POLYPEPTIDES HAVING HYDROLYTIC ACTIVITY ON 1-KESTOSE IN THE PRESENCE OF SUCROSE BUT LACKING SUCRASE (INVERTASE) ACTIVITY, POLYNUCLEOTIDES ENCODING SAME AND METHODS OF PRODUCTING AND USING SAME IN INDUSTRIAL SUCROSE PRODUCTION FROM 1-KESTOSE

Abstract

An industrial process using kestose hydrolases to enable the use of sugar mixtures containing 1-kestose in the industrial production of sucrose (as crystallized sugar) by providing a method for the conversion of 1-kestose into sucrose and fructose in a sugar solution, containing kestose and more than 10 mM sucrose (3 g/L), and comprising the enzymatic hydrolysis (preferably using 1-FEH enzymesEC 3.2.1.153) of 1-kestose. The process further provides a method of producing a polypeptide having 1-kestose hydrolase activity and a composition comprising the polypeptide.

Claims

1. A method for conversion of 1-kestose into sucrose and fructose in a sugar solution containing kestose and more than 10 mM sucrose (3g/L), wherein said method comprises the enzymatic hydrolysis of 1-kestose.

2. The method of claim 1 in which said sugar solution is derived from sugarcane, sugar beet or other crop plants with increased storage carbohydrate content in the form of fructans, particularly 1-kestose.

3. The method of claim 2 in which the hydrolysis of 1-kestose is performed by 1-FEH enzymes (EC 3.2.1.153).

4. A method for producing a polypeptide having Kestose Hydrolase activity, comprising: (a) cultivating a recombinant host cell transformed with a heterologous nucleic acid construct encoding at least one hydrolase acting on glycosidic compounds under conditions conducive for production of polypeptides; and (b) recovering the polypeptides.

5. (canceled)

6. The method of claim 4 wherein the recombinant host cell of step (a) is transformed by a vector comprising a polynucleotide sequence encoding at least one 1-FEH enzyme (EC 3.2.1.153).

7. A composition comprising a polypeptide having Kestose Hydrolase activity, and produced by: (a) cultivating a recombinant host cell transformed with a heterologous nucleic acid construct coding for hydrolases acting on glycosidic compounds under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

8. The composition of claim 7, further comprising polypeptides having Dextranase activity.

9. The composition of claim 7, further comprising polypeptides having Dextranase activity and polypeptides having Amylase activity.

10. The composition of claim 7, further comprising a stabilizer.

11. A method of improving sucrose crystal recovery comprising contacting a sugar solution containing kestose and more than 10 mM sucrose (3 g/L) with an enzyme having a kestose hydrolase activity, wherein the enzyme is a 1-FEH enzyme (EC 3.2.1.153).

Description

BRIEF DESCRIPTON OF THE DRAWINGS

[0022] FIG. 1 is the suggested mechanism of 1-FEH inhibition by sucrose, with part A) showing Catalytic site of chicory 1-FEHIIa with sucrose as ligand (PDB: 2ADE) and part B) showing Catalytic site of chicory 1-FEHIIa with 1-kestose as ligand (PDB: 2AEZ).

[0023] FIG. 2 is the Homology modeling of chicory (Cichorium intybus) 1-FEH IIa (PDB: 2ADE) and 1-FEH I complexed with sucrose (in the center) with residues highlighted in red corresponding to 1-FEH I and those highlighted in blue corresponding to 1-FEH IIa.

[0024] FIG. 3 are HPAEC-PAD Dionex Chromatograms illustrating the validation of 1-kestose hydrolysis by 1-FEH enzyme A (Campanula rapunculoides). The remaining target enzymes tested showed a similar reaction product pattern, with only fructose and sucrose being produced. The upper panel of FIG. 3 indicates the chromatogram of control reaction containing the substrate 1-kestose without enzyme and the lower panel of FIG. 3 shows the formation of fructose and sucrose after hydrolysis of 1-kestose by 1-FEH A, at the concentration of 0.75 mg/mL, for 1 hour, at 30 C. Initial concentration of 1-kestose was 10 mM in all experiments.

