Use of glycoside phosphorylases for the production of isomelezitose

Abstract

The present invention relates to glycoside phosphorylases which are useful for the production of isomelezitose (α-D-glucopyranosyl-(1,6)-β-D-fructofuranosyl-(2,1)-α-D-glucopyranoside). More specifically, the invention relates to sucrose 6(F)-phosphate phosphorylases (EC 2.4.1.329), which catalyze, among other things, the synthesis of isomelezitose from a suitable glycoside donor and isomaltulose, where said glucosyl donor can be sucrose or α-D-glucose 1- phosphate. The present invention further relates to a method for producing isomelezitose using said sucrose 6(F)-phosphate phosphorylases.

Description

[0001] USE OF GLYCOSIDE PHOSPHORYLASES FOR THE PRODUCTION

[0002] OF ISOMELEZITOSE

[0003] TECHNICAL FIELD OF INVENTION

[0004] The present invention relates to glycoside phosphorylases which are useful for the production of isomelezitose (a-D-glucopyranosyl-(l,6)-P-D-fructofuranosyl-(2, l)-a-D-glucopyranoside). More specifically, the invention relates to sucrose 6(F)-phosphate phosphorylases (EC 2.4.1.329), which catalyze, among other things, the synthesis of isomelezitose from a suitable glycoside donor and isomaltulose, where said glucosyl donor can be sucrose or a-D-glucose 1- phosphate. The present invention further relates to a method for producing isomelezitose using said sucrose 6(F)-phosphate phosphorylases.

[0005] BACKGROUND ART

[0006] Isomelezitose (a-D-glucopyranosyl-(l,6)-P-D-fructofuranosyl-(2,l)-a-D-glucopyranoside) is a rare trisaccharide naturally present in honey and first reported as a minor transglycosylation product of a-glucosidase from brewer's yeast.1Studies have shown that this non-reducing trisaccharide is non-cariogenic, low in calories and could be a promising candidate for diabetic foods.2-5Moreover, in vitro studies have demonstrated that isomelezitose supports the growth of probiotic bacteria such as Lactobacillus and Enterococcus.4The presence of this trisaccharide in bacterial cultures enhanced the production of butyrate, acetate, and lactate, which are metabolites crucial for gastrointestinal health4. Additionally, isomelezitose exhibits potential as an osmolyte as it significantly reduces losses in cell viability and activity during air drying and storage.6Its osmoprotective properties have even been found to surpass those of melezitose and trehalose.6

[0007] Nevertheless, the potential of isomelezitose in applications such as pharmaceutical excipients, osmoprotectants and diabetic foods is limited by its high cost and low abundance. Its scarcity in nature makes extraction economically unfeasable. However, a number of biocatalytic production processes have been developed starting from sucrose which involve altemansucrases , dextransucrases or glucansucrases.2,3,7Such processes typically result In low yields (7-11%), even when using engineered variants (57%).3In addition, such syntheses often results in the formation of additional oligosaccharides which can further complicate downstream processing.

[0008] Another synthesis approach involves an isomelezitose synthase, engineered from a sucrose isomerase from Protaminobacter rubrum, which was able to achieve yields up to 70% when calculated based on the sucrose that is converted.5However, in this approach, the reaction is stopped before all sucrose is converted in order to limit the synthesis of unwanted side products. Therefore, if the yield were calculated in the same manner as in other reported processes, by considering the total sucrose content rather than just the converted fraction, the actual yield would be substantially lower.3,5

[0009] Finally, several synthesis approaches using a-glucosidases or cellular systems have been described.3, 8 10Notably, the synthesis of isomelezitose was achieved using cell extracts from Metschnikowia reukaufii in the presence of high sucrose concentrations, yielding 16.1% isomelezitose relative to the total sugar content of the mixture.11

[0010] Some rare sugars or glycosides can be synthesized using glycoside phosphorylases. Glycoside phosphorylases catalyze the phosphorolysis of carbohydrates or glycosides. However, due to the reversible nature of the phosphorolysis reaction, these enzymes can also be used for synthesis reactions. Notably, some glycoside phosphorylases are known to be promiscuous, allowing them to transfer the glycosyl group of a glycosyl donor to a variety of alternative glycosyl acceptors. For example, sucrose phosphorylases (EC 2.4.1.7; SP) can efficiently use certain monosaccharides as glycosyl acceptor to produce rare disaccharides.12,13As another example, sucrose 6(F)-phosphate phosphorylase (EC 2.4.1.329; SPP) and variants of SPP can efficiently use certain phenolic molecules such as resorcinol or resveratrol as glycosyl acceptor to produce the corresponding glucosides.14However, for numerous interesting carbohydrate or glycoside products, a suitable promiscuous glycoside phosphorylase that enables their high- yielding synthesis has not been discovered. Previous studies have evaluated the activity of SP, SPP, and other evolutionarily related glycoside phosphorylases from carbohydrate-active enzyme subfamily GH13 18 on a wide range of candidate glycosyl acceptors. However, those studies have reported that these glycoside phosphorylases are either unable to use disaccharides as glycosyl acceptor to synthesize the corresponding tri saccharides, or the activities on these acceptors are low in comparison to the competing hydrolytic activity.15-18More specifically, earlier work on the characterization of SPs or SPPs did not report their ability to catalyze the synthesis of isomelezitose.15,16,19-21

[0011] It can be concluded that there is a need to develop more efficient methods to produce isomelezitose. There is also a need to find appropriate glycoside phosphorylases for the synthesis of this tri saccharide.

[0012] BRIEF DESCRIPTION OF FIGURES

[0013] Figure 1 The effect of (a) pH and (b) temperature on the initial activity of A / rSPP. The pH profile was determined at 37°C, the temperature profile was determined at pH 5.5. All reactions were performed using 100 mM a-glucose 1 -phosphate and 50 mM glycosyl acceptor.

[0014] Figure 2 (a) Reactions catalyzed by by Sphaerochaeta globosa str. Buddy sucrose 6(F)- phosphate phosphorylase and related glycoside phosphorylases, (a) Transglycosylation reaction with sucrose and isomaltulose to form isomelezitose, (b) Transglycosylation reaction with a-D- glucose 1 -phosphate and isomaltulose to form isomelezitose.

[0015] Figure 3 Production process of isomelezitose with a 2:1 ratio of glycosyl donor (sucrose) and glycosyl acceptor (isomaltulose). The reaction was performed using 0.02 mg.mL'1A / rSPP, 400 mM sucrose, 200 mM isomaltulose and incubated at 37°C and pH 5.5.

