D-allulose 3-epimerases and methods of producing allulose

Abstract

The present invention discloses a variant D-allulose 3-epimerase (DAEase) that is distinguished from a wild-type DAEase derived from Spirochaetales bacterium (spDAEase) by at least one amino acid sequence variation comprising substitution of an 5 amino acid residue corresponding to position 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1; or a variant DAEase that is distinguished from a wild-type DAEase derived from Clostridium cellulolyticum (ccDAEase) by an amino acid sequence variation comprising substitution of an amino acid at position 46 and / or a truncation of C-terminal end of in SEQ ID NO. 2.

Description

[0001] D-Allulose 3-Epimerases and Methods of Producing Allulose

[0002] Technical Field

[0003] The present invention relates, in general terms, to D-allulose 3-epimerases and methods of producing allulose from fructose.

[0004] Background

[0005] D-allulose, also known as D-psicose, is a natural sugar with 70% of the sweetness of sucrose but only 10% of the calories. The FDA has classified it as safe. Compared to artificial sweeteners, D-allulose has various health benefits, including anti-inflammatory and anti-obesity properties, and neuroprotection effects.

[0006] D-allulose production primarily involves the Izumoring strategy, which utilizes the epimerase enzyme family for the bioproduction of all hexose sugars. However, to make production cost-efficient, enzymes need to be stable under the conditions of more than 45 °C. The current optimal pH condition is 7.5 - 9.0 for the enzymes, and high reaction temperatures cause the non-enzymatic browning of D-allulose, reducing the yield and further complicating the purification process.

[0007] In addition to enzyme stability, the conversion of fructose to allulose has a relatively low yield, typically around 30-40%. This is because the conversion is an equilibrium reaction. This means a significant portion of the fructose feedstock is not converted to the desired allulose product.

[0008] There are also purification difficulties. Separating and purifying allulose from the reaction mixture can be technically challenging. Allulose has similar physical and chemical properties to fructose, making it difficult to achieve high purity levels.

[0009] Further, the overall production process, including the conversion reaction and purification steps, can be costly.

[0010] It would be desirable to overcome or ameliorate at least one of the above-described problems.

[0011] Summary The present disclosure provides a variant D-allulose 3-epimerase (DAEase) that is distinguished from a wild-type DAEase derived from Spirochaetales bacterium (spDAEase) by at least one amino acid sequence variation comprising substitution of an amino acid residue corresponding to position 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1; or a variant DAEase that is distinguished from a wild-type DAEase derived from Clostridium cellulolyticum (ccDAEase) by an amino acid sequence variation comprising substitution of an amino acid at position 46 and / or a truncation of C-terminal end of in SEQ ID NO. 2.

[0012] In some embodiments, the variant DAEase distinguished from wild-type DAEase derived from Spirochaetales bacterium comprises an amino acid substitution at at least two positions of 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1.

[0013] In some embodiments, the variant spDAEase comprises an amino acid substitution at position 240, and at least another amino acid substitution at positions 69, 119, 138, 175, 203, 208, 229, 265 of SEQ ID NO. 1.

[0014] In some embodiments, the variant spDAEase comprises an amino acid substitution at position 175, and at least another amino acid substitution at positions 69, 119, 138, 203, 208, 229, 240, or 265 of SEQ ID NO. 1.

[0015] In some embodiments, the variant spDAEase comprises an amino acid substitution at position 69 or 138, and at least another amino acid substitution at positions 119, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1.

[0016] In some embodiments, the variant spDAEase comprises an amino acid substitution at position 203 or 265, and at least another amino acid substitution at positions 69, 119, 138, 175, 208, 229, 240 of SEQ ID NO. 1.

[0017] In some embodiments, the variant spDAEase comprises an amino acid substitution at position 119 or 229, and at least another amino acid substitution at positions 69, 138, 175, 203, 208, 240, 265 of SEQ ID NO. 1.

[0018] In some embodiments, the variant spDAEase comprises amino acid substitutions at positions corresponding to one of the following sets of positions of SEQ ID NO. 1 : a) 69 and at least one of 138, 175, 203, 240, 265; b) 138 and at least one of 69, 175, 203, 240, 265; c) 175 and at least one of 69, 138, 203, 240, 265; d) 203 and at least one of 69, 138, 175, 240, 265; e) 240 and at least one of 69, 138, 175, 203, 265; and f) 265 and at least one of 69, 138, 175, 203, 240.

[0019] In some embodiments, the variant spDAEase comprises amino acid substitutions at at least three positions of 69, 119, 138, 175, 203, 208, 229, 240, or 265-of SEQ ID NO.

[0020] 1.

[0021] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with a charged amino acid residue.

[0022] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with a positively charged amino acid residue.

[0023] In some embodiments, the variant spDAEase comprises one or more amino acid substitutions selected from the following: a) A substitution to S at position 69 of SEQ ID NO. 1; b) A substitution to T at position 119 of SEQ ID NO. 1; c) A substitution to K at position 138 of SEQ ID NO. 1; d) A substitution to H at position 175 of SEQ ID NO. 1; e) A substitution to S at position 203 of SEQ ID NO. 1; f) A substitution to L at position 208 of SEQ ID NO. 1; g) A substitution to G at position 229 of SEQ ID NO. 1; h) A substitution to S at position 240 of SEQ ID NO. 1; and i) A substitution to N at position 265 of SEQ ID NO. 1;

[0024] In some embodiments, the variant spDAEase comprise at least one amino acid substitution selected from N69S, P119T, R138K, I175H, P203S, F208L, R229G, T240S, and H265N.

[0025] In some embodiments, variant spDAEase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 1.

[0026] In some embodiments, variant ccDAEase comprises an amino acid substitution to glutamine (Q) at position 46 of SEQ ID NO. 2. In some embodiments, variant ccDAEase comprises an amino acid substitution of isoleucine (I) to glutamine (Q) at position 46 of SEQ ID NO. 2.

[0027] In some embodiments, variant ccDAEase comprises 1-12 amino acid deletions at the C- terminus.

[0028] In some embodiments, variant ccDAEase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 2.

[0029] The present disclosure provides an immobilized enzyme, wherein the enzyme is a variant DAEase as disclosed herein and / or a wild-type DAEase derived from Spirochaetales bacterium (SEQ ID NO. 1).

[0030] In some embodiments, the enzyme comprises an affinity tag and is immobilised on a resin comprising metal ions.

[0031] In some embodiments, the enzyme is inter-enzyme cross-linked with a cross-linker.

[0032] In some embodiments, the immobilised enzyme is characterised by an activity of more than 90% for at least 50 days.

[0033] The present disclosure provides a method of converting fructose to allulose using a variant DAEase as disclosed herein, an immobilised enzyme as disclosed herein, and / or a wild-type DAEase derived from Spirochaetales bacterium (SEQ ID NO. 1).

[0034] Brief description of the drawings

[0035] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0036] Figure 1 shows comparison of activity of known DAEases. Each x-axis item consists of a unique combination of gene (as annotated) and solubility tag. The reaction mixture (200 pL final volume) for D-allulose conversion contained 100 g / L D-fructose as substrate in 50 mM NazHPC / NaHzPC buffer (pH 6.5), 100 pmol / L Co2+and 20 pL out of 300 pL cell pellet resuspension. The reaction was incubated at 60 °C for 10 minutes. The allulose conversion was quantified by high performance liquid chromatography (HPLC) with refractive index detector (RID). Figure 2 shows the identification of new candidates. Activity comparison of DAEases, ccDAEase (DPC19) was used as a control. Each x-axis item consists of a unique combination of gene (as annotated by their respective Genbank IDs) and solubility tag. The reaction mixture (200 pL final volume) for D-allulose conversion contained 100 g / L D-fructose as substrate in 50 mM Na2HPO4 / NaH2PO4 buffer (pH 6.5), 100 pmol / L Co2+and 50 pL of eluate suspension. The reaction was incubated at 60 °C for 10 minutes.