[0025] FIG. 4 shows the Sucrose inhibition assay of four 1-FEH targets, with the Target ID letters corresponding to Table 4.

DETAILED DESCRIPTION OF THE INVENTION

[0026] One embodiment according to the present invention refers to a method of producing a polypeptide having Kestose Hydrolase activity, comprising (a) cultivating a recombinant host cell transformed with a heterologous nucleic acid construct coding for hydrolases acting on glycosidic compounds under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0027] According to the method of producing a polypeptide having Kestose Hydrolase activity of the present invention the recombinant host cell is a prokaryotic or eukaryotic cell. In a preferred aspect of the present invention, said cell is a yeast cell or filamentous fungal cell. In a most preferred aspect of the present invention, said cell is a Pichia, a Myceliophthora, a Thielavia, a Trichoderma, an Aspergillus or a Saccharomyces cell.

[0028] In one aspect, the recombinant host cell of the above method is transformed by a vector comprising a polynucleotide sequence encoding at least one hydrolase acting on glycosidic compounds. In a preferred aspect, the abovementioned polynucleotide sequence encodes at least one 1-FEH enzyme (EC 3.2.1.153), wherein said 1-FEH enzyme is listed at Table 1. In another aspect, the 1-FEH enzyme is engineered to show increased performance for the conversion of 1-kestose into sucrose and fructose in the presence of sucrose (more than 10 mM).

[0029] In a further aspect, the invention relates to a composition comprising the polypeptide having Kestose Hydrolase activity according to the present invention and a stabilizer. In one embodiment, the composition comprises polypeptides having Kestose Hydrolase activity according to the present invention and polypeptides having Dextranase activity. In another embodiment, the composition comprises polypeptides having Kestose Hydrolase activity, polypeptides having Dextranase activity and polypeptides having Amylase activity.

1-FEH Inhibition by Sucrose

[0030] Among the 1-FEH enzymes characterized so far in the state of the art, the vast majority is completely inhibited by sucrose at low mM concentration (Verhaest et al. 2007; Le Roy et al. 2008). While it is not straightforward to compare enzyme inhibition data from different research groups, a few 1-FEH enzymes can be identified as being less inhibited by sucrose: 1-FEH I from Chicory Roots (Claessens, Van Laere, and De Proft 1990), 1-FEH I from Helianthus (Xu et al. 2014) and (6 and 1)-FEH from Arabidopsis thaliana (De Coninck et al. 2005). All of them were reported to retain at least 50% of their activity in the presence of 10 mM of sucrose.

[0031] The present invention relates to fructosyl hydrolysis, particularly the hydrolysis of terminal, non-reducing (2.fwdarw.1)- beta-D-fructofuranose residues in oligosaccharides, and more particularly to the selective hydrolysis of small fructans like 1-kestose by 1-FEH enzymes in the presence of substantial amounts (above 10 mM) of sucrose.

[0032] In this way, one aspect of the present invention consists in a method for conversion of 1-kestose into sucrose and fructose in a sugar solution containing more than 10 mM sucrose, wherein said method comprises the enzymatic hydrolysis of 1-kestose.

Structural Determinants for Sucrose Inhibition in 1-FEH Enzymes

[0033] While consistent enzyme kinetics data is essential for an in deep understanding of substrate specificity, specific activity and inhibition profile of 1-FEH candidates, three-dimensional structures, protein alignments and molecular modeling has the potential to shed some light on the structural determinants for sucrose inhibition in 1-FEH enzymes.

[0034] As discussed by Verhaest and colleagues (2007), sucrose inhibition could be related to conserved protein regions among plant cell wall invertases and FEHs known as GSAT and YTG. Enzymes reported as strongly inhibited by sucrose contain a serine or a glycine residue in the YTG region while plant invertases and some FEHs that are reported as not, or only very weakly, inhibited by sucrose contain a W82 homologue (using Cichorium intybus 1-FEH IIa protein as reference) stacking with a hydrophobic amino acid. Interestingly, in chicory 1-FEH I, which is described as very weakly inhibited by sucrose, a leucine instead of tryptophan (W82) is observed in the GSAT region.