[0016] DESCRIPTION OF THE INVENTION

[0017] The present invention relates to the use of glycoside phosphorylases for the production of isomelezitose. More specifically, the invention relates to the use of sucrose 6(F)-phosphate phosphorylases (SPP, EC 2.4.1.329) for the production of isomelezitose from sucrose or alpha- D glucose 1 phosphate as glycosyl donor and isomaltulose as glycosyl acceptor. The term “sucrose 6(F)-phosphate phosphorylases” relates to enzymes of the EC 2.4.1.329 class. Sucrose 6(F)-phosphate phosphorylases catalyze the reversible conversion of sucrose 6(F)- phosphate and phosphate into a-glucose 1 -phosphate and fructose 6-phosphate. An accepted alternative name of this enzyme is “sucrose 6’-phosphate phosphorylase”. A person skilled in the art can determine whether an enzyme catalyzes the reaction of a sucrose 6(F)-phosphate phosphorylase by, for example, incubating said enzyme with a-glucose 1 -phosphate as glycosyl donor and fructose 6-phosphate as glycosyl acceptor and monitoring the synthesis of sucrose 6(F)-phosphate by high-performance anion exchange chromatography, or monitoring the release of phosphate from a-glucose 1 -phosphate using the colorimetric phosphomolybdate assay according to Gawronski et al. (2004).22

[0018] The present invention further relates to the use of a sucrose 6(F)-phosphate phosphorylase for the production of isomelezitose, where said sucrose 6(F)-phosphate phosphorylase originates from Lachnosporaceae bacterium, Lachnospiraceae bacterium, Clostridia bacterium, Clostridiales bacterium, Clostridium sp. chh4-2, Hungatella sp., Lacrimispora sp. 210928- DFI.3.58, Treponema socranskii. Treponema socranskii subsp. paredis ATCC 35535, Treponema sp. Marseille-Q4523, Roseburia inulinivorans. Spirochaetaceae bacterium, Spirochaetae bacterium HGW-Spirochaetae-8, Sphaerochaetaceae bacterium, Sphaerochaeta globosa str. Buddy, Candidatus Choladousia inleslinigallinarum. Candidatus Eisenbergiella merdigallinariim. Candidatus Anaerofdum excrementigallinarum, Blautia sp. OF03-15BH or Thermoanaerobacterium thermosaccharolyticum .

[0019] More specifically, the present invention relates to the usage of a sucrose 6(F)-phosphate phosphorylase for the production of isomelezitose as described above wherein said sucrose 6(F)-phosphate phosphorylase has an amino acid sequence corresponding to SEQ ID N° 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 or 35.

[0020] The latter SEQ ID N° correspond to the following amino acid sequences:

[0021] 1) Sucrose 6(F)-phosphate phosphorylase originating from Sphaerochaeta globosa str. Buddy and having the following amino acid sequence (SEQ ID N° 1): MSQKITNRIMLITYPDCFGQGLKDLRMVLDTDFKGLFGGLHILPFFPSTADRGFSPTTY TEVDPRFGDWDDIMALGEQYYLMYDYMINHLSSESDAFKDFVEKKDASLYRDFFIRY KDFWSNGEPTKQDLERMYRRKQIPWITVQFNDGSQEKLWTTFSDYQVDINQNSRVA KQFHADTISFLASHGGTLIRLDAVAYAAKREGTSCFFAEPEIWELLQQCADVLKGSNS VVLPEIHENYFLQQKVEENGYYVYDFQLPMLVLNALYFGKSRYLKNWLRICPRKQFT TLDTHDGIGVVDARYLMPDEELLATRRRCFEMNPDVYAMYAHSGIKIDLDAFDTYQI NCTYYSACGADDWQYYIARAIQFFAPGIPQVYYVGLLAGENDFELYNQTQQNRDVN RSYYSLLDVKENMKRPIVQKLMRLMAFRNTHPAFNGDFEVLPSGVSELDLCWRNGS EYAILYVDFSTFECSIVYTEQGVEKEL

[0022] 2) Sucrose 6(F)-phosphate phosphorylase originating from Lachnosporaceae bacterium and having the following amino acid sequence (SEQ ID N° 2, SEQ ID N° 2 is 54% identical to SEQ ID N° 1):

[0023] MSFKKVSNQIMLITYADSMGKNLKELEEILTTQVKGAIGGLHILPFFPSSADRGFAPMT YREVDPAFGDWEDIERLASI<YYLMYDYMINHVSAHSPEYLDYLEI<I<DESEYRDFFIR FQDFWENGYPTQEQLEKIYKRK AGGPS VKAEFADGS SELIWSTF S SEQIDLDCTQPKV KEFIRKNLEFLAKHGAALIRLDAFGYATKKPGTNCFFLEPEVWDLLKETQDMMAPYG nCALPEIHETYFTQLKLDARGYDVYDFALPLLVLQALYFGDAIYLKNWMKICPKHQFT TLDTHDGIGVMDTYHLLPDDEIDRTLERLYEISPVTKGISTKSSHTYFDAYQVNCSYFS ALDEDENVYLLARAIQFFTPGIPMVYYMGMLAGVNDLELLKKTGGGRDINRRYYTK EQAEEAFMQPVVQKLLRMMELRNTHPAFDGELRILDSDPYTLSVCWSKEQEWAQLD ADLRTKEWKIVYTEQGKEKTFA

[0024] 3) Sucrose 6(F)-phosphate phosphorylase originating from Clostridium sp. chh4-2 and having the following amino acid sequence (SEQ ID N° 3, SEQ ID N° 3 is 54% identical to SEQ ID N° 1):

[0025] MFQKVKNQIMLITYADSMGHNLKELEYVLDTYVKDAIGGIHVLPFFPSSADRGFAPV TYREVDQSFGSWEDVERLASI<YYLMYDYMINHVSAHSPEYLDYLEI<I<DNSAYRNFF IRYRDFWKDGEPTQEELEKIYKRKDGGPSVMAEFADGSRELIWSTFSSEQIDVDVNQP EVKQFIRKNLEFLSQHGAALIRLDAFGYATKKPGTSCFFLEPEIWSLLEEIQDILTAHGIR SLAEIHETYFTQLKLSERGYEVYDFALPLLVLQALYFGDAIYLKNWMKICPRNQFTTL DTHDGIGVMD AYHLLPDEEIRRTLNELYKISP VSKEISTKS SHTYFD AYQVNC S YYS AL SEDDEKYLTARAIQFFTPGIPMVYYQGMLIGENDYELAEKSGSGRDINRHYYTLDQV GEAMGRPAVQKQLRMMRLRNSHPAFQGELEILDSDPHTLLLRWVNGGEWAKLQADL KKGSCEITYTDQGMIQRFE

[0026] 4) 6(F)-phosphate phosphorylase originating from Hungatella sp.and having the following amino acid sequence (SEQ ID N° 4, SEQ ID N° 4 is 54% identical to SEQ ID N° 1):

[0027] MFQKVKNQIMLIAYADCMGHDLKDLEKVLKTYVGKAIGGLHVLPFFPSSADRGFAPI TYDEVDPAFGSWEDIERLASEYYLMYDYMINHVSAHSPEYLDYLEKKDGSPYKSFFI RFRDFWEGGAPTEEQLEKIYKRKEGGPS VMAEFEDGSKELVWSTF S SEQIDLDVNDP GVKEFIRKNLEALAKHGAALIRLDAFGYATKKPGTGCFFLEPEVWPLLEESRNILAPY GVRALPEIHETYFTQLKLDARGYDVYDFALPLLVLQALYFGDSIYLKNWMRICPRHQF TTLDTHDGIGVMDTYHLLPDEEIRRTLEHLYTLSPVSKGISTKSSHTYFDAYQINATYY SALGEDDDKYLTARAIQFFTPGIPMVYYVGMLAGSNDYKHLEETAHGRDINRGYYTL EQVDESMKKPVVRRLLQMMEFRNTHPAFDGELHILDSSPETLKVCWRLGEEWARLD

