

[0038] In another embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 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, and preferably the pair of two amino acid positions is selected from the group consisting of L114 and I118, L114 and A304, L114 and G357, L114 and P383, L114 and D590, L114 and L712, I118 and A304, I118 and G357, I118 and P383, I118 and S556, I118 and T564, I118 and D590, I118 and L712, N225 and A304, N225 and P383, N225 and Q487, N225 and V550, N225 and S556, N225 and D590, A304 and T323, A304 and G357, A304 and P383, A304 and Q487, A304 and S556, A304 and T564, A304 and D590, A304 and L712, T323 and G357, T323 and Q487, T323 and S556, T323 and T564, T323 and D590, T323 and A649, F349 and P383, F349 and D590, G357 and P383, G357 and D590, G357 and K705, G357 and L712, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and L712, Q487 and T564, Q487 and D590, Q487 and A649, V550 and D590, S556 and T564, S556 and D590, S556 and A649, T564 and D590, T564 and L712, D590 and A649, D590 and L712, and A649 and L712.

[0039] In another preferred embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 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, 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 712, 487 and 564, 487 and 590, 550 and 590, 556 and 564, 556 and 590, 564 and 590, 564 and 712, and 590 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of L114 and I118, L114 and A304, L114 and G357, L114 and P383, L114 and D590, L114 and L712, I118 and A304, I118 and G357, I118 and P383, I118 and S556, I118 and T564, I118 and D590, I118 and L712, N225 and A304, N225 and P383, N225 and Q487, N225 and V550, N225 and S556, N225 and D590, A304 and T323, A304 and G357, A304 and P383, A304 and Q487, A304 and S556, A304 and T564, A304 and D590, A304 and L712, T323 and G357, T323 and Q487, T323 and S556, T323 and T564, T323 and D590, F349 and P383, F349 and D590, G357 and P383, G357 and D590, G357 and K705, G357 and L712, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and L712, Q487 and T564, Q487 and D590, V550 and D590, S556 and T564, S556 and D590, T564 and D590, T564 and L712, and D590 and L712.

[0040] In another embodiment of the first aspect, the pair of two amino acid positions is 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, and preferably the pair of two amino acid positions is selected from the group consisting of I118 and P383, I118 and S556, I118 and T564, I118 and D590, N225 and A304, N225 and P383, N225 and Q487, N225 and V550, N225 and S556, N225 and D590, A304 and T323, A304 and P383, A304 and Q487, A304 and S556, A304 and T564, A304 and D590, T323 and G357, T323 and Q487, T323 and S556, T323 and T564, T323 and D590, T323 and A649, F349 and P383, F349 and D590, G357 and P383, G357 and D590, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and L712, Q487 and T564, Q487 and D590, Q487 and A649, V550 and D590, S556 and T564, S556 and D590, S556 and A649, T564 and D590, T564 and L712, D590 and A649, D590 and L712, A649 and L712, and K705 and L712.

[0041] In another preferred embodiment of the first aspect, the pair of two amino acid positions is 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, 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 712, 487 and 564, 487 and 590, 550 and 590, 556 and 564, 556 and 590, 564 and 590, 564 and 712, 590 and 712, and 705 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of I118 and P383, I118 and S556, I118 and T564, I118 and D590, N225 and A304, N225 and P383, N225 and Q487, N225 and V550, N225 and S556, N225 and D590, A304 and T323, A304 and P383, A304 and Q487, A304 and S556, A304 and T564, A304 and D590, T323 and G357, T323 and Q487, T323 and S556, T323 and T564, T323 and D590, F349 and P383, F349 and D590, G357 and P383, G357 and D590, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and L712, Q487 and T564, Q487 and D590, V550 and D590, S556 and T564, S556 and D590, T564 and D590, T564 and L712, D590 and L712, and K705 and L712.

[0042] In another embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 114 and 118, 114 and 383, 114 and 712, 118 and 383, 118 and 556, 118 and 590, 118 and 712, 225 and 304, 225 and 383, 225 and 550, 225 and 590, 304 and 383, 304 and 556, 304 and 590, 323 and 556, 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, 556 and 564, 556 and 590, 564 and 590, 564 and 712, 590 and 649, and 590 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of L114 and I118, L114 and P383, L114 and L712, I118 and P383, I118 and S556, I118 and D590, I118 and L712, N225 and A304, N225 and P383, N225 and V550, N225 and D590, A304 and P383, A304 and S556, A304 and D590, T323 and S556, T323 and D590, T323 and A649, F349 and P383, F349 and D590, G357 and P383, G357 and D590, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and L712, S556 and T564, S556 and D590, T564 and D590, T564 and L712, D590 and A649, and D590 and L712.

[0043] In another preferred embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 114 and 118, 114 and 383, 114 and 712, 118 and 383, 118 and 556, 118 and 590, 118 and 712, 225 and 304, 225 and 383, 225 and 550, 225 and 590, 304 and 383, 304 and 556, 304 and 590, 323 and 556, 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 712, 556 and 564, 556 and 590, 564 and 590, 564 and 712, and 590 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of L114 and I118, L114 and P383, L114 and L712, I118 and P383, I118 and S556, I118 and D590, I118 and L712, N225 and A304, N225 and P383, N225 and V550, N225 and D590, A304 and P383, A304 and S556, A304 and D590, T323 and S556, T323 and D590, F349 and P383, F349 and D590, G357 and P383, G357 and D590, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and L712, S556 and T564, S556 and D590, T564 and D590, T564 and L712, and D590 and L712.

[0044] In another embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 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 590, 118 and 712, 225 and 383, 304 and 357, 304 and 383, 304 and 590, 304 and 712, 323 and 590, 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, 556 and 590, 564 and 590, 590 and 649, and 590 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of L114 and I118, L114 and A304, L114 and G357, L114 and P383, L114 and D590, L114 and L712, I118 and A304, I118 and G357, I118 and P383, I118 and S556, I118 and D590, I118 and L712, N225 and P383, A304 and G357, A304 and P383, A304 and D590, A304 and L712, T323 and D590, F349 and P383, F349 and D590, G357 and P383, G357 and D590, G357 and K705, G357 and L712, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and L712, S556 and D590, T564 and D590, D590 and A649, and D590 and L712.

[0045] In another preferred embodiment of the first aspect, the pair of two amino acid positions is selected from the group consisting of 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 590, 118 and 712, 225 and 383, 304 and 357, 304 and 383, 304 and 590, 304 and 712, 323 and 590, 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 712, 556 and 590, 564 and 590, and 590 and 712, and preferably the pair of two amino acid positions is selected from the group consisting of L114 and I118, L114 and A304, L114 and G357, L114 and P383, L114 and D590, L114 and L712, I118 and A304, I118 and G357, I118 and P383, I118 and S556, I118 and D590, I118 and L712, N225 and P383, A304 and G357, A304 and P383, A304 and D590, A304 and L712, T323 and D590, F349 and P383, F349 and D590, G357 and P383, G357 and D590, G357 and K705, G357 and L712, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and L712, S556 and D590, T564 and D590, and D590 and L712.

[0046] In another preferred embodiment of the first aspect, the pair of two amino acid positions is 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 and preferably the pair of two amino acid positions is selected from the group consisting of I118 and P383, I118 and D590, N225 and P383, N225 and D590, A304 and P383, A304 and D590, T323 and D590, F349 and P383, F349 and D590, G357 and P383, G357 and D590, P383 and Q487, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and L712, Q487 and D590, V550 and D590, S556 and D590, T564 and D590, D590 and A649, D590 and L712.

[0047] In another embodiment of the first aspect, in addition to the substitution at the two amino acid positions, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at at least one or more additional amino acid positions, wherein the one or more additional amino acid position is 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, preferably, in addition to the substitution at the two amino acid positions, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at at least one or more additional amino acid positions, wherein the one or more additional amino acid position is independently and individually 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.

[0048] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO:1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO:1.

[0049] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions 712, 383, 10, 114, 118, 220, 225, 304, 318, 323, 349, 357, 481, 487, 488, 506, 511, 550, 556, 564, 590, 649, 703 and 705 of SEQ ID NO: 1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions 712, 383, 10, 114, 118, 220, 225, 304, 318, 323, 349, 357, 481, 487, 488, 506, 511, 550, 556, 564, 590, 649, 703 and 705 of SEQ ID NO:1.

[0050] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, Y220, N225, A304, P318, T323, F349, G357, E481, Q487, K488, A506, A511, V550, S556, T564, D590, A649, A703 and K705, of SEQ ID NO:1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, Y220, N225, A304, P318, T323, F349, G357, E481, Q487, K488, A506, A511, V550, S556, T564, D590, A649, A703 and K705 of SEQ ID NO:1.

[0051] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions 712, 383, 10, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO:1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions 712, 383, 10, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO:1.

[0052] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO:1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions L712, P383, V10, L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO:1.

[0053] In another embodiment of the first aspect, wherein the one or more additional amino acid position is selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

[0054] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions L712, P383, L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions L712, P383, L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1.

[0055] In another embodiment of the first aspect, the one or more additional amino acid position selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 357, 487, 556, and 590 of SEQ ID NO: 1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 357, 487, 556, and 590 of SEQ ID NO: 1.

[0056] In another embodiment of the first aspect, the one or more additional amino acid position is selected from the group consisting of amino acid positions L712, P383, L114, I118, N225, A304, G357, Q487, S556, and D590 of SEQ ID NO: 1, preferably the one or more additional amino acid position is independently and individually selected from the group consisting of amino acid positions L712, P383, L114, I118, N225, A304, G357, Q487, S556, and D590 of SEQ ID NO: 1.

[0057] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at amino acid positions 383, and 590 of SEQ ID NO: 1, preferably at amino acid positions P383, and D590 of SEQ ID NO: 1, and wherein the amino acid sequence of the trehalose phosphorylase comprises further amino acid substitutions at the following amino acid positions of SEQ ID NO: 1 (i) 114, 118, 304, 357, and 712, preferably L114, I118, A304, G357, and L712, or (ii) 114, 118, 304, 357, 550, 556, and 712, preferably L114, I118, A304, G357, V550, S556, and L712, or (iii) 114, 118, 225, 304, 357, 487, 564, and 712, preferably L114, I118, N225, A304, G357, Q487, T564, and L712, or (iv) 114, 118, 304, 323, 357, 550, 564, and 712, preferably L114, I118, A304, T323, G357, V550, T564, and L712, or (v) 114, 118, 225, 304, 323, 357, 487, and 712, preferably L114, I118, N225, A304, T323, G357, Q487, and L712, or (vi) 114, 118, 225, 304, 357, 487, 550, 556, and 712, preferably L114, I118, N225, A304, G357, Q487, V550, S556, and L712, or (vii) 114, 118, 225, 304, 357, 487, 550, 564, and 712, preferably L114, I118, N225, A304, G357, Q487, V550, T564, and L712, or (viii) 114, 118, 225, 304, 357, 550, 556, 564, and 712, preferably L114, I118, N225, A304, G357, V550, S556, T564, and L712, or (ix) 114, 118, 225, 304, 357, 550, 564, and 712, preferably L114, I118, N225, A304, G357, V550, T564, and L712, or (x) 114, 118, 304, 323, 357, 550, 556, 564, and 712, preferably L114, I118, A304, T323, G357, V550, S556, T564, and L712, or (xi) 114, 118, 304, 323, 357, 487, 550, 649, and 712, preferably L114, I118, A304, T323, G357, Q487, V550, A649, and L712, or (xii) 114, 118, 225, 304, 357, 487, 550, 556, 564, and 712, preferably L114, I118, N225, A304, G357, Q487, V550, S556, T564, and L712, or (xiii) 114, 118, 225, 304, 357, 550, 556, 564, 649, and 712, preferably L114, I118, N225, A304, G357, V550, S556, T564, A649, and L712, or (xiv) 114, 118, 225, 304, 323, 357, 487, 550, 564, and 712, preferably L114, I118, N225, A304, T323, G357, Q487, V550, T564, and L712, or (xv) 114, 118, 225, 304, 323, 357, 487, 550, 649, and 712, preferably L114, I118, N225, A304, T323, G357, Q487, V550, A649, and L712, or (xvi) 114, 118, 225, 304, 323, 357, 487, 550, 556, and 712, preferably L114, I118, N225, A304, T323, G357, Q487, V550, S556, and L712, or (xvii) 114, 118, 225, 304, 323, 357, 550, 556, 564, and 712, preferably L114, I118, N225, A304, T323, G357, V550, S556, T564, and L712, or (xviii) 114, 118, 304, 323, 349, 357, 487, 550, 649, and 712, preferably L114, I118, A304, T323, F349, G357, Q487, V550, A649, and L712, or (xix) 114, 118, 304, 323, 357, 487, 550, 556, 564, and 712, preferably L114, I118, A304, T323, G357, Q487, V550, S556, T564, and L712, or (xx) 114, 118, 304, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, A304, G357, Q487, V550, S556, T564, A649, and L712, or (xxi) 114, 118, 225, 304, 323, 349, 357, 550, 556, 649, and 712, preferably L114, I118, N225, A304, T323, F349, G357, V550, S556, A649, and L712, or (xxii) 114, 118, 225, 304, 323, 357, 487, 550, 556, 649, and 712, preferably L114, I118, N225, A304, T323, G357, Q487, V550, S556, A649, and L712, or (xxiii) 114, 118, 225, 304, 349, 357, 487, 550, 556, 564, and 712, preferably L114, I118, N225, A304, F349, G357, Q487, V550, S556, T564, and L712, or (xxiv) 114, 118, 225, 304, 349, 357, 487, 550, 564, 649, and 712, preferably L114, I118, N225, A304, F349, G357, Q487, V550, T564, A649, and L712, or (xxv) 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, A649, and L712, or (xxvi) 114, 118, 225, 304, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, N225, A304, G357, Q487, V550, S556, T564, A649, and L712, or (xxvii) 114, 118, 304, 323, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, A304, T323, G357, Q487, V550, S556, T564, A649, and L712, or (xxviii) 114, 118, 225, 304, 323, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, N225, A304, T323, G357, Q487, V550, S556, T564, A649, and L712, or (xxix) 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, A649, and L712, or (xxx) 114, 225, 304, 323, 357, 487, 550, 556, 564, 649, and 712, preferably L114, N225, A304, T323, G357, Q487, V550, S556, T564, A649, and L712, or (xxxi) 114, 118, 225, 323, 357, 487, 550, 556, 564, 649, and 712, preferably L114, I118, N225, T323, G357, Q487, V550, S556, T564, A649, and L712, or (xxxii) 114, 118, 225, 304, 323, 349, 487, 550, 556, 564, 649, and 712, preferably L114, I118, N225, A304, T323, F349, Q487, V550, S556, T564, A649, and L712, or (xxxiii) 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, and 649, preferably L114, I118, N225, A304, T323, F349, G357, Q487, V550, S556, T564, and A649.

[0058] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises one or more additional amino acid substitution at amino acid positions selected from the group consisting of positions 712, 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, 649, 667, 703 and 705 of SEQ ID NO: 1, preferably selected from the group consisting of positions L712, V10, L114, I118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, A649, R667, A703 and K705 of SEQ ID NO: 1.

[0059] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 114, 118, 225, 304, 357, 487, 556, and 590.

[0060] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of L712, P383, L114, I118, N225, A304, G357, Q487, S556, and D590.

[0061] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 114, 118, 304 and 357.

[0062] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of L712, P383, L114, I118, A304 and G357.

[0063] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at four amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 114, 118, 304 and 357.

[0064] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at four amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of L712, P383, L114, I118, A304 and G357.

[0065] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at five amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 114, 118, 304 and 357.

[0066] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at five amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of L712, P383, L114, I118, A304 and G357.

[0067] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 114, 118, 304 and 357.

[0068] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of L712, P383, L114, I118, A304 and G357.

[0069] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 118, 225, 304, 349, 357, 487, 556, 564, and 590.

[0070] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 712, 383, 225, 304, 349, 556, and 590.

[0071] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 383, 225, 304, 556, and 590.

[0072] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of 383, 556, and 590.

[0073] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at three amino acid positions of SEQ ID NO: 1, preferably at four amino acid positions of SEQ ID NO: 1, more preferably at five amino acid positions of SEQ ID NO: 1, and most preferably at six amino acid positions of SEQ ID NO: 1, wherein each and any of the amino acid positions of SEQ ID NO: 1 is selected from the group consisting of L712, P383, I118, N225, A304, F349, G357, Q487, S556, T564, and D590.

[0074] In another embodiment or of any other of the previous embodiments of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises (a) 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; or (b) 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; or (c) 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; or (d) an amino acid substitution at the three amino acid positions 383, 556, and 590 of SEQ ID NO: 1.

[0075] In another embodiment or of any other of the previous embodiments of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises (a) 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, and preferably at the four amino acid positions L712, P383, L114 and I118 of SEQ ID NO: 1; and / or P383, Q487, S556, and D590 of SEQ ID NO: 1; and / or P383, N225, S556, and D590 of SEQ ID NO: 1; or (b) an amino acid substitution at the five amino acid positions 712, 383, 114, I118 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, preferably at five amino acid positions L712, P383, L114, I118 and A304 of SEQ ID NO: 1; and / or L712, P383, L114, I118 and G357 of SEQ ID NO: 1; and / or P383, N225, A304, S556, and D590 of SEQ ID NO: 1; and / or P383, N225, Q487, S556, and D590 of SEQ ID NO: 1; or (c) an amino acid substitution 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, preferably at the six amino acid positions L712, P383, L114, I118, A304 and G357 of SEQ ID NO: 1; and / or P383, N225, A304, Q487, S556, and D590 of SEQ ID NO: 1, or (d) an amino acid substitution at the three amino acid positions 383, 556, and 590, preferably at the three amino acid positions P383, S556, and D590 of SEQ ID NO: 1.

[0076] In another embodiment or of any other of the previous embodiments of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid sequence of SEQ ID NO: 14.

[0077] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase consists of an amino acid sequence of SEQ ID NO: 14.

[0078] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more further amino acid positions, wherein the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions 10, 114, 118, 192, 197, 220, 225, 304, 306, 318, 323, 339, 349, 357, 383, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 590, 649, 667, 703, 705 and 712 of SEQ ID NO: 1.

[0079] In another preferred embodiment of this aspect, the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions V10, L114, I118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703, K705, and L712 of SEQ ID NO: 1.

[0080] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more further amino acid positions, wherein the one or more further amino acid positions are selected from the group consisting of amino acid positions 10, 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 the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions 10, 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.

[0081] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions V10, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions V10, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO: 1.

[0082] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions 10, 220, 225, 304, 318, 323, 349, 357, 481, 487, 488, 506, 511, 550, 556, 564, 590, 649, 703 and 705 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions 10, 220, 225, 304, 318, 323, 349, 357, 481, 487, 488, 506, 511, 550, 556, 564, 590, 649, 703 and 705 of SEQ ID NO: 1.