[0035] In general, the terminal fructosyl unit is positioned in a very similar way at the 1 subsite on proteins belonging to the family GH 32. In this orientation, the anomeric C2 of fructose (i.e., the one that will suffer the nucleophilic attack and will be covalently attached to the enzyme during hydrolysis) is correctly positioned between the two catalytic active amino acids Glutamate and Aspartate, (respectively D22 and E201 on 1-FEH IIa). Contrariwise, glucosyl unit of sucrose is found in a slightly altered position in 1-FEH IIa enzyme from Cichorium intybus. In this position, the active site E201 forms a short H-linkage with the 02 of the glucosyl part of sucrose, impairing its action as acid-base catalyst. Consequently, the glycosidic oxygen O1 will not be protonated and an enzymatic reaction (i.e., the nucleophilic attack and the sucrose hydrolysis) will not occur. When the substrate 1-kestose is bound in the active site, is the second fructosyl residue that lies in the S1 subsite and the oxygen O3 forms a close intramolecular hydrogen bond with the glycosidic oxygen O1 between glucose and fructose. In this way, the acid-base catalyst E201 can fulfil its function as proton donor to the glycosidic oxygen O1.

[0036] Verhaest and colleagues (2007) speculate that sucrose bind in the so called inhibitor configuration because the high conserved GH32 residue W82 is shifted further away from the active site in 1-FEH IIa enzyme, probably because it does not stack with an aromatic residue as observed in the microbial exo-inulinases enzymes. Most probably, the smaller residues such as glycine and serine at YTG region allow a different position of W82 and the binding of sucrose in the inhibitor configuration. Interestingly, all plant invertases contain a W82 homologue in the GSAT region that stacks with a hydrophobic amino acid at YTG region, which probably prevents the binding of sucrose in the inhibitor configuration.

[0037] A probable mechanism for inhibition considers the position of sucrose, compared to 1-kestose, in chicory 1-FEH IIa catalytic site (FIG. 1). This figure shows that the fructosyl moiety of 1-kestose (B) bound to 1-FEH locates roughly in the same position as the glucosyl moiety of sucrose (A). However, because of the longer binding distance between two fructosyl units compared with the binding distance between the fructosyl and glucosyl moieties in sucrose, this last sugar binds in an orientation that prevents it from hydrolysis.

[0038] In order to elucidate the residues that might be responsible for sucrose binding at the catalytic site, a homology model was generated in which the crystal structure of chicory 1-FEHIIa (PDB: 2ADE) was superimposed with the chicory 1-FEHI (FIG. 2). The main differences observed were: i) the presence of a W82 residue in 1-FEHIIa, instead of the L163 homologue in 1-FEHI; ii) the presence of a S101 residue in 1-FEHIIa, instead of the N182 homologue in 1-FEHI. The tryptophan present in position 82 of 1-FEHII apparently leads to a higher interaction with glucosyl moiety of sucrose than its Leucine homologue in 1-FEHI (FIG. 2). This interaction stabilizes sucrose binding to the active site, preventing 1-kestose to enter. Residues modifications that decrease the interaction of glucosyl moiety of sucrose to the active site might decrease enzymatic inhibition by the sugar. Highlighting this view, mutagenesis experiments in the literature showed that W82L and S101L mutants of 1-FEH IIa enzyme were no longer inhibited by 10 mM sucrose, against a 63% inhibition of the wild type (Verhaest et al., 2007).

[0039] The present invention relates to fructosyl hydrolysis, particularly the hydrolysis of terminal, non-reducing (2.fwdarw.1)- beta-D-fructofuranose residues in oligosaccharides, and more particularly to the selective hydrolysis of small fructans like 1-kestose by native or engineered/mutated 1-FEH enzymes in the presence of sucrose. Some of the Kestose Hydrolases disclosed by the present invention, although not limiting its scope, are listed in Table 2.