[0028] ADLKQSCFEITYTDKGEVRRF

[0029] 5) Sucrose 6(F)-phosphate phosphorylase originating from Lacrimispora sp. 210928-DFI.3.58 and having the following amino acid sequence (SEQ ID N° 5, SEQ ID N° 5 is 54% identical to SEQ ID N° 1):

[0030] MFQKVSNQIMLITYADSMGKNLKELEEILEKYVKDAIGGLHILPFFPSSADRGFAPITY RMVDPKFGDWDDIRRLASRYYLMYDCMINHVSAHSPEYLDYLEKKDASEYRDFFIR YRDFWEGGAPTGEQMEKIYKRKDGGPS VEAQFADGSKELIWSTF S SEQIDLDCTRPE VRRFIKDNLEFLAGQGAALIRLDAFGYATKAPDTSCFFLEPQVWELLKEAQDVLEPHG VRALAEIHETYFTQLKLAARGYSVYDFALPLLVLQALYFGDGIYLKNWMRICPRNQF TTLDTHDGIGVMDTYHLLPDDEIRRTLDRLFEISPVSRNISTKSSHTYFDAYQVNCTYY SALGGDDETYLAARAIQFFTPGIPMVYYVGMLAGENDYEILKKADSGRDINRGYYTK EQAEEAFAKPVVQKLLRMMELRNSHPAFGGELEILESDPYTLYVCWRLEEEWAKLW

[0031] VDLRSK AWEIT YTDQGSVRTF S 6) Sucrose 6(F)-phosphate phosphorylase originating from Clostridia bacterium and having the following amino acid sequence (SEQ ID N° 6, SEQ ID N° 6 is 55% identical to SEQ ID N° 1):

[0032] MLITYADSLGHNLRDLEQTLNGPFDGAFGGVHILPFFPSSGDRGFSPITYREVDPAFGT

[0033] WEDVDRLGKRYYLMFDYMINHISVRSPEYQDFLKNKDASRYRDFFIRFKDFWKNGE

[0034] PTREEDEAIYRRRIGKGTYIIANFADGTSEKVWCTFSDEQVDINCQRSEEAKRFLKDNL

[0035] AFLSEHGCSLIRLDAFAYATKKAGTNCFFVEPDVWQLMAESRDAASAAGVDILPEIHE

[0036] NYFIQMKLSEKGYYVYDFQLPMLLLNALFTGKTLYLKNWLKICSPHQFTTLDTHDGI

[0037] GVVDVRYLMPDEEVQDTKMKVYEMNPGVAKIFTELRGPLEKSFNTYQINCSYYSAV

[0038] GESDERYLLTRAVQMFAPGIPQIHYQGLLAGKNDFELFEKTQQPRDVNRHNYTLEEIA

[0039] AEVI<RPVVQI<LLELLRFRNTHPAFGGSFALI<DCGENELVIRRENGDAWAELRADFVN

[0040] MTYEISCS

[0041] 7) Sucrose 6(F)-phosphate phosphorylase originating from Clostridia bacterium and having the following amino acid sequence (SEQ ID N° 7, SEQ ID N° 7 is 58% identical to SEQ ID N° 1):

[0042] MSHKNLNHTAMLITYPDSLGNNLQDLHKVLQGPFRGAFGGLHILPFVPSSGDRGFSPI

[0043] TYREVDPRFGTWDDIDAIGQDYYLMYDYMINHISVQSPEYQDFLHNKDASRYRDFFI

[0044] RFKDFWNGGEPTKEEDEAIYRRRIGKGTYITAHFDDGTEEKVWCTFSDEQVDINCQR

[0045] SEEAKRFLKDNLAFLSAHGCSLVRLDAFAYATKKAGTNCFFVEPEVWDLMRECHDV

[0046] VKPLNVELLPEIHENYFTQMKLSERGYWVYDFQLPMLLLNAIFTGKTMYLKNWLRIC

[0047] SPRQFTTLDTHDGIGVVDARYLLPDYELQDTKMHVYEMNPGTAKIFSELRGPLNRTF

[0048] NTYQINCSYFDACGADEKAYEMARAIQIFAPGIPQVYYQGLLAGTNDFALFEKTKQPR

[0049] DVNRRFYSLSDIEKEVQRPVVRAVLELLRLRNTHPAFSGTYHLLASSDHEVTIRWENG

[0050] LSWAELHADF SDHS YS VTHT

[0051] 8) Sucrose 6(F)-phosphate phosphorylase originating from Clostridia bacterium and having the following amino acid sequence (SEQ ID N° 8, SEQ ID N° 8 is 56% identical to SEQ ID N° 1):

[0052] MPIKNKAMLITYADSLGKDLKDLEKVLDGPLAGAFGGVHILPFFPSSGDRGFSPITYRE

[0053] VDPVFGTWEDIDRIGSRYYLMYDYMINHISVRSPEYQDFLAKKDASRYHDFFIRFKDF

[0054] WENGEPTEEEDKAIYRRRIGKGTYITANFADGTSEKVWCTFSDEQVDINCQRSEEAKR

[0055] FLRDTLAFLGDHGCSLIRLDAFAYATKKAGTNCFFVEPDVWELMAEARDAAMARGV DILPEIHENYFIQMKLSEKGYYVYDFQLPMLLLNAIFTGKTHYLKNWLKICSPHQFTT LDTHDGIGVVDVRYLMPDDEVQETKMKVYELNPGTAKIFSELRGPLDRNFNTYQINC SYYSAVGEDDDRYLLTRAVQMFAPGIPQIHYQGLLAGKNDFELYEKTKQPRDVNRHN YTLEEIRKETERPVVQKLLDLLRFRNTHPAFDGSFRMEDCGENELVIRRENGAAWAQL

[0056] RADFEKMTFEISCS

[0057] 9) Sucrose 6(F)-phosphate phosphorylase originating from Clostridia bacterium and having the following amino acid sequence (SEQ ID N° 9, SEQ ID N° 9 is 54% identical to SEQ ID N° 1):

[0058] MSHRPVENKAMLITYPNSLGHDLRDLEQVLEGPLSGAFGGVHILPFFPSSGDRGFSPIT YREVDPSFGTWEDIDRIGSRYYLMFDYMINHISVRSPEFQDFLAKKDASRYHNFFIRFK DFWKNGEPTQEEDAAIYRRRIGKGTYILAQFADGTSEKVWCTFSDEQVDINCLHSEE ARRFLRDTLSFLAGHGCSLIRLDAFAYAAKRAGTNCFFVEPEVWQLMGEARETAGAL

[0059] GVEILPEIHENYFTQMKLWEKGYYVYDFQLPMLLLNAVFTGKTRYIKNWLKICSPHQ FTTLDTHDGIGVVDVRYLMPDNEVQDTKMKVYALNPGTAKIFSELRGPLDRSFNTYQ INCSYYSAVGESDDRYLLTRAVQIFAPGIPQIHYQGLLAGKNDFERFEKTKEPRDVNRH NYTLEEIADEVKRPAVRKLLEMLRFRNTHPAFDGACVLEDCGENELVIRREKGDDWA

[0060] RLHADFEKMTWEITC S

[0061] 10) Sucrose 6(F)-phosphate phosphorylase originating from Clostridia bacterium and having the following amino acid sequence (SEQ ID N° 10, SEQ ID N° 10 is 52% identical to SEQ ID N° 1):