[0083] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions V10, Y220, N225, A304, P318, T323, F349, G357, E481, Q487, K488, A506, A511, V550, S556, T564, D590, A649, A703 and K705 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions V10, Y220, N225, A304, P318, T323, F349, G357, E481, Q487, K488, A506, A511, V550, S556, T564, D590, A649, A703 and K705 of SEQ ID NO: 1.

[0084] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions 10, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions 10, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

[0085] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions V10, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions V10, N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1.

[0086] In another embodiment of the first aspect, wherein the one or more further amino acid positions are selected from the group consisting of amino acid positions 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

[0087] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions N225, A304, T323, F349, G357, Q487, V550, S556, T564, D590 and A649 of SEQ ID NO: 1.

[0088] In another embodiment of the first aspect, wherein the one or more further amino acid positions are selected from the group consisting of amino acid positions 304, 357 and 590 of SEQ ID NO: 1, preferably wherein the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions 304, 357 and 590 of SEQ ID NO: 1.

[0089] In another embodiment of the first aspect, the one or more further amino acid positions are selected from the group consisting of amino acid positions A304, G357 and D590 of SEQ ID NO: 1, preferably the one or more further amino acid positions are independently and individually selected from the group consisting of amino acid positions A304, G357 and D590 of SEQ ID NO: 1.

[0090] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid sequence according to SEQ ID NO: 44.

[0091] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase consists of an amino acid sequence according to SEQ ID NO: 44.

[0092] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more still further amino acid positions, wherein the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions 10, 114, 118, 192, 197, 220, 225, 304, 306, 318, 323, 339, 349, 357, 383, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 590, 649, 667, 703, 705 and 712 of SEQ ID NO: 1.

[0093] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more still further amino acid positions, wherein the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions V10, L114, I118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703, K705, and L712 of SEQ ID NO: 1.

[0094] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more still further amino acid positions, wherein the one or more still further amino acid positions are selected from the group consisting of amino acid positions 10, 192, 197, 220, 225, 306, 318, 323, 339, 349, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 649, 667, 703 and 705 of SEQ ID NO: 1, preferably the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more still further amino acid positions, wherein the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions 10, 192, 197, 220, 225, 306, 318, 323, 339, 349, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 649, 667, 703 and 705 of SEQ ID NO: 1.

[0095] In another embodiment of the first aspect , the one or more still further amino acid positions are selected from the group consisting of amino acid positions V10, S192, S197, Y220, N225, D306, P318, T323, L339, F349, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, A649, R667, A703 and K705 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions V10, S192, S197, Y220, N225, D306, P318, T323, L339, F349, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, A649, R667, A703 and K705 of SEQ ID NO: 1.

[0096] In another embodiment of the first aspect , the one or more still further amino acid positions are selected from the group consisting of amino acid positions 10, 220, 225, 318, 323, 349, 481, 487, 488, 506, 511, 550, 556, 564, 649, 703 and 705 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions 10, 220, 225, 318, 323, 349, 481, 487, 488, 506, 511, 550, 556, 564, 649, 703 and 705 of SEQ ID NO: 1.

[0097] In another embodiment of the first aspect , the one or more still further amino acid positions are selected from the group consisting of amino acid positions V10, Y220, N225, P318, T323, F349, E481, Q487, K488, A506, A511, V550, S556, T564, A649, A703 and K705 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions V10, Y220, N225, P318, T323, F349, E481, Q487, K488, A506, A511, V550, S556, T564, A649, A703 and K705 of SEQ ID NO: 1.

[0098] In another embodiment of the first aspect , the one or more still further amino acid positions are selected from the group consisting of amino acid positions 10, 225, 323, 349, 487, 550, 556, 564 and 649 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions 10, 225, 323, 349, 487, 550, 556, 564 and 649 of SEQ ID NO: 1.

[0099] In another embodiment of the first aspect , the one or more still further amino acid positions are selected from the group consisting of amino acid positions V10, N225, T323, F349, Q487, V550, S556, T564 and A649 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions V10, N225, T323, F349, Q487, V550, S556, T564 and A649 of SEQ ID NO: 1.

[0100] In another embodiment of the first aspect, the one or more still further amino acid positions are selected from the group consisting of amino acid positions 225, 323, 349, 487, 550, 556, 564 and 649 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions 225, 323, 349, 487, 550, 556, 564 and 649 of SEQ ID NO: 1.

[0101] In another embodiment of the first aspect, the one or more still further amino acid positions are selected from the group consisting of amino acid positions N225, T323, F349, Q487, V550, S556, T564 and A649 of SEQ ID NO: 1, preferably the one or more still further amino acid positions are individually and independently selected from the group consisting of amino acid positions N225, T323, F349, Q487, V550, S556, T564 and A649 of SEQ ID NO: 1.

[0102] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at amino acid positions 712, 383, 114, 118, 304, 357 and 590 of SEQ ID NO: 1, preferably at amino acid positions L712, P383, L114, I118, A304, G357 and D590 of SEQ ID NO: 1, and the amino acid sequence of the trehalose phosphorylase comprises further amino acid substitutions at the following amino acid positions of SEQ ID NO: 1 (i) 550 and 556, preferably V550 and S556, or (ii) 225, 487 and 564, preferably N225, Q487and T564, or (iii) 323, 550 and 564, preferably T323, V550 and T564, or (iv) 225, 323 and 487, preferably N225, T323 and Q487, or (v) 225, 487, 550 and 556, preferably N225, Q487, V550 and S556, or (vi) 225, 487, 550 and 564, preferably N225, Q487, V550 and T564, or (vii) 225, 550, 556 and 564, preferably N225, V550, S556 and T564, or (viii) 225, 550, 564 and 649, preferably N225, V550, T564, A649, or (ix) 323, 550, 556 and 564, preferably T323, V550, S556, T564, or (x) 323, 487, 550 and 649, preferably T323, Q487, V550 and A649, or (xii) 225, 487, 550, 556 and 564, preferably N225, Q487, V550, S556 and T564, or (xiii) 225, 550, 556, 564 and 649, preferably N225, V550, S556, T564 and A649, or (xiv) 225, 323, 487, 550 and 564, preferably N225, T323, Q487, V550, T564, or (xv) 225, 323, 487, 550 and 649, preferably N225, T323, Q487, V550 andA649, or (xvi) 225, 323, 487, 550 and 556, preferably N225, T323, Q487, V550 and S556, or (xvii) 225, 323, 550, 556, 564, preferably N225, T323, V550, S556, T564, or (xviii) 323, 349, 487, 550 and 649, preferably T323, F349, Q487, V550 and A649, or (ixx) 323, 487, 550, 556 and 564, preferably T323, Q487, V550, S556 and T564, or (xx) 487, 550, 556, 564 and 649, preferably Q487, V550, S556, T564 and A649, or (xxi) 225, 323, 349, 550, 556 and 649, preferably N225, T323, F349, V550, S556 and A649, or (xxii) 225, 323, 487, 550, 556, 649, preferably N225, T323, Q487, V550, S556 and A649, or (xxiiii) 225, 349, 487, 550, 556 and 564, preferably N225, F349, Q487, V550, S556 and T564, and / or (xxiv) 225, 349, 487, 550, 564 and 649, preferably N225, F349, Q487, V550, T564, A649, or (xxv) 225, 487, 550, 556, 564 and 649, preferably N225, Q487, V550, S556, T564 and A649, or (xxvi) 323, 487, 550, 556, 564 and 649, preferably T323, Q487, V550, S556, T564 and A649, or (xxvii) 225, 323, 487, 550, 556, 564 and 649, preferably N225, T323, Q487, V550, S556, T564 and A649.

[0103] In another embodiment of the first aspect, the substitution at any of the amino acid positions is selected from the group of amino acids consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V, preferably the substitution at any of the amino acid positions is individually and independently selected from the group of amino acids consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V, wherein when the amino acid substitution comprises a substitution at amino acid position 649, then said amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, preferably 649E; and wherein when the amino acid substitution comprises a substitution at amino acid position 10, then said amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and, or V10H, preferably V10R.

[0104] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase comprises one or more substitutions, wherein the substitution is selected from the group consisting of an amino acid substitution at position V10 of SEQ ID NO: 1 with the substitution being V10R, or V10H, preferably V10R; an amino acid substitution at position L114 of SEQ ID NO: 1 with the substitution being L114A, L114G, L1141, L114M, L114P or L114V, preferably L114I; an amino acid substitution at position I118 of SEQ ID NO: 1 with the substitution being I118A, I118G, I118L, I118M, I118P or I118V, preferably I118V; an amino acid substitution at position S192 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 S197 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 Y220 of SEQ ID NO: 1 with the substitution being Y220F or Y220W, preferably Y220F; an amino acid substitution at position N225 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 A304 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 D306 of SEQ ID NO: 1 with the substitution being D306R, D306H or D306K, preferably D306H; an amino acid substitution at position P318 of SEQ ID NO: 1 with the substitution being P318R, P318H or P318K, preferably P318H; an amino acid substitution at position T323 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 L339 of SEQ ID NO: 1 with the substitution being L339A, L339G, L339I, L339L, L339M, L339P or L339V, preferably L339I; an amino acid substitution at position F349 of SEQ ID NO: 1 with the substitution being F349W or F349Y, preferably F349Y; an amino acid substitution at position G357 of SEQ ID NO: 1 with the substitution being G357A, G357I, G357L, G357M, G357P or G357V, preferably G357A; an amino acid substitution at position P383 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 A459 of SEQ ID NO: 1 with the substitution being A459N, A459C, A459Q or A459S, A459T, preferably A459S; an amino acid substitution at position Q476 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 E481 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 A484 of SEQ ID NO: 1 with the substitution being A484N, A484C, A484Q, A484S or A484T, preferably A484S; an amino acid substitution at position Q487 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 K488 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 A506 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 R526 of SEQ ID NO: 1 with the substitution being R526D or R526E preferably R526E; an amino acid substitution at position E530 of SEQ ID NO: 1 with the substitution being E530A, E530G, E530I, E530L, E530M, E530P, E530V, preferably E530V; an amino acid substitution at position G532 of SEQ ID NO: 1 with the substitution being G532R, G532H or G532K, preferably G532R; an amino acid substitution at position D533 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 D537 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 V550 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 S556 of SEQ ID NO: 1 with the substitution being S556N, S556C, S556Q or S556T, preferably S556T; an amino acid substitution at position T564 of SEQ ID NO: 1 with the substitution being T564D or T564E, preferably T564E; an amino acid substitution at position D590 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 A649 of SEQ ID NO: 1 with the substitution being A649D or A649E, preferably A649E; an amino acid substitution at position R667 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 A703 of SEQ ID NO: 1 with the substitution being A703D or A703E, preferably A703E; an amino acid substitution at position K705 of SEQ ID NO: 1 with the substitution being K705N, K705C, K705Q, K705S or K705T, preferably K705N; and an amino acid substitution at position L712 of SEQ ID NO: 1 with the substitution being L712A, L712G, L712I, L712M, L712P or L712V, preferably L712M.

[0105] In another preferred embodiment of the first aspect, the trehalose phosphorylase comprises one or more substitutions, wherein the substitution is selected from the group consisting of amino acid positions L712M, P383G, P383V, P383C, P383S, P383T, V10R, L114I, I118V, S192V, S197G, Y220F, N225I, N225L, N225M, N225V, A304L, A304I, D306H, P318H, T323I, T323V, L339I, F349Y, G357A, A459S, Q476G, E4811, 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, preferably 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, E4811, 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.

[0106] In another embodiment of the first aspect, 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%.

[0107] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to the amino acid sequence 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, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 190.

[0108] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to the amino acid sequence 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.

[0109] In a preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to the amino acid sequence of SEQ ID NO: 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, 155, 156, 157, or 190.

[0110] In another embodiment of the first aspect , the amino acid sequence of the trehalose phosphorylase is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, 7, 9, 10, 12, 13, 15, 20, 29, 44, 45, 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, 98, 104, 110, 113, 115, 121, 125, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 147, 152, 154, 155, 156, 157, 158, 159, or 190.

[0111] In a preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to the amino acid sequence of SEQ ID NO: 44, 45, 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, or 190.

[0112] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is least 85% identical to two or more of the 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, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 190.

[0113] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to two or more of the 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.

[0114] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to two or more of the amino acid sequences of SEQ ID NO: 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, 155, 156, 157, or 190

[0115] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to two or more of the amino acid sequences of SEQ ID NO: 3, 7, 9, 10, 12, 13, 15, 20, 29, 44, 45, 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, 98, 104, 110, 113, 115, 121, 125, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 147, 152, 154, 155, 156, 157, 158, 159, or 190.

[0116] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% identical to two or more of the amino acid sequences of SEQ ID NO: 44, 45, 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, or 190.

[0117] In another embodiment of the first aspect, the identity of the amino acid sequence is at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, 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%.

[0118] In another embodiment of the first aspect, the homology 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%.

[0119] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to the amino acid sequence 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, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, or 159.

[0120] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to the amino acid sequence 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.

[0121] In another preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to the amino acid sequence of SEQ ID NO: ID NO: 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, 155, 156, 157, or 190.

[0122] In another embodiment of the first aspect the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to the amino acid sequence of SEQ ID NO: 3, 7, 9, 10, 12, 13, 15, 20, 29, 44, 45, 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, 98, 104, 110, 113, 115, 121, 125, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 147, 152, 154, 155, 156, 157, 158, 159, or 190.

[0123] In a preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to the amino acid sequences of SEQ ID NO: 44, 45, 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, or 190.

[0124] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is least 85% homologous to two or more of the 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, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 190.

[0125] In another embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to two or more of the 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.

[0126] In a preferred embodiment of the first aspect, the amino acid sequence of the trehalose phosphorylase is at least 85% homologous to two or more of the amino acid sequences of SEQ ID NO: 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, 155, 156, 157, or 190.

[0127] In another embodiment of the first aspect, the problem underlying the present invention is solved by a polypeptide, wherein the polypeptide comprises an amino acid sequence 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, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 190. The polypeptide is a trehalose phosphorylase. Whenever in this disclosure it is referred to the trehalose phosphorylase of the first aspect and any embodiment thereof, such first aspect includes any subaspects thereof, including any embodiment thereof.

[0128] In another embodiment of the first aspect, the the trehalose phosphorylase comprises or consists of an amino acid sequence 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.

[0129] In another embodiment of the first aspect, the trehalose phosphorylase, comprises or consists of an amino acid sequence of SEQ ID NO: 3, 7, 9, 10, 12, 13, 15, 20, 29, 44, 45, 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, 98, 104, 110, 113, 115, 121, 125, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 147, 152, 154, 155, 156, 157, 158, 159, or 190.

[0130] In another embodiment of the first aspect, the trehalose phosphorylase, is capable of catalyzing the reaction of a glycosyl monosaccharide and alpha-D-glucose-1 phosphate.

[0131] In another embodiment of the first aspect, 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.

[0132] In another embodiment of the first aspect, the conversion is a reversible conversion.

[0133] In another embodiment of the first aspect, the trehalose phosphorylase is a trehalose phosphorylase according to EC number EC 2.4.1.231.