TABLE-US-00002 TABLE2 Kestosehydrolasesfor selectivehydrolysisofsmallfructans. GenBank GSAT YTG Gene accession Sequence Sequence 1-FEHI AJW31155.1 GC TGSST LYTA DA [Helianthus tuberosus] 1-FEHI[Cichorium CAC19366.1 GC SGSAT ILYTG DVN intybus] 1-FEHIIaWT CAC37922.1 SC SGSAT MLYTG DSK [Cichorium intybus] 1-FEHIIa(W82L) SC SGSAT MLYTG DSK [Cichorium intybus] 1-FEHIIa(S101L) SC SGSAT MLYTG DSK [Cichorium intybus] 1-FEHIIa SC SGSAT MLYTG DSK (W82L/S101L) [Cichorium intybus] 1-FEH[Campanula CAD49079.1 SC SGSAT LYTG DT rapunculoides] 1-FEH[Brassica AFO66503.1 SC SGSAT ILYTG EE napus] 1-FEHIIa SC SGSAT MLYTG DSK (W82L/S101L) 1-FEHw1 CAD56806.1 GC TGSVT IIYTG DKD [Triticum aestivum] 1-FEHw2 CAD48199.1 GC TGSVT IIYTG DID [Triticum aestivum] 1-FEHw1 GC TGSVT IIYTG DKD [Triticum aestivum] (W84L) 1-FEHw1 GC TGSVT IIYTG DKD [Triticum aestivum] (G103L) 1-FEHw1 GC TGSVT IIYTG DKD [Triticum aestivum] (W84L/G103L) 1-FEHw2 GC TGSVT IIYTG DID [Triticum aestivum] (W84L) 1-FEHw2 GC TGSVT IIYTG DID [Triticum aestivum] (G103L) 1-FEHw2 GC TGSVT IIYTG DID [Triticum aestivum] (W84L/G103L) (6and1)-FEH AAL31183.1 SC SGSAT ILYTG DVN [Arabidopsis thaliana] *aminoacid substitution
Process: The use of the Enzyme for higher yield sugar production

[0040] In a particular embodiment according to the present invention, an enzyme having Kestose Hydrolase activity is intended for use in the sugar industry for conversion of 1-kestose into sucrose, in various sugar solutions. The term Kestose hydrolase means enzymes capable of performing the hydrolysis of 1-kestose into sucrose and fructose in the presence of substantial amounts (above 10 mM) of sucrose.

[0041] The Kestose Hydrolase enzyme used herein may be produced by any means known in the art.

[0042] In one aspect, the invention relates to the application of an enzyme having Kestose Hydrolase activity of the invention for conversion of 1-kestose into sucrose and fructose in a sugar solution.

[0043] In a further aspect, the invention relates to a method for conversion of 1-kestose into sucrose and fructose in a sugar solution comprising contacting the sugar solution with an enzyme having Kestose Hydrolase activity according to the invention. In particular, the enzyme having Kestose Hydrolase activity is a 1-FEH enzyme (EC 3.2.1.153).

[0044] In a preferred aspect of the present invention, the 1-FEH enzyme shows increased performance for the conversion of 1-kestose into sucrose and fructose in the presence of sucrose (more than 10 mM). Table 3 demonstrates a comparison of sucrose inhibition profiles for some of the Kestose Hydrolases disclosed by the present invention.