[0062] MLITYPDCMGNDLRDLYTVLTGPLNGAFSGVHILPFFPSSGDRGFAPITYREVDPRFGT

[0063] WEDVDRLGDKYYLMYDYMINHLSVRSQEYQDFLQNKDQSPYHDFFIRYKDFWKNG EPTADESAKIYRRRVQGGTYITAHFADGSEEKVWCTFSDEQVDINCQHSAMAQHFLE DNLAFLASHGCSLVRLDAFAYATKKAGTNCFFVEPDVWKLMGDCRDVVRKLGVDLL

[0064] PEIHENYFTQMKLWEKGYYVYDFQLPMLTLHALFSGQTMYLKNWLRIRSPHAFTTLD THDGIGVVDVRYLMPDDEVQETKMHVYRLNPEAAKIYTELRGPLNKTFNTYQINCSF FSAVGEDAESYVLARALQLFVPGIPQIYYQGLLAGKNDFSLFAKTREPRDVNRHFYTV EEVRSAVLTPTVSRILELVRLRNTHPAFQGTCSFPACSEQELNIHWKNGSAFAELHINFA SRRFTVEHSSN 11) Sucrose 6(F)-phosphate phosphorylase originating from Clostridiales bacterium and having the following amino acid sequence (SEQ ID N° 11, SEQ ID N° 11 is 56% identical to SEQ ID N° 1):

[0065] MPHLPIKNKAMLITYADSLGKDLKDLERVLDGPLAGTFGGVHILPFFPSSGDRGFSPIT

[0066] YREVDPVFGTWEDIDRIGSRYYLMYDYMINHISVRSPEYQDFLAKKDASRYHDFFIRF KDFWENGEPTEEEDKAIYRRRIGKGTYITANFADGTSEKVWCTFSDEQVDINCQRSEE AKRFLRDTLAFLGDHGCSLIRLDAFAYATKKAGTNCFFVEPDVWELMAEARDAAMA RGVEILPEIHENYFIQMKLSEKGYYVYDFQLPMLLLNAIFTGKTHYLKNWLKICSPHQ

[0067] FTTLDTHDGIGVVDVRYLMPDDEVQETKMKVYELNPGTAKIFSELRGPLDRNFNTYQ INCSYYSAVGEDDDRYLLTRAVQMFAPGIPQIHYQGLLAGKNDFELYEKTKQPRDVNR HNYTLEEIRKETERPVVQKLLDLLRFRNTHPAFDGSFRMEDCGENELVIRRENGAAWA QLRADFEKMTFEITCS

[0068] 12) Sucrose 6(F)-phosphate phosphorylase originating from Clostridiales bacterium and having the following amino acid sequence (SEQ ID N° 12, SEQ ID N° 12 is 56% identical to SEQ ID N° 1):

[0069] MAHRPIENKAMLITYADSLGKDLKDLEQVLDGPFSGAFGGVHILPFFPSSGDRGFSPIT YREVDPVFGTWEDIDRIGSRYYLMYDYMINHISVRSPEYRDFLEKKDESRYRDFFIRF KDFWENGEPTEEEDKAIYRRRIGKGTYIIANFADGTSEKVWCTFSDEQVDINCQRSEE ARRFLRDTMEFLGEHGCSLIRLDAFAYATKKAGTNCFFVEPEVWDLMAEARAAAAA

[0070] HGVEILPEIHENYFIQMKLSEKGYYVYDFQLPMLLLNALFTGKTRYLKNWLKICSPHQ FTTLDTHDGIGVVDVRYLMPDDEVQETKMKVYELNPGTAKIFSELRGPLDRNFNTYQ INCSYYSAVGEDDDRYLLTRAVQMFAPGIPQIHYQGLLAGKNDFDLFEKTKQPRDVN RHNYTMEEVRTETERPVVQALLNLLRFRNTHPAFDGTFRLEDCGENELVIRRENGAA

[0071] WAQLRADFEKMTYEITCS

[0072] 13) Sucrose 6(F)-phosphate phosphorylase originating from Treponema socranskii and having the following amino acid sequence (SEQ ID N° 13, SEQ ID N° 13 is 65% identical to SEQ ID N° 1): MPQKI ANKIMLIT YADCMGNDLKDIQ YVLERYLDGAVGGLHILPFFP S S ADRGFAPLT YKEVDPRFGTWDDIASLGKKYYLMYDYMINHLSSASTMYKDFLAKKDASRYRDFFI RYKDFWTKGEPTAEDIDRLYKRKNIPYIEAEFADGSKEKLWTTFSDYQIDINQYSEEAK KFLKDNLIFLSEHGASIVRLDAVAYASKREGTDCFFVEPEIWRLLGECDEILSPRGVAVL PEIHEHYFIQKKTEARGYYTYDFQLPMLTLNALYFGKSMYLKNWLLVCPRKQFTTLD THDGIGVVDARYLMSDEDLLATRRKCFELNPGTFELYEKFGIRMDLARFDTYQINCTY YSACGNDDNKYFIARALQFFAPGIPQVYYVGVWGAKNDFELCNKTGVHRDINRHYY PLDEIDRVSASSLAQKLVALMRFRNSHPAFDGEFRLLPSDEHSLRILRKNGADYAYLFI DFLTYSCDIAYSEDGGERRFPCGF

[0073] 14) Sucrose 6(F)-phosphate phosphorylase originating from Lachnospiraceae bacterium and having the following amino acid sequence (SEQ ID N° 14, SEQ ID N° 14 is 58% identical to SEQ ID N° 1):

[0074] MKKNVSNKIMLITYPDSLGKNLKDLDFTLSQYLDKAVGGVHILPFFPSSGDRGFAPIT YEQVEPAFGDWSDIKTLSEKYYLMCDYMINHMSVRSEIYQDYMEKHEASRYNDFFIK WNKFWDGEPTEEEENKLYKRQEIPYIDARFKDGTTERLWTTFSEEQIDIDCLHSEEAK KFLAQQLRNLADRGISLIRTDAMAYAAKRKGTSCFFVEPEIWNLLKQCQDALEGTGV EVLPEIHENYFIQQKLQEKDVYTYDFQLPMLILNAVYFGRTLYLKNWLRLCPKKQFTT LDTHDGIGVVDVRYLLPDEEVLETKQRVFEQNPEIYQLYTVRNLKVNFSKFDTYQINC TYYDALGADDNRYLMARAVQFFTPGIPQVYYVGLFAGRNDFDYFRETGQARDVNRH NYSLEEIEEAFKRPVVKKMNRLMILRNSHPAFEGKFTLLDTDEHTLGLKWENGKEWA SLTVNFQTFVWEILYTQNGEAVRFE

[0075] 15) Sucrose 6(F)-phosphate phosphorylase originating from Roseburia inulinivorans and having the following amino acid sequence (SEQ ID N° 15, SEQ ID N° 15 is 63% identical to SEQ ID N° 1):

[0076] MGKSVNNEIMLITYADCMGNDLNDLENVLNKYLEGAVGGLHILPFFPSSADRGFAPT TYKEVDPVFGTWENIERLSKKYYLMYDYMINHLSIESDIFKDFIQKKDDSKYKDFFIR YKDFWDGGEPTEEQLGKLYKRKNSPYITVTFADGSQEKLWTTFSEYQIDINCQHSEVA KQFMRDNLTFLCEHGASLIRLDAFAYASKKAGTNCFFIEPDVWNLMDECKEVLESYH AELLPEIHEHYFIQKKLEEKSYYTYDFQLPMLLLNAVHFGKTMYLKNWLKICPRKQF TTLDTHDGIGVVDVRYLMPDEEVLATKQKVFELNPDITEVYIKADFEINFKKFDTYQI