[0134] In another embodiment of the first aspect, 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 i) from 30% to 90%, preferably from 39% to 90%, more preferably from 42% to 90%, more preferably from 54% to 90%, more preferably from 55% to 90%, and even more preferably from 63% to 90%, even more preferably from 64% to 90%, even more preferably from 68% to 90%, and most preferably from 64% to 86%, and / or ii) from 30% to 90%, preferably from 31% to 90%, preferably from 32% to 90%, preferably from 33% to 90%, preferably from 34% to 90%, preferably from 35% to 90%, preferably from 36% to 90%, preferably from 37% to 90%, preferably from 38% to 90%, preferably from 39% to 90%, more preferably from 40% to 90%, more preferably from 41% to 90%, more preferably from 42% to 90%, more preferably from 43% to 90%, more preferably from 44% to 90%, more preferably from 45% to 90%, more preferably from 46% to 90%, more preferably from 47% to 90%, more preferably from 48% to 90%, more preferably from 49% to 90%, even more preferably from 50% to 90%, even more preferably from 51% to 90%, even more preferably from 52% to 90%, even more preferably from 53% to 90%, even more preferably from 54% to 90%, even more preferably from 55% to 90%, even more preferably from 60% to 90%, even more preferably from 61% to 90%, even more preferably from 65% to 90%, even more preferably from 70% to 90%, even more preferably from 75% to 90%, and most preferably from 72% to 81%, and / or iii) from 55% to 100%, preferably from 60% to 100%, preferably from 70% to 100%, preferably from 75% to 100%, preferably from 76% to 100%, preferably from 77% to 100%, preferably from 78% to 100%, preferably from 79% to 100%, more preferably from 80% to 100%, more preferably from 81% to 100%, more preferably from 82% to 100%, more preferably from 83% to 100%, more preferably from 84% to 100%, more preferably from 85% to 100%, more preferably from 86% to 100%, more preferably from 87% to 100%, more preferably from 88% to 100%, more preferably from 89% to 100%, even more preferably from 90% to 100%, even more preferably from 91% to 100%, even more preferably from 92% to 100%, even more preferably from 93% to 100%, even more preferably from 94% to 100%, even more preferably from 95% to 100%, even more preferably from 96% to 100%, even more preferably from 97% to 100%, even more preferably from 98% to 100%, even more preferably from 99% to 100%, and most preferably 100%, and wherein 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, and wherein characteristic (C) is thermal stability characterized by i) a Tm30-value between 52 °C and 90°C, preferably between 52 °C and 80 °C, preferably between 52,5 °C and 80°C, preferably between 53 °C and 80°C, preferably between 53,5 °C and 80°C, more preferably between 54 °C and 80°C, preferably between 54,5 °C and 80°C, even more preferably between 55 °C and 80°C, preferably between 55,5 °C and 80°C, preferably between 56 °C and 80°C, preferably between 56,5 °C and 80°C, preferably between 57 °C and 80°C, preferably between 57,5 °C and 80°C, preferably between 58 °C and 80°C, preferably between 58,5 °C and 80°C, even more preferably between 52 °C and 70 °C, even more preferably between 52,5 °C and 70°C, even more preferably between 53 °C and 70°C, even more preferably between 53,5 °C and 70°C, more even more preferably between 54 °C and 70°C, even more preferably between 54,5 °C and 70°C, even more even more preferably between 55 °C and 70°C, even more preferably between 55,5 °C and 70°C, even more preferably between 56 °C and 70°C, even more preferably between 56,5 °C and 70°C, even more preferably between 57 °C and 70°C, even more preferably between 57,5 °C and 70°C, even more preferably between 58 °C and 70°C, even more preferably between 58,5 °C and 70°C, even more preferably between 52 °C and 65 °C, even more preferably between 52,5 °C and 65°C, even more preferably between 53 °C and 65°C, even more preferably between 53,5 °C and 65°C, more even more preferably between 54 °C and 65°C, even more preferably between 54,5 °C and 65°C, even more even more preferably between 55 °C and 65°C, even more preferably between 55,5 °C and 65°C, even more preferably between 56 °C and 65°C, even more preferably between 56,5 °C and 65°C, even more preferably between 57 °C and 65°C, even more preferably between 57,5 °C and 65°C, even more preferably between 58 °C and 65°C, even more preferably between 58,5 °C and 65°C, even more preferably between 52 °C and 60 °C, even more preferably between 52,5 °C and 60°C, even more preferably between 53 °C and 60°C, even more preferably between 53,5 °C and 60°C, more even more preferably between 54 °C and 60°C, even more preferably between 54,5 °C and 60°C, even more even more preferably between 55 °C and 60°C, even more preferably between 55,5 °C and 60°C, even more preferably between 56 °C and 60°C, even more preferably between 56,5 °C and 60°C, even more preferably between 57 °C and 60°C, even more preferably between 57,5 °C and 60°C, even more preferably between 58 °C and 60°C, even more preferably between 58,5 °C and 60°C, and most preferably between 53,5 °C and 58,5°C and / or ii) a Tm50-value between 52 °C and 90°C, preferably between 52 °C and 80 °C, preferably between 52,5 °C and 80°C, preferably between 53 °C and 80°C, preferably between 53,5 °C and 80°C, more preferably between 54 °C and 80°C, preferably between 54,5 °C and 80°C, even more preferably between 55 °C and 80°C, preferably between 55,5 °C and 80°C, preferably between 56 °C and 80°C, preferably between 56,5 °C and 80°C, preferably between 57 °C and 80°C, preferably between 57,5 °C and 80°C, even more preferably between 52 °C and 70 °C, even more preferably between 52,5 °C and 70°C, even more preferably between 53 °C and 70°C, even more preferably between 53,5 °C and 70°C, more even more preferably between 54 °C and 70°C, even more preferably between 54,5 °C and 70°C, even more even more preferably between 55 °C and 70°C, even more preferably between 55,5 °C and 70°C, even more preferably between 56 °C and 70°C, even more preferably between 56,5 °C and 70°C, even more preferably between 57 °C and 70°C, even more preferably between 57,5 °C and 70°C, even more preferably between 52 °C and 65 °C, even more preferably between 52,5 °C and 65°C, even more preferably between 53 °C and 65°C, even more preferably between 53,5 °C and 65°C, more even more preferably between 54 °C and 65°C, even more preferably between 54,5 °C and 65°C, even more even more preferably between 55 °C and 65°C, even more preferably between 55,5 °C and 65°C, even more preferably between 56 °C and 65°C, even more preferably between 56,5 °C and 65°C, even more preferably between 57 °C and 65°C, even more preferably between 57,5 °C and 65°C, even more preferably between 52 °C and 60 °C, even more preferably between 52,5 °C and 60°C, even more preferably between 53 °C and 60°C, even more preferably between 53,5 °C and 60°C, more even more preferably between 54 °C and 60°C, even more preferably between 54,5 °C and 60°C, even more even more preferably between 55 °C and 60°C, even more preferably between 55,5 °C and 60°C, even more preferably between 56 °C and 60°C, even more preferably between 56,5 °C and 60°C, even more preferably between 57 °C and 60°C, even more preferably between 57,5 °C and 60°C, and most preferably between 52 °C and 57,5°C and wherein 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, preferably of from 24 hours to 9 days or more, preferably of from 39 hours to 9 days or more, preferably of from 2 days to 9 days or more, more preferably of from 4 days to 9 days or more, more preferably of from 5.5 days to 9 days or more, more preferably of from least 7 days to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days; ii) a process stability, characterized by a half-life at 45 °C of from 24 hours to 9 days or more, more preferably of from 39 hours to 9 days or more, more preferably of from 2 days to 9 days or more, more preferably of from 4 days to 9 days or more, more preferably of from 5.5 days to 9 days or more, more preferably of from 7 days to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days; iii) a process stability, characterized by a half-life at 45 °C of 4 days to 9 days or more, preferably of from 5.5 days up to 9 days or more, more preferably of from 7 days up to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days; and wherein characteristic (E) is relative activity expressed as 100 / 500-ratio of i) between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between at least 0.7 and 1.0; ii) between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between 0.8 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].

[0135] In another embodiment of the first aspect, the trehalose phosphorylase, has thermal stability after incubation at 52°C for 15 minutes defined by a residual activity of from 30% to 90%, preferably from 39% to 90%, more preferably from 42% to 90%, more preferably from 54% to 90%, more preferably from 55% to 90%, and even more preferably from 63% to 90%, even more preferably from 64% to 90%, even more preferably from 68% to 90%, and most preferably from 64% to 86%; and / or from 30% to 90%, preferably from 31% to 90%, preferably from 32% to 90%, preferably from 33% to 90%, preferably from 34% to 90%, preferably from 35% to 90%, preferably from 36% to 90%, preferably from 37% to 90%, preferably from 38% to 90%, preferably from 39% to 90%, more preferably from 40% to 90%, more preferably from 41% to 90%, more preferably from 42% to 90%, more preferably from 43% to 90%, more preferably from 44% to 90%, more preferably from 45% to 90%, more preferably from 46% to 90%, more preferably from 47% to 90%, more preferably from 48% to 90%, more preferably from 49% to 90%, even more preferably from 50% to 90%, even more preferably from 51% to 90%, even more preferably from 52% to 90%, even more preferably from 53% to 90%, even more preferably from 54% to 90%, even more preferably from 55% to 90%, even more preferably from 60% to 90%, even more preferably from 61% to 90%, even more preferably from 65% to 90%, even more preferably from 70% to 90%, even more preferably from 75% to 90%, and most preferably from 72% to 81%, and / or from 55% to 100%, preferably from 60% to 100%, preferably from 70% to 100%, preferably from 75% to 100%, preferably from 76% to 100%, preferably from 77% to 100%, preferably from 78% to 100%, preferably from 79% to 100%, more preferably from 80% to 100%, more preferably from 81% to 100%, more preferably from 82% to 100%, more preferably from 83% to 100%, more preferably from 84% to 100%, more preferably from 85% to 100%, more preferably from 86% to 100%, more preferably from 87% to 100%, more preferably from 88% to 100%, more preferably from 89% to 100%, even more preferably from 90% to 100%, even more preferably from 91% to 100%, even more preferably from 92% to 100%, even more preferably from 93% to 100%, even more preferably from 94% to 100%, even more preferably from 95% to 100%, even more preferably from 96% to 100%, even more preferably from 97% to 100%, even more preferably from 98% to 100%, even more preferably from 99% to 100%, and most preferably 100%.

[0136] In another embodiment of the first aspect, the trehalose phosphorylase, has thermal stability characterized by a Tm30-value of at least 52 °C.

[0137] In another embodiment of the first aspect, the trehalose phosphorylase, has a half-life at 45 °C of from 3 hours to 9 days or more, preferably of from 24 hours to 9 days or more, preferably of from 39 hours to 9 days or more, preferably of from 2 days to 9 days or more, more preferably of from 4 day to 9 days or more, more preferably of from 5.5 days to 9 days or more, more preferably of from 7 days to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days.

[0138] In another embodiment of the first aspect, the trehalose phosphorylase, has relative activity expressed as 100 / 500-ratio of between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between 0.7 and 1.0.

[0139] In another embodiment of the first aspect, the trehalose phosphorylase, has thermal stability after incubation at 52°C for 15 minutes defined by a residual activity of from 30% to 90%, preferably from 31% to 90%, preferably from 32% to 90%, preferably from 33% to 90%, preferably from 34% to 90%, preferably from 35% to 90%, preferably from 36% to 90%, preferably from 37% to 90%, preferably from 38% to 90%, preferably from 39% to 90%, more preferably from 40% to 90%, more preferably from 41% to 90%, more preferably from 42% to 90%, more preferably from 43% to 90%, more preferably from 44% to 90%, more preferably from 45% to 90%, more preferably from 46% to 90%, more preferably from 47% to 90%, more preferably from 48% to 90%, more preferably from 49% to 90%, even more preferably from 50% to 90%, even more preferably from 51% to 90%, even more preferably from 52% to 90%, even more preferably from 53% to 90%, even more preferably from 54% to 90%, even more preferably from 55% to 90%, even more preferably from 60% to 90%, even more preferably from 61% to 90%, even more preferably from 65% to 90%, even more preferably from 70% to 90%, even more preferably from 75% to 90%, and most preferably from 72% to 81%, and / or from 55% to 100%, preferably from 60% to 100%, preferably from 70% to 100%, preferably from 75% to 100%, preferably from 76% to 100%, preferably from 77% to 100%, preferably from 78% to 100%, preferably from 79% to 100%, more preferably from 80% to 100%, more preferably from 81% to 100%, more preferably from 82% to 100%, more preferably from 83% to 100%, more preferably from 84% to 100%, more preferably from 85% to 100%, more preferably from 86% to 100%, more preferably from 87% to 100%, more preferably from 88% to 100%, more preferably from 89% to 100%, even more preferably from 90% to 100%, even more preferably from 91% to 100%, even more preferably from 92% to 100%, even more preferably from 93% to 100%, even more preferably from 94% to 100%, even more preferably from 95% to 100%, even more preferably from 96% to 100%, even more preferably from 97% to 100%, even more preferably from 98% to 100%, even more preferably from 99% to 100%, and most preferably 100%.

[0140] In another embodiment of the first aspect, the trehalose phosphorylase, has thermal stability characterized by a Tm30-value of at least 52 °C and / or a Tm50-value of at least 52°C.

[0141] In another embodiment of the first aspect, the trehalose phosphorylase, has a half-life at 45 °C of from 1 day to 9 days, more preferably of from 2 days to 9 days, more preferably of from 4 days to 9 days, more preferably of from 5.5 days to 9 days, more preferably of from 7 days to 9 days, most preferably of 9 days.

[0142] In another embodiment of the first aspect, the trehalose phosphorylase, has relative activity expressed as 100 / 500-ratio of between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between 0.7 and 1.0.

[0143] In another embodiment of the first aspect, the trehalose phosphorylase, has thermal stability after incubation at 52°C for 15 minutes defined by a residual activity from 55% from to 100%, preferably from 60% to 100%, preferably from 70% to 100%, preferably from 75% to 100%, preferably from 76% to 100%, preferably from 77% to 100%, preferably from 78% to 100%, preferably from 79% to 100%, more preferably from 80% to 100%, more preferably from 81% to 100%, more preferably from 82% to 100%, more preferably from 83% to 100%, more preferably from 84% to 100%, more preferably from 85% to 100%, more preferably from 86% to 100%, more preferably from 87% to 100%, more preferably from 88% to 100%, more preferably from 89% to 100%, even more preferably from 90% to 100%, even more preferably from 91% to 100%, even more preferably from 92% to 100%, even more preferably from 93% to 100%, even more preferably from 94% to 100%, even more preferably from 95% to 100%, even more preferably from 96% to 100%, even more preferably from 97% to 100%, even more preferably from 98% to 100%, even more preferably from 99% to 100%, and most preferably of 100%.

[0144] In another embodiment of the first aspect, the trehalose phosphorylase has thermal stability characterized by a Tm30-value of at least 52 °C and / or a Tm50-value of at least 52°C, and which is preferably characterized by a Tm30-value of a at least 52 °C and a Tm50-value of at least 52°C.

[0145] In another embodiment of the first aspect, the trehalose phosphorylase has thermal stability characterized by i) a Tm30-value between 52 °C and 90°C, preferably between 52 °C and 80 °C, preferably between a 52.5 °C and 80°C, preferably between 53 °C and 80°C, preferably between 53.5 °C and 80°C, more preferably between 54 °C and 80°C, preferably between 54.5 °C and 80°C, even more preferably between 55 °C and 80°C, preferably between 55.5 °C and 80°C, preferably between 56 °C and 80°C, preferably between 56.5 °C and 80°C, preferably between 57 °C and 80°C, preferably between 57.5 °C and 80°C, preferably between 58 °C and 80°C, preferably between 58.5 °C and 80°C, even more preferably between 52 °C and 70 °C, even more preferably between 52.5 °C and 70°C, even more preferably between 53 °C and 70°C, even more preferably between 53.5 °C and 70°C, more even more preferably between 54 °C and 70°C, even more preferably between 54.5 °C and 70°C, even more even more preferably between 55 °C and 70°C, even more preferably between 55.5 °C and 70°C, even more preferably between 56 °C and 70°C, even more preferably between 56.5 °C and 70°C, even more preferably between 57 °C and 70°C, even more preferably between 57.5 °C and 70°C, even more preferably between 58 °C and 70°C, even more preferably between 58.5 °C and 70°C, even more preferably between 52 °C and 65 °C, even more preferably between 52.5 °C and 65°C, even more preferably between 53 °C and 65°C, even more preferably between 53.5 °C and 65°C, more even more preferably between 54 °C and 65°C, even more preferably between 54.5 °C and 65°C, even more even more preferably between 55 °C and 65°C, even more preferably between 55.5 °C and 65°C, even more preferably between 56 °C and 65°C, even more preferably between 56.5 °C and 65°C, even more preferably between 57 °C and 65°C, even more preferably between 57.5 °C and 65°C, even more preferably between 58 °C and 65°C, even more preferably between 58.5 °C and 65°C, even more preferably between 52 °C and 60 °C, even more preferably between 52.5 °C and 60°C, even more preferably between 53 °C and 60°C, even more preferably between 53.5 °C and 60°C, more even more preferably between from 54 °C and 60°C, even more preferably between from 54.5 °C and 60°C, even more even more preferably between 55 °C and 60°C, even more preferably between 55.5 °C and 60°C, even more preferably between 56 °C and 60°C, even more preferably between 56.5 °C and 60°C, even more preferably between 57 °C and 60°C, even more preferably between 57.5 °C and 60°C, even more preferably between 58 °C and 60°C, even more preferably between 58.5 °C and 60°C, and most preferably between 53.5 °C and 58.5°C and / or ii) a Tm50-value between 52 °C and 90°C, preferably between 52 °C and 80 °C, preferably between 52.5 °C and 80°C, preferably between 53 °C and 80°C, preferably between 53.5 °C and 80°C, more preferably between 54 °C and 80°C, preferably between 54.5 °C and 80°C, even more preferably between 55 °C and 80°C, preferably between 55.5 °C and 80°C, preferably between 56 °C and 80°C, preferably between 56.5 °C and 80°C, preferably between 57 °C and 80°C, preferably between 57.5 °C and 80°C, even more preferably between 52 °C and 70 °C, even more preferably between 52.5 °C and 70°C, even more preferably between 53 °C and 70°C, even more preferably between 53.5 °C and 70°C, more even more preferably between 54 °C and 70°C, even more preferably between 54.5 °C and 70°C, even more even more preferably between 55 °C and 70°C, even more preferably between 55.5 °C and 70°C, even more preferably between 56 °C and 70°C, even more preferably between 56.5 °C and 70°C, even more preferably between 57 °C and 70°C, even more preferably between 57.5 °C and 70°C, even more preferably between 52 °C and 65 °C, even more preferably between 52.5 °C and 65°C, even more preferably between 53 °C and 65°C, even more preferably between 53.5 °C and 65°C, more even more preferably between 54 °C and 65°C, even more preferably between 54.5 °C and 65°C, even more even more preferably between 55 °C and 65°C, even more preferably between 55.5 °C and 65°C, even more preferably between 56 °C and 65°C, even more preferably between 56.5 °C and 65°C, even more preferably between 57 °C and 65°C, even more preferably between 57.5 °C and 65°C, even more preferably between 52 °C and 60 °C, even more preferably between 52.5 °C and 60°C, even more preferably between 53 °C and 60°C, even more preferably between 53.5 °C and 60°C, more even more preferably between 54 °C and 60°C, even more preferably between 54.5 °C and 60°C, even more even more preferably between 55 °C and 60°C, even more preferably between 55.5 °C and 60°C, even more preferably between 56 °C and 60°C, even more preferably between 56.5 °C and 60°C, even more preferably between 57 °C and 60°C, even more preferably between 57.5 °C and 60°C, and most preferably between 52 °C and 57.5°C.

[0146] In another embodiment of the first aspect, wherein the trehalose phosphorylase has a half-life at 45 °C of from 4 days to 9 days or more, preferably of from 5.5 days to 9 days or more, more preferably of from 7 days up to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days.

[0147] In another embodiment of the first aspect, the trehalose phosphorylase has a relative activity expressed as 100 / 500-ratio of between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between 0.8 and 1.0.