TABLE-US-00003 TABLE3 SucroseinhibitionprofilesforsomeoftheKestosehydrolases GSAT YTG Sucrose %inhibition Gene Sequence Sequence inhibition bysucrose Reference 1-FEHI GC TGSST LYTA DA Weak 13%with (Xuetal.2014) [Helianthus 10mMsucrose tuberosus] 49%with 100mM sucrose 1-FEHI GC SGSAT ILYTG DVN Weak 25%with (Claessensetal. [Cichorium 20mMsucrose 1990;Verhaestet intybus] al.2007) 1-FEHIIa SC SGSAT MLYTG DSK Strong 63%with (Verhaestetal. [Cichorium 10mMsucrose 2007) intybus] 1-FEHIIa SC SGSAT MLYTG DSK Weak 0%with10mM (Verhaestetal. (W82L) sucrose 2007) [Cichorium intybus] 1-FEHIIa SC SGSAT MLYTG DSK Weak 0%with10mM (Verhaestetal. (S101L) sucrose 2007) [Cichorium intybus] 1-FEHw1 GC TGSVT IIYTG DKD Strong 50%with1mM (VanDenEndeet [Triticum sucrose al.2003) aestivum] 1-FEHw2 GC TGSVT IIYTG DID Strong 50%with1mM (VanDenEndeet [Triticum sucrose al.2003) aestivum] *aminoacid substitution

[0045] Alternatively, the 1-FEH enzyme is engineered to show increased performance for the conversion of 1-kestose into sucrose and fructose in the presence of sucrose (more than 10 mM).

[0046] In the present context, the contact of the Kestose Hydrolase enzyme with the sugar solution may be performed by any means known in the art.

[0047] In the present context a sugar solution means any solution comprising sugar derived from sugarcane, sugar beet or other crop plants with an increased storage carbohydrate content in the form of fructans, particularly 1-kestose.

[0048] In particular, the sugar solution is selected from the group comprising any juice (including primary juice, secondary juice, mixed juice, sulphited juice, limed juice, decanted juice, filtered juice, evaporated juice, concentrated juice, or juices derived from unit operations of sugarcane mills, beet sugar mills or sugar refinery), any syrup (concentrated syrup, sulphited sugar, floated syrup, limed syrup, syrup derived from unit operation of sugarcane mills, sugar beet industry or sugar refinery), any massacuite (massacuite A, massacuite B, massacuite C), any molasses, any magma, raw sugar solution, and/or VVHP/VHP sugar solution, also affinated sugar, melted sugar, clarified sugar, carbonated sugar, phosphated sugar, (including any sugar solution derived from unit operation of sugar refinery).

[0049] The Kestose Hydrolase may be added at any suitable step during the raw sugar, white sugar or refined sugar process. In particular, the Kestose Hydrolase may be added to the sugar cane juice before or during clarification. Other suitable points to add Kestose Hydrolase could be to the holding sugar juice tanks or to syrup tanks. In another particular embodiment the Kestose Hydrolase is added during the evaporation step, e.g., to the molasses stream between evaporators, more particularly prior to the last evaporator.

[0050] The Kestose Hydrolase may be also used in a composition with polypeptides having dextranase activity and polypeptides having amylase activity, since dextran and starch are common contaminants in sugar production processes.

EXAMPLERESULTS

[0051] As a proof-of-concept, four kestose hydrolase targets were selected, cloned, expressed in Pichia pastoris and tested for hydrolysis of 1-kestose to sucrose and fructose, in the presence of sucrose. The targets, depicted in Table 4, comprise one wild type 1-FEH from Campanula rapunculoides, mutated versions of 1-FEH w1 and w1 from Triticum aestivum and a mutated version of 1-FEH IIa from Cichorium intybus.

[0052] Targets B, C and D from Table 4 are described in the literature as strongly inhibited by sucrose (Verhaest et al. 2007; Van Den Ende et al. 2003). In order to obtain enzymes able to hydrolyze 1-kestose in the presence of high amounts of sucrose (e.g. 380 mM) point mutations were performed to substitute W82 homologue Tryptophan residue present within the GSAT region for a Leucine residue. These substitutions are supposed to decrease the interaction of glucosyl moiety of sucrose to the active site of the enzymes, leading to destabilization of sucrose binding and, consequently, less inhibition.