[0077] NCTYYSALGEDDNKYLISRAVQFFAPGIPQVYYVGLFAGKNDFELYHKTEQHRDINRH YYSLDEIETEFERPVVQKMNRLMKFRNTHPAFGGQFMLLNSDDHSLHIRWEKDVEYT ELYVDFETFKWNIIYSENGEEKMFI

[0078] 16) Sucrose 6(F)-phosphate phosphorylase originating from Spirochaetaceae bacterium and having the following amino acid sequence (SEQ ID N° 16, SEQ ID N° 16 is 67% identical to SEQ ID N° 1):

[0079] MQQTIPNTIMLITYADCMGKNLKDLSSVLDKHLQGAIGGLHILPFFPSTADRGFAPTTY

[0080] KEVDPAFGTWDDIKDLGSRYYLMYDYMINHLSSGSAIYQDFLEKKDHSKYKDFFIRY

[0081] KNFWSKGEPSAHDLEIMYRRKEQPWIEATFADGTKEKLWTTFSDHQIDINQKSQAAW

[0082] NFHTDNLRFLAAHGASIIRLDAVAYAAKREGTSCFFAEPEIWELLRRCRSILDQEGTLL

[0083] LSEIHEQYFLEKRLEEHGYWTYDFQLPMLLLNAIYKGHTAYLKDWLRICPRTQFTTLD

[0084] THDGIGVVDARYLMPDEELLDTRRECLKRNPGVYEMYARAGMHMDLDKFDTYQIN

[0085] CTYYSACDADDDKYFLARAVQFFTPGIPQVYYVGMLAGRNDWDLYNKTKANRDIN RHYYSTQEVAQEVERPIVRKLLALMRLRNTHPAFDGTFHLLPSDSHTLAIMWTKGVE YVSLHADF STFQ AGIVYSEAGTEKRFEG

[0086] 17) Sucrose 6(F)-phosphate phosphorylase originating from Spirochaetaceae bacterium and having the following amino acid sequence (SEQ ID N° 17, SEQ ID N° 17 is 63% identical to SEQ ID N° 1):

[0087] MSQKITNRVMLISYPDCIGHGLKDLKAVLDTDFRELFGGLHILPFFPSTADRGFSPTTY

[0088] KEVDPRFGDWNDITALGETYYLMFDYMINHVSSESEAFKDFVEKKDASVYRDFFIRY

[0089] KDFWSNGMPAEKDLDIMYRRKRIPWTIVRFKDRSEEKLWTTFSDDQIDINQHSLVAQQ

[0090] FHKDNISFLASHGGTLIRLDAIAYATKRKGTSCFFIEPEIWELFERCDEILKNTDSVVLPE

[0091] IHENYFLQQKVEEKGYYVYDFQLPMLLLNALYFGNSRYLKNWLKICPRKQFTTLDTH

[0092] DGIGVVDARYLMPDEELLATRTRCFEMNPEVYAMYARAGINIELSKFDTYQINCTYYS ACGADDQKYYIARAVQFF 18) Sucrose 6(F)-phosphate phosphorylase originating from Candidates Ornithospirochaeta stercorigallinarum and having the following amino acid sequence (SEQ ID N° 18, SEQ ID N° 18 is 58% identical to SEQ ID N° 1):

[0093] MQQKVANKIMLITYPDSIGKDFHELDYFLTRYYEGAIGGLHILPFFPSSADRGFSPITYK EVDSAFGDWKDIMNFAQKYYLMYDYMINHISDESEVFKDFVKNKDKSKYWDFFIKY KDFWKNGNPTKEEDAILYKRKEHPYVDVRFEDGSVERIWSTFSDHQMDINIQRSEVA KDFMKENLSFLGQHGAALIRLDAFAYATKKAGTDCFFVEPDVWDLIDECRKFLAPYG VEVLPEIHENYFIQKKLEERGYWTYDFQLPMLIINAFFTGRTMYLKNWMTFCPRKQF TTLDTHDGIGVVD VRYLLPDDELLKTKQHVFEINPNITEVYAKTNRAITF S SFDTYQIN CTYFSALGEDERKYLISRAVQFFAPGIPQVYYCGLFASRNDFDLFHKTGEPRNVNRHY YSLDDIEKEMERPVVKALKRLMLFRNNHPSFNGTFALLASDEHSVRIRWQNGVEYSE LYANFKSLEYRIIYSDNAEEKVLEVEE

[0094] 19) Sucrose 6(F)-phosphate phosphorylase originating from Treponema sp. Marseille-Q4523 and having the following amino acid sequence (SEQ ID N° 19, SEQ ID N° 19 is 65% identical to SEQ ID N° 1):

[0095] MPQKITNKIMLITYADCMGNDLKDLAHVLDRYLDGAVGGLHILPFFPSSADRGFAPLT YKEVDPQFGTWDDIALLGKKYYLMYDYMINHLSSASAVYKDFLAKKEASRYRDFFI RWKDFWTNGEPTAEDFDKLYKRKKIPYIEAEFADGSKEKLWTTFSDHQIDINQYSEEA KNFLKDNLTFLSEHGASIVRLDAVAYASKREGTNCFFVEPEIWQLLDGCNEILFPRGVA VLPEIHEQYFMQKKVEAHGYYTYDFQLPMLTLNALYFGSSLYLKNWLLVCPRKQFTT LDTHDGIGVIDARYLMSDEDLLATRRKCFETNPGTYELYEKFGVHMDLDRFDTYQIN CTYYSACGNDDKKYFIARALQFFAPGIPQVYYVGLWGGKNDFELCNKTGVHRDINR HYYPLSEIDSVSASPLVKKILALMRFRNTHPAFDGEFHLLPSDEHSLRILRKNGAEYAY LF VDFTSF SC AIAYSEDGGERHFPC SF

[0096] 20) Sucrose 6(F)-phosphate phosphorylase originating from Sphaerochaetaceae bacterium and having the following amino acid sequence (SEQ ID N° 20, SEQ ID N° 20 is 65% identical to SEQ ID N° 1): MSQKITNRVMLISYPDCIGHGLKDLKAVLDTDFRELFGGLHILPFFPSTADRGFSPTTY KEVDPRFGDWNDITALGETYYLMFDYMINHVSSESEAFKDFVEKKDASVYRDFFIRY KDFWSNGMPAEKDLDIMYRRKRIPWTIVRFKDRSEEKLWTTFSDDQIDINQHSLVAQQ FHKDNISFLASHGGTLIRLDAIAYATKRKGTSCFFIEPEIWELFERCDEILKNTDSVVLPE IHENYFLQQKVEEKGYYVYDFQLPMLLLNALYFGNSRYLKNWLKICPRKQFTTLDTH DGIGVVDARYLMPDEELLATRTRCFEMNPEVYAMYARAGINIELSKFDTYQINCTYYS ACGADDQKYYIARAVQFFAPGIPQVYY