[0148] In an embodiment of the first aspect, wherein the trehalose phosphorylase, compared to a trehalose phosphorylase of SEQ ID NO:1, is characterized by an increase in thermal stability, whereby the increase in thermal stability is i) an increase of the Tm30-value of at least 2 °C up to 40.5°C, preferably 2 °C up to 30.5°C, preferably of at least 2.5°C up to 30.5°C, preferably of at least 3°C up to 30.5°C, preferably of at least 3.5°C up to 30.5°C, preferably of at least 4°C up to 30.5°C, preferably of at least 4.5°C up to 30.5°C, preferably of at least 5°C up to 30.5°C, preferably of at least 5.5°C up to 30.5°C, preferably of at least 6°C up to 30.5°C, preferably of at least 6.5°C up to 30.5°C, preferably of at least 7°C up to 30.5°C, preferably of at least 7.5°C up to 30.5°C, preferably of at least 8°C up to 30.5°C, preferably of at least 8.5°C up to 30.5°C, preferably of at least 9°C up to 30.5°C, more preferably 2 °C up to 20.5°C, more preferably of at least 2.5°C up to 20.5°C, more preferably of at least 3°C up to 20.5°C, more preferably of at least 3.5°C up to 20.5°C, more preferably of at least 4°C up to 20.5°C, more preferably of at least 4.5°C up to 20.5°C, more preferably of at least 5°C up to 20.5°C, more preferably of at least 5.5°C up to 20.5°C, more preferably of at least 6°C up to 20.5°C, more preferably of at least 6.5°C up to 20.5°C, more preferably of at least 7°C up to 20.5°C, more preferably of at least 7.5°C up to 20.5°C, more preferably of at least 8°C up to 20.5°C, more preferably of at least 8.5°C up to 20.5°C, more preferably of at least 9°C up to 20.5°C, even more preferably 2 °C up to 15.5°C, even more preferably of at least 2.5°C up to 15.5°C, even more preferably of at least 3°C up to 15.5°C, even more preferably of at least 3.5°C up to 15.5°C, even more preferably of at least 4°C up to 15.5°C, even more preferably of at least 4.5°C up to 15.5°C, even more preferably of at least 5°C up to 15.5°C, even more preferably of at least 5.5°C up to 15.5°C, even more preferably of at least 6°C up to 15.5°C, even more preferably of at least 6.5°C up to 15.5°C, even more preferably of at least 7°C up to 15.5°C, even more preferably of at least 7.5°C up to 15.5°C, even more preferably of at least 8°C up to 15.5°C, even more preferably of at least 8.5°C up to 15.5°C, even more preferably of at least 9°C up to 15.5°C, even more preferably 2 °C up to 10.5°C, even more preferably of at least 2.5°C up to 10.5°C, even more preferably of at least 3°C up to 10.5°C, even more preferably of at least 3.5°C up to 10.5°C, even more preferably of at least 4°C up to 10.5°C, even more preferably of at least 4.5°C up to 10.5°C, even more preferably of at least 5°C up to 10.5°C, even more preferably of at least 5.5°C up to 10.5°C, even more preferably of at least 6°C up to 10.5°C, even more preferably of at least 6.5°C up to 10.5°C, even more preferably of at least 7°C up to 10.5°C, even more preferably of at least 7.5°C up to 10.5°C, even more preferably of at least 8°C up to 10.5°C, even more preferably of at least 8.5°C up to 10.5°C, even more preferably of at least 9°C up to 10.5°C, and most preferably by at least 4°C up to 9°C; and / or ii) an increase of the Tm50-value of at least 2 °C up to 42.5°C, preferably 2 °C up to 32.5°C, preferably of at least 2.5°C up to 32.5°C, preferably of at least 3°C up to 32.5°C, preferably of at least 3.5°C up to 32.5°C, preferably of at least 4°C up to 32.5°C, preferably of at least 4.5°C up to 32.5°C, preferably of at least 5°C up to 32.5°C, preferably of at least 5.5°C up to 32.5°C, preferably of at least 6°C up to 32.5°C, preferably of at least 6.5°C up to 32.5°C, preferably of at least 7°C up to 32.5°C, preferably of at least 7.5°C up to 32.5°C, preferably of at least 8°C up to 32.5°C, preferably of at least 8.5°C up to 32.5°C, preferably of at least 9°C up to 32.5°C, preferably of at least 9.5°C up to 32.5°C, preferably of at least 10°C up to 32.5°C, more preferably 2 °C up to 22.5°C, more preferably of at least 2.5°C up to 22.5°C, more preferably of at least 3°C up to 22.5°C, more preferably of at least 3.5°C up to 22.5°C, more preferably of at least 4°C up to 22.5°C, more preferably of at least 4.5°C up to 22.5°C, more preferably of at least 5°C up to 22.5°C, more preferably of at least 5.5°C up to 22.5°C, more preferably of at least 6°C up to 22.5°C, more preferably of at least 6.5°C up to 22.5°C, more preferably of at least 7°C up to 22.5°C, more preferably of at least 7.5°C up to 22.5°C, more preferably of at least 8°C up to 22.5°C, more preferably of at least 8.5°C up to 22.5°C, more preferably of at least 9°C up to 22.5°C, more preferably of at least 9.5°C up to 22.5°C, more preferably of at least 10°C up to 22.5°C, even more preferably 2 °C up to 17.5°C, even more preferably of at least 2.5°C up to 17.5°C, even more preferably of at least 3°C up to 17.5°C, even more preferably of at least 3.5°C up to 17.5°C, even more preferably of at least 4°C up to 17.5°C, even more preferably of at least 4.5°C up to 17.5°C, even more preferably of at least 5°C up to 17.5°C, even more preferably of at least 5.5°C up to 17.5°C, even more preferably of at least 6°C up to 17.5°C, even more preferably of at least 6.5°C up to 17.5°C, even more preferably of at least 7°C up to 17.5°C, even more preferably of at least 7.5°C up to 17.5°C, even more preferably of at least 8°C up to 17.5°C, even more preferably of at least 8.5°C up to 17.5°C, even more preferably of at least 9°C up to 17.5°C, even more preferably of at least 9.5°C up to 17.5°C, even more preferably of at least 10°C up to 17.5°C, even more preferably 2 °C up to 12.5°C, even more preferably of at least 2.5°C up to 12.5°C, even more preferably of at least 3°C up to 12.5°C, even more preferably of at least 3.5°C up to 12.5°C, even more preferably of at least 4°C up to 12.5°C, even more preferably of at least 4.5°C up to 12.5°C, even more preferably of at least 5°C up to 12.5°C, even more preferably of at least 5.5°C up to 12.5°C, even more preferably of at least 6°C up to 12.5°C, even more preferably of at least 6.5°C up to 12.5°C, even more preferably of at least 7°C up to 12.5°C, even more preferably of at least 7.5°C up to 12.5°C, even more preferably of at least 8°C up to 12.5°C, even more preferably of at least 8.5°C up to 12.5°C, even more preferably of at least 9°C up to 12.5°C, even more preferably of at least 9.5°C up to 12.5°C, even more preferably of at least 10°C up to 12.5°C, and most preferably by at least 4°C up to 9°C; and / or iii) an improved process stability, characterized by an increased half-life at 45 °C of i) at least 3-fold up to 216-fold or more, preferably of at least 24-fold up to 216-fold or more, preferably of at least 39-fold up to 216-fold or more, preferably of at least 48-fold up to 216-fold or more, preferably of at least 96-fold up to 216-fold or more, preferably of at least 132-fold up to 216-fold or more, more preferably of at least 216-fold or more, and most preferably of 216-fold; ii) at least 24-fold up to 216-fold or more, preferably of at least 39-fold up to 216-fold or more, preferably of at least 48-fold up to 216-fold or more, preferably of at least 96-fold up to 216-fold or more, preferably of at least 132-fold up to 216-fold or more, more preferably of at least 216-fold or more, and most preferably of 216-fold; iii) at least 96-fold up to 216-fold or more, preferably of at least 132-fold up to 216-fold or more, more preferably of at least 216-fold or more, and most preferably of 216-fold.

[0149] More specifically, these and other problems are solved in a second aspect, by a trehalose phosphorylase as defined in the claims, comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase, is at least 80% identical 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 i) from 30% to 90%, preferably from 39% to 90%, more preferably from 42% to 90%, more preferably from 54% to 90%, more preferably from 55% to 90%, and even more preferably from 63% to 90%, even more preferably from 64% to 90%, even more preferably from 68% to 90%, and most preferably from 64% to 86%, and / or ii) from 30% to 90%, preferably from 31% to 90%, preferably from 32% to 90%, preferably from 33% to 90%, preferably from 34% to 90%, preferably from 35% to 90%, preferably from 36% to 90%, preferably from 37% to 90%, preferably from 38% to 90%, preferably from 39% to 90%, more preferably from 40% to 90%, more preferably from 41% to 90%, more preferably from 42% to 90%, more preferably from 43% to 90%, more preferably from 44% to 90%, more preferably from 45% to 90%, more preferably from 46% to 90%, more preferably from 47% to 90%, more preferably from 48% to 90%, more preferably from 49% to 90%, even more preferably from 50% to 90%, even more preferably from 51% to 90%, even more preferably from 52% to 90%, even more preferably from 53% to 90%, even more preferably from 54% to 90%, even more preferably from 55% to 90%, even more preferably from 60% to 90%, even more preferably from 61% to 90%, even more preferably from 65% to 90%, even more preferably from 70% to 90%, even more preferably from 75% to 90%, and most preferably from 72% to 81%, and / or iii) from 55% to 100%, preferably from 60% to 100%, preferably from 70% to 100%, preferably from 75% to 100%, preferably from 76% to 100%, preferably from 77% to 100%, preferably from 78% to 100%, preferably from 79% to 100%, more preferably from 80% to 100%, more preferably from 81% to 100%, more preferably from 82% to 100%, more preferably from 83% to 100%, more preferably from 84% to 100%, more preferably from 85% to 100%, more preferably from 86% to 100%, more preferably from 87% to 100%, more preferably from 88% to 100%, more preferably from 89% to 100%, even more preferably from 90% to 100%, even more preferably from 91% to 100%, even more preferably from 92% to 100%, even more preferably from 93% to 100%, even more preferably from 94% to 100%, even more preferably from 95% to 100%, even more preferably from 96% to 100%, even more preferably from 97% to 100%, even more preferably from 98% to 100%, even more preferably from 99% to 100%, and most preferably 100%, and wherein 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, and wherein characteristic (C) is thermal stability characterized by i) a Tm30-value between 52 °C and 90°C, preferably between 52 °C and 80 °C, preferably between 52.5 °C and 80°C, preferably between 53 °C and 80°C, preferably between 53.5 °C and 80°C, more preferably between 54 °C and 80°C, preferably between 54.5 °C and 80°C, even more preferably between 55 °C and 80°C, preferably between 55.5 °C and 80°C, preferably between 56 °C and 80°C, preferably between 56.5 °C and 80°C, preferably between 57 °C and 80°C, preferably between 57.5 °C and 80°C, preferably between 58 °C and 80°C, preferably between 58.5 °C and 80°C, even more preferably between 52 °C and 70 °C, even more preferably between 52.5 °C and 70°C, even more preferably between 53 °C and 70°C, even more preferably between 53.5 °C and 70°C, more even more preferably between 54 °C and 70°C, even more preferably between 54.5 °C and 70°C, even more even more preferably between 55 °C and 70°C, even more preferably between 55.5 °C and 70°C, even more preferably between 56 °C and 70°C, even more preferably between 56.5 °C and 70°C, even more preferably between 57 °C and 70°C, even more preferably between 57.5 °C and 70°C, even more preferably between 58 °C and 70°C, even more preferably between 58.5 °C and 70°C, even more preferably between 52 °C and 65 °C, even more preferably between 52.5 °C and 65°C, even more preferably between 53 °C and 65°C, even more preferably between 53.5 °C and 65°C, more even more preferably between 54 °C and 65°C, even more preferably between 54.5 °C and 65°C, even more even more preferably between 55 °C and 65°C, even more preferably between 55.5 °C and 65°C, even more preferably between 56 °C and 65°C, even more preferably between 56.5 °C and 65°C, even more preferably between 57 °C and 65°C, even more preferably between 57.5 °C and 65°C, even more preferably between 58 °C and 65°C, even more preferably between 58.5 °C and 65°C, even more preferably between 52 °C and 60 °C, even more preferably between 52.5 °C and 60°C, even more preferably between 53 °C and 60°C, even more preferably between 53.5 °C and 60°C, more even more preferably between 54 °C and 60°C, even more preferably between 54.5 °C and 60°C, even more even more preferably between 55 °C and 60°C, even more preferably between 55.5 °C and 60°C, even more preferably between 56 °C and 60°C, even more preferably between 56.5 °C and 60°C, even more preferably between 57 °C and 60°C, even more preferably between 57.5 °C and 60°C, even more preferably between 58 °C and 60°C, even more preferably between 58.5 °C and 60°C, and most preferably between 53.5 °C and 58.5°C and / or ii) a Tm50-value between between 52 °C and 90°C, preferably between 52 °C and 80 °C, preferably between 52.5 °C and 80°C, preferably between 53 °C and 80°C, preferably between 53.5 °C and 80°C, more preferably between 54 °C and 80°C, preferably between 54.5 °C and 80°C, even more preferably between 55 °C and 80°C, preferably between 55.5 °C and 80°C, preferably between 56 °C and 80°C, preferably between 56.5 °C and 80°C, preferably between 57 °C and 80°C, preferably between 57.5 °C and 80°C, even more preferably between 52 °C and 70 °C, even more preferably between 52.5 °C and 70°C, even more preferably between 53 °C and 70°C, even more preferably between 53.5 °C and 70°C, more even more preferably between 54 °C and 70°C, even more preferably between 54.5 °C and 70°C, even more even more preferably between 55 °C and 70°C, even more preferably between 55.5 °C and 70°C, even more preferably between 56 °C and 70°C, even more preferably between 56.5 °C and 70°C, even more preferably between 57 °C and 70°C, even more preferably between 57.5 °C and 70°C, even more preferably between 52 °C and 65 °C, even more preferably between 52.5 °C and 65°C, even more preferably between 53 °C and 65°C, even more preferably between 53.5 °C and 65°C, more even more preferably between 54 °C and 65°C, even more preferably between 54.5 °C and 65°C, even more even more preferably between 55 °C and 65°C, even more preferably between 55.5 °C and 65°C, even more preferably between 56 °C and 65°C, even more preferably between 56,5 °C and 65°C, even more preferably between 57 °C and 65°C, even more preferably between 57.5 °C and 65°C, even more preferably between 52 °C and 60 °C, even more preferably between 52.5 °C and 60°C, even more preferably between 53 °C and 60°C, even more preferably between 53.5 °C and 60°C, more even more preferably between 54 °C and 60°C, even more preferably between 54.5 °C and 60°C, even more even more preferably between 55 °C and 60°C, even more preferably between 55.5 °C and 60°C, even more preferably between 56 °C and 60°C, even more preferably between 56.5 °C and 60°C, even more preferably between 57 °C and 60°C, even more preferably between 57.5 °C and 60°C, and most preferably between 52 °C and 57.5°C; and wherein 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, preferably of from 24 hours to 9 days or more, preferably of from 39 hours to 9 days or more, preferably of from 2 days to 9 days or more, more preferably of from 4 days to 9 days or more, more preferably of from 5.5 days to 9 days or more, more preferably of from least 7 days to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days; ii) a process stability, characterized by a half-life at 45 °C of from 24 hours to 9 days or more, more preferably of from 39 hours to 9 days or more, more preferably of from 2 days to 9 days or more, more preferably of from 4 days to 9 days or more, more preferably of from 5.5 days to 9 days or more, more preferably of from 7 days to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days; iii) a process stability, characterized by a half-life at 45 °C of 4 days to 9 days or more, preferably of from 5.5 days up to 9 days or more, more preferably of from 7 days up to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days; and wherein characteristic (E) is relative activity expressed as 100 / 500-ratio of (i) between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between at least 0.7 and 1.0; (ii) between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between 0.8 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].

[0150] In a second embodiment of the second aspect, the trehalose phosphorylase has thermal stability after incubation at 52°C for 15 minutes defined by a residual activity of from 30% to 90%, preferably from 39% to 90%, more preferably from 42% to 90%, more preferably from 54% to 90%, more preferably from 55% to 90%, and even more preferably from 63% to 90%, even more preferably from 64% to 90%, even more preferably from 68% to 90%,and most preferably from 64% to 86%; and / or from 30% to 90%, preferably from 31% to 90%, preferably from 32% to 90%, preferably from 33% to 90%, preferably from 34% to 90%, preferably from 35% to 90%, preferably from 36% to 90%, preferably from 37% to 90%, preferably from 38% to 90%, preferably from 39% to 90%, more preferably from 40% to 90%, more preferably from 41% to 90%, more preferably from 42% to 90%, more preferably from 43% to 90%, more preferably from 44% to 90%, more preferably from 45% to 90%, more preferably from 46% to 90%, more preferably from 47% to 90%, more preferably from 48% to 90%, more preferably from 49% to 90%, even more preferably from 50% to 90%, even more preferably from 51% to 90%, even more preferably from 52% to 90%, even more preferably from 53% to 90%, even more preferably from 54% to 90%, even more preferably from 55% to 90%, even more preferably from 60% to 90%, even more preferably from 61% to 90%, even more preferably from 65% to 90%, even more preferably from 70% to 90%, even more preferably from 75% to 90%, and most preferably from 72% to 81%, and / or from 55% to 100%, preferably from 60% to 100%, preferably from 70% to 100%, preferably from 75% to 100%, preferably from 76% to 100%, preferably from 77% to 100%, preferably from 78% to 100%, preferably from 79% to 100%, more preferably from 80% to 100%, more preferably from 81% to 100%, more preferably from 82% to 100%, more preferably from 83% to 100%, more preferably from 84% to 100%, more preferably from 85% to 100%, more preferably from 86% to 100%, more preferably from 87% to 100%, more preferably from 88% to 100%, more preferably from 89% to 100%, even more preferably from 90% to 100%, even more preferably from 91% to 100%, even more preferably from 92% to 100%, even more preferably from 93% to 100%, even more preferably from 94% to 100%, even more preferably from 95% to 100%, even more preferably from 96% to 100%, even more preferably from 97% to 100%, even more preferably from 98% to 100%, even more preferably from 99% to 100%, and most preferably 100%.

[0151] In a further thembodiment of the second aspect, the trehalose phosphorylase has thermal stability characterized by a Tm30-value of at least 52 °C.

[0152] In a further embodiment of the second aspect, the trehalose phosphorylase has a half-life at 45 °C of from 3 hours to 9 days or more, preferably of from 24 hours to 9 days or more, preferably of from 39 hours to 9 days or more, preferably of from 2 days to 9 days or more, more preferably of from 4 day to 9 days or more, more preferably of from 5.5 days to 9 days or more, more preferably of from 7 days to 9 days or more, even more preferably of at least 9 days or more, and most preferably of 9 days.

[0153] In a further embodiment of the second aspect, the trehalose phosphorylase has relative activity expressed as 100 / 500-ratio of between 0.65 and 1.0, preferably of between 0.7 and 1.0, preferably of between 0.75 and 1.0, preferably of between 0.8 and 1.0, preferably of between 0.85 and 1.0, preferably of between 0.9 and 1.0, preferably of between 0.95 and 1.0, and more preferably of between 0.7 and 1.0.

[0154] The trehalose phosphorylase has thermal stability characterized by a Tm30-value of at least 52 °C and / or a Tm50-value of at least 52°C; and the trehalose phosphorylase is defined as in any one of the claims.

[0155] More specifically, these and other problems are solved in a third aspect, by a thermally stable trehalose phosphorylase variant as described in the claims, 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.

[0156] In an embodiment of the third aspect, 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.

[0157] More specifically, these and other problems are solved in a fourth aspect, by a thermally stable variant of trehalose phosphorylase as described in the claims, which is derived from the organism Schizophyllum commune, wherein the variant has a residual activity of at least 30% after incubation at 52° C for 15 minutes, 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.

[0158] More specifically, these and other problems are solved in a 16 th< aspect, by the trehalose phosphorylase as described in the claims, wherein the trehalose phosphorylase is an enzymatically active fragment of the trehalose phosphorylase of the invention, as defined in any aspect or in any of the preferred embodiments of an aspect described herein and / or above.

[0159] .

[0160] More specifically, these and other problems are solved in a ninth aspect, by 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 as described in the claims, 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,

[0161] In an embodiment of the ninth aspect, the reaction is performed at from 40 to 55° C.

[0162] In an embodiment of the ninth aspect, the reaction is performed at from 45 to 55° C.

[0163] In a further embodiment the only stabilizing agent contained in the reaction is sucrose.