[0037] Figure 3 shows temperature and pH profiles of purified spDAEase (HOV93914.1, DPC47) (A) and ccDAEase (DM1) (B). Effects of temperature on the activity of enzymes were determined between 50 °C and 80 °C in 50 mM Na2HPO4 / NaH2PO4 buffer (pH 6.5), 100 pmol / L Co2+, 100 g / L D-fructose and 12 pg of purified enzyme. The enzyme activity was measured after incubation at respective temperatures for 5 minutes in thermomixer. The allulose conversion was quantified by high performance liquid chromatography (HPLC) with refractive index detector (RID). The optimal pH value of enzyme activity was assayed at different values from 6.0-8.5 using Na2HPO4 / NaH2PO4 buffer. The reaction mixture (200 pL final volume) for D-allulose conversion contained 100 g / L D-fructose as substrate in 50 mM Na2HPO4 / NaH2PO4 buffer at respective pH, 100 pmol / L Co2+and 12 pg of purified enzyme. The reaction was incubated at 60 °C for 5 minutes in thermomixer and was then stopped by boiling for 5 minutes. The allulose conversion was quantified by high performance liquid chromatography (HPLC) with refractive index detector (RID).

[0038] Figure 4 shows comparison of spDAEase stability. Residual activity comparison of ccDAEase (DPC19), spDAEase (DPC47) and crosslinked spDAEase (DPC47 crosslinked with BS(PEG)5 in phosphate buffer at various pH containing different amount of Co2+. The immobilized enzyme was treated at 70 °C for 90 minutes, followed by at 75 °C for 60 minutes in 50 mM Na2HPO4 / NaHzPO4 buffer at various pH (6.5 - 8.0) containing different amounts of Co2+(0.1 mM - 0.5 mM). The immobilized enzyme after heat treatment was reacted with 100 g / L D-fructose as substrate in 50 mM NazHPC / NaHzPCU buffer at respective pH and Co2+at 45 °C for 20 minutes. The allulose conversion was quantified by high performance liquid chromatography (HPLC) with refractive index detector (RID). The original activity before heat treatment was taken as 100%.

[0039] Figure 5 shows comparison of variants. (A) Sequence alignment, (B) Phylogentic tree and (C) Crystal structure of ccDAEase (PDB ID: 3VNI) with the possible residues indicated. Sequences were aligned by Clustal Omega (Sievers et al 2011) and depicted by ESPript 3. Ox (Robert & Gouet, 2014). Black arrows indicate the current mutations in ccDAEase (H56Q / Q277R / S293R). Black triangles indicated K46, K138 and H175 positions.

[0040] Figure 6 shows comparison of mutants (DPC19, 87, 88) against DM1 (ccDAEase). Residual activity comparison of ccDAEase (DM1), spDAEase (DPC47) and ccDAEase mutants (DPC19, 87, 88) in phosphate buffer at pH 6.9 (A) and pH 8 (B). The immobilized enzyme was treated at 70 °C for 180 minutes in 50 mM Na2HPO4 / NaH2PC>4 buffer at pH 6.9 or 8 containing 0.1 mM Co2+. The immobilized enzyme after heat treatment every hour was reacted with 100 g / L D-fructose as substrate in 50 mM Na2HPO4 / NaH2PC>4 buffer at respective pH and 100 pmol / L Co2+at 45 °C for 20 minutes. Allulose conversion was quantified by high performance liquid chromatography (HPLC) with refractive index detector (RID). The activity after heat treatment for 60 minutes was taken as 100%.

[0041] Figure 7 shows new mutation sites for engineering (annotated) as directed from comparison of spDAEase and ccDAEase. Mutated sequences of tdDAEase, AsDAEase, ccDAEase and DoraDAEase (Table 1) aligned with NCA98512.1 (from Clostridum, CDAEase) and HOV93914.1 (from Spirochaetales bacterium, spDAEase).

[0042] Figure 8 shows residual activity comparison of mutants against spDAEase (DPC47). The immobilized enzyme was treated at 70 °C for 180 minutes in 50 mM Na2HPO4 / NaH2PO4 buffer at pH 6.9 containing 0.1 mM Co2+. The immobilized enzyme after heat treatment was reacted with 100 g / L D-fructose as substrate in 50 mM Na2HPO4 / NaH2PO4 buffer at pH 6.9 and 100 pmol / L Co2+at 45 °C for 8 minutes. The allulose conversion was quantified by high performance liquid chromatography (HPLC) with refractive index detector (RID). The figure shows the % of residual activity after heat treatment of the enzymes. The original activity before heat treatment was taken as 100%.

[0043] Figure 9 shows long term stability of immobilized spDAEase (DPC47) under continuous flow of fructose substrate. Activity is shown by % conversion to allulose. 100 g / L D- fructose in 50 mM Na2HPO4 / NaH2PO4 buffer at pH 6.9 and 300 pmol / L Co2+was passed through the column containing 1 mL of immobilized enzyme continuously at 0.50 mL / min and 45 °C. The liquid flowed out of the column was collected daily for allulose conversion measured by high performance liquid chromatography (HPLC) with refractive index detector (RID). At 73 days, activity of both the cross-linked and native enzymes are retained at >99%.

[0044] Figure 10 shows long term stability of immobilized spDAEase (DPC47) under continuous flow of fructose substrate. Residual activity of allulose conversion is shown whereby the original activity at day 0 was taken as 100%. 100 g / L D-fructose in 50 mM Na2HPO4 / NaH2PC>4 buffer at pH 6.9 and 300 pmol / L Co2+was passed through the column containing 1 mL of immobilized enzyme continuously at 0.07 miymin and 45 °C. The liquid flowed out of the column was collected daily for allulose conversion measured by high performance liquid chromatography (HPLC) with refractive index detector (RID). Both the cross-linked and native enzymes maintained more than 90% of their initial activity for 151 days. However, the residual activity of the native enzymes dropped below 60% after 175 days, while the cross-linked enzymes experienced a similar decline after 179 days.

[0045] Detailed description

[0046] The present disclosure is predicated on the understanding the ideal biocatalyst should be stable under a pH of less than 7.5. Accordingly, one way to advance the industrial production of D-allulose is by enhancing and refining existing DAEases. In particular, additional variations of the enzyme from diverse sources can deepen our understanding of their mechanisms and also provide new opportunities for enzyme engineering in industrial settings.

[0047] It was found that thermal stable biocatalysts for conversion of fructose to allulose may be obtained by mutating D-allulose 3-epimerases (DAEase) at specific sites. By comparing these variants, mutations for improving activity and stability of DAEases may also be identified.

[0048] In particular, D-allulose 3-epimerase Spirochaetales bacterium (HOV93914.1, spDAEase) demonstrates significant thermal stability (50-80 °C) while maintaining activity across a range of pH from 6-8.5. In this regard, SpDAEase is stable under the conditions of more than 45 °C. The biocatalyst is stable in the pH range of 6-8.5. spDAEase may be immobilized on a resin; the immobilized biocatalyst has significant stability, where it is able to maintain more than 99% activity for at least 68 days.

[0049] This is in contrast to DAEases in general, which have an optimal pH preference (pH>7). The optimal pH for the activity of DAEases can vary depending on the specific source and conditions.

[0050] By comparative analysis of spDAEase, new sites affecting activity and stability were also uncovered. For example, spDAEase may comprise at least one mutation selected from N69S, P119T, R138K, I175H, P203S, F208L, R229G, T240S, or H265N. Further, comparison of differences between spDAEases and DAEase from C. cellulolyticum (ccDAEase) identified 2 new mutation sites for improved stability of ccDAEase at position I46Q and truncation of C-terminal end.

[0051] In this regard, a cost efficient and low pH biocatalyst for conversion of allulose is disclosed. Accordingly, the present disclosure provides a variant D-allulose 3-epimerase (DAEase) that is distinguished from a wild-type DAEase derived from Spirochaetales bacterium (spDAEase) by at least one amino acid sequence variation comprising substitution of an amino acid residue corresponding to position 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1; or a variant DAEase that is distinguished from a wild-type DAEase derived from Clostridium cellulolyticum (ccDAEase) by an amino acid sequence variation comprising substitution of an amino acid at position 46 and / or a truncation of C-terminal end of in SEQ ID NO. 2.