[0097] 21) Sucrose 6(F)-phosphate phosphorylase originating from Candidates Choladousia intestinigallinarum and having the following amino acid sequence (SEQ ID N° 21, SEQ ID N° 21 is 59% identical to SEQ ID N° 1):

[0098] MNKEVSNKIMLITYADSLGDNLKDLQTVLEKYLEGAVYGLHILPFFPSSADRGFAPVT YDVVDPQFGSWEDIEKLSEKYYLMYDYMINHISAKSEIYKDFLEKKDDSEYRDFFIRF KDFWKNGEPTEEQIAKIYLRRPLPYIEAEFADGSREKLWCTFSEEQIDINCLGSEKAKE YLREKLLFF S SHGASLIRLD AL AYATKREDTRCFFIEPEIWELIRQCQEILEGTGTEILPEI HENYFIQKKLEEKDVYTYDFQLPMLLLNAVYFGRTLYLKNWLKLCPRKQFTTLDTHD GIGVVDVRYLMPDEEVLETKKKVFDANPGIMDIHMRQDAQVNFSKFDTYQICCTYY DALGGEDVRYLMARAVQFFAPGIPQVYYVGLFAGRNDYEFYNTTLQSRDINRHYYKL DEIEEEFKRPVVQKMNELMKFRNSHPAFDGEFELLNTDEHTLGIRWSKGEEFAQLTAD FEKLSYSITYSKDGECLEL

[0099] 22) Sucrose 6(F)-phosphate phosphorylase originating from Blautia sp. OF03-15BH having the following amino acid sequence (SEQ ID N° 22, SEQ ID N° 22 is 59% identical to SEQ ID N° 1):

[0100] MKKVSNKIMLITYADCMGHDLKDLNTVLDKYLDGAVYGLHILPFFPSSADRGFAPTT YDIVDPQFGDWDDMKKLSEKYYLMYDYMINHISAHSEIYHDFLEKKDASEYRDFFIR FKDFWKNGEPTEEQISKIYLRKEQPYIEAEFADGTKEKLWCTFSPEQIDINCLGSEKAK EYLEEKLKFLSDHGASLIRLDALAYASKREDTNCFFVEPEIWKIVDQCEKALEGTDSEI LPEIHENYFIQKKLEEKNVYTYDFQLPMLILNAVYFGRTLYLKDWMKLCPRKQFTTLD THDGIGVVDVRYLMPDEEVLATKKKVFDANPDITEVHTHGNTKVNFSKFDTYQICCT YYDALGSEDERYLIARAVQFFAPGIPQVYYVGLFAGRNDWDFYRETRQPRDINRHYY GMDEIEEEFKRPVVQKMNRLMKLRNSHPAFDGEFVLLDTDEHTLGLRWEKDGEYAQ LVVDFETLTYEIDYSENGENIKFK

[0101] 23) Sucrose 6(F)-phosphate phosphorylase originating from Treponema socranskii subsp. paredis ATCC 35535 having the following amino acid sequence (SEQ ID N° 23, SEQ ID N° 23 is 66% identical to SEQ ID N° 1):

[0102] MPQKIANKIMLITYADCMGGNLKDLSYVLDSYLDGAVGGVHILPFFPSSADRGFAPLT YKEVDPQFGTWDDIASLGKKYYLMYDYMINHLSSASSIYKDFLEKKDASRYRDFFIR YKDFWTKGEPTAEDIDRLYKRKKIPYIEAEFADGSKEKLWTTFSDYQIDINQYSEEAK

[0103] NFLKDNLTFLSEHGASIIRLDAVAYASKREGTDCFFVEPEIWHLLGECDEILSPRGVAVL PEIHEHYFIQKKTEARGYYTYDFQLPMLTLNALYFGKSMYLKNWLLVCPRKQFTTLD THDGIGVVDARYLMSDEDLLATRRKCFELNPGTFELYEKFGIRMDLDRFDTYQINCTY

[0104] YSACGNDDKKYFIARALQFFAPGIPQVYYVGVWGGKNDFELCNKTGVHRDINRHYY PLGEIESVSASPLVQKLIALMRFRNAHPAFDGEFHLLPSDEHSLRILRKNGADYASLFV DFTSFSCDIAYSESGAERKFPCGF

[0105] 24) Sucrose 6(F)-phosphate phosphorylase originating from Treponema socranskii having the following amino acid sequence (SEQ ID N° 24, SEQ ID N° 24 is 66% identical to SEQ ID N° 1):

[0106] MPQRIANKIMLITYADCMGGNLKDLSYVLDRCLDGAVGGVHILPFFPSSADRGFAPLT YKEVDPQFGTWDDIASLGKKYYLMYDYMINHLSSASSIYKDFLEKKDASRYRDFFIR YKDFWTKGEPTAEDIDRLYKRKNIPYIEAQFADGSKEKLWTTFSDYQIDINQYSEEAK

[0107] NFLKDNLVFLSEHGASIIRLDAVAYASKREGTDCFFVEPEIWRLLGECDEILSPRGVAVL PEIHEHYFIQKKTEARGYYTYDFQLPMLTLNALYFGKSMYLKNWLLVCPRKQFTTLD THDGIGVVDARYLMSDEDLLATRRKCFELNPGTFELYEKFGIRMDLDRFDTYQINCTY YSACGNDDKKYFIARVLQFFAPGIPQVYYVGVWGGKNDFELCNKTGVHRDINRHYY PLGEIESVSASPLAQKLIALMRFRNAHPAFDGEFHLLPSDEHSLRILRKNGADYASLFV DFLTYSCDIAYSENGTERRFPCGF 25) Sucrose 6(F)-phosphate phosphorylase originating from Clostridiales bacterium having the following amino acid sequence (SEQ ID N° 25, SEQ ID N° 25 is 59% identical to SEQ ID N° 1):

[0108] MSKKVTNQIMLITYADSLGNNLKDLDRVLSDYLEGAVYGLHILPFFPSSADRGFAPVT YKEVSPEFGTWEDILSLADRYYMMYDYMINHLSAQSEIYKDFLAKKDESKYRDFFIR FKDFWPEGEPTQEQISQLYLRRQVPYIEAEFADGSKEKLWCTFSPEQIDIDCLHSETAK EYMRENMEFLSSHGASLIRLDAVAYASKRRNTNCFFVEPEIWQIMGDCQKALEGTGV EILPEIHENYFIQKKLEEKNVYTYDFQLPMLILNAVYFGRTLYLKDWMKLCPRKQFTT

[0109] LDTHDGIGVVD VRYLMPDEEVLETKKK VFE ANPEINRLNGKVNF SKFDT YQICC S YY DALGSDDNRYLIARAIQFFAPGIPQVYYVGLFAGRNDFDLYEKTKQSRDINRHYYSLA EIEEEFRRPVVQKMNRLMKLRNSHPAFNGDFVLMDTDEYTLGLRWEKGGEYAQLTV RFDTFDWEIRYTEGGEEKVFE

[0110] 26) Sucrose 6(F)-phosphate phosphorylase originating from Candidates Eisenbergiella merdigallinarum having the following amino acid sequence (SEQ ID N° 26, SEQ ID N° 26 is 59% identical to SEQ ID N° 1):

[0111] MSKKVTNQIMLITYADSLGNNLKDLDRVLSDYLEGAVSGLHILPFFPSSADRGFAPVT YREVSPEFGNWDDIMSLADRYYMMYDYMINHLSAHSEIYRDFLAKKDESRYRDFFIR