[0164] In a further embodiment the trehalose phosphorylase is present in non-immobilized form.

[0165] More specifically, these and other problems are solved in an eleventh aspect, by 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 said 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.

[0166] In an embodiment of the eleventh aspect, the method further comprises testing whether the substituted 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, results in increased thermal stability of the second trehalose phosphorylase compared to the thermal stability of the first trehalose phosphorylase, preferably thermal stability is determined as defined in the description herein.

[0167] In another embodiment of the eleventh aspect, the 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, is substituted such that the substituted amino acid residue results in increased thermal stability of the second trehalose phosphorylase.

[0168] In another embodiment of the eleventh aspect, the homology between the amino acid sequence of the first trehalose phosphorylase and the amino acid sequence of the second trehalose phosphorylase is at least 50 %.

[0169] In another embodiment of the eleventh aspect, the homology is at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70,%, 72%, 73 % , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably the homology is at least 55%, 60%, 63%, 65%, 68%, 70%, 75%, 77% or 80%.

[0170] In another embodiment of the eleventh aspect, the identity between the amino acid sequence of the first trehalose phosphorylase and the amino acid sequence of the second trehalose phosphorylase is at least 50 %.

[0171] In another embodiment of the eleventh aspect, the identity is at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70,%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably, the identity is at least 55%, 60%, 63%, 65%, 68%, 70%, 75%, 77% or 80%, and more preferably the identity is at least 68%, 73 %, 75 %, 76 %, 77% or 78%.

[0172] In another embodiment of the eleventh aspect, the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is 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, preferably the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is each and individually 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, preferably selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 more preferably selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 487, 550, 556, 564, 590, and 649 SEQ ID NO: 1, even more preferably selected from the group consisting of amino acid positions of 383, 114, 118, 225, 304, 323, 487, 550, 556, and 564 SEQ ID NO: 1, and even more preferably selected from the group consisting of amino acid positions 383, 114, 225, 323, 487, 556, and 564 of SEQ ID NO: 1.

[0173] In another embodiment of the eleventh aspect, the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is selected from the group consisting of amino acid positions L712, P383, V10, L114, 1118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO: 1, preferably the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is each and individually selected from the group consisting of amino acid positions L712, P383, V10, L114, 1118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705 of SEQ ID NO: 1, preferably selected from the group consisting of amino acid positions L712, P383, L114, 1118, N225, A304, T323, F349, Q487, V550, S556, T564, D590, and A649 SEQ ID NO: 1, more preferably selected from the group consisting of P383, L114, 1118, N225, A304, T323, Q487, V550, S556, and T564 of SEQ ID NO: 1, and even more preferably selected from the group consisting of amino acid positions P383, L114, N225, A323, Q487, S556, and T564 of SEQ ID NO: 1. In another embodiment of the eleventh aspect w, the substituted amino acid residue is: A, G, I, M, P, V, preferably M for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 712 of SEQ ID NO:1 of the first trehalose phosphorylase; A, G, I, L, M, V, N, C, Q, S, T, preferably A, G, M, V, N, C, Q, S, T, more preferably G, V, S or T, even more preferably V or T, and most, preferably V, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 383 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H or K, preferably R for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 10 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, M, P, V, preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 114 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 118 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 192 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably G for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 197 of SEQ ID NO: 1 of the first trehalose phosphorylase; F, W, preferably F for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 220 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I, L, M or V, and most preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 225 of SEQ ID NO: 1 of the first trehalose phosphorylase; G, I, L, M, P, V, preferably I or L, and more preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 304 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H, K, preferably H for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 306 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H, K, preferably H for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 318 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I or V, more preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 323 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 339 of SEQ ID NO: 1 of the first trehalose phosphorylase; W or Y, preferably Y for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 349 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, I, L, M, P, V, preferably A for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 357 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 459 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably G for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 476 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 481 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 484 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably A, M, G, V or L, more preferably A, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 487 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably A for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 488 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 506 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 511 of SEQ ID NO: 1 of the first trehalose phosphorylase; D or E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 526 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 530 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H, K, preferably R for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 532 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably G for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 533 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably M for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 537 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, preferably I or P, more preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 550 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, T, preferably T for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 556 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 564 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, A, G, I, L, M, P, V, preferably N or A, more preferably N, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 590 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 649 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, R, H, K, preferably E or K, more preferably E, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 667 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 703 of SEQ ID NO: 1 of the first trehalose phosphorylase; and N, C, Q, S, T, preferably N for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 705 of SEQ ID NO: 1 of the first trehalose phosphorylase.

[0174] In another embodiment of the eleventh aspect, if the amino acid residue of the second trehalose phosphorylase corresponding to one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase, is the same as the substituted amino acid residue of the first trehalose phosphorylase, the amino acid residue of the second trehalose phosphorylase is not substituted.

[0175] More specifically, these and other problems are solved in a twelfth aspect, by a method for increasing the relative activity of a trehalose phosphorylase expressed as 100 / 500-ratio, 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 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 100 / 500-ratio activity 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 said 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.

[0176] In an embodiment of the twelfth aspect, the method comprises testing whether the substituted 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, results in increased activity of the second trehalose phosphorylase compared to the activity of the first trehalose phosphorylase.

[0177] In a further embodiment of the twelfth aspect, the 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, is substituted such that the substituted amino acid residue results in increased activity of the second trehalose phosphorylase.

[0178] In a further embodiment of the twelfth aspect, the homology between the amino acid sequence of the first trehalose phosphorylase and the amino acid sequence of the second trehalose phosphorylase is at least 50 %.

[0179] In a further embodiment of the twelfth aspect, the homology is at least 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70,%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably the homology is at least 55%, 60%, 65%, 70%, 75% or 80%.

[0180] In a further embodiment of the twelfth aspect, the identity between the amino acid sequence of the first trehalose phosphorylase and the amino acid sequence of the second trehalose phosphorylase is at least 50%.

[0181] In a further embodiment of the twelfth aspect, the identity is at least 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70,%, 72%, 73 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably the identity is at least 55%, 60%, 65%, 70%, 75% or 80%, and more preferably the identity is at least 73 %, 75 %, 76 %, or 78%.

[0182] In a further embodiment of the twelfth aspect, the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is 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, preferably the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is each and individually 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, and even more preferably the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is each and individually selected from the group consisting of amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649 of SEQ ID NO: 1.

[0183] In a further embodiment of the twelfth aspect, the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is selected from the group consisting of amino acid positions of SEQ ID NO: 1 L712, P383, V10, L114, 1118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705, preferably the one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase is each and individually selected from the group consisting of amino acid positions of SEQ ID NO: 1 L712, P383, V10, L114, 1118, S192, S197, Y220, N225, A304, D306, P318, T323, L339, F349, G357, A459, Q476, E481, A484, Q487, K488, A506, A511, R526, E530, G532, D533, D537, V550, S556, T564, D590, A649, R667, A703 and K705.

[0184] In a further embodiment of the twelfth aspect, the substituted amino acid residue is A, G, I, M, P, V, preferably M for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 712 of SEQ ID NO:1 of the first trehalose phosphorylase; A, G, I, L, M, V, N, C, Q, S, T, preferably A, G, M, V, N, C, Q, S or T, more preferably G, V, C, S or T, even more preferably V or T, and most preferably V, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 383 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H or K, preferably R for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 10 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, M, P, V, preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 114 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 118 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 192 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably G for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 197 of SEQ ID NO: 1 of the first trehalose phosphorylase; F, W, preferably F for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 220 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably, I, L, M or V, and more preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 225 of SEQ ID NO: 1 of the first trehalose phosphorylase; G, I, L, M, P, V, preferably I or L, and more preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 304 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H, K, preferably H for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 306 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H, K, preferably H for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 318 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I or V, and more preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 323 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 339 of SEQ ID NO: 1 of the first trehalose phosphorylase; W or Y, preferably Y for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 349 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, I, L, M, P, V, preferably A for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 357 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 459 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably G for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 476 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 481 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 484 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably A, G, V or L, more preferably A, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 487 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably A for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 488 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 506 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, preferably S for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 511 of SEQ ID NO: 1 of the first trehalose phosphorylase; D or E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 526 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably V for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 530 of SEQ ID NO: 1 of the first trehalose phosphorylase; R, H, K, preferably R for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 532 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably G for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 533 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, V, preferably M for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 537 of SEQ ID NO: 1 of the first trehalose phosphorylase; A, G, I, L, M, P, preferably I or P, and more preferably I for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 550 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, T, preferably T for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 556 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 564 of SEQ ID NO: 1 of the first trehalose phosphorylase; N, C, Q, S, T, A, G, I, L, M, P, V, preferably N, G or A, and more preferably N, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 590 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 649 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, R, H, K, preferably E or K, more preferably E, for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 667 of SEQ ID NO: 1 of the first trehalose phosphorylase; D, E, preferably E for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 703 of SEQ ID NO: 1 of the first trehalose phosphorylase; and N, C, Q, S, T, preferably N for the amino acid position of the second trehalose phosphorylase corresponding to amino acid position 705 of SEQ ID NO: 1 of the first trehalose phosphorylase.

[0185] In a further embodiment of the twelfth aspect, if the amino acid residue of the second trehalose phosphorylase corresponding to one or more amino acid positions of the amino acid sequence of the first trehalose phosphorylase, is the same as the substituted amino acid residue of the first trehalose phosphorylase, the amino acid residue of the second trehalose phosphorylase is not substituted.

[0186] In a further embodiment of the twelfth aspect, activity of trehalose phosphorylase is phosphorolysis activity and synthesis activity as defined in the description.

[0187] It will be understood that the trehalose phosphorylase described herein in its various aspects and embodiments may also be referred to as the trehalose phosphorylase of the present invention.

[0188] The present invention is based on the surprising finding that trehalose phosphorylase of EC number EC 2.4.1.231, which is also referred to as "TP", may be improved by enzyme engineering. As a starting point for such enzyme engineering trehalose phosphorylase having an amino acid sequence according to SEQ ID NO: 1 was used. Said trehalose phosphorylase having an amino acid sequence according to SEQ ID NO: 1 may be prepared from Shizophyllum commune which was characterized by Eis et al. (FEBS Letters 440, 440-443 (1998)). UniProt: D8PWQ7 (GenBank: EFJ00254.1) discloses the wild type protein sequence of an individual Schizophyllum commune isolate H4-8 (99.1 % identity). The sequence deviates from SEQ ID NO: 1 in seven positions of SEQ ID NO: 1, wherein the isolate H4-8 carries a lysine amino acid residue in the position homologous to position 10 of SEQ ID NO: 1, a serine amino acid residue in the position homologous to position 32 of SEQ ID NO: 1, a valine amino acid residue in the position homologous to position 374 of SEQ ID NO: 1, a glutamic acid amino acid residue in the position homologous to position 535 of SEQ ID NO: 1, a proline amino acid residue in the position homologous to position 536 of SEQ ID NO: 1, an isoleucine amino acid residue in the position homologous to position 616 of SEQ ID NO:1and a valine amino acid residue in the position homologous to position 649 of SEQ ID NO: 1. UniProt: A0A151VW19 (GenBank: KYQ39707.1) discloses the wild type protein sequence of a trehalose phosphorylase from the organism Hypsizygus marmoreus, which is to 77.9 % identical to SEQ ID NO: 1. Besides Schizophyllum commune TP, other TPs from fungal origin have been described, e.g. Lentinus sajor-caju (73.2 % homology), Pleurotus ostreatus (75.8 % homology), or Grifola frondosa (76.7 % homology). For Grifola frondosa, protein sequences derived from different isolates have been described, amongst them the sequences SEQ ID NO: 160 (UniProtKB / Swiss-Prot: Accession No: 075003.1) and SEQ ID NO: 81 (Genbank Accession No: ADM15725.1).

[0189] The present inventors have surprisingly found that replacing an amino acid residue at one or several of the following amino acid positions of the amino acid sequence of SEQ ID NO: 1 is suitable for improving the reaction characteristics of the trehalose phosphorylase 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. Among these amino acid positions of SEQ ID NO: 1 amino acid positions 712, 383, 10, 114, 118, 220, 225, 304, 318, 323, 349, 357, 481, 487, 488, 506, 511, 550, 556, 564, 590, 649, 703 and 705 are particularly suitable, with amino acid positions 712, 383, 114, 118, 225, 304, 323, 349, 357, 487, 550, 556, 564, 590 and 649, each of SEQ ID NO: 1 being even more suitable.

[0190] In accordance therewith, the present invention relates in a first aspect to a polypeptide comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical 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, wherein when the amino acid substitution comprises a substitution at amino acid position 649, then said amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, preferably 649E; and wherein when the amino acid substitution comprises a substitution at amino acid position 10, then said amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and, or V10H , preferably V10R, 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]; or wherein the trehalose phosphorylase is characterized by a thermal stability after incubation at 52.5°C for 15 minutes defined by a residual activity of 15% to 100%, or wherein the trehalose phosphorylase compared to the trehalose phosphorylase of SEQ ID NO:1, is characterized by an increase in thermal stability, whereby the increase in thermal stability is an increase of the Tm30-value of at least 2 °C up to 40.5°C, or an increase of the Tm50-value of at least 2 °C up to 42.5°C, or an improved process stability, characterized by an increased half-life at 45 °C of at least 3-fold up to 216-fold or more, or at least 24-fold up to 216-fold or more.

[0191] The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of a polypeptide, whereby the polypeptide is a trehalose phosphorylase.

[0192] In accordance therewith, the present invention equally relates in a first aspect to a trehalose phosphorylase comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical 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, wherein when the amino acid substitution comprises a substitution at amino acid position 649, then said amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, preferably 649E; and wherein when the amino acid substitution comprises a substitution at amino acid position 10, then said amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and, or V10H , preferably V10R, 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]; or wherein the trehalose phosphorylase is characterized by a thermal stability after incubation at 52.5°C for 15 minutes defined by a residual activity of 15% to 100%, or wherein the trehalose phosphorylase compared to the trehalose phosphorylase of SEQ ID NO:1, is characterized by an increase in thermal stability, whereby the increase in thermal stability is an increase of the Tm30-value of at least 2 °C up to 40.5°C, or an increase of the Tm50-value of at least 2 °C up to 42.5°C, or an improved process stability, characterized by an increased half-life at 45 °C of at least 3-fold up to 216-fold or more, or at least 24-fold up to 216-fold or more.

[0193] In an embodiment, the polypeptide of the first aspect is a trehalose phosphorylase having one or several of the characteristics disclosed herein. In accordance therewith, the disclosure of the trehalose phosphorylase of the instant invention and in particular according to each and any aspect equally applies to the polypeptide of the instant invention, and vice versa. Furthermore, in an embodiment the trehalose phosphorylase of the invention has trehalose phosphorylase activity. Preferably, trehalose activity is one which is defined herein.

[0194] The wording that a trehalose phosphorylase comprises 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, has, in an embodiment, the same meaning as the wording that a trehalose phosphorylase has at least 80% homology to SEQ ID NO: 1, wherein the trehalose phosphorylase comprises a substitution at one or more positions selected from 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, where each position corresponds to a position of the amino acid sequence of SEQ ID NO: 1.

[0195] Insofar, the trehalose phosphorylase of the present invention, preferably including any aspect of the present invention, is a mutant trehalose phosphorylase and more specifically a mutant trehalose phosphorylase of a trehalose phosphorylase having an amino acid sequence of SEQ ID NO: 1. In an embodiment, the trehalose phosphorylase of the present invention is a trehalose phosphorylase having an amino acid sequence different from the amino acid sequence of SEQ ID NO: 1. In a further embodiment, the trehalose phosphorylase of the present invention is a trehalose phosphorylase which is different from a trehalose phosphorylase consisting of an amino acid sequence of SEQ ID NO: 1.

[0196] In accordance with the first aspect of the present invention the trehalose phosphorylase of the present invention comprises at least one amino acid substitution at at least one amino acid position of SEQ ID NO: 1. It is of note that, in principle, both the amino acid position where such substitution is made and the kind of substituted amino acid are of importance. In accordance therewith, in an embodiment of the trehalose phosphorylase of the present invention the amino acid substitution is made at one of the specific, indicated amino acid positions of SEQ ID NO: 1, whereby, optionally, the very amino acid residue present at such position of SEQ ID NO: 1 does not have any bearing on the kind of substituted amino acid residue present in the trehalose phosphorylase of the present invention; alternatively, and again optionally, the very amino acid residue present at such position of SEQ ID NO:1 has a bearing on the kind of substituted amino acid residue present in the trehalose phosphorylase of the present invention. The substitution may be non-conservative or conservative. For the purposes of the present invention, conservative means an exchange of the amino acid G to A; A to G, S; V to I, L,A,T,S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R.

[0197] Also in accordance therewith, in an embodiment of the trehalose phosphorylase of the present invention the amino acid substitution is one where the very amino acid residue of any of the indicated amino acid positions of SEQ ID NO: 1 is substituted, whereby, optionally, the amino acid position of the substituted amino acid residue is not exactly the one as indicated by reference to SEQ ID NO: 1; rather, in an embodiment, the amino acid position of the substituted amino acid residue differs from the corresponding position of SEQ ID NO: 1, preferably by one to five, preferably one to three, more preferably one to two and most preferably one amino acid position.

[0198] To the extent the trehalose phosphorylase of the present invention is characterized as comprising an amino acid substitution at one or more specified amino acid positions it will be understood that the trehalose phosphorylase of the present invention is one which has a single substitution at one of the indicated amino acid positions; it will, however, also be understood that the thus described trehalose phosphorylase of the present invention also encompasses those trehalose phosphorylases where more than one amino acid residue is substituted, such as two, three etc. amino acid residues. In the latter case, the number of amino acid residues which is substituted is any integer between one and the number of amino acid residues indicated and defined as being substituted. In accordance therewith, the trehalose phosphorylase of the present invention is any trehalose phosphorylase which has or realizes one amino acid substitution or any combination, actually each and any permutation, of the amino acid residues indicated and defined as being substituted.

[0199] In accordance therewith, the trehalose phosphorylase may comprise an amino acid substitution at one or any combination and, respectively, permutation of amino acid position of 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. Accordingly, in an embodiment the trehalose phosphorylase of the present invention comprises at least an amino acid substitution at one of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least two of said amino acid positions of SEQ ID NO: 1, at least one amino acid one amino acid substitution at at least three of said amino acid positions of SEQ ID NO: 1, at least one amino acid one amino acid substitution at at least four of said amino acid positions of SEQ ID NO: 1, at least one amino acid one amino acid substitution at at least five of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least six of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least seven of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least eight of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least nine of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least ten of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least eleven of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least twelve of said amino acid positions of SEQ ID NO: 1, at least one amino acid substitution at at least thirteen of said amino acid positions of SEQ ID NO: 1, or at least one amino acid substitution at at least fourteen of said amino acid positions of SEQ ID NO: 1.