TABLE-US-00004 TABLE4 Kestosehydrolasetargetstestedforkestosehydrolaseactivityandsucroseinhibition. GenBank %Inhibition accession ofWT (WT GSAT YTG enzymeby ID Gene SourceOrganism version) region region Sucrose A 1-FEH Campanula CAD49079.1 SC SGSAT LYTG DT - (Wild rapunculoides Type) B 1-FEHw1 Triticumaestivum CAD56806.1 GC TGSVT IIYTG DKD 50%with (W135L) 1mM sucrose C 1-FEHw2 Triticumaestivum CAD48199.1 GC TGSVT IIYTG DID 50%with (W134L) 1mM sucrose D 1-FEHIIa Cichoriumintybus CAC37922.1 SC SGSAT MLYTG DSK 63%with (W120L) 10mM sucrose *aminoacid substitution

[0053] The enzymes A, B, C and D were expressed in Pichia pastoris KM71H strain with a fusion GFP protein at C-terminal in 300 mL of culture media. After 48-hour induction with 0.5% (v/v) mM of methanol, the recombinant proteins were partially purified by precipitation with ammonium sulphate at 70% of saturation, followed by solubilization with sodium phosphate buffer 50 mM pH6.0. The partially purified proteins were concentrated and analyzed by SDS-PAGE and blue-light transluminator for GFP fluorescence. The enzymatic activity assays of the partially purified enzymes were evaluated using 10 mM 1-kestose as substrate, 50 uL of enzyme and sucrose as inhibitor ranging from zero to 380 mM, in a 100 L-reaction buffered with sodium phosphate 50 mM pH6.0. The reactions were carried for 1 hour at 30 C. and stopped by heating at 95 C. for 5 minutes. The reactions were than diluted to 4 mL with ultrapure water and analyzed high-pressure anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex, Sunnyvale, Calif., USA). Enzyme relative activity was determined by the amount of fructose released.

[0054] HPAEC-PAD analysis showed that all enzymes tested are kestose hydrolases without sucrase activity, since the only reaction products were fructose and sucrose, but no glucose was detected. An example of such HPAEC-PAD chromatograms can be seen in FIG. 3.

[0055] After identifying that the chosen targets are active against 1-kestose but not sucrose, we performed sucrose inhibition studies in which the amount of enzyme and substrate were fixed and increasing concentrations of sucrose were added to the reaction. Inhibition percentage was calculated by the amount of fructose released in each reaction, compared to the fructose released in the reaction without added sucrose (FIG. 3).

[0056] The 1-FEH enzyme from Campanula rapunculoides (line A in Table 4) was selected as a potential target because of its natural substitution of the W82 homologue in the GSAT region by a phenylalanine residue (Table 4). One could expect a low inhibition profile by sucrose, but our data shows that the enzyme is more than 70% inhibited in the presence of 100 mM sucrose. This probably happens because phenylalanine has a similar role as tryptophan in interacting to the glucosyl moiety of sucrose in the active site of the enzyme. This is reasonable, since both amino acids have aromatic side-chains.

[0057] The Triticum aestivum enzymes 1-FEH w1 and 1-FEH w2 (lines B and C, respectively in FIG. 4) were point-mutated to substitute the W82 homologue of GSAT region for a leucine residue, generating the (W135L) and (W134L) versions, respectively (Table 4). These mutations were expected to cause a decrease in the interaction of sucrose to the active site, preventing inhibition. However, as seen in FIG. 4, the enzyme B had a similar inhibition pattern of enzyme A and the enzyme C showed a lower inhibition, but higher than 75% at 380 mM of sucrose. These results are probably explained by the presence of a threonine residue adjacent to the leucine substitution (Table 4), that may also be interacting and stabilizing sucrose in the active site. Thus, further point mutations in these threonine residues have a high potential to decrease enzyme inhibition by sucrose.

[0058] The target enzyme 1-FEH IIa from Cichorium intybus is described as strongly inhibited by sucrose (Verhaest et al. 2007). The same authors showed that the substitution of the W82 homologue residue of GSAT region by a leucine (W120L version, Table 4) decreased enzymatic inhibition from 63% to 0% in the presence of 10 mM sucrose. Since sucrose concentrations usually reach 380 mM in the industrial processes from sugarcane, inhibition data of kestose hydrolases in such sucrose concentrations are necessary. In this sense, the same W120L mutation of 1-FEH IIa from Cichorium intybus was tested and the inhibition reached 52% at 380 mM sucrose (line D in FIG. 4). These results are representative examples of using this enzyme for industrial application for conversion of 1-kestose into sucrose and fructose. Other mutations may have additional effects, like the S139L substitution, which was also demonstrated by Verhaest et al., 2007 to reduce sucrose inhibition in the same enzyme. The double mutation (W120L and S139L) has also a high potential to decrease inhibition in high sucrose concentrations (e.g. 380 Mm).

FULL CITATIONS FOR NON-PATENT LITERATURE CITED WITHIN

[0059] Claessens, Guy, Andr Van Laere, and Maurice De Proft. 1990. Purification and Properties of an Inulinase from Chicory Roots (Cichorium Intybus L.). Journal of Plant Physiology 136 (1): 35-39. doi:10.1016/S0176-1617(11)81611-1.

[0060] De Coninck, Barbara, Katrien Le Roy, Isolde Francis, Stefan Clerens, Rudy Vergauwen, Aileen M. Halliday, Steven M. Smith, Andr Van Laere, and Wim Van Den Ende. 2005. Arabidopsis AtcwINV3 and 6 Are Not Invertases but Are Fructan Exohydrolases (FEHs) with Different Substrate Specif WO96/01904, WO96/21023, WO98/39460, WO99/24593, WO2006066969 and WO2009152285icities. Plant, Cell and Environment 28 (4): 432-43. doi:10.1111/j.1365-3040.2004.01281.x.

[0061] Le Roy, Katrien, Willem Lammens, Andr Van Laere, and Wim Van Den Ende. 2008. Influencing the Binding Configuration of Sucrose in the Active Sites of Chicory Fructan 1-Exohydrolase and Sugar Beet Fructan 6-Exohydrolase. New Phytologist 178 (November 2015): 572-80. doi:10.1111/j.1469-8137.2008.02386.x.

[0062] Sainz-Polo, M. A., M. Ramirez-Escudero, A. Lafraya, B. Gonzalez, J. Marin-Navarro, J. Polaina, and J. Sanz-Aparicio. 2013. Three-Dimensional Structure of Saccharomyces Invertase: ROLE OF A NON-CATALYTIC DOMAIN IN OLIGOMERIZATION AND SUBSTRATE SPECIFICITY. Journal of Biological Chemistry 288 (14): 9755-66. doi:10.1074/jbc.M112.446435.

[0063] Van Den Ende, Wim, Stefan Clerens, Rudy Vergauwen, Liesbet Van Riet, Andr Van Laere, Midori Yoshida, and Akira Kawakami. 2003. Fructan 1-Exohydrolases. Beta-(2,1)-Trimmers during Graminan Biosynthesis in Stems of Wheat? Purification, Characterization, Mass Mapping, and Cloning of Two Fructan 1-Exohydrolase Isoforms. Plant Physiology 131 (2): 621-31. doi:10.1104/pp.015305.

[0064] Verhaest, Maureen, Willem Lammens, Katrien Le Roy, Camiel J. De Ranter, Andr Van Laere, Anja Rabijns, and Wim Van Den Ende. 2007. Insights into the Fine Architecture of the Active Site of Chicory Fructan 1-Exohydrolase: 1-Kestose as Substrate vs Sucrose as Inhibitor. New Phytologist 174 (1): 90-100. doi:10.1111/j.1469-8137.2007.01988.x.

[0065] Xu, Huanhuan, Mingxiang Liang, Li Xu, Hui Li, Xi Zhang, Jian Kang, Qingxin Zhao, and Haiyan Zhao. 2014. Cloning and Functional Characterization of Two Abiotic Stress-Responsive Jerusalem Artichoke (Helianthus Tuberosus) Fructan 1-Exohydrolases (1-FEHs). Plant Molecular Biology 87 (1-2): 81-98. doi:10.1007/s11103-014-0262-1.