[0112] FKDFWPEGEPTQEQISQLYLRREVPYIEAEFADGSTEKLWCTFSPEQIDIDCLHSETAKE YLRENLQFLSAHGASLIRLDALAYASKRRNTSCFFVEPEIWQIMGDCQKALEGTGVEI LPEIHENYFIQKKLEEKDVYTYDFQLPMLILNAVYFGRTLYLKDWMKLCPRKQFTTLD THDGIGVVD VRYLMPDEEVLETKKK VFEANPDITRLHKKVNF SKFDT YQICC S YYD A LGSDDERYLIARAVQFFAPGIPQVYYVGLFAGRNDYELYDKTQQPRDINRHYYSLEEIE

[0113] AEFKRPVVQRMNRLMKLRNSHPAFNGNFVLMDTDEVTLGLRWEAGEEYAQLTVRFD TFQWEIRYTQGGEEKVFE

[0114] 27) Sucrose 6(F)-phosphate phosphorylase originating from Lachnospiraceae bacterium having the following amino acid sequence (SEQ ID N° 27, SEQ ID N° 27 is 56% identical to SEQ ID N° 1): MIKNVSNKILLITSPDSLGQNLKDLNFVLSEYLDKAVSGIHILPFFPSSGDRGFAPVTYD

[0115] QVEPAFGDWDDIQVLSEKYYLMCDYMINHMSVQSAIYQDYLEKHEASRYHDFFIKW NKFWDGEPTQEDESKLYKRQDVPYINARFKDGTTERLWTTFSAEQIDIDCLHSEEAKK FLAQQLRNLAGRGISLIRADAMAYAAKRKGTSCFFVEPEMWDLMKQCQDALDGTGV EVLPEIHENYFIQQKLQEKDIYTYDFQLPMLILNAVYFGRTLYLKNWMKLCPRKQFTT

[0116] LDTHDGIGVVDVRYLLPDEEVLETKQRVFEQNPEIHQLYTVRNLKVNFSKFDTYQINC TYYDALGSDDNKYLMARAVQFFTPGIPQVYYVGLFAGRNDFDYFHETGQSRDVNRH NYTLEEIEEAFRRPVVQKMNRLMKLRNNHPAFDGQFILLDTDGHTLGLKWQNKEAW ASLTVNFQTFDWEILYTQNDEIVRFE

[0117] 28) Sucrose 6(F)-phosphate phosphorylase originating from Treponema socranskii having the following amino acid sequence (SEQ ID N° 28, SEQ ID N° 28 is 65% identical to SEQ ID N° 1):

[0118] MPRKIANKIMLITYADCMGGNLKDLSYVLDRYLDGAVGGLHILPFFPSSADRGFAPLT

[0119] YKEVDPQFGTWDDIASLGKKYYLMYDYMINHLSSTSAVYKDFLEKKDASRYRDFFIR

[0120] YKDFWTKGEPSAEDIDRLYKRKKIPYIEAEFADGSKEKLWTTFSDYQIDINQYSEEAK NFLKDNLIFLSEHGASIIRLDAVAYASKREGTDCFFVEPEIWQLLGECDEILSPRGVAVL PEIHEHYFIQKKTEARGYYTYDFQLPMLTLNALYFGKSLYLKNWLLVCPRKQFTTLDT HDGIGVVDARYLMSDEDLLATRRKCFELNPGTFELYEKFGIRMDLSRFDTYQINCTYY

[0121] SACGNDDKKYFIARALQFFAPGIPQVYYVGVWGGKNDFELCKKTGVHRDINRHYYP LSEIESVSASPLAQKLIALMRFRNSHPAFDGEFHLLPSDEHSLRILRKNGADYAYLFVD FLTYSCDIAYSENGTECRFPCGF

[0122] 29) Sucrose 6(F)-phosphate phosphorylase originating from Spirochaetae bacterium HGW- Spirochaetae-8 having the following amino acid sequence (SEQ ID N° 29, SEQ ID N° 29 is 74% identical to SEQ ID N° 1):

[0123] MTQKITNRILLITYPDCIGDGLKDLKAVLDTEFKGLFGGLHILPFYPSTADRGFSPTTYK

[0124] EVDPRFGDWNDIMALGEKYYLMFDYMINHLSSESDAFKDFIEKKDASVYRDFFIRYK DFWTNGEPTEKDLKRMYRRKKNPWIT VQFKDGSKEKLWTTF SEYQ VDINQC SP VAE RFHKEVIAFLASHGGTLIRLDAVAYAAKREGTSCFFAEPEIWELLHRCTEILKDTDSVV LPEIHENYFFQQKIEEKGYYVYDFQLPMLLLNALYFGESRYLKNWLRICPRKQFTTLD THDGIGVVDARYLMPDTELLATRARCFAMNPDVYSMYARGGIKIELDTFDTYQINCT YYSLEDVAKNLKRPIVQRLMGLIVFRNTHPAFNGEFEELPSSPSELALCWRNGSEYAL LRVDFSTSACSIIYTENGVEKELLP

[0125] 30) Sucrose 6(F)-phosphate phosphorylase originating from Lachnospiraceae bacterium having the following amino acid sequence (SEQ ID N° 30, SEQ ID N° 30 is 57% identical to SEQ ID N° 1):

[0126] MPSTGDHMIKKISNRIMLITYPDSMGNNLKDLEQVLSKYYGDAIGGIHILPFFPSSGDR GFAPITYDVVDPRFGDWDDIKRLSEKYYLMCDYMINHMSAQSAIYQDYLKKHNKSK YRDFFIKWNEFWDGEPSSEDFQKLYLRKQIPYIYAQFADGSKEKIWTTFSDEQIDINCL

[0127] GSEEAKKYLAEQLLKLSDRGMTLIRTDAMAYAAKRKGENCFFVEPEMWTLIDQCKE

[0128] ALKGTDVEILPEIHENYFYQKKLEEQDVWTYDFQLPYLILNAVYFGRTLYLKNWLKIC

[0129] PRKQFTTLDTHDGIGVVDTRYLMPDEEVLETKRKVFEANPDLNVLYKDSRVKVNFSK FDTYQIICCYYDALGSDDNRYLIARAVQFFAPGIPQVYYVGLFAGRNDFKLFESTRKG RDVNRHYYALSEIEEEVKRPVVRNMYRLMRLRNNHEAFNGEFKLLDSDMYSLHIRW ENGGSFAELF VNFKEMRFKII YTENDKVEEF S VDG

[0130] 31) Sucrose 6(F)-phosphate phosphorylase originating from Sphaerochaetaceae bacterium having the following amino acid sequence (SEQ ID N° 31, SEQ ID N° 31 is 80% identical to SEQ ID N° 1):

[0131] MSQKITNRVMLISYPDCIGHGLKDLKAVLDTDFRELFGGLHILPFFPSTADRGFSPTTY KEVDPRFGDWNDITALGETYYLMFDYMINHVSSESEAFKDFVEKKDASVYRDFFIRY