[0200] It is, however, also within the present invention that the trehalose phosphorylase of the present invention comprises even more than the above indicated substitutions at the indicated amino acid positions of SEQ ID NO: 1, preferably under the provision that the amino acid sequence of the trehalose phosphorylase meets at least one of the minimum structural feature and / or minimum functional features of the trehalose phosphorylase of the present invention. Such minimum structural feature is that the trehalose phosphorylase comprises an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical 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 each and independently 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. Such minimum functional feature is one or any combination of any one of characteristics (A), (B), (C), (D) and (E) including any further specifications thereof disclosed herein, whereby characteristic (A) is thermal stability after incubation at 52°C for 15 minutes defined by a residual activity of (i) at least 30% up to 90 %; or (ii) at least 55% up 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 a Tm50-value of at least 52 °C, characteristic (C) is thermal stability characterized by a Tm30-value between 52°C and 90°C and / or a Tm50-value between 52°C and 90°C, characteristic (D) is thermal stability characterized by (i) process stability characterized by a half-life at 45°C of at least 3 hours up to 9 days, (ii) process stability characterized by a half-life at 45°C of at least 24 hours up to 9 days and / or process stability characterized by a half-life at 45°C of at least 4 days up to 9 days and characteristic (E) is relative activity expressed as 100 / 500-ratio of between at least 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]. It is within the present invention that the trehalose phosphorylase of the present invention displays characteristic (A), characteristic (B), characteristic (C), characteristic (D), characteristic (E), characteristics (A) and (B), characteristics (A) and (C), characteristics (A) and (D), characteristics (A) and (E), characteristics (B) and (C), characteristics (B) and (D), characteristics (B) and (E), characteristics (C) and (D), characteristics (C) and (E), characteristics (D) and (E), characteristics (A), (B) and (C), characteristics (A), (B) and (D), characteristics (A), (B) and (E), characteristics (B), (C) and (D), characteristics (B), (C) and (E), characteristics (C), (D) and (E), characteristics (A), (B), (C) and (D), characteristics (A), (B), (C) and (E), characteristics (B), (C), (D) and (E), characteristics (A), (C), (D) and (E), or characteristics (A), (B), (C), (D) and (E).

[0201] In an embodiment of the trehalose phosphorylase of the present invention, the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at at least two amino acid positions of the amino acid sequence of SEQ ID NO: 1. These at least two amino acid positions are also referred to herein as a pair of two amino acid positions. In an embodiment of the trehalose phosphorylase of the present invention the pair of two amino acid positions is selected from the group consisting of V10 and L114, V10 and 1118, V10 and Y220, V10 and N225, V10 and A304, V10 and T323, V10 and F349, V10 and G357, V10 and P383, V10 and Q487, V10 and A506, V10 and V550, V10 and S556, V10 and T564, V10 and D590, V10 and A649, V10 and K705, V10 and L712, L114 and 1118, L114 and Y220, L114 and N225, L114 and A304, L114 and T323, V10 and F349, L114 and G357, L114 and P383, L114 and Q487, L114 and A506, L114 and V550, L114 and S556, L114 and T564, L114 and D590, L114 and A649, L114 and K705, L114 and L712, 1118 an Y220, 1118 and N225, 1118 and A304, 1118 and T323, V10 and F349, 1118 and G357, 1118 and P383, 1118 and Q487, 1118 and A506, 1118 and V550, 1118 and S556, 1118 and T564, 1118 and D590, 118 and A649, L118 and K705, 1118 and L712, Y220 and N225, Y220 and A304, Y220 and T323, V10 and F349, Y220 and G357, Y220 and P383, Y220 and Q487, Y220 and A506, Y220 and V550, Y220 and S556, Y220 and T564, Y220 and D590, Y220 and A649, Y220 and K705, Y220 and L712, N225 and A304, N225 and T323, V10 and F349, N225 and G357, N225 and P383, N225 and Q487, N225 and A506, N225 and V550, N225 and S556, N225 and T564, N225 and D590, N225 and A649, N225 and K705, N225 and L712, A304 and T323, V10 and F349, A304 and G357, A304 and P383, A304 and Q487, A304 and A506, A304 and V550, A304 and S556, A304 and T564, A304 and D590, A304 and A649, A304 and K705, A304 and L712, T323 and F349, T323 and G357, T323 and P383, T323 and Q487, T323 and A506, T323 and V550, T323 and S556, T323 and T564, T323 and D590, T323 and A649, T323 and K705, T323 and L712, F349 and G357, F349 and P383, F349 and Q487, F349 and A506, F349 and V550, F349 and S556, F349 and T564, F349 and D590, F349 and A649, F349 and K705, F349 and L712, G357 and P383, G357 and Q487, G357 and A506, G357 and V550, G357 and S556, G357 and T564, G357 and D590, G357 and A649, G357 and K705, G357 and L712, P383 and Q487, P383 and A506, P383 and V550, P383 and S556, P383 and T564, P383 and D590, P383 and A649, P383 and K705, P383 and L712, Q487 and A506, Q487 and V550, Q487 and S556, Q487 and T564, Q487 and D590, Q487 and A649, Q487 and K705, Q487 and L712, A506 and V550, A506 and S556, A506 and T564, A506 and D590, A506 and A649, A506 and K705, A506 and L712, V550 and S556, V550 and T564, V550 and D590, V550 and A649, V550 and K705, V550 and L712, S556 and T564, S556 and D590, S556 and A649, S556 and K705, S556 and L712, T564 and D590, T564 and A649, T564 and K705, T564 and L712, D590 and A649, D590 and K705, D590 and L712, A649 and L712, A649 and K705, and K705 and L712. Further embodiments of the trehalose phosphorylase of the present invention are those where the pair of two amino acid positions is selected from a more limited group. It is within the present invention that a trehalose phosphorylase comprises an amino acid substitution at one of these pairs of two amino acid positions of SEQ ID NO: 1.

[0202] As disclosed herein, in a further embodiment the trehalose phosphorylase comprises in addition to amino acid substitution at the pair of two amino acid positions of SEQ ID NO: 1 an amino acid substitution at at least one or more additional amino acid positions of SEQ ID NO: 1. Such one or more additional amino acid positions of SEQ ID NO: 1 are individually and independently 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, each of SEQ ID NO: 1. Further embodiments of the trehalose phosphorylase of the present invention are those where the one or more additional amino acid position is selected from a more limited group. It is within the present invention that a trehalose phosphorylase comprises an amino acid substitution at one of these pairs of two amino acid positions of SEQ ID NO: 1 and at one of said additional amino acid sequences. In accordance therewith, the trehalose phosphorylase of the present invention is, each in an embodiment, a trehalose phosphorylase having an amino acid substitution at at least three amino acid positions, namely at the pair of two amino acid position and at one of said additional amino acid positions of SEQ ID NO: 1, a trehalose phosphorylase having an amino acid substitution at at least four amino acid positions, namely at the pair of two amino acid position and at two of said additional amino acid positions of SEQ ID NO: 1, a trehalose phosphorylase having an amino acid substitution at at least five amino acid positions, namely at the pair of two amino acid position and at three of said additional amino acid positions of SEQ ID NO: 1, or a trehalose phosphorylase having an amino acid substitution at at least six amino acid positions, namely at the pair of two amino acid position and at four of said additional amino acid positions of SEQ ID NO: 1. An embodiment of the trehalose phosphorylase of the present invention wherein the trehalose phosphorylase comprises an amino acid substitution at at least four amino acid positions, is a trehalose phosphorylase comprising an amino acid substitution at amino acid positions 712, 383, 114 and 118, each of SEQ ID NO: 1. An embodiment of the trehalose phosphorylase of the present invention wherein the trehalose phosphorylase comprises an amino acid substitution at at least five amino acid positions, is a trehalose phosphorylase comprising an amino acid substitution at amino acid positions 712, 383, 114, 118 and 304, each of SEQ ID NO: 1, and / or at amino acid positions 712, 383, 114, 118 and 357, each of SEQ ID NO: 1. An embodiment of the trehalose phosphorylase of the present invention wherein the trehalose phosphorylase comprises an amino acid substitution at at least six amino acid positions, is a trehalose phosphorylase comprising an amino acid substitution at amino acid positions 712, 383, 114, 118, 304 and 357, each of SEQ ID NO: 1.

[0203] In a further embodiment, the trehalose phosphorylase comprises in addition to the amino acid substitution at the pair of two amino acid positions of SEQ ID NO:1 and the amino acid substitution at at least one more additional amino acid positions of SEQ ID NO: 1, with the one or more additional amino acid position of SEQ ID NO: 1 being preferably one, two, three or four amino acid positions of SEQ ID NO: 1 as disclosed herein, an amino acid substitution at one or more still further amino acid positions of SEQ ID NO:1. Such one or more still further amino acid positions of SEQ ID NO: 1 are individually and independently selected from the group consisting of amino acid positions 10, 192, 197, 220, 225, 306, 318, 323, 339, 349, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 649, 667, 703 and 705, each of SEQ ID NO: 1. Further embodiments of the trehalose phosphorylase of the present invention are those where the one or more still further amino acid position is selected from a more limited group.

[0204] As to specific substitutions disclosed herein it is to be noted that in accordance with common practice in the field of amino acid substitutions the substitution refers to the number of the amino acid position where a substitution is made, with the left number being flanked on both sides by the indication of an amino acid residue with the amino acid residue on the left side being the amino acid residue as present in the non-substituted amino acid sequence and the amino acid residue on the right side being the amino acid residue as present in the substituted amino acid sequence. Accordingly and for illustrative purposes only, substitution V10R indicated that at amino acid position 10 the valine reside is replaced by arginine.

[0205] It is within the present invention that any of the mutations at an amino acid positions of SEQ ID NO: 1 as disclosed herein is one which results, either alone or in any combination, in a protein which is still active as a trehalose phosphorylase, more specifically as a trehalose phosphorylase in accordance with EC 2.4.1.213. Preferably, any of said mutations results, either alone or in any combination, in a trehalose phosphorylase of EC number EC 2.4.1.213 having at least one of characteristics (A), (B), (C), (D) and (E), or any combination of such characteristics, as disclosed herein.

[0206] It will be understood that in connection with the trehalose phosphorylase of the present invention apart from the substitutions specifically discloses herein, preferably the substitutions at the amino acid positions of SEQ ID NO:1 disclosed herein, further and / or different substitution may be made. Preferably, such further substitutions are made at positions different from amino acid positions D379, H403, R507 and K512, each of SEQ ID NO: 1, which positions are described to be essential for proper reaction catalysis.

[0207] It is known how the identity and homology, respectively, of a polymer of amino acid residues is determined. Homology is preferably calculated as identity using BLASTP (see, for example, Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; or Stephen F. Altschul, John C. Wootton, E. Michael Gertz, Richa Agarwala, Aleksandr Morgulis, Alejandro A. Schäffer, and Yi-Kuo Yu (2005) "Protein database searches using compositionally adjusted substitution matrices." FEBS J. 272:5101-5109).

[0208] In an embodiment, the trehalose of the present invention is a phosphorylase classified as EC 2.4.1.231 which catalyzes phosphorolytic cleavage of trehalose with net retention of the anomeric configuration using inorganic phosphate as glucosyl acceptor into glucose and alpha-D-glucose-1 phosphate (aG1P). Such reaction is reversible and, accordingly, the trehalose phosphorylase of the present invention converts glucose and alpha-D-glucose-1 phosphate (aG1P) into trehalose and inorganic phosphate. Trehalose is the disaccharide alpha-d-glucopyranosyl alpha-d-glucopyranoside (alpha,alpha-trehalose), characterized as the molecular entity with the molecular structure as defined by IUPAC as (2R,3S,4S,5R,6R)-2-(Hydroxymethyl) -6-[(2R,3R,4S,5S,6R)-3,4, 5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-3,4,5-triol in any physical form.

[0209] In a further embodiment, based on amino acid sequence similarity, the trehalose phosphorylase of the present invention is classified as a member of the glycosyltransferase family GT4 in the Carbohydrate-Active Enzymes database (CAZy, Lombard et al (2014) Nucleic Acids Res 42: D490-D495, http: / / www.cazy.org / ).

[0210] In an embodiment, the trehalose phosphorylase of the present invention reacts a glucosyl monosaccharide and alpha-D-glucose-1 phosphate. In a preferred embodiment, the glucosyl monosaccharide is glucose, whereby the reaction product is preferably trehalose.

[0211] In a further embodiment, trehalose phosphorylase of the present invention reacts monosaccharides different from glucose. Such monosaccharides different from glucose are preferably selected from the group comprising D-mannose, 2-deoxy D-glucose, 2-fluoro D-glucose 2-deoxy-2-fluoro-D-glucose, 5-thio-D-glucose and 2-keto-D-glucose, which are described as suitable monosaccharides for phosphorylase classified as EP 2.4.1.231see, for example, (Schwarz et al. J Biotechnol 129, 140-150 (2007), Nidetzky 2001 et al Biochem J (2001) 360, 727-736)).

[0212] In a still further embodiment, the trehalose phosphorylase of the present invention does not react beta-D glucose 1-phosphate, alpha-D-galactose 1- phosphate, alpha-D-mannose 1- phosphate, or alpha-D-xylose 1-phosphate as alternative glycosyl donors instead of aG1P (Schwarz et al. J Biotechnol 129, 140-150 (2007), Saito et al Appl Microbiol Biotechnol 64: 4340-4345 (1998)).

[0213] In accordance with another embodiment, the trehalose phosphorylase of the present invention preferably being a phosphorylase classified as EC 2.4.1.231, does not phosphorolytically cleave other disaccharides such as alpha,beta-trehalose, beta,beta-trehalose, sucrose, cellobiose, lactose, maltose, isomaltulose, isomaltose, lactulose or melibiose (see, for example, Schwarz et al. J Biotechnol 129, 140-150 (2007), Eis et al FEBS Letters 440 (1998) 440-443, Saito et al Appl Microbiol Biotechnol 64: 4340-4345 (1998)).

[0214] In a second aspect, the present invention is related to a polypeptide as described in the claims having certain functional characteristics. Preferably, the polypeptide is a polypeptide having trehalose phosphorylase activity, wherein the polypeptide is the trehalose phosphorylase

[0215] In a preferred embodiment of the second aspect, and which is also an additional aspect of the invention, the polypeptide comprises an amino acid sequence, wherein the amino acid sequence of the polypeptide, preferably of the trehalose phosphorylase, is at least 80% identical to an amino acid sequence of SEQ ID NO: 1, wherein the polypeptide, preferably trehalose phosphorylase, has at least one of characteristics (A), (B), (C), (D) and (E), or any combination thereof, with characteristic (A) being thermal stability after incubation at 52°C for 15 minutes defined by a residual activity, characteristic (B) being thermal stability after incubation at 52°C for 15 minutes, whereby such thermal stability is defined by a minimum Tm30 value and / or a minimum Tm50 value, whereby the Tm30 value and the Tm50 value are as defined herein, characteristic (C) being thermal stability defined by a Tm30 value and / or a Tm50 value between a certain temperature range, characteristic (D) being thermal stability, wherein thermal stability is defined by process stability with process stability being defined as half-life, preferably half-life of trehalose phosphorylase activity at 45°C for a certain period of time, and characteristic (E) is relative activity expressed as 100 / 500 ratio .

[0216] In another preferred embodiment of the second aspect, w the invention is related to 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 wherein the trehalose variant is a trehalose phosphorylase according to the first aspect of the present invention.

[0217] In another preferred embodiment of the second aspect, the present invention is related to a thermally stable variant of trehalose phosphorylase from Schizophyllum commune, wherein the variant has a residual activity of at least 30% after incubation at 52° C for 15 minutes, and wherein such trehalose variant is a trehalose phosphorylase according to the first aspect of the present invention.

[0218] In another preferred embodiment of the second aspect, the present invention is related to a variant of a trehalose phosphorylase comprising an amino acid sequence of SEQ ID NO: 1, wherein the variant has a residual activity of at least 30% after incubation at 52° C for 15 minutes, and wherein such trehalose variant is a trehalose phosphorylase according to the first aspect of the present invention.

[0219] In another preferred embodiment of the second aspect, the present invention is related to a polypeptide, preferably of the trehalose phosphorylase, which is an enzymatically active fragment of the claimed polypeptide.

[0220] In a further aspect, the present invention is related to 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 of the present invention, preferably a trehalose phosphorylase according to the first and / or second aspect.

[0221] In a further aspect, the present invention is related to a method for converting glucose and alpha-D-glucose-1 phosphate into trehalose and inorganic phosphate, wherein the method comprises reacting glucose and alpha-D-glucose-1 phosphate to trehalose and inorganic phosphate with a trehalose phosphorylase of the present invention, preferably a trehalose phosphorylase according to the first and / or second aspect.

[0222] In an further aspect, the present invention is related to a method for converting trehalose and inorganic phosphate into glucose and alpha-D-glucose-1 phosphate, and wherein the method comprises reacting trehalose and inorganic phosphate to glucose and alpha-D-glucose-1 phosphate with a trehalose phosphorylase of the present invention, preferably a trehalose phosphorylase according to the first and / or second aspect.

[0223] In a further aspect, the present invention is related to the use of a trehalose phosphorylase according to the first and / or second aspect for producing trehalose.

[0224] In a further aspect, the present invention is related to 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 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 (ii) has a ratio of activity at 100 mM glucose to catalytic activity at 500 mM glucose of at least 0.65, and wherein the trehalose phosphorylase is a trehalose phosphorylase according to the first and / or second aspect.

[0225] In a further aspect, the present invention is related to 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 said 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.

[0226] It is within the present invention that the trehalose phosphorylase of the invention is present as full-length enzyme. It is also within the present invention that the trehalose phosphorylase of the invention is present as a fragment, which retains trehalose phosphorylase activity, preferably a trehalose phosphorylase activity as defined and, respectively disclosed herein for the trehalose phosphorylase of the invention.

[0227] In a further aspect, the present invention is related to a nucleic acid molecule encoding the trehalose phosphorylase of the present invention. It is generally known to derive such nucleic acid molecule based on the amino acid sequence discloses herein. Preferably, the nucleic acid sequence depends on the expression system used for the expression of the trehalose phosphorylase of the present invention. Preferred expression systems used for the expression of trehalose phosphorylase of the invention are E. coli, Bacillus sp, P. pastoris and fungal expression systems like Aspergillus sp.

[0228] In a still further aspect, the present invention is related to a vector containing the nucleic acid molecule encoding the trehalose phosphorylase of the present invention. Preferably, the vector is an expression vector. Suitable vectors for the expression of enzymes have been described in the state of the art.

[0229] In a further aspect, the present invention is related to a host organism containing the vector of the invention. Suitable hosts for hosts containing vectors for the expression of enzymes have been described, and preferably, the host organism is are E. coli, Bacillus sp, P. pastoris or a fungal expression system like Aspergillus sp., preferably E.coli and P. pastoris. Also known are methods to incorporate such vector into the host organism.

[0230] In another aspect, the present invention is related a method for the expression of a trehalose phosphorylase. Such method comprises cultivating a host organism disclosed in the description, wherein the host organism comprises an expression vector, wherein the expression vector comprises a nucleic acid molecule encoding a trehalose phosphorylase according to the present invention, under conditions which allow expression of said nucleic acid molecule, and harvesting the trehalose phosphorylase.

[0231] In an embodiment of each and any aspect of the invention, including any embodiment thereof, the Tm30-value is determined using Assay I as disclosed herein.

[0232] In an embodiment of each and any aspect of the invention, including any embodiment thereof, the Tm30-value is determined using Assay II as disclosed herein.

[0233] In an embodiment of each and any aspect of the invention, including any embodiment thereof, the Tm50-value is determined using Assay I as disclosed herein.

[0234] In an embodiment of each and any aspect of the invention, including any embodiment thereof, the Tm50-value is determined using Assay II as disclosed herein.

[0235] It is within the present invention that the trehalose phosphorylase of each and any aspect of the invention, including any embodiment thereof, is present in one of the following forms: a liquid solution, a dry powder, a freeze-dried powder, in an immobilized form.

[0236] In embodiments of each and any aspect of the present invention, including any embodiment thereof, the following definitions apply.

[0237] Definition thermal stability: Thermal stability is the ability of an enzyme to resist irreversible inactivation after exposure to a specified elevated temperature over a given period of time. Residual activity of an enzyme incubated at the elevated temperature for a certain time is calculated as relative of the enzyme activity to a sample of the enzyme that has not been incubated at the elevated temperature. The enzyme activity can be determined in principle by using any activity assay. For the purpose of this invention Assay 1 or Assay 2 as described below, have been used as specified in the examples.

[0238] There are many ways of measuring and describing thermal stability. For the purpose of this invention thermal stability was determined by measuring and describing one or more of the following characteristics: Tm30-value: For the purpose of this invention, the Tm30-value is the temperature at which the enzyme possesses 30 % of its initial activity after incubation for 15 min at this temperature in a buffer containing 1 M sucrose. The initial activity is the activity of the respective enzyme without temperature treatment, i.e. with 15 min incubation at room temperature. The enzyme activity can be determined in principle by using any activity assay; for the purpose of this invention Assay 1 or Assay 2 as described below have been used, as specified in the examples. Tm50-value: For the purpose of this invention, the Tm50-value is the temperature at which the enzyme possesses 50 % of its initial activity after incubation for 15 min at this temperature in a buffer containing 1 M sucrose. The initial activity is the activity of the respective enzyme without temperature treatment, i.e. with 15 min incubation at room temperature. The enzyme activity can be determined in principle by using any activity assay; for the purpose of this invention Assay 1 or Assay 2 as described below have been used, as specified in the examples. process stability / half-life: For the purpose of this invention, long-term stability was determined in 50 mM potassium phosphate buffer pH 7 with 1 M sucrose at 40 or 45 °C, respectively, as indicated in the examples. The half-life is defined as the duration of time after which the enzyme possesses 50 % of the activity at t=0min. The enzyme activity can be determined in principle by using any activity assay; for the purpose of this invention Assay 1 or Assay 2 as described below have been used, as specified in the examples.

[0239] Definition of phosphorolysis activity and synthesis activity: As TPs catalyze the reversible phosphorolytic cleavage of trehalose, activity can be determined either in the direction of trehalose phosphorolysis or synthesis. For the purpose of this invention, phosphorolysis activity is defined as the activity for trehalose cleavage in the presence of inorganic phosphate to aG1P and glucose at the conditions described below as Assay 1. Synthesis activity is defined as the activity for trehalose synthesis from aG1P and glucose at the conditions described below as Assay 2. It is within the present invention that any activity and any activity of the trehalose phosphorylase is in an embodiment a catalytic activity.

[0240] 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 µM glucose 1,6-bisphosphate, 10 mM MgCl2, 225 mM trehalose, 3 U / mL phosphoglucomutase and 3.4 U / mL glucose 6-phosphate dehydrogenase.

[0241] 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 µL of sample are mixed with 250 µL 0.5 M HCl and 500 µL 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.

[0242] Definition S / P-ratio: For the purpose of this invention, the S / P-ratio is defined as the ratio between phosphorolysis activity and synthesis activity measured by Assay 1 and Assay 2, respectively, using 500mM glucose as described in the examples.

[0243] Definition 100 / 500-ratio: For the purpose of this invention, the 100 / 500-ratio is the ratio between activity of a TP variant using 100 mM glucose and activity of a TP variant using 500 mM glucose with 100 mM aG1P, respectively, according to Assay 2. An increase in 100 / 500-ratio of an enzyme variant compared to the wild-type enzyme is indicative of a decreased K M -value for glucose.

[0244] In an embodiment, if not indicated to the contrary any activity, enzymatic activity, phosphorolysis activity and synthesis activity displayed or to be displayed by the polypeptide and, preferably any trehalose phosphorylase, of the present invention is defined and, respectively, determined by the methods and assays, respectively, disclosed herein. Table 2: Overview over the Sequence IDs: Wild type Trehalose PhosphorylasesSEQ ID source SEQ ID NO: 1wild-type, S. communeSEQ ID NO: 80wild type, Hypholoma sublateritium FD-334 SS-4, Genbank: KJA27491.1SEQ ID NO: 81wild type, Grifola frondosa, Genbank: ADM15725SEQ ID NO: 82wild type, Pleurotus ostreatus, Genbank: KDQ33172.1SEQ ID NO: 83wild type, Lentinus sajor-caju, UniProtKB / Swiss-Prot: Q9UV63.1SEQ ID NO: 160wild type, Grifola frondosa, UniProtKB / Swiss-Prot: O75003.1SEQ ID NO: 191Schizophyllum commune H4-8, NCBI Reference Sequence: XP_003035156.1SEQ ID NO: 192Trametes cinnabarina, Genbank: CDO74881.1SEQ ID NO: 193Hypsizygus marmoreus, Genbank: KYQ39707.1SEQ ID NO: 194Trametes versicolor FP-101664 SS1, NCBI Reference Sequence: XP_008036133.1SEQ ID NO: 195Pleurotus pulmonarius, UniProtKB / Swiss-Prot: A6YRN9.1SEQ ID NO: 196Agaricus bisporus var. bisporus H97, NCBI Reference Sequence: XP_006458503.1SEQ ID NO: 197Agaricus bisporus var. bumettii JB137-S8, NCBI Reference Sequence: XP_007326883.1SEQ ID NO: 198Laetiporus sulphureus 93-53, Genbank: KZT11205.1SEQ ID NO: 199Gloeophyllum trabeum ATCC 11539, NCBI Reference Sequence: XP_007863746.1SEQ ID NO: 200Grifola frondosa, Genbank: OBZ75413.1SEQ ID NO: 201Trametes pubescens, Genbank: OJT04097.1 Table 3: Overview over the Sequence IDs: Variants of Trehalose Phosphorylase of SEQ ID NO: 1 SEQ ID source number of mutations in variant mutations to SEQ ID NO: 1 SEQ ID NO: 2variant of SEQ ID NO:11K705NSEQ ID NO: 3variant of SEQ ID NO:12P383S, L712MSEQ ID NO: 4variant of SEQ ID NO:12K705N, L712MSEQ ID NO: 5variant of SEQ ID NO:13V10R, A506S, L712MSEQ ID NO: 6variant of SEQ ID NO:14V10R, Y220F, A506S, L712MSEQ ID NO: 7variant of SEQ ID NO:14V10R, L114I, Y220F, L712MSEQ ID NO: 8variant of SEQ ID NO:14Y220F, A506S, K705N, L712MSEQ ID NO: 9variant of SEQ ID NO:16V10R, L114I, Y220F, A506S, K705N, L712MSEQ ID NO: 10variant of SEQ ID NO:12P383V, L712MSEQ ID NO: 11variant of SEQ ID NO:18L114I, I118V, P383V, Q476G, K488A, A506S, A511S, L712MSEQ ID NO: 12variant of SEQ ID NO:19L114I, I118V, A304I, F349Y, G357A, P383V, A506S, A511S, L712MSEQ ID NO: 13variant of SEQ ID NO:17L114I, I118V, A304I, F349Y, P383V, E481I, L712MSEQ ID NO: 14variant of SEQ ID NO:16L114I, I118V, A304I, G357A, P383V, L712MSEQ ID NO: 15variant of SEQ ID NO:18L114I, I118V, F349Y, G357A, P383V, E481I, K488A, L712MSEQ ID NO: 16variant of SEQ ID NO:18L114I, I118V, A304I, P383V, E481I, A506S, A511S, L712MSEQ ID NO: 17variant of SEQ ID NO:17L114I, I118V, P383V, K488A, A506S, A511S, L712MSEQ ID NO: 18variant of SEQ ID NO:19L114I, I118V, A304I, G357A, P383V, K488A, A506S, A511S, L712MSEQ ID NO: 19variant of SEQ ID NO:15L114I, I118V, P383V, E481I, L712MSEQ ID NO: 20variant of SEQ ID NO:17L114I, I118V, A304I, F349Y, P383V, K488A, L712MSEQ ID NO: 21variant of SEQ ID NO:17L114I, I118V, P383V, E481I, A506S, A511S, L712MSEQ ID NO: 22variant of SEQ ID NO:17L114I, I118V, S192V, A304I, G357A, P383V, L712MSEQ ID NO: 23variant of SEQ ID NO:17L114I, I118V, S197G, A304I, G357A, P383V, L712MSEQ ID NO: 24variant of SEQ ID NO:17L114I, I118V, N225V, A304I, G357A, P383V, L712MSEQ ID NO: 25variant of SEQ ID NO:17L114I, I118V, A304I, D306H, G357A, P383V, L712MSEQ ID NO: 26variant of SEQ ID NO:17L114I, I118V, A304I, P318H, G357A, P383V, L712MSEQ ID NO: 27variant of SEQ ID NO:17L114I, I118V, A304I, T323I, G357A, P383V, L712MSEQ ID NO: 28variant of SEQ ID NO:17L114I, I118V, A304I, L339I, G357A, P383V, L712MSEQ ID NO: 29variant of SEQ ID NO:17L114I, I118V, A304I, F349Y, G357A, P383V, L712MSEQ ID NO: 30variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, A459S, L712MSEQ ID NO: 31variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, E481I, L712MSEQ ID NO: 32variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, A484S, L712MSEQ ID NO: 33variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, Q487V, L712MSEQ ID NO: 34variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, Q487A, L712MSEQ ID NO: 35variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, Q487L, L712MSEQ ID NO: 36variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, R526E, L712MSEQ ID NO: 37variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, E530V, L712MSEQ ID NO: 38variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, G532R, L712MSEQ ID NO: 39variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, D533G, L712MSEQ ID NO: 40variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, D537M, L712MSEQ ID NO: 41variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, V550I, L712MSEQ ID NO: 42variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, S556T, L712MSEQ ID NO: 43variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, T564E, L712MSEQ ID NO: 44variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, D590N, L712MSEQ ID NO: 45variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, D590A, L712MSEQ ID NO: 46variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, A649E, L712MSEQ ID NO: 47variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, R667E, L712MSEQ ID NO: 48variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, R667K, L712MSEQ ID NO: 49variant of SEQ ID NO:17L114I, I118V, A304I, G357A, P383V, A703E, L712MSEQ ID NO: 50variant of SEQ ID NO:19L114I, I118V, A304I, G357A, P383V, V550I, S556T, D590N, L712MSEQ ID NO: 51variant of SEQ ID NO:110L114I, I118V, N225V, A304I, G357A, P383V, Q487L, T564E, D590N, L712MSEQ ID NO: 52variant of SEQ ID NO:110L114I, I118V, A304I, T323I, G357A, P383V, V550I, T564E, D590N, L712MSEQ ID NO: 53variant of SEQ ID NO:110L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, D590N, L712MSEQ ID NO: 54variant of SEQ ID NO:111L114I, I118V, N225V, A304I, G357A, P383V, Q487L, V550I, S556T, D590N, L712MSEQ ID NO: 55variant of SEQ ID NO:111L114I, I118V, N225V, A304I, G357A, P383V, Q487L, V550I, T564E, D590N, L712MSEQ ID NO: 56variant of SEQ ID NO:111L114I, I118V, N225V, A304I, G357A, P383V, V550I, S556T, T564E, D590N, L712MSEQ ID NO: 57variant of SEQ ID NO:111L114I, I118V, N225V, A304I, G357A, P383V, V550I, T564E, D590N, A649E, L712MSEQ ID NO: 58variant of SEQ ID NO:111L114I, I118V, A304I, T323I, G357A, P383V, V550I, S556T, T564E, D590N, L712MSEQ ID NO: 59variant of SEQ ID NO:111L114I, I118V, A304I, T323I, G357A, P383V, Q487L, V550I, D590N, A649E, L712MSEQ ID NO: 60variant of SEQ ID NO:112L114I, I118V, N225V, A304I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, L712MSEQ ID NO: 61variant of SEQ ID NO:112L114I, I118V, N225V, A304I, G357A, P383V, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 62variant of SEQ ID NO:112L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, T564E, D590N, L712MSEQ ID NO: 63variant of SEQ ID NO:112L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, D590N, A649E, L712MSEQ ID NO: 64variant of SEQ ID NO:112L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, D590N, L712MSEQ ID NO: 65variant of SEQ ID NO:112L114I, I118V, N225V, A304I, T323I, G357A, P383V, V550I, S556T, T564E, D590N, L712MSEQ ID NO: 66variant of SEQ ID NO:112L114I, I118V, A304I, T323I, F349Y, G357A, P383V, Q487L, V550I, D590N, A649E, L712MSEQ ID NO: 67variant of SEQ ID NO:112L114I, I118V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, L712MSEQ ID NO: 68variant of SEQ ID NO:112L114I, I118V, A304I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 69variant of SEQ ID NO:112L114I, I118V, A304I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 70variant of SEQ ID NO:113L114I, I118V, N225V, A304I, T323I, F349Y, G357A, P383V, V550I, S556T, D590N, A649E, L712MSEQ ID NO: 71variant of SEQ ID NO:113L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, D590N, A649E, L712MSEQ ID NO: 72variant of SEQ ID NO:113L114I, I118V, N225V, A304I, F349Y, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, L712MSEQ ID NO: 73variant of SEQ ID NO:113L114I, I118V, N225V, A304I, F349Y, G357A, P383V, Q487L, V550I, T564E, D590N, A649E, L712MSEQ ID NO: 74variant of SEQ ID NO:113L114I, I118V, N225V, A304I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 75variant of SEQ ID NO:113L114I, I118V, N225V, A304I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 76variant of SEQ ID NO:113L114I, I118V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 77variant of SEQ ID NO:113L114I, I118V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 78variant of SEQ ID NO:114L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 79variant of SEQ ID NO:114L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487G, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 84variant of SEQ ID NO:11N225VSEQ ID NO: 85variant of SEQ ID NO:11A304ISEQ ID NO: 86variant of SEQ ID NO:11T323ISEQ ID NO: 87variant of SEQ ID NO:11P383VSEQ ID NO: 88variant of SEQ ID NO:11Q487ASEQ ID NO: 89variant of SEQ ID NO:11S556TSEQ ID NO: 90variant of SEQ ID NO:11T564ESEQ ID NO: 91variant of SEQ ID NO:11D590NSEQ ID NO: 92variant of SEQ ID NO:11N225ISEQ ID NO: 93variant of SEQ ID NO:11N225LSEQ ID NO: 94variant of SEQ ID NO:11N225MSEQ ID NO: 95variant of SEQ ID NO:11A304LSEQ ID NO: 96variant of SEQ ID NO:11T323VSEQ ID NO: 97variant of SEQ ID NO:11P383ASEQ ID NO: 98variant of SEQ ID NO:11P383GSEQ ID NO: 99variant of SEQ ID NO:11P383MSEQ ID NO: 100variant of SEQ ID NO:11P383NSEQ ID NO: 101variant of SEQ ID NO:11P383CSEQ ID NO: 102variant of SEQ ID NO:11P383QSEQ ID NO: 103variant of SEQ ID NO:11P383SSEQ ID NO: 104variant of SEQ ID NO:11P383TSEQ ID NO: 105variant of SEQ ID NO:11Q487GSEQ ID NO: 106variant of SEQ ID NO:11Q487LSEQ ID NO: 107variant of SEQ ID NO:11Q487MSEQ ID NO: 108variant of SEQ ID NO:11V550PSEQ ID NO: 109variant of SEQ ID NO:11D590GSEQ ID NO: 110variant of SEQ ID NO:12I118V, P383VSEQ ID NO: 111variant of SEQ ID NO:12I118V, S556TSEQ ID NO: 112variant of SEQ ID NO:12I118V, T564ESEQ ID NO: 113variant of SEQ ID NO:12I118V, D590NSEQ ID NO: 114variant of SEQ ID NO:12N225V, A304ISEQ ID NO: 115variant of SEQ ID NO:12N225V, P383VSEQ ID NO: 116variant of SEQ ID NO:12N225V, Q487ASEQ ID NO: 117variant of SEQ ID NO:12N225V, V550ISEQ ID NO: 118variant of SEQ ID NO:12N225V, S556TSEQ ID NO: 119variant of SEQ ID NO:12N225V, D590NSEQ ID NO: 120variant of SEQ ID NO:12A304I, T323ISEQ ID NO: 121variant of SEQ ID NO:12A304I, P383VSEQ ID NO: 122variant of SEQ ID NO:12A304I, Q487ASEQ ID NO: 123variant of SEQ ID NO:12A304I, S556TSEQ ID NO: 124variant of SEQ ID NO:12A304I, T564ESEQ ID NO: 125variant of SEQ ID NO:12A304I, D590NSEQ ID NO: 126variant of SEQ ID NO:12T323I, G357ASEQ ID NO: 127variant of SEQ ID NO:12T323I, Q487ASEQ ID NO: 128variant of SEQ ID NO:12T323I, S556TSEQ ID NO: 129variant of SEQ ID NO:12T323I, T564ESEQ ID NO: 130variant of SEQ ID NO:12T323I, D590NSEQ ID NO: 131variant of SEQ ID NO:12T323I, A649ESEQ ID NO: 132variant of SEQ ID NO:12F349Y, P383VSEQ ID NO: 133variant of SEQ ID NO:12F349Y, D590NSEQ ID NO: 134variant of SEQ ID NO:12G357A, P383VSEQ ID NO: 135variant of SEQ ID NO:12G357A, D590NSEQ ID NO: 136variant of SEQ ID NO:12P383V, Q487ASEQ ID NO: 137variant of SEQ ID NO:12P383V, V550ISEQ ID NO: 138variant of SEQ ID NO:12P383V, S556TSEQ ID NO: 139variant of SEQ ID NO:12P383V, T564ESEQ ID NO: 140variant of SEQ ID NO:12P383V, D590NSEQ ID NO: 141variant of SEQ ID NO:12P383V, A649ESEQ ID NO: 142variant of SEQ ID NO:12Q487A, T564ESEQ ID NO: 143variant of SEQ ID NO:12Q487A, D590NSEQ ID NO: 144variant of SEQ ID NO:12Q487A, A649ESEQ ID NO: 145variant of SEQ ID NO:12V550I, D590NSEQ ID NO: 146variant of SEQ ID NO:12S556T, T564ESEQ ID NO: 147variant of SEQ ID NO:12S556T, D590NSEQ ID NO: 148variant of SEQ ID NO:12S556T, A649ESEQ ID NO: 149variant of SEQ ID NO:12T564E, D590NSEQ ID NO: 150variant of SEQ ID NO:12T564E, L712MSEQ ID NO: 151variant of SEQ ID NO:12D590N, A649ESEQ ID NO: 152variant of SEQ ID NO:12D590N, L712MSEQ ID NO: 153variant of SEQ ID NO:12A649E, L712MSEQ ID NO: 154variant of SEQ ID NO:113L114I, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 155variant of SEQ ID NO:113L114I, I118V, N225V, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 156variant of SEQ ID NO:113L114I, I118V, N225V, A304I, T323I, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 157variant of SEQ ID NO:113L114I, I118V, N225V, A304I, T323I, G357A, Q487A, V550I, S556T, T564E, D590N, A649E, L712MSEQ ID NO: 158variant of SEQ ID NO:113L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, A649E, L712MSEQ ID NO: 159variant of SEQ ID NO:113L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649ESEQ ID NO: 190variant of SEQ ID NO:114L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712M Table 4: Overview over the Sequence IDs: Variant Trehalose Phosphorylase of SEQ ID NO: 160 SEQ ID source number of mutations in variant mutations to SEQ ID NO: 160 SEQ ID NO: 161variant of SEQ ID NO: 1601L108ISEQ ID NO: 162variant of SEQ ID NO: 1601V112ISEQ ID NO: 163variant of SEQ ID NO: 1601N221VSEQ ID NO: 164variant of SEQ ID NO: 1601A300ISEQ ID NO: 165variant of SEQ ID NO: 1601T319ISEQ ID NO: 166variant of SEQ ID NO: 1601P379VSEQ ID NO: 167variant of SEQ ID NO: 1601S550TSEQ ID NO: 168variant of SEQ ID NO: 1601Q558ESEQ ID NO: 169variant of SEQ ID NO: 1601A643ESEQ ID NO: 170variant of SEQ ID NO: 1601L707MSEQ ID NO: 171variant of SEQ ID NO: 1602L108I,N221VSEQ ID NO: 172variant of SEQ ID NO: 1602L108I,A300ISEQ ID NO: 173variant of SEQ ID NO: 1602L108I,T319ISEQ ID NO: 174variant of SEQ ID NO: 1602L108I,V483ASEQ ID NO: 175variant of SEQ ID NO: 1602L108I,S550TSEQ ID NO: 176variant of SEQ ID NO: 1602P379V,L108ISEQ ID NO: 177variant of SEQ ID NO: 1602P379V,V112ISEQ ID NO: 178variant of SEQ ID NO: 1602P379V,N221VSEQ ID NO: 179variant of SEQ ID NO: 1602P379V,A300ISEQ ID NO: 180variant of SEQ ID NO: 1602P379V,T319ISEQ ID NO: 181variant of SEQ ID NO: 1602P379V,F345YSEQ ID NO: 182variant of SEQ ID NO: 1602P379V,V483ASEQ ID NO: 183variant of SEQ ID NO: 1602P379V,V544ISEQ ID NO: 184variant of SEQ ID NO: 1602P379V,S550TSEQ ID NO: 185variant of SEQ ID NO: 1603P379V,V483A,Q558ESEQ ID NO: 186variant of SEQ ID NO: 1603P379V,V544I,S550TSEQ ID NO: 187variant of SEQ ID NO: 1603P379V,V544I,Q558ESEQ ID NO: 188variant of SEQ ID NO: 1603P379V,S550T,Q558ESEQ ID NO: 189variant of SEQ ID NO: 1604P379V,V483A,V544I,Q558E Table 5: Amino acids and corresponding positions after alignment with SEQ ID NO: 1 of different wild type TPs at the positions 114, 118, 225, 304, 323, 349, 383, 487, 550, 556, 564, 590, 649, 712 of SEQ ID NO: 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 Jul, 38 Suppl: W695-9). SEQ ID Source Genbank / Uniprot SEQ ID NO: 1Schizophyllum communeABC84380.1postition114118225304323349383487550556564590649712amino acidLINATFPQVSTDALSEQ ID NO: 191Schizophyllum commune H4-8XP_ 003035156.1postition114118225304323349383487550556564590649712amino acidLINATFPQVSTDVLSEQ ID NO: 80Hypholoma sublateritium FD-334 SS-4KJA27491.1postition7074183262281307341445509515523550609672amino acidLVNATFPTVSQNAMSEQ ID NO: 192Trametes cinnabarinaCDO74881.1postition108112221300319345379483546552560587646709amino acidLVNATFPAVSQNAMSEQ ID NO: 193Hypsizygus marmoreusKYQ39707.1postition26115194213239273377438444452478537601amino acidPSNLTFPVVSQNALSEQ ID NO: 160Grifola frondosa075003.1postition108112221300319345379483544550558584643707amino acidLVNATFPVVSQNALSEQ ID NO: 81Grifola frondosaADM15725.1postition108112221300319345379483544550558584643707amino acidLVNATFPIVSQNALSEQ ID NO: 194Trametes versicolor FP-101664 SS1XP_ 008036133.1postition110114223302321347381485549555563590649712amino acidLINATFPAVSANDLSEQ ID NO: 82Pleurotus ostreatus PC15KDQ33172.1postition112116225304323349383487551557565592651714amino acidLINATFPAVSANDLSEQ ID NO: 195Pleurotus pulmonariusA6YRN9.1postition111115224303322348382486547553561587646710amino acidLVNLTFPVVSQNALSEQ ID NO: 196Agaricus bisporus var. bisporus H97XP_ 006458503.1postition110114223302321347381485549555563590649712amino acidLVNATFPAVSQNELSEQ ID NO: 197Agaricus bisporus var. burnettii JB137-S8XP_007326883.1postition110114223302321347381485549555563590649712amino acidLVNATFPAVSQNELSEQ ID NO: 198Laetiporus sulphureus 93-53KZT11205.1postition108112221300319345380484545551559585644708amino acidLVNATFPAVSANEMSEQ ID NO: 83Lentinus sajor-caju (Pleurotus sajor-caju)Q9UV63.1postition112116225304323349383499563569577604663726amino acidLINATFPAVSANDFSEQ ID NO: 199Gloeophyllum trabeum ATCC 11539XP_007863746.1postition106110227306325351385489552558566592651715amino acidLVNLTFPVVSGNELSEQ ID NO: 200Grifola frondosaOBZ75413.1postition108112221300319345379483505511519545604668amino acidLVNVTFPSVSQNALSEQ ID NO: 201Trametes pubescensOJT04097.1postition7074183263297401462468476502561625amino acidLVN--FPVVSQNAL

[0245] The present invention is further illustrated by the figures, examples and the sequence listing from which further features, embodiments and advantages may be taken, wherein 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 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 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 Jul, 38 Suppl: W695-9).

[0246] 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

[0247] 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.

[0248] Molecular biology methods: Mutants of the TP enzymes were created by standard site-directed mutagenesis technologies as known in the state of the art.

[0249] Expression of recombinant TPs: Recombinant TPs were routinely expressed by inoculating Medium (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 µg / 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.

[0250] 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.

[0251] 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: 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 µM glucose 1,6-bisphosphate, 10 mM MgCl2, 225 mM trehalose, 3 U / mL phosphoglucomutase and 3.4 U / mL glucose 6-phosphate dehydrogenase. 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 µL of sample are mixed with 250 µL 0.5 M HCl and 500 µL 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

[0252] 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 µg / 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.

[0253] 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.

[0254] Determination of denaturation profile: 50 µL 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 1. Figure 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 15min

[0255] 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 µg / 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.

[0256] 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.

[0257] Heat-inactivation and activity measurement: A 50 µL 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 6: Residual activity of TP variants after incubation at 52 °C for 15 minSEQ ID residual activity in % after 15min incubation at 52°C [%] SEQ ID NO: 119SEQ ID NO: 230SEQ ID NO: 364SEQ ID NO: 430SEQ ID NO: 539SEQ ID NO: 654SEQ ID NO: 755SEQ ID NO: 842SEQ ID NO: 963SEQ ID NO: 1068SEQ ID NO: 1139SEQ ID NO: 1255SEQ ID NO: 1361SEQ ID NO: 1430SEQ ID NO: 1575SEQ ID NO: 1643SEQ ID NO: 1750SEQ ID NO: 1841SEQ ID NO: 1948SEQ ID NO: 2075SEQ ID NO: 2152SEQ ID NO: 2237SEQ ID NO: 2336SEQ ID NO: 2447SEQ ID NO: 2541SEQ ID NO: 2645SEQ ID NO: 2742SEQ ID NO: 2831SEQ ID NO: 2955SEQ ID NO: 3038SEQ ID NO: 3133SEQ ID NO: 3239SEQ ID NO: 3331SEQ ID NO: 3453SEQ ID NO: 3551SEQ ID NO: 3634SEQ ID NO: 3737SEQ ID NO: 3839SEQ ID NO: 3935SEQ ID NO: 4046SEQ ID NO: 4151SEQ ID NO: 4243SEQ ID NO: 4342SEQ ID NO: 4455SEQ ID NO: 4532SEQ ID NO: 4643SEQ ID NO: 4741SEQ ID NO: 4835SEQ ID NO: 4953SEQ ID NO: 5096SEQ ID NO: 51106SEQ ID NO: 5299SEQ ID NO: 53105SEQ ID NO: 54105SEQ ID NO: 5577SEQ ID NO: 56106SEQ ID NO: 5784SEQ ID NO: 5888SEQ ID NO: 5986SEQ ID NO: 60113SEQ ID NO: 61117SEQ ID NO: 62101SEQ ID NO: 6379SEQ ID NO: 64101SEQ ID NO: 6599SEQ ID NO: 66105SEQ ID NO: 6799SEQ ID NO: 6892SEQ ID NO: 69104SEQ ID NO: 7076SEQ ID NO: 7197SEQ ID NO: 7290SEQ ID NO: 73105SEQ ID NO: 74108SEQ ID NO: 75107SEQ ID NO: 7697SEQ ID NO: 78109SEQ ID NO: 7997SEQ ID NO: 8429SEQ ID NO: 8523SEQ ID NO: 8628SEQ ID NO: 8738SEQ ID NO: 8838SEQ ID NO: 8942SEQ ID NO: 9028SEQ ID NO: 9140SEQ ID NO: 9225SEQ ID NO: 9335SEQ ID NO: 9431SEQ ID NO: 9535SEQ ID NO: 9629SEQ ID NO: 9738SEQ ID NO: 9864SEQ ID NO: 9922SEQ ID NO: 10022SEQ ID NO: 10154SEQ ID NO: 10226SEQ ID NO: 10341SEQ ID NO: 10478SEQ ID NO: 10533SEQ ID NO: 10629SEQ ID NO: 10730SEQ ID NO: 10826SEQ ID NO: 10933SEQ ID NO: 11056SEQ ID NO: 11143SEQ ID NO: 11225SEQ ID NO: 11363SEQ ID NO: 11432SEQ ID NO: 11572SEQ ID NO: 11624SEQ ID NO: 11737SEQ ID NO: 11825SEQ ID NO: 11932SEQ ID NO: 12029SEQ ID NO: 12172SEQ ID NO: 12227SEQ ID NO: 12332SEQ ID NO: 12427SEQ ID NO: 12555SEQ ID NO: 12625SEQ ID NO: 12723SEQ ID NO: 12830SEQ ID NO: 12923SEQ ID NO: 13051SEQ ID NO: 13131SEQ ID NO: 13264SEQ ID NO: 13367SEQ ID NO: 13461SEQ ID NO: 13559SEQ ID NO: 13663SEQ ID NO: 13756SEQ ID NO: 13870SEQ ID NO: 13962SEQ ID NO: 14069SEQ ID NO: 14162SEQ ID NO: 14226SEQ ID NO: 14328SEQ ID NO: 14423SEQ ID NO: 14529SEQ ID NO: 14634SEQ ID NO: 14755SEQ ID NO: 14826SEQ ID NO: 14943SEQ ID NO: 15034SEQ ID NO: 15140SEQ ID NO: 15256SEQ ID NO: 15326SEQ ID NO: 15481SEQ ID NO: 15568SEQ ID NO: 15694SEQ ID NO: 15769SEQ ID NO: 15874SEQ ID NO: 15987SEQ ID NO: 190100 Example 4: 100 / 500-ratio of TP variants

[0258] 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. Table 7: 100 / 500 ratio of TP variantsSEQ ID 100 / 500 ratio SEQ ID NO: 10.6SEQ ID NO: 50.7SEQ ID NO: 60.8SEQ ID NO: 80.7SEQ ID NO: 220.7SEQ ID NO: 240.8SEQ ID NO: 260.7SEQ ID NO: 270.9SEQ ID NO: 290.7SEQ ID NO: 340.7SEQ ID NO: 360.7SEQ ID NO: 370.9SEQ ID NO: 380.8SEQ ID NO: 411.0SEQ ID NO: 420.8SEQ ID NO: 520.8SEQ ID NO: 530.9SEQ ID NO: 560.7SEQ ID NO: 570.9SEQ ID NO: 601.0SEQ ID NO: 611.0SEQ ID NO: 620.8SEQ ID NO: 630.9SEQ ID NO: 640.8SEQ ID NO: 650.8SEQ ID NO: 660.9SEQ ID NO: 680.7SEQ ID NO: 690.7SEQ ID NO: 750.9SEQ ID NO: 760.7SEQ ID NO: 1900.9 Example 5: Denaturation profiles of TP variants

[0259] 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 8: Tm30- and Tm50-values of TP-variants in 50 mM potassium phosphate buffer pH 7 containing 1 M sucroseSEQ ID Tm30 value Tm50 value SEQ ID NO: 149.547.5SEQ ID NO: 4453.552SEQ ID NO: 5054.553.5SEQ ID NO: 5455.554.5SEQ ID NO: 5755.554.5SEQ ID NO: 585654.5SEQ ID NO: 6257.556SEQ ID NO: 6457.556SEQ ID NO: 6557.556SEQ ID NO: 715756SEQ ID NO: 725856.5SEQ ID NO: 7358.557.5SEQ ID NO: 7457.556SEQ ID NO: 785856SEQ ID NO: 7957.556 Example 6: S / P-ratio

[0260] 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 9: S / P-ratioSEQ ID S / P-ratio SEQ ID NO: 10.3SEQ ID NO: 140.5SEQ ID NO: 240.4SEQ ID NO: 270.5SEQ ID NO: 290.7SEQ ID NO: 340.6SEQ ID NO: 410.5SEQ ID NO: 420.9SEQ ID NO: 430.9SEQ ID NO: 440.5SEQ ID NO: 460.8SEQ ID NO: 500.6SEQ ID NO: 540.5SEQ ID NO: 570.4SEQ ID NO: 580.5SEQ ID NO: 620.5SEQ ID NO: 640.5SEQ ID NO: 650.5SEQ ID NO: 710.5SEQ ID NO: 720.5SEQ ID NO: 730.6SEQ ID NO: 740.5SEQ ID NO: 780.5SEQ ID NO: 790.5 Example 7: Process stability at 45 °C

[0261] 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 Figure 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

[0262] 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.

[0263] 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, 1118, 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, 1118, 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 10: Residual activity after incubation at 52.5 °C for 15 minSEQ ID residual activity in % after 15min incubation at 52.5 °C [%] SEQ ID NO: 1609SEQ ID NO: 16114SEQ ID NO: 16222SEQ ID NO: 16333SEQ ID NO: 16426SEQ ID NO: 16537SEQ ID NO: 16637SEQ ID NO: 16720SEQ ID NO: 16834SEQ ID NO: 16912SEQ ID NO: 17015SEQ ID NO: 17137SEQ ID NO: 17229SEQ ID NO: 17342SEQ ID NO: 17412SEQ ID NO: 17525SEQ ID NO: 17657SEQ ID NO: 17742SEQ ID NO: 17856SEQ ID NO: 17938SEQ ID NO: 18074SEQ ID NO: 18116SEQ ID NO: 18228SEQ ID NO: 18321SEQ ID NO: 18443SEQ ID NO: 18558SEQ ID NO: 18642SEQ ID NO: 18749SEQ ID NO: 18870SEQ ID NO: 18941

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 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, wherein the trehalose phosphorylase has at least one of characteristics (A), (B), (C), (D) and (E), or any combination thereof, wherein when the amino acid substitution comprises a substitution at amino acid position 649, then said amino acid substitution at position 649 is selected from the group consisting of the substitutions 649D and 649E, preferably 649E; and wherein when the amino acid substitution comprises a substitution at amino acid position 10, then said amino acid substitution at position 10 is selected from the group consisting of the substitutions V10R and V10H, preferably V10R; 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]; or wherein the trehalose phosphorylase is characterized by a thermal stability after incubation at 52.5°C for 15 minutes defined by a residual activity of 15% to 100%, or wherein the trehalose phosphorylase compared to the trehalose phosphorylase of SEQ ID NO:1, is characterized by an increase in thermal stability, whereby the increase in thermal stability is an increase of the Tm30-value of at least 2 °C up to 40.5°C, or an increase of the Tm50-value of at least 2 °C up to 42.5°C, or an improved process stability, characterized by an increased half-life at 45 °C of at least 3-fold up to 216-fold or more, or at least 24-fold up to 216-fold or more.

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, 649, 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 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.

5. 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 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.

6. 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, I118 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.

7. 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.

8. 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.

9. The trehalose phosphorylase variant of any one of claims 1 to 8 , 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.

10. The trehalose phosphorylase variant of any one of claims 1 to 8, wherein the variant is a thermally stable variant of trehalose phosphorylase from Schizophyllum commune , and 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.

11. 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 10.

12. A method for increasing thermal stability and / or 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 thermal stability and / or 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 thermal stability and / or 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, wherein said 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.

13. Use of a trehalose phosphorylase according to any one of claims 1 to 10 for producing trehalose, preferably for producing trehalose according to claim 11.