[0052] As used herein, the variant DAEase distinguished from wild-type Spirochaetales bacterium DAEase is abbreviated as "variant spDAEase". The Spirochaetales are an order of spirochete bacteria.

[0053] In some embodiments, variant spDAEase comprises an amino acid substitution at at least two positions of 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1.

[0054] It was found that these amino acid substitutions (individually or in combination) confers stability to the DAEase, either alone or when supported on a resin, and further improves the enzyme activity.

[0055] In some embodiments, variant spDAEase comprises an amino acid substitution at position 240, and at least another amino acid substitution at positions 69, 119, 138, 175, 203, 208, 229, 265 of SEQ ID NO. 1.

[0056] In some embodiments, variant spDAEase comprises an amino acid substitution at position 175, and at least another amino acid substitution at positions 69, 119, 138, 203, 208, 229, 240, or 265 of SEQ ID NO. 1.

[0057] In some embodiments, variant spDAEase comprises an amino acid substitution at position 138, and at least another amino acid substitution at positions 69, 119, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1.

[0058] In some embodiments, variant spDAEase comprises an amino acid substitution at position 69, and at least another amino acid substitution at positions 119, 138, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1. In some embodiments, variant spDAEase comprises an amino acid substitution at position 69 or 138, and at least another amino acid substitution at positions 119, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1.

[0059] In some embodiments, variant spDAEase comprises an amino acid substitution at position 265, and at least another amino acid substitution at positions 69, 119, 138, 175, 203, 208, 229, 240 of SEQ ID NO. 1.

[0060] In some embodiments, variant spDAEase comprises an amino acid substitution at position 203, and at least another amino acid substitution at positions 69, 119, 138, 175, 208, 229, 240, 265 of SEQ ID NO. 1.

[0061] In some embodiments, variant spDAEase comprises an amino acid substitution at position 203 or 265, and at least another amino acid substitution at positions 69, 119, 138, 175, 208, 229, 240 of SEQ ID NO. 1.

[0062] In some embodiments, variant spDAEase comprises an amino acid substitution at position 229, and at least another amino acid substitution at positions 69, 119, 138, 175, 203, 208, 240, 265 of SEQ ID NO. 1.

[0063] In some embodiments, variant spDAEase comprises an amino acid substitution at position 119, and at least another amino acid substitution at positions 69, 138, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1.

[0064] In some embodiments, variant spDAEase comprises an amino acid substitution at position 119 or 229, and at least another amino acid substitution at positions 69, 138, 175, 203, 208, 240, 265 of SEQ ID NO. 1.

[0065] In some embodiments, variant spDAEase comprises an amino acid substitution at position 208, and at least another amino acid substitution at positions 69, 119, 138, 175, 203, 229, 240, 265 of SEQ ID NO. 1.

[0066] In some embodiments, variant spDAEase comprises an amino acid substitution at at least one position of 69, 138, 175, 203, 240, 265, and at least another amino acid substitution at positions 119, 208, 229 of SEQ ID NO. 1.

[0067] In some embodiments, variant spDAEase comprises amino acid substitutions at at least two positions of 69, 138, 175, 203, 240, 265 of SEQ ID NO. 1. In some embodiments, variant spDAEase comprises amino acid substitutions at positions 240 and 175 of SEQ ID NO. 1.

[0068] In some embodiments, variant spDAEase comprises an amino acid substitution at positions corresponding to one of the following sets of positions of SEQ ID NO. 1 : a) 69 and at least one of 138, 175, 203, 240, 265; b) 138 and at least one of 69, 175, 203, 240, 265; c) 175 and at least one of 69, 138, 203, 240, 265; d) 203 and at least one of 69, 138, 175, 240, 265; e) 240 and at least one of 69, 138, 175, 203, 265; and f) 265 and at least one of 69, 138, 175, 203, 240.

[0069] In some embodiments, variant spDAEase comprises amino acid substitutions at at least three positions of 69, 119, 138, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1.

[0070] In some embodiments, variant spDAEase comprises amino acid substitution at at least two position selected from 69, 138, 175, 203, 240, 265, and at least another amino acid substitution at positions 119, 208, 229 of SEQ ID NO. 1.

[0071] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with a charged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with a positively charged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with H. This was found to be advantageous from comparative analysis and experimental validation.

[0072] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 240 of SEQ ID No. 1 is substituted with a polar amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 240 of SEQ ID No. 1 is substituted with a polar uncharged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 240 of SEQ ID No. 1 is substituted with S. This was found to be advantageous from comparative analysis.

[0073] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 69 of SEQ ID No. 1 is substituted with a polar amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 69 of SEQ ID No. 1 is substituted with a polar uncharged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 69 of SEQ ID No. 1 is substituted with S. This was found to be advantageous from comparative analysis.

[0074] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 119 of SEQ ID No. 1 is substituted with a polar amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 119 of SEQ ID No. 1 is substituted with a polar uncharged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 119 of SEQ ID No. 1 is substituted with T. This was found to be advantageous from comparative analysis.

[0075] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 138 of SEQ ID No. 1 is substituted with a charged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 138 of SEQ ID No. 1 is substituted with a positively charged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 138 of SEQ ID No. 1 is substituted with K. This was found to be advantageous from comparative analysis.

[0076] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 203 of SEQ ID No. 1 is substituted with a polar amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 203 of SEQ ID No. 1 is substituted with a polar uncharged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 203 of SEQ ID No. 1 is substituted with S. This was found to be advantageous from comparative analysis.

[0077] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 208 of SEQ ID No. 1 is substituted with a hydrophobic amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 208 of SEQ ID No. 1 is substituted with L. This was found to be advantageous from comparative analysis.

[0078] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 229 of SEQ ID No. 1 is substituted with a charged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 229 of SEQ ID No. 1 is substituted with a positively charged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 229 of SEQ ID No. 1 is substituted with L. This was found to be advantageous from comparative analysis.

[0079] In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 265 of SEQ ID No. 1 is substituted with a polar amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 265 of SEQ ID No. 1 is substituted with a polar uncharged amino acid residue. In some embodiments, the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 265 of SEQ ID No. 1 is substituted with N. This was found to be advantageous from comparative analysis.

[0080] In some embodiments, the variant spDAEase comprises one or more amino acid substitution selected from the following : a) A substitution to S at position 69 of SEQ ID NO. 1; b) A substitution to T at position 119 of SEQ ID NO. 1; c) A substitution to K at position 138 of SEQ ID NO. 1; d) A substitution to H at position 175 of SEQ ID NO. 1; e) A substitution to S at position 203 of SEQ ID NO. 1; f) A substitution to L at position 208 of SEQ ID NO. 1; g) A substitution to G at position 229 of SEQ ID NO. 1; h) A substitution to S at position 240 of SEQ ID NO. 1; and i) A substitution to N at position 265 of SEQ ID NO. 1.

[0081] In some embodiments, the amino acid at position 46 is Q. In some embodiments, the amino acid at position 56 is Q. In some embodiments, the amino acid at position 277 is R. In some embodiments, the amino acid at position 293 is R.

[0082] In some embodiments, the variant spDAEase comprises at least one mutation selected from N69S, P119T, R138K, I175H, P203S, F208L, R229G, T240S, and H265N.

[0083] In some embodiments, the variant spDAEase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the variant spDAEase has a homology of at least 70% identical to an amino acid sequence set forth in SEQ ID NO. 1, or at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0084] In some embodiments, the variant spDAEase comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 1, and at least one mutation selected from N69S, P119T, R138K, I175H, P203S, F208L, R229G, T240S, and H265N.

[0085] As used herein, the variant DAEase distinguished from wild-type DAEase derived from Clostridium cellulolyticum is abbreviated as variant ccDAEase.

[0086] In some embodiments, the variant ccDAEase comprises mutations at position 46 and a truncation of C-terminal end of SEQ ID NO. 2.

[0087] In some embodiments, the variant ccDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 46 of SEQ ID No. 2 is substituted with a charged amino acid residue. In some embodiments, the variant ccDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 46 of SEQ ID No. 2 is substituted with a charged amino acid residue. In some embodiments, the variant ccDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 46 of SEQ ID No. 2 is substituted with Q.

[0088] It is believed that this mutation improves residual enzyme activity. In some embodiments, the variant ccDAEase comprises an amino acid substitution of isoleucine (I) to glutamine (Q) at position 46 of SEQ ID NO. 2.

[0089] Variant ccDAEase may already comprise mutations known for conferring thermal stability. For example, the amino acid at position 56 may be glutamine (Q), the amino acid at position 277 may be arginine (R) and the amino acid at position 293 may be arginine (R).

[0090] In some embodiments, the variant ccDAEase comprises 1-12 amino acid deletions at the C-terminus. From Figure 5, in the comparison of the DAEases, the C-terminus equivalent to be removed can be up to 12 residues. In some embodiments, the variant ccDAEase comprises 1-10, or 1-8 amino acid deletions at the C-terminus.

[0091] In some embodiments, the variant ccDAEase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 2. In some embodiments, the variant ccDAEase has a homology of at least 70% identical to an amino acid sequence set forth in SEQ ID NO. 2, or at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0092] In some embodiments, the variant ccDAEase comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 2, and a mutation I46Q and / or a truncation of C-terminal end, or at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0093] The terms "polypeptide", "proteinaceous molecule", "peptide", "protein" and "biocatalyst" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non- naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

[0094] The polypeptide may comprise an amino acid sequence having at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 1-23. Sequence variations herein include amino acid substitutions, insertions, deletions and sequence inversions, and may be natural or engineered. It is to be understood that the sequence variations herein (such as the up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30% sequence variation) are variations which do not substantially affect any biological activity of the polypeptide. For example, the variations may be conservative amino acid substitutions and / or are located in segments of the polypeptide which do not contain the following : an active site, an allosteric site, a chelating site, a site for protein modification (e.g., a phosphorylation, acetylation, glycosylation or cleavage site), a site for intramolecular interaction (e.g., a site of a disulphide or other covalent or non-covalent bond), a binding site for a receptor, ligand, antigen, nucleic acid, protein, lipid, ion or metabolite, a site for an intermolecular covalent or non-covalent interaction, or a multimerisation site (including a dimerisation site). A skilled person can identify appropriate segments and sites which minimally affect a biological activity of a polypeptide using, for example, structural or homology data for the polypeptide.

[0095] As used herein "sequence identity" refers to the number (or fraction expressed as a percentage %) of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global, but for purposes herein alignment is generally a global alignment where the full-length of each sequence is compared. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. Sequence identity can be determined by taking into account gaps as the number of identical residues / length of the shortest sequencexlOO. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalised). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions / length of the total aligned sequencex lOO.

[0096] Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known to those skilled in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0097] As used herein, the phrase "consisting essentially of" in the context of a recited subunit sequence (e.g., amino acid sequence) indicates that the sequence may comprise at least one additional upstream subunit (e.g., 1, 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 or more upstream subunits; e.g., amino acids) and / or at least one additional downstream subunit (e.g., 1, 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 or more upstream subunits; e.g., amino acids), wherein the number of upstream subunits and the number of downstream subunits are independently selectable.

[0098] The terms "wild-type protein" or "parent protein" are used interchangeably herein and refer to the non-mutated version of a polypeptide as it appears naturally. The terms "mutant", "variant", "engineered polypeptide" and "engineered protein" are used interchangeably herein to refer to a polypeptide derived from a wild-type protein and comprising one or more amino acid modifications, e.g., an amino acid substitution, insertion and / or deletion. The variants may be obtained by various techniques well known in the art, e.g., site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.

[0099] The term "modification" or "alteration" as used herein in relation to a position in a polypeptide sequence or an amino acid means that the amino acid in the particular position has been modified compared to the amino acid of the wild-type protein.

[0100] A "substitution" means that an amino acid residue is replaced by another amino acid residue. An amino acid residue may be replaced by another selected from the naturally- occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N- ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign "+" indicates a combination of substitutions. The following terminology is used herein to designate a substitution: N202C denotes that the amino acid residue at position 202 (asparagine, N) of the parent sequence is changed to a cysteine (C). F207V / I denotes that the amino acid residue at position 207 (phenylalanine, F) of the parent sequence is substituted with either a valine (V) or an isoleucine (I).

[0101] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows: a) Amino acid sub-classification

[0102] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in the table below under the heading of exemplary substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. b) Exemplary Amino Acid Substitutions

[0103] The term "deletion", used in relation to an amino acid, means that the amino acid has been removed or is absent. The term "insertion" means that one or more amino acids have been added.

[0104] The present disclosure also provides a polynucleotide encoding a polypeptide or mutant DAEase as disclosed herein. The present disclosure also relates to isolated polynucleotides encoding the polypeptide or mutant DAEase as disclosed herein. The present disclosure also relates to nucleic acid constructs comprising a polynucleotide encoding the polypeptide or mutant DAEase as disclosed herein. The polynucleotide may be operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0105] The present disclosure provides an expression vector comprising the polynucleotide encoding the mutant DAEase as disclosed herein.

[0106] The present disclosure provides a host cell comprising the polynucleotide or expression vector as disclosed herein.

[0107] The term "polynucleotide" or "nucleic acid" is used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA. Polynucleotides can be isolated from natural sources, synthesised in vitro, or prepared from a combination of natural and synthetic molecules using methods known in the art.

[0108] The codon usage in a polynucleotide disclosed herein may be adapted according to the host cell in which the nucleic acids will be transcribed. These steps may be carried out according to methods well known to one skilled in the art. A polynucleotide herein may further comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, secretion peptides and the like that can be used to cause or regulate expression of the polypeptide in a selected host cell or system.

[0109] The term "expression", as used herein, refers to any step involved in the production of a polypeptide including, but being not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0110] The term "expression cassette" denotes a polynucleotide comprising a coding region, i.e., a polynucleotide encoding a polypeptide as defined herein, and a regulatory region, i.e., comprising one or more control sequences, operably linked. The regulatory region may comprise a promoter, e.g., a transcriptional promoter or transcription terminator. The regulatory region may include a promoter that is recognised by a host cell or an in vitro expression system for expression of a nucleic acid encoding a polypeptide as defined herein. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The regulatory region may also contain a transcription terminator, which is recognised by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the nucleic acid encoding the protease. Any terminator that is functional in the host cell may be used in the present invention. Typically, an expression cassette comprises, or consists of, a polynucleotide as defined herein operably linked to a transcriptional promoter and a transcription terminator.

[0111] It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

[0112] By "expression vector" or "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art. The present disclosure provides a host cell comprising a polynucleotide, expression cassette or expression vector as disclosed herein.

[0113] The host cell may be transformed, transfected or transduced with the polynucleotide, expression cassette or expression vector in a transient or stable manner. The expression cassette or vector disclosed herein may be maintained in the host cell as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term "host cell" also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. The host cell may be any cell useful in the production of a polypeptide or polypeptide variant disclosed herein, e.g., a prokaryote or a eukaryote. The prokaryotic host cell may be any Gram-positive or Gramnegative bacterium. The host cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell.

[0114] The polynucleotide, expression cassette or expression vector disclosed herein may be introduced into the host cell by any method known by the skilled person, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation. More than one copy of a polynucleotide, cassette or vector disclosed herein may be inserted into a host cell to increase production of a polypeptide or polypeptide variant disclosed herein. The host cell may be engineered to have an improved capacity to convert fructose to allulose. For instance, an amino acid sequence disclosed herein may be used to complement a wild-type strain of fungus or bacterium already known as able to produce DAEase, in order to improve and / or increase the strain capacity.

[0115] In some embodiments, the host cell is selected from the group consisting of Grampositive bacteria, Gram-negative bacteria, filamentous fungi, yeast and algae. Nonlimiting examples of host cells include Spirochaetales bacterium, Clostridium cellulolyticum, Escherichia coli, Lactococcus lactis, Bacillus sp., Pichia sp., Streptomyces sp. and Actinobacteria.

[0116] The present disclosure provides a method of producing a polypeptide or mutant DAEease as disclosed herein, comprising culturing a host cell defined herein under conditions suitable for expressing the polypeptide. In some embodiments, the method further comprises a step of recovering the polypeptide or mutant DAEease.

[0117] In some embodiments, the polypeptide or mutant DAEease is secreted extracellularly. In some embodiments, the polypeptide is recovered from the cell culture.

[0118] The host cells may be cultivated by methods well known in art, e.g., by shake flask cultivation or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated.

[0119] Where the polypeptide is secreted extracellularly into the nutrient medium, the mutant DAEase can be recovered directly from the culture supernatant. Conversely, the polypeptide can be recovered from cell lysates or after permeabilisation. The polypeptide may be recovered using any method known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spraydrying, evaporation, or precipitation. Alternatively, the polypeptide may be partially or totally purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides.

[0120] The biocatalyst may be immobilized on a resin. Accordingly, the present disclosure provides an immobilized enzyme, wherein the enzyme is a variant DAEase as disclosed herein and / or a wild-type DAEase derived from Spirochaetales bacterium (SEQ ID NO. 1).

[0121] The enzyme may be immobilised on the resin by any means known in the art. Immobilization is a technical process in which enzymes are fixed to or within solid supports, creating a heterogeneous immobilized enzyme system. As compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. In addition, heterogeneous immobilized enzymes systems allow the easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. For example, enzyme immobilization may occur via (1) non-covalent adsorption and deposition, (2) physical entrapment, (3) covalent attachment, and (4) bio-conjugation. Support binding can be physical or chemical, involving weak or covalent bonds. The support can be a synthetic resin, an inorganic polymer such as zeolite or silica, or a biopolymer. Entrapment involves inclusion of an enzyme in a polymer network (gel lattice) such as an organic polymer or a silica sol-gel, or a membrane device such as a hollow fiber or a microcapsule. Entrapment requires the synthesis of the polymeric network in the presence of the enzyme. The last category involves cross-linking of enzyme aggregates or crystals, using a bifunctional reagent, to prepare carrier-free macroparticles.

[0122] Non-covalent methods of protein immobilization involve either passive adsorption onto hydrophobic surfaces or electrostatic interactions with charged surfaces. Nitrocellulose membranes or polylysine-coated slides for electrostatic binding may be used. The major advantage of this kind of immobilization is that neither additional coupling reagents nor modification of the protein of interest is required.

[0123] Enzyme immobilization may be by absorption on mesoporous silicates with pore sizes in the range of 20-300 A. Mesoporous silicates provide a sheltered protected environment in which reactions with selected substrates could proceed. Mesoporous structures have been synthesized using cationic, neutral, and block copolymer surfactants. These copolymers contain organic functional groups and metals located within their framework or grafted onto their surface and have been used as a scaffold to develop mesoporous carbon materials. It is possible to chemically modify their surfaces with various functional groups, enabling electrostatic attraction or repulsion between the mesoporous silicate support and the biological molecule of interest. As a result of their silicate inorganic framework, mesoporous silicates are chemically and mechanically stable and are resistant to microbial attack.

[0124] Enzyme immobilization may be on polyketone polymer by hydrogen bonds. Polyketone polymer -[-CO-CH2CH2-]n- immobilization procedure may be carried out in diluted aqueous buffer, gently mixing the proteins with the polymer. No bi-functional agents or spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain.

[0125] For more stable attachment, the formation of covalent bonds is required, and these are generally formed through reaction with functional groups present on the protein surface. In common with non-covalent adsorption, these methods can be used on unmodified proteins since they rely only on naturally present functional groups. For example, the exposed amine groups of lysine residues readily react with supports bearing active esters, with the most common being N-hydroxysuccinimide (NHS) esters, to form stable amide bonds.

[0126] Cysteine residues, bearing the thiol group, may be employed for protein immobilization and readily undergo conjugate addition with unsaturated carbonyls (e.g., maleimides) to form stable thioether bonds. Maleimide groups strongly favor conjugate addition with thiols at physiological pH (6.5-7.5) since under these conditions amines are predominantly protonated and un-reactive. As proteins generally have very few surface- exposed cysteine residues, it is possible to achieve site-selective immobilization, especially if the protein of interest can be engineered to remove all but one surface cysteine residue or to insert a single cysteine on the surface where none previously existed. The nucleophilicity of the thiol group also means that it can react with epoxides and NHS esters, although in practical terms this latter reaction is relatively slow and the resultant thioester moiety is susceptible to hydrolysis.

[0127] As regard aspartic and glutamic acid residues, the generic method in which they can be used for immobilization is by conversion to their corresponding active esters in situ with a carbodiimide coupling agent and an auxiliary nucleophile. The most commonly used example of the former is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, while NHS is widely used to generate the NHS ester on the protein. This active ester can then react with amine-bearing supports.

[0128] Oxidative cleavage of the 1,2-diols on the oligosaccharides (usually with periodate) generates aldehyde moieties that can then be used for attachment in a semispecific manner to hydrazine or hydroxylamine functionalized supports via their respective hydrazone or oxime.

[0129] The enzyme may also be labelled with an azide moiety for conjugation to the resin. In the Staudinger ligation, the reaction of an azide with a phosphine forms an intermediate iminophosphorane (aza-ylide) that can then react with electrophiles to give a variety of products. The generation of an iminophosphorane is typically followed by reaction with an ester to form a stable amide bond. In the first version of this approach, the electrophilic ester is incorporated into the phosphine to give a final product with the phosphine oxide attached to the linkage. Another method of selective immobilization is derived from the Huisgen 1,3-dipolar cycloaddition of azides with an alkyne, where the covalent link is formed through the formation of a 1,4-disubstituted 1,2,3-triazole. This reaction has been popularized as "click" chemistry, and in the most well-known version, a terminal alkyne and azide are reacted with Cu(I) catalysts to give near-quantitative conversions to the triazole. The alkyne moiety is also rarely present in biological pathways and adds further versatility to this reaction since the alkyne may be introduced to the biomolecule instead of the azide.

[0130] Another related family of reactions is "photoclick chemistry," which relies on photoirradiation to trigger pyrazoline formation between tetrazoles and alkenes. An added advantage of this method is that the newly formed heterocycle is fluorescent, enabling the monitoring of the reaction progress.

[0131] If the enzyme is extremely fragile and easily aggressed by external agent such as proteases, one way to avoiding any negative influence on the structure of an enzyme is to encapsulate it. Encapsulation of these fragile macromolecules is a possible strategy for preventing their aggression and denaturation. A technique to encapsulate biological species such as enzymes, antibodies, and other proteins in a functional state is based on the sol-gel chemistry method.

[0132] Sol-gels are silica materials that are highly porous. The synthesis of sol-gels is relatively mild for enzymes. In the first step, a tetra-alkoxysilane is hydrolyzed via acid catalysis. Hydrolysis is followed by condensation where the sol is formed. This is a mixture of partially hydrolyzed and partially condensed. All the pores of this gel are filled with water and alcohol; it is therefore known as aquagel. When the aquagel is dried by evaporation, a xerogel is obtained. Due to the action of capillary forces during the evaporation process, the aquagel shrinks and part of the structure collapses. The xerogel consequently does not have the same structure as the aquagel. To avoid such capillary action, the water in the aquagel can be exchanged with acetone and then with supercritical carbon dioxide. On evaporation of the carbon dioxide, the structure of the aquagel is maintained and a brittle aerogel is obtained. In this manner, hydrophilic aqua-, xero-, and aerogels are made. By adding alkyltrialkoxysilanes to the synthesis mixture, sol-gels with a hydrophobic surface can be obtained. Overall the sol-gel method can generate gels with different properties.

[0133] Lipid vesicles (also called liposomes) may also be used for encapsulation, which are polymolecular aggregates formed in aqueous solution on the dispersion of certain bilayer-forming amphiphilic molecules, such as phospholipids. Under osmotically balanced conditions, the vesicles are spherical in shape and contain one or more (concentric) lamellae that are composed of amphiphilic molecules. These shells are curved and self-closed molecular bilayers in which the hydrophobic part of the amphiphiles forms the hydrophobic interior of the bilayer and the hydrophilic part (the polar head group) is in contact with the aqueous phase. The interior of the lipid vesicles is an aqueous core, the chemical composition of which corresponds in a first approximation to the chemical composition of the aqueous solution in which the vesicles are prepared. Depending on the method of preparation, lipid vesicles can be multi-, oligo-, or unilamellar, containing many, a few, or one bilayer shell(s), respectively. The diameter of the lipid vesicles may vary between about 20 nm and a few hundred micrometers. Lipid vesicles are generally not a thermodynamically stable state of amphiphiles and do not form "spontaneously" (without input of external energy); they are only kinetically stable, kinetically trapped systems. The physical properties of lipid vesicles depend on how and under which conditions lipid vesicles of a certain amphiphile (or of a mixture of amphiphiles) are prepared. The mean size, the lamellarity, and the physical stability of the vesicles not only depend on the chemical structure of the amphiphiles used, but in general particularly on the method of vesicle preparation.

[0134] Biodegradable polymers nanosystems are an attractive alternative to liposomes since they have the advantages of longer circulation in the blood stream and generally higher drug carrying capacity. Polymers such as poly(lactic acid) and poly(lactic-co-glycolic acid) may be used. These polymers are degradable by bulk erosion through hydrolysis of the ester bonds. To improve the performance of these polymer nanoparticles, polysaccharides such as alginate and chitosan may be applied.

[0135] Affinity tags may be used to conjugate enzymes to the resin. The most well-known genetically encoded affinity tag is the polyhistidine tag (His-tag). The His-tag may be attached to a protein at its N- or C-terminus, or to the internal polypeptide sequence of a protein. This small tag, usually consisting of six sequential histidine residues, chelates transition metals including Cu(II), Co(II), Zn(II), or Ni(II), although the latter is most commonly employed. A support bearing a chelating moiety such as nitrilotriacetic acid or imino-diacetic acid is treated with a solution of the relevant metal salt to produce a support presenting the metal ions. This metal-activated support is then used for protein immobilization through chelation with the Histag.

[0136] Antibodies, may also be used as a means of immobilizing other proteins due to the selectivity of their binding interactions. In order to achieve uniform immobilization, a well-defined monoclonal antibody is needed; polyclonal antibodies are unsuitable since they are not a single species but a heterogeneous population of antibodies that bind their epitope in a variety of conformations.

[0137] The non-covalent interaction of either avidin or streptavidin to proteins functionalized with biotin may be used. The interaction between biotin and (strept) avidin is extremely strong (Kd « 1015M), and this combined with the fact that these proteins are unusually stable to heat, denaturants, extremes of pH, and proteolysis means that the binding is essentially irreversible. The widespread availability of supports such as microtiter plates, microarray substrates, and magnetic particles that are coated with these proteins has also greatly contributed to the popularity of this method as a means of protein immobilization. In order to exploit this attachment for protein immobilization, the protein of interest must first be labeled with biotin. Classical ly, this can be achieved with a number of nonselective chemical biotinylation reagents such as biotin NHS ester.

[0138] One method of protein immobilization is through the use of enzymatically active fusion proteins. In this case, a protein of interest is fused to an enzyme (capture protein) that reacts selectively with an immobilized substrate analogue or inhibitor. This reaction forms a covalent bond between the enzyme and the substrate on the surface.

[0139] There are also a number of other site-selective immobilization methods that do not require enzymatically mediated attachment. These rely on the recognition of specific functional groups present on amino acids in the protein or an associated peptide tag. One example of this employs phenolic oxidative cross-linking, a phenomenon that is widely observed in nature, which includes protein cross-linking through tyrosine residues. Such dityrosine crosslinks occur in structural proteins such as elastin and silk and are catalyzed by metalloenzymes, although a simple complex of Ni(II) with the tripeptide glycine-glycine-histidine can also catalyze these reactions. Since IMAC and immobilization with Ni(II) via six-histidine tags and NTA-functionalized supports are already widely used, it was rationalized that this complex could be used as the crosslinking catalyst instead. By bringing two tyrosine residues, one from the protein to be immobilized and another from the support, into close proximity to the Ni(II) complex, it would be possible to cross-link the two and form a covalent bond between the support and a specific location on the protein. Once the cross-linking had been achieved, the nickel was removed.

[0140] In some embodiments, the resin comprises metal ions. The metal ions allows for coordination of peptides and / or proteins via coordination chemistry. For example, the resin may be configured to coordinate with peptides and / or proteins comprising an affinity tag. For example, the resin may be Ni-NTA (nickel-nitrilotriacetic acid) resin. Ni- NTA resin comprises immobilized nickel ions, which allows for coordination of peptides and / or proteins comprising a His tag. Histidine residues in the His tag may bind to the vacant positions in the coordination sphere of the Ni ions with high specificity and affinity.

[0141] In some embodiments, the resin is Co-NTA (cobalt-nitrilotriacetic acid) resin. Co-NTA resin has an affinity to His-tag, HA-Tag or flag-tag.

[0142] Ni2+resin may result in higher levels of non-specific binding, leading to co-purification of contaminant proteins. This is due to the strong metal-protein interactions that may also attract non-His-tagged proteins with histidine-rich sequences. As a result, additional steps may be required in the purification process, such as increased wash stringencies or secondary purification methods. On the other hand, Co2+resins may form a slightly weaker complex with the His tag compared to nickel ions, but this reduced binding strength may translate into higher specificity for His-tagged proteins. Consequently, Co2+resins may exhibit lower levels of non-specific binding.

[0143] Imidazole may be added into buffers to facilitate competitive binding. A low imidazole concentration (e.g., 10-25 mM) in the binding and wash buffers may help to minimize the binding of non-specifically interacting proteins. Washing may remove some impurities such as non-specific binding of other proteins, carbohydrates, and / or lipids.

[0144] For example, the washing may be performed using 20 mM imidazole phosphate buffer (pH7.0). The washing may be performed using an aqueous medium. The term 'aqueous medium' used herein refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or non-polar, and / or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both 'solvents' and 'solvent systems' can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetra hydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also falls within this definition.

[0145] In some embodiments, the immobilised enzyme is characterised by an enzyme to resin loading of about 0.01 mg / g to about 50 mg / g. In other embodiments, the enzyme to resin loading is about 0.1 mg / g to about 50 mg / g, about 0.5 mg / g to about 50 mg / g, about 1 mg / g to about 50 mg / g, about 12 mg / g to about 50 mg / g, about 14 mg / g to about 50 mg / g, about 16 mg / g to about 50 mg / g, about 18 mg / g to about 50 mg / g, about 20 mg / g to about 50 mg / g, about 22 mg / g to about 50 mg / g, or about 24 mg / g to about 50 mg / g.

[0146] In some embodiments, the immobilised enzyme is characterised by an average resin particle size of about 1 pm to about 300 pm. This size range may allows for higher enzyme activity compared to a size range of more than 300 pm. In other embodiments, the average resin particle size is about 10 pm to about 300 pm, about 50 pm to about 300 pm, about 100 pm to about 300 pm, about 150 pm to about 300 pm, or about 200 pm to about 300 pm.

[0147] In some embodiments, the immobilised enzyme is characterised by an activity of more than 90% for at least 50 days. In some embodiments, the activity is more than 92%, 94%, 96%, 98%, or 99%. In some embodiments, the duration is for at least 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 110 days, 120 days, 130 days, 150 days, or 200 days. In some embodiments, the immobilised enzyme is characterised by an activity of more than 99% for at least 70 days. In some embodiments, the immobilised enzyme is characterised by an activity of more than 90% for at least 150 days. In some embodiments, the immobilised enzyme is characterised by an activity of more than 90% for at least 170 days.

[0148] Compared with free enzymes, the presently disclosed immobilised enzyme show higher activity and, generally, lower apparent Michaelis constants because of a relative ease in accessing the substrate.

[0149] In some embodiments, the enzyme is cross-linked. The enzyme may be intra crosslinked within the enzyme, or may be inter cross-linked between enzymes. The crosslinking of the enzyme may be performed prior to immobilisation. In some embodiments, cross-linker comprises at least one N-hydroxysuccinimide ester (NHS ester) moiety or sulfo-NHS ester moiety. For example, the cross-linker may comprise at least one succinimidyl moiety or sulfo-succinimidyl moiety. For example, the crosslinker may be bis(succinimidyl penta(ethylene glycol) (BS(PEG)s), bis(succinimidyl nona(ethylene glycol) (BS(PEG)g), disuccinimidyl suberate, or bis(sulfoisuccinimidyl) suberate. The cross-linker thus formed an amide linkage with the enzyme with the release of NHS.

[0150] The present disclosure provides a method of converting fructose to allulose using a variant DAEase as disclosed herein, an immobolised enzyme as disclosed herein, and / or a wild-type DAEase derived from Spirochaetales bacterium (SEQ ID NO. 1). The variant DAEase may be variant spDAEase and / or variant ccDAEase.

[0151] The method comprises catalysing the epimerisation of fructose to allulose. Epimerization is a chemical reaction that converts one epimer into another epimer or its chiral partner, meaning it involves a change in the configuration of only one chiral center. This process creates a diastereomer, a stereoisomer that is not a mirror image of the original molecule. This may be facilitated by an enzyme.

[0152] In some embodiments, the DAEase or variant thereof is characterised by a protein stability at more than 45 °C. In some embodiments, the DAEase or variant thereof is characterised by a protein stability at about 45 °C to about 80 °C. In some embodiments, the DAEase or variant thereof is characterised by a protein stability at about 45 °C to about 75 °C, about 45 °C to about 70 °C, about 45 °C to about 65 °C, about 45 °C to about 60 °C, about 45 °C to about 55 °C, or about 45 °C to about 50 °C. In some embodiments, the DAEase or variant thereof is characterised by a protein stability at about 50 °C to about 80 °C, about 55 °C to about 80 °C, about 60 °C to about 80 °C, about 65 °C to about 80 °C, or about 70 °C to about 80 °C.

[0153] In some embodiments, the method is performed at a temperature of about 40 °C to about 80 °C. In other embodiments, the temperature is about 40 °C to about 75 °C, about 40 °C to about 70 °C, about 40 °C to about 65°C, about 40 °C to about 60 °C, about 40 °C to about 55°C, about 40 °C to about 50 °C, about 40 °C to about 45 °C, about 45 °C to about 80 °C, about 45 °C to about 75 °C, about 45 °C to about 70 °C, about 45 °C to about 65 °C, about 45 °C to about 60 °C, about 45 °C to about 55 °C, about 45 °C to about 50 °C, about 50 °C to about 80 °C, about 50 °C to about 75 °C, about 50 °C to about 70 °C, about 50 °C to about 65 °C, about 50 °C to about 60 °C, about 50 °C to about 55 °C, about 55 °C to about 80 °C, about 55 °C to about 75 °C, about 55 °C to about 70 °C, about 55 °C to about 65 °C, about 55 °C to about 60 °C, about 60 °C to about 80 °C, about 60 °C to about 75 °C, about 60 °C to about 70 °C, about 60 °C to about 65 °C, about 65 °C to about 80 °C, about 65 °C to about 75 °C, about 65 °C to about 70 °C, about 70 °C to about 80 °C, or about 70 °C to about 75 °C.

[0154] In some embodiments, the DAEase or variant thereof is characterised by a protein stability at about pH 6 to about pH 8.5. In some embodiments, the DAEase or variant thereof is characterised by a protein stability at about pH 6 to about pH 8, about pH 6 to about pH 7.5, about pH 6 to about pH 7, about pH 6.5 to about pH 8, about pH 7 to about pH 8, or about pH 7.5 to about pH 8.

[0155] In some embodiments, the method is performed at a pH of about 6 to about 8.5. In other embodiments, the method is performed at pH of about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.5, about 6.5 to about 8.5, about 6.5 to about 8, about 6.5 to about 7.5, about 6.5 to about 7, about 7 to about 8.5, about 7 to about 8, about 7 to about 7.5, about 7.5 to about 8.5, about 7.5 to about 8, or about 8 to about 8.5.

[0156] Examples

[0157] To establish a control for DAEases, we started by comparing known stable DAEases (Table 1). Our initial screening (Figure 1) revealed that C. cellulolyticum DAEase (ccDAEase) consistently displayed higher activity. This prompted us to use ccDAEase as our starting point to search for other DAEase variants. The numbering of residues (mutations) follows SEQ ID NO. 1 and SEQ ID NO. 2 of the wild-type sequences as listed in Table 1.

[0158] Table 1 - list of ID to their AA sequences

[0159] Starting from ccDAEase, we identified 8 DAEases with homology ranging from 95-70% (Figure 5A). These DAEases were expressed in E. coli using various optimized tag expression systems. The recombinant DAEases were then tested for activity (Figure 2). Interestingly, DAEase variants with high homology demonstrated low activity. Out of the 8, only NCA98512.1 (71% homology, from Clostridum; SEQ ID NO. 24) and HOV93914.1 (75% homology, from Spirochaetales bacterium, spDAEase; SEQ ID NO. 1) had activity. In particular, spDAEase had showed significant activity compared to ccDAEase (H56Q / Q277R / S293R / I46Q, DPC19) (Figure 2).

[0160] We further characterized the temperature and pH profiles of triple mutant ccDAEase (H56Q / Q277R / S293R, DM1) and spDAEase (Figure 3). We observed differences in temperature profiles, with spDAEase maintaining or even improving activity at 80 °C (Figure 3). In contrast, ccDAEase's activity decreased at temperatures >70 °C. spDAEase's activity remained consistent over pH ranges 6-8.5, while ccDAEase's activity decreased at pH <7.5.

[0161] An important application of these biocatalysts is as immobilized enzymes for manufacturing, and stability is a critical parameter related to the lifetime of immobilized enzymes. Here, we further confirmed the stability of spDAEase with enhanced ccDAEase (DPC19) through assays utilizing immobilized enzymes. We used residual enzyme activity as a means to correlate with stability; this examine the retention of enzyme activity after heat treatment. In our study, we observed significant differences between ccDAEase (DPC19) and spDAEase with spDAEase retaining its activity much better under various conditions, particularly when subjected to heat treatment and lower pH levels (pH <7) (Figure 4). Furthermore, we also noticed further improvement of residual enzyme activity with the use of crosslinking (see Figure 4). The immobilized enzyme under continuous fructose feed was able to maintain at >99% activity for >73 days. (Figure 9). The immobilized enzyme under continuous fructose feed was also able to maintain more than 90% activity for more than 150 days (Figure 10). The activity declined below 60% after 179 days.

[0162] Finally, to examine why spDAEase was so stable, we compared protein sequences and verified this with experimental improvement of ccDAEases. Interestingly, mutations that were earlier observed to confer thermal stability to ccDAEase are already "corrected" in spDAEase (Figure 5A, black arrows). For example, spDAEase has a truncated C-terminal instead of S293R (DPC88 - truncated C-terminal), which we verified in ccDAEase. Additionally, we examined two other possible sites for improvement in ccDAEase. (Figure 5- DPC19-I46Q, DPC87-H175I). With these mutations in addition to triple mutant ccDAEase (H56Q / Q277R / S293R, DM1), in particular I46Q and truncated C- terminus, we observe a significant improvement in residual enzyme activity, indicating their impact on stability (Figure 6). Due to the conservation of the enzyme family (Figure 7), we further hypothesized these are new potential sites for thermal stability.

[0163] Through comparative analyses of the DAEases sequences, we identified new sites of interest that were subsequently tested on DPC47 (see Figure 8). In particular, single mutations of N69S, P119T, R138K, P203S, F208L, R229G, T240S, or H265N have demonstrated improved stability over the wild-type DPC47. To validate our previous findings about the non-beneficial mutation of H175I (Figure 5 - DPC87), converting I175H on DPC47 also resulted in an enzyme with significant improved stability.

[0164] Table 2 - Amino acid sequences of mutant DAEases

[0165] Materials and methods

[0166] Plasmid construction

[0167] Codon-optimized DNA sequences for Escherichia coli (E. coli) were synthesized from Twist Biosciences. These inserts were cloned with E. coli Omnimax competent cells (Thermo Fisher), after it was assembled via golden gate assembly into pET28a (+). These constructs were subsequently transformed into E. coli for protein expression.

[0168] Protein expression

[0169] Method for high-throughput screening of heterologous expression constructs follows the protocol as described in Tiong et al, 2023.

[0170] Shake Flask Culture

[0171] A single colony of transformed E. coli was inoculated into 5 mL of lysogeny broth (LB) containing 50 pg / mL kanamycin and cultured overnight at 37 °C with shaking. The overnight culture was transferred into fresh LB media with kanamycin at a 1 : 100 ratio and incubated at the above conditions until OD600 reaches 0.4-0.6. Then, isopropyl-3- D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM for the induction of the DAEase at 20 °C overnight. The cells were then harvested by centrifugation at 9,000 g for 10 minutes.

[0172] Purification of protein

[0173] The bacteria pellet was resuspended with lysis buffer (50 mM NaaPCU, 100 mM NaCI, pH 7.0) and sonicated with an ultrasonication probe to lyse, while kept on ice. The lysate was centrifuged at 12,000 g for 1 hour at 4 °C. The clarified lysate was added to preequilibrated Ni-NTA beads and incubated with shaking at 4 °C for at least 1 hour. The Ni-NTA resin was washed with wash buffer (50 mM NaaPCU, 100 mM NaCI, 60 mM imidazole, pH 7.0) and finally the proteins were eluted with elution buffer (50 mM NasPCU, 250 mM NaCI, 370 mM imidazole, pH 7.0). The eluate was de-salted using a Cytiva PD10 column and the yields of the resultant proteins were determined.

[0174] Quantification of protein The proteins were quantified through Bradford assay using a protein concentration standard plotted using known concentrations of bovine serum albumin (BSA). The OD595nm reading for the proteins were fitted into the protein concentration standard curve to obtain concentration of proteins and determine volumetric yield of the enzymes.

[0175] Analytical method

[0176] The content of D-fructose and D-allulose was determined by HPLC (Agilent Technologies, 1200 series) with RID detector using Hi-Plex Ca column (300 x 7.7 mm) and ultrapure water as mobile phase with flow rate of 0.6 mL / min at 80 °C.

[0177] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0178] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0179] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0180] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. A variant D-allulose 3-epimerase (DAEase) that is distinguished from a wild-type DAEase derived from Spirochaetales bacterium (spDAEase) by at least one amino acid sequence variation comprising substitution of an amino acid residue corresponding to position 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1; or a variant DAEase that is distinguished from a wild-type DAEase derived from Clostridium cellulolyticum (ccDAEase) by an amino acid sequence variation comprising substitution of an amino acid at position 46 and / or a truncation of C-terminal end of in SEQ ID NO.2.

2. The variant DAEease according to claim 1, wherein the variant spDAEase comprises an amino acid substitution at at least two positions of 69, 119, 138, 175, 203, 208, 229, 240, or 265 of SEQ ID NO. 1.

3. The variant DAEease according to claim 1 or 2, wherein the variant spDAEase comprises an amino acid substitution at position 240, and at least another amino acid substitution at positions 69, 119, 138, 175, 203, 208, 229, 265 of SEQ ID NO. 1.

4. The variant DAEease according to claim 1 or 2, wherein the variant spDAEase comprises an amino acid substitution at position 175, and at least another amino acid substitution at positions 69, 119, 138, 203, 208, 229, 240, or 265 of SEQ ID NO. 1.

5. The variant DAEease according to claim 1 or 2, wherein the variant spDAEase comprises an amino acid substitution at position 69 or 138, and at least another amino acid substitution at positions 119, 175, 203, 208, 229, 240, 265 of SEQ ID NO. 1.

6. The variant DAEease according to claim 1 or 2, wherein the variant spDAEase comprises an amino acid substitution at position 203 or 265, and at least another amino acid substitution at positions 69, 119, 138, 175, 208, 229, 240 of SEQ ID NO. 1.

7. The variant DAEease according to claim 1 or 2, wherein the variant spDAEase comprises an amino acid substitution at position 119 or 229, and at least another amino acid substitution at positions 69, 138, 175, 203, 208, 240, 265 of SEQ ID NO. 1.

8. The variant DAEease according to any one of claims 1 to 7, wherein the variant spDAEase comprises amino acid substitutions at positions corresponding to one of the following sets of positions of SEQ ID NO. 1 :a) 69 and at least one of 138, 175, 203, 240, 265; b) 138 and at least one of 69, 175, 203, 240, 265; c) 175 and at least one of 69, 138, 203, 240, 265; d) 203 and at least one of 69, 138, 175, 240, 265; e) 240 and at least one of 69, 138, 175, 203, 265; and f) 265 and at least one of 69, 138, 175, 203, 240.

9. The variant DAEease according to any one of claims 1 to 8, wherein the variant spDAEase comprises amino acid substitutions at at least three positions of 69, 119, 138, 175, 203, 208, 229, 240, or 265-of SEQ ID NO. 1.

10. The variant DAEease according to any one of claims 1 to 9, wherein the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with a charged amino acid residue.

11. The variant DAEease according to any one of claims 1 to 10, wherein the variant spDAEase comprises an amino acid substitution in which the amino acid residue corresponding to position 175 of SEQ ID No. 1 is substituted with a positively charged amino acid residue.

12. The variant DAEease according to any one of claims 1 to 11, wherein the variant spDAEase comprises one or more amino acid substitutions selected from the following : a) A substitution to S at position 69 of SEQ ID NO. 1; b) A substitution to T at position 119 of SEQ ID NO. 1; c) A substitution to K at position 138 of SEQ ID NO. 1; d) A substitution to H at position 175 of SEQ ID NO. 1; e) A substitution to S at position 203 of SEQ ID NO. 1; f) A substitution to L at position 208 of SEQ ID NO. 1; g) A substitution to G at position 229 of SEQ ID NO. 1; h) A substitution to S at position 240 of SEQ ID NO. 1; and i) A substitution to N at position 265 of SEQ ID NO. 1;13. The variant DAEease according to any one of claims 1 to 12, wherein the variant spDAEase comprise at least one amino acid substitution selected from N69S, P119T, R138K, I175H, P203S, F208L, R229G, T240S, and H265N.

14. The variant DAEease according to any one of claims 1 to 13, wherein the variant spDAEase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 1.

15. The variant DAEease according to claim 1 or 2, wherein the variant ccDAEase comprises an amino acid substitution to glutamine (Q) at position 46 of SEQ ID NO. 2.

16. The variant DAEease according to any one of claims 1, 2, and 15, wherein the variant ccDAEase comprises an amino acid substitution of isoleucine (I) to glutamine (Q) at position 46 of SEQ ID NO. 2.

17. The variant DAEease according to any one of claims 1, 2, 15 and 16, wherein variant ccDAEase comprises 1-12 amino acid deletions at the C-terminus.

18. The variant DAEease according to any one of claims 1, 2, and 15 to 17, wherein the variant ccDAEase has at least 70% sequence identity to an amino acid sequence set forth in SEQ ID NO. 2.

19. An immobilized enzyme, wherein the enzyme is a variant DAEase according to any one of claims 1 to 18 and / or a wild-type DAEase derived from Spirochaetales bacterium (SEQ ID NO. 1).

20. The immobilised enzyme according to claim 19, wherein the enzyme comprises an affinity tag and is immobilised on a resin comprising metal ions.

21. The immobilised enzyme according to claim 19 or 20, wherein the enzyme is inter-enzyme cross-linked with a cross-linker.

22. The immobilised enzyme according any one of claims 19 to 21, wherein the immobilised enzyme is characterised by an activity of more than 90% for at least 50 days.

23. A method of converting fructose to allulose using a variant DAEase according to any one of claims 1 to 18, an immbolised enzyme according to any one of claims 19 to 22, and / or a wild-type DAEase derived from Spirochaetales bacterium (SEQ ID NO. 1).

24. The method according to claim 23, wherein the method is performed at a temperature of about 40 °C to about 80 °C.

25. The method according to claim 23 or 24, wherein the method is performed at a pH of about 6 to about 8.5.

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