[0132] KDFWSNGMPAEKDLDIMYRRKRIPWTIVRFKDRSEEKLWTTFSDDQIDINQHSLVAQQ

[0133] FHKDNISFLASHGGTLIRLDAIAYATKRKGTSCFFIEPEIWELFERCDEILKNTDSVVLPE

[0134] IHENYFLQQKVEEKGYYVYDFQLPMLLLNALYFGNSRYLKNWLKICPRKQFTTLDTH

[0135] DGIGVVDARYLMPDEELLATRTRCFEMNPEVYAMYARAGINIELSKFDTYQINCTYYS

[0136] ACGADDQKYYIARAVQFFAPGIPQVYYVGLLAGENDFELFNQTHLNRDVNRTHYSLE DVAKNLQRPIVQRLMKLIEFRSTHTAFHGEFNVLPSSASDLSLVWKNGSEYALLHVDF STLACSITYTEMGVEKVLLP 32) Sucrose 6(F)-phosphate phosphorylase originating from Candidates Anaerofdum excrementigallinarum having the following amino acid sequence (SEQ ID N° 32, SEQ ID N° 32 is 61% identical to SEQ ID N° 1):

[0137] MSKQVTNQIMLITYADCMGNNLKDLDYVLTHYLQGAVSGLHILPFFPSSGDRGFAPLT YI<EVDPAFGGWEEVLSLADRYYMMYDYMINHLSAESDIYI<DFLAI<I<DDSEYRDFFI

[0138] RYKDFWPENEPTEEQISKMYLRRAQPYIEAQFADGSTERLWCTFSPTQIDINCLGSEKA

[0139] KEFMEDNLRFLSSHGASLIRLDALAYATKRRNTNCFFVEPEIWQLINQCQQALEGTGS

[0140] EILPEIHENYFIQKKLEEKDVYTYDFQLPMLILNAVYFGRTLYLKDWMKLCPRKQFTT

[0141] LDTHDGIGVVDVRYLMPDEEVLETKRKVFEANPGIRQLYKDSNTKVNFSKFDTYQIC CTYYDALGGDDERYLIARAVQFFAPGIPQVYYVGLFAGRNDFDFYNQTKQSRDINRH

[0142] YYTLEEIEQEFKRPVVQRMNRLMKLRNSHPAFNGQFSLLDTDEYTLGLRWQQGEEYA QLTVDFRTFAWDIVYTEAGQEKHF

[0143] 33) Sucrose 6(F)-phosphate phosphorylase originating from Lachnospiraceae bacterium having the following amino acid sequence (SEQ ID N° 33, SEQ ID N° 33 is 56% identical to SEQ ID N° 1):

[0144] MKKNITNQVMLITYSDSIGSNLKELYEVLTRNFKGAIGGIHILPFFPSSGDRGFAPITYD

[0145] VVDPAFGDWDDITKFSEEYYLMCDYMINHMSAQSEIYQDYLKNHNDSRYKDFFIKW

[0146] NEFWDGEPSEEDFQKLYLRKQTPYVNAEFADGTTEKVWSTFSNEQIDIDCLHSEEAK

[0147] KYLYEQLKKLAGRGMALIRTDAMAYAAKRKGENCFFVEPEMWTLVDQCRDALRGT GVEILPEIHENYFYQKKLEEQGVWTYDFQLPFLILNAVHFGRTMYLKNWLKICPRKQF TTLDTHDGIGVVDARYLMPDEEVLETKRAVFDANPDINEIYKDRQMRVNFSKFDTYQ lACTYYDALGSEDDRYLIARAVQFFAPGIPMVYYVGLFAGRNDFDFYHQSKQGRDINR

[0148] HYYTLPEIEEEMKRPVVQKMYRLMCFRNSHEAFNGEFRLLTSDEHSLHIRWEKGAAY AELFVDFEKLTYDIRYTEDGKVKRFE

[0149] 34) Sucrose 6(F)-phosphate phosphorylase originating from Spirochaetia bacterium having the following amino acid sequence (SEQ ID N° 34, SEQ ID N° 34 is 63% identical to SEQ ID N° 1): MPQKLTNKIMLITYADCMGKDLKDLSYVLDHYLQDAVGGIHLLPFFPSSADRGFTPIT YI<EVDPAFGSWDDIQDLGENYYLMFDYMINHLSAESDIYI<DFII<I<I<DNSVYI<DFFIR YKDFWKHGGEPTQEEYEKLYKRKEVPYIEAEFADGTKEKLWTTFSDHQIDINQDSLA AKRFMKENLSFLAEHAAAVIRLDALGYASKREDTRCFFIEPEVWDILAECDKVLEPYD VTILPEVHEHYFLQKKLEERNYYTYDFQLPMLLLNAFYFGKTLYLKNWLKICPRKQF TTLDTHDGIGVVDARYLMPDEELLKTKRKVFEINPGISEVYVKSNMKIDFNTFDTYQI NCTYFSALGEDEQKYFIARAVQFFTPGIPQVYYVGLFAGKNDFELYHRTAVNRDINRH YYSLEEIDQLSQASIVQRLNRLMKFRNSHPAFNGQFLLLLSDTQSLHMRWENDMDYA DLSVDFISLECQITYSCKGKEKAFV

[0150] 35) Sucrose 6(F)-phosphate phosphorylase originating from Thermoanaerobacterium thermosaccharolyticum (77SPP) having the following amino acid sequence (SEQ ID N° 35, SEQ ID N° 35 is 40% identical to SEQ ID N° 1):

[0151] MGGSHHHHHHGMASMALKNKVQLITYPDSLGGNLKTLNDVLEKYFSDVFGGVHILP PFPSSGDRGFAPITYSEIEPKFGTWYDIKKMAENFDILLDLMVNHVSRRSIYFQDFLKK GRKSEYADMFITLDKLWKDGKPVKGDIEKMFLRRTLPYSTFKIEETGEEEKVWTTFG KTDPSEQIDLDVNSHLVREFLLEVFKTFSNFGVKIVRLDAVGYVIKKIGTSCFFVEPEIY EFLDWAKGQAASYGIELLLEVHSQFEVQYKLAERGFLIYDFILPFTVLYTLINKSNEML YHYLKNRPINQFTMLDCHDGIPVKPDLDGLIDTKKAKEVVDICVQRGANLSLIYGDK YKSEDGFDVHQINCTYYSALNCDDDAYLAARAIQFFTPGIPQVYYVGLLAGVNDFEA VKKTKEGREINRHNYGLKEIEESVQKNWQRLLKLIRFRNEYEAFNGEFFIEDCRKDEI RLTWKKDDKRCSLFIDLKTYKTTIDYINENGEEVKYLV

[0152] The present invention further relates to the use of a sucrose 6(F)-phosphate phosphorylases as described above wherein the enzyme is a homologue or a modified homologue or a functional fragment of a sucrose 6(F)-phosphate phosphorylase obtainable from Lachnosporaceae bacterium, Lachnospiraceae bacterium, Clostridia bacterium, Clostridiales bacterium, Clostridium sp. chh4-2, Hungatella sp., Lacrimispora sp. 210928-DFI.3.58, Treponema socranskii. Treponema socranskii subsp. paredis ATCC 35535, Treponema sp. Marseille-Q4523, Roseburia inulinivorans. Spirochaetaceae bacterium, Spirochaetae bacterium HGW- Spirochaetae-8, Sphaerochaetaceae bacterium, Sphaerochaeta globosa str. Buddy, Candidatus Choladousia inleslinigallinarum. