TREHALOSE PHOSPHORYLASE

Abstract

The present invention is related to a trehalose phosphorylase comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 80% identical to and/or at least 80% homologous to an amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions of SEQ ID NO: 1712, 383, 10, 114, 118, 192, 197, 220, 225, 304, 306, 318, 323, 339, 349, 357, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 590, 649, 667, 703 and 705.

Claims

1-51. (canceled)

52. A trehalose phosphorylase comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 87% identical to an amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 160, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein (a) the one or more amino acid positions is selected from the group consisting of amino acid positions 108, 112, 221, 300, 319, 345, 379, 483, 544, 550, 558, 584, 643, and 707 of SEQ ID NO:81 or SEQ ID NO: 160; (b) if the one or more amino acid substitutions comprises a substitution at amino acid position I483 of SEQ ID NO: 81, then the amino acid substitution at position 483 is selected from the group consisting of I483A, I483G, I483L, I483M, and I483P; and (b) if the one or more amino acid substitutions comprises a substitution at amino acid position V483 of SEQ ID NO: 160, then the amino acid substitution at position 483 is selected from the group consisting of V483A, V483G, V483L, V483M, and V483P.

53. The trehalose phosphorylase of claim 52, comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 87% identical to an amino acid sequence of SEQ ID NO: 160, wherein the one or more amino acid positions is selected from the group consisting of amino acid positions of L108, V112, N221, A300, T319, F345, P379, V483, V544, S550, Q558, N584, A643, and L707 of SEQ ID NO: 160.

54. The trehalose phosphorylase of claim 52, comprising an amino acid sequence, wherein the amino acid sequence of the trehalose phosphorylase is at least 87% identical to an amino acid sequence of SEQ ID NO: 81, wherein the one or more amino acid positions is selected from the group consisting of amino acid positions of L108, V112, N221, A300, T319, F345, P379, I483, V544, S550, Q558, N584, A643, and L707 of SEQ ID NO:81.

55. The trehalose phosphorylase of claim 52, wherein the trehalose phosphorylase comprises amino acid substitutions at two amino acid positions, wherein the two amino acid positions are selected from the group consisting of 108 and 112, 108 and 221, 108 and 300, 108 and 319, 108 and 345, 108 and 379, 108 and 483, 108 and 544, 108 and 550, 108 and 558, 108 and 584, 108 and 643, 108 and 707, 112 and 221, 112 and 300, 112 and 319, 112 and 345, 112 and 379, 112 and 483, 112 and 544, 112 and 550, 112 and 558, 112 and 584, 112 and 643, 112 and 707, 221 and 300, 221 and 319, 221 and 345, 221 and 379, 221 and 483, 221 and 544, 221 and 550, 221 and 558, 221 and 584, 221 and 643, 221 and 707, 300 and 319, 300 and 345, 300 and 379, 300 and 483, 300 and 544, 300 and 550, 300 and 558, 300 and 584, 300 and 643, 300 and 707, 319 and 345, 319 and 379, 319 and 483, 319 and 544, 319 and 550, 319 and 558, 319 and 584, 319 and 643, 319 and 707, 345 and 379, 345 and 483, 345 and 544, 345 and 550, 345 and 558, 345 and 584, 345 and 643, 345 and 707, 379 and 483, 379 and 544, 379 and 550, 379 and 558, 379 and 584, 379 and 643, 379 and 707, 483 and 544, 483 and 550, 483 and 558, 483 and 584, 483 and 643, 483 and 707, 544 and 550, 544 and 558, 544 and 584, 544 and 643, 544 and 707, 550 and 558, 550 and 584, 550 and 643, 550 and 707, 558 and 584, 558 and 643, 558 and 707, 584 and 643, 584 and 707, and 643 and 707.

56. The trehalose phosphorylase of claim 52, wherein the trehalose phosphorylase comprises amino acid substitutions at two amino acid positions, wherein the two amino acid positions are selected from the group consisting of (i) L108 and T319, L108 and P379, L108 and V483, L108 and S550, L108 and Q558, N221 and T319, N221 and P379, N221 and V483, N221 and V544, N221 and S550, N221 and Q558, T319 and P379, T319 and V483, T319 and V544, T319 and S550, T319 and Q558, P379 and V483, P379 and V544, P379 and S550, P379 and Q558, V483 and V544, V483 and S550, V483 and Q558, and S550 and Q558 of SEQ ID NO: 160; and (ii) L108 and T319, L108 and P379, L108 and I483, L108 and S550, L108 and Q558, N221 and T319, N221 and P379, N221 and I483, N221 and V544, N221 and S550, N221 and Q558, T319 and P379, T319 and I483, T319 and V544, T319 and S550, T319 and Q558, P379 and I483, P379 and V544, P379 and S550, P379 and Q558, I483 and V544, I483 and S550, I483 and Q558, and S550 and Q558 of SEQ ID NO:81.

57. The trehalose phosphorylase of claim 52, wherein the one or more substitution is selected from the group consisting of an amino acid substitution at position L108 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being L108A, L108G, L108I, L108M, L108P or L108V; an amino acid substitution at position V112 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being V112A, V112G, V112L, V112M, V112P or V112I; an amino acid substitution at position N221 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being N221A, N221G, N221I, N221L, N221M, N221P or N221V; an amino acid substitution at position A300 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being A300G, A300I, A300L, A300M, A300P or A300V; an amino acid substitution at position T319 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being T319A, T319G, T319I, T319L, T319M, T319P, or T319V; an amino acid substitution at position P379 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being P379A, P379G, P379I, P379L, P379M, P379V, P379N, P379C, P379Q, P379S or P379T; an amino acid substitution at position I483 of SEQ ID NO: 81 with the substitution being I483A, I483G, I483I, I483L, I483M, or I483P; an amino acid substitution at position V483 of SEQ ID NO: 160 with the substitution being V483A, V483G, V483I, V483L, V483M, or V483P; an amino acid substitution at position V544 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being V544A, V544G, V544I, V544L, V544M or V544P; an amino acid substitution at position S550 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being S550N, S550C, S550Q or S550T; an amino acid substitution at position Q558 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being Q558D or Q558E; an amino acid substitution at position D558 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being D558N, D558C, D558Q, D558S, D558T, D558A, D558G, D558I, D558L, D558M, D558P or D558V; an amino acid substitution at position A643 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being A643D or A643E; and an amino acid substitution at position L707 of SEQ ID NO: 81 or SEQ ID NO: 160, with the substitution being L707A, L707G, L707I, L707M, L707P or L707V.

58. The trehalose phosphorylase of claim 52, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid sequence according to any one of SEQ ID NO: 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, and 189.

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

60. The trehalose phosphorylase of claim 52, wherein the trehalose phosphorylase has a residual activity of at least 30% after incubation at 52° C. for 15 minutes, in a buffer containing 1 M sucrose, and the initial activity is determined after incubation for 15 minutes at room temperature.

61. 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 the trehalose phosphorylase of claim 52.

62. 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 of claim 52, 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.

Description

[0704] 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

[0705] 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

[0706] 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

[0707] FIG. 3 shows an alignment of the wild type TPs from Grifola frondosa (UniProtKB/Swiss-Prot: Accession No: 075003.1 and Genebank Accession No: ADM15725.1), Pleurotus ostreatus (Genebank Accession No: KDQ33172.1), Lentinus sajor-caju (Synonym: Pleurotus sajor-caju, Genebank Accession No: Q9UV63.1). The alignment was done using Clustal omega (Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R Nucleic acids research 2010 July, 38 Suppl: W695-9).

[0708] 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

[0709] 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 pLElA17 (derivative of pRSF-1b, Novagen). The resulting plasmids were used for transformation of E. coli BL21(DE3) cells.

[0710] Molecular biology methods: Mutants of the TP enzymes were created by standard site-directed mutagenesis technologies as known in the state of the art.

[0711] Expression of recombinant TPs: Recombinant TPs were routinely expressed by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone, 25 mM Na2HPO4*12H2O, 25 mM KH2PO4, 50 mM NH4Cl.sub.2, 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.

[0712] 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.

[0713] 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:

[0714] 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.

[0715] 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 μF 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/F 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

[0716] Expression of recombinant TPs: The wild-type enzyme SEQ ID NO: 1 was expressed in shaking flasks by inoculating Medium I (4.6 g/F yeast extract, 9.3 g/F peptone, 25 mM Na2HPO4*12H2O, 25 mM KH2PO4, 50 mM NH4Cl2, Na2SO4, 5 g/F glycerol, 0.5 g/F glucose*1H2O, 2 mM MgSO4, 50 μg/mF 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.

[0717] 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.

[0718] 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 I. FIG. 1 is a denaturing profile of SEQ ID NO: 1 with and without 1 M sucrose as a stabilizing agent showing the obtained residual activities compared to the enzyme preparations without heat inactivation. The addition of 1 M sucrose results in an increase of Tm50 from approx. 40° C. to 47.5° C. 1 M sucrose was therefore chosen as a stabilizing agent for TP.

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

[0719] 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.

[0720] 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.

[0721] 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-US-00006 TABLE 6 Residual activity of TP variants after incubation at 52° C. for 15 min residual activity in % after 15 min incubation at 52° C. SEQ ID [%] SEQ ID NO: 1 19 SEQ ID NO: 2 30 SEQ ID NO: 3 64 SEQ ID NO: 4 30 SEQ ID NO: 5 39 SEQ ID NO: 6 54 SEQ ID NO: 7 55 SEQ ID NO: 8 42 SEQ ID NO: 9 63 SEQ ID NO: 10 68 SEQ ID NO: 11 39 SEQ ID NO: 12 55 SEQ ID NO: 13 61 SEQ ID NO: 14 30 SEQ ID NO: 15 75 SEQ ID NO: 16 43 SEQ ID NO: 17 50 SEQ ID NO: 18 41 SEQ ID NO: 19 48 SEQ ID NO: 20 75 SEQ ID NO: 21 52 SEQ ID NO: 22 37 SEQ ID NO: 23 36 SEQ ID NO: 24 47 SEQ ID NO: 25 41 SEQ ID NO: 26 45 SEQ ID NO: 27 42 SEQ ID NO: 28 31 SEQ ID NO: 29 55 SEQ ID NO: 30 38 SEQ ID NO: 31 33 SEQ ID NO: 32 39 SEQ ID NO: 33 31 SEQ ID NO: 34 53 SEQ ID NO: 35 51 SEQ ID NO: 36 34 SEQ ID NO: 37 37 SEQ ID NO: 38 39 SEQ ID NO: 39 35 SEQ ID NO: 40 46 SEQ ID NO: 41 51 SEQ ID NO: 42 43 SEQ ID NO: 43 42 SEQ ID NO: 44 55 SEQ ID NO: 45 32 SEQ ID NO: 46 43 SEQ ID NO: 47 41 SEQ ID NO: 48 35 SEQ ID NO: 49 53 SEQ ID NO: 50 96 SEQ ID NO: 51 106 SEQ ID NO: 52 99 SEQ ID NO: 53 105 SEQ ID NO: 54 105 SEQ ID NO: 55 77 SEQ ID NO: 56 106 SEQ ID NO: 57 84 SEQ ID NO: 58 88 SEQ ID NO: 59 86 SEQ ID NO: 60 113 SEQ ID NO: 61 117 SEQ ID NO: 62 101 SEQ ID NO: 63 79 SEQ ID NO: 64 101 SEQ ID NO: 65 99 SEQ ID NO: 66 105 SEQ ID NO: 67 99 SEQ ID NO: 68 92 SEQ ID NO: 69 104 SEQ ID NO: 70 76 SEQ ID NO: 71 97 SEQ ID NO: 72 90 SEQ ID NO: 73 105 SEQ ID NO: 74 108 SEQ ID NO: 75 107 SEQ ID NO: 76 97 SEQ ID NO: 78 109 SEQ ID NO: 79 97 SEQ ID NO: 84 29 SEQ ID NO: 85 23 SEQ ID NO: 86 28 SEQ ID NO: 87 38 SEQ ID NO: 88 38 SEQ ID NO: 89 42 SEQ ID NO: 90 28 SEQ ID NO: 91 40 SEQ ID NO: 92 25 SEQ ID NO: 93 35 SEQ ID NO: 94 31 SEQ ID NO: 95 35 SEQ ID NO: 96 29 SEQ ID NO: 97 38 SEQ ID NO: 98 64 SEQ ID NO: 99 22 SEQ ID NO: 100 22 SEQ ID NO: 101 54 SEQ ID NO: 102 26 SEQ ID NO: 103 41 SEQ ID NO: 104 78 SEQ ID NO: 105 33 SEQ ID NO: 106 29 SEQ ID NO: 107 30 SEQ ID NO: 108 26 SEQ ID NO: 109 33 SEQ ID NO: 110 56 SEQ ID NO: 111 43 SEQ ID NO: 112 25 SEQ ID NO: 113 63 SEQ ID NO: 114 32 SEQ ID NO: 115 72 SEQ ID NO: 116 24 SEQ ID NO: 117 37 SEQ ID NO: 118 25 SEQ ID NO: 119 32 SEQ ID NO: 120 29 SEQ ID NO: 121 72 SEQ ID NO: 122 27 SEQ ID NO: 123 32 SEQ ID NO: 124 27 SEQ ID NO: 125 55 SEQ ID NO: 126 25 SEQ ID NO: 127 23 SEQ ID NO: 128 30 SEQ ID NO: 129 23 SEQ ID NO: 130 51 SEQ ID NO: 131 31 SEQ ID NO: 132 64 SEQ ID NO: 133 67 SEQ ID NO: 134 61 SEQ ID NO: 135 59 SEQ ID NO: 136 63 SEQ ID NO: 137 56 SEQ ID NO: 138 70 SEQ ID NO: 139 62 SEQ ID NO: 140 69 SEQ ID NO: 141 62 SEQ ID NO: 142 26 SEQ ID NO: 143 28 SEQ ID NO: 144 23 SEQ ID NO: 145 29 SEQ ID NO: 146 34 SEQ ID NO: 147 55 SEQ ID NO: 148 26 SEQ ID NO: 149 43 SEQ ID NO: 150 34 SEQ ID NO: 151 40 SEQ ID NO: 152 56 SEQ ID NO: 153 26 SEQ ID NO: 154 81 SEQ ID NO: 155 68 SEQ ID NO: 156 94 SEQ ID NO: 157 69 SEQ ID NO: 158 74 SEQ ID NO: 159 87 SEQ ID NO: 190 100

Example 4: 100/500-Ratio of TP Variants

[0722] 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-US-00007 TABLE 7 100/500 ratio of TP variants SEQ ID 100/500 ratio SEQ ID NO: 1 0.6 SEQ ID NO: 5 0.7 SEQ ID NO: 6 0.8 SEQ ID NO: 8 0.7 SEQ ID NO: 22 0.7 SEQ ID NO: 24 0.8 SEQ ID NO: 26 0.7 SEQ ID NO: 27 0.9 SEQ ID NO: 29 0.7 SEQ ID NO: 34 0.7 SEQ ID NO: 36 0.7 SEQ ID NO: 37 0.9 SEQ ID NO: 38 0.8 SEQ ID NO: 41 1.0 SEQ ID NO: 42 0.8 SEQ ID NO: 52 0.8 SEQ ID NO: 53 0.9 SEQ ID NO: 56 0.7 SEQ ID NO: 57 0.9 SEQ ID NO: 60 1.0 SEQ ID NO: 61 1.0 SEQ ID NO: 62 0.8 SEQ ID NO: 63 0.9 SEQ ID NO: 64 0.8 SEQ ID NO: 65 0.8 SEQ ID NO: 66 0.9 SEQ ID NO: 68 0.7 SEQ ID NO: 69 0.7 SEQ ID NO: 75 0.9 SEQ ID NO: 76 0.7 SEQ ID NO: 190 0.9

Example 5: Denaturation Profiles of TP Variants

[0723] 14 TP variants, which had shown high improvements in thermal stability in Example 3, were selected for the determination of their Tm30- and Tm50-values. Denaturation profiles were determined in 50 mM potassium phosphate buffer pH 7 containing 1 M sucrose as described in Example 2. The following Tm30- and Tm-50-values were extrapolated from the denaturation profiles:

TABLE-US-00008 TABLE 8 Tm30- and Tm50-values of TP-variants in 50 mM potassium phosphate buffer pH 7 containing 1M sucrose SEQ ID Tm30 value Tm50 value SEQ ID NO: 1 49.5 47.5 SEQ ID NO: 44 53.5 52 SEQ ID NO: 50 54.5 53.5 SEQ ID NO: 54 55.5 54.5 SEQ ID NO: 57 55.5 54.5 SEQ ID NO: 58 56 54.5 SEQ ID NO: 62 57.5 56 SEQ ID NO: 64 57.5 56 SEQ ID NO: 65 57.5 56 SEQ ID NO: 71 57 56 SEQ ID NO: 72 58 56.5 SEQ ID NO: 73 58.5 57.5 SEQ ID NO: 74 57.5 56 SEQ ID NO: 78 58 56 SEQ ID NO: 79 57.5 56

Example 6: S/P-Ratio

[0724] The wild-type enzyme and 23 TP-variants were selected for the determination of the S/P-ratio. Cell extracts were prepared as described in Example 2. The activity was determined in the direction of trehalose phosphorolysis (Assay I) and trehalose synthesis (Assay II, 500 mM glucose). The ratio between synthesis and phosphorolysis activity (S/P-ratio) was 0.3 for the wild-type enzyme, which means that the enzyme shows higher reaction rates in the direction of trehalose cleavage. The tested TP variants all showed S/P-ratios above 0.3. SEQ ID NO: 42 and SEQ ID NO: 53 showed the highest improvements with an over 3-fold higher S/P-ratio compared to the wild-type enzyme.

TABLE-US-00009 TABLE 9 S/P-ratio SEQ ID S/P-ratio SEQ ID NO: 1 0.3 SEQ ID NO: 14 0.5 SEQ ID NO: 24 0.4 SEQ ID NO: 27 0.5 SEQ ID NO: 29 0.7 SEQ ID NO: 34 0.6 SEQ ID NO: 41 0.5 SEQ ID NO: 42 0.9 SEQ ID NO: 43 0.9 SEQ ID NO: 44 0.5 SEQ ID NO: 46 0.8 SEQ ID NO: 50 0.6 SEQ ID NO: 54 0.5 SEQ ID NO: 57 0.4 SEQ ID NO: 58 0.5 SEQ ID NO: 62 0.5 SEQ ID NO: 64 0.5 SEQ ID NO: 65 0.5 SEQ ID NO: 71 0.5 SEQ ID NO: 72 0.5 SEQ ID NO: 73 0.6 SEQ ID NO: 74 0.5 SEQ ID NO: 78 0.5 SEQ ID NO: 79 0.5

Example 7: Process Stability at 45° C.

[0725] Process stability of the wild-type enzyme and the TP variants SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 78 was determined at 45° C. in 50 mM potassium phosphate buffer pH 7 containing 1M sucrose. Cell extracts were prepared as described in Example 2. Samples were incubated at 45° C. for 16 days. Samples were taken over time and the activity was measured using Assay I. The results are shown in FIG. 2. SEQ ID NO: 1 showed a rapid activity loss within the first 3 hours and a half-life of approx. 1 hour. As expected from the Tm50-values, the new variants showed greatly improved process stability. SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 78 showed the highest improvements with half-lives of approx. 8.8 days. This constitutes an over 200-fold improvement compared to the wild-type enzyme.

Example 8: Alternative TP Enzymes

[0726] 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 NOT 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.

[0727] Variants of alternative wild-type trehalose phosphorylases are created using the methods described in Example 1. The variants contain one or more mutations at the positions corresponding to L114, I118, G357, P383, N225, A304, T323, S556, T564, A649 and L712 in SEQ ID NOT.

[0728] Variants are tested for improved thermal stability as described in Example 3. The heat inactivation step is carried out at the temperature at which the corresponding wild-type retains approximately 20% residual activity after incubation for 15 min. It is expected, that the new variants will show similar improvements to the variants in Example 3. Further mutations corresponding to positions L114, I118, G357, P383, N225, A304, T323, F349, Q487, V550, S556, T564, A649 and L712 in SEQ ID NO: 1 may be added. It is expected, that the addition of one or more of these mutations will lead to a further improvement in thermal stability.

TABLE-US-00010 TABLE 10 Residual activity after incubation at 52.5° C. for 15 min residual activity in % after 15 min incubation at SEQ ID 52.5° C. [%] SEQ ID NO: 160 9 SEQ ID NO: 161 14 SEQ ID NO: 162 22 SEQ ID NO: 163 33 SEQ ID NO: 164 26 SEQ ID NO: 165 37 SEQ ID NO: 166 37 SEQ ID NO: 167 20 SEQ ID NO: 168 34 SEQ ID NO: 169 12 SEQ ID NO: 170 15 SEQ ID NO: 171 37 SEQ ID NO: 172 29 SEQ ID NO: 173 42 SEQ ID NO: 174 12 SEQ ID NO: 175 25 SEQ ID NO: 176 57 SEQ ID NO: 177 42 SEQ ID NO: 178 56 SEQ ID NO: 179 38 SEQ ID NO: 180 74 SEQ ID NO: 181 16 SEQ ID NO: 182 28 SEQ ID NO: 183 21 SEQ ID NO: 184 43 SEQ ID NO: 185 58 SEQ ID NO: 186 42 SEQ ID NO: 187 49 SEQ ID NO: 188 70 SEQ ID NO: 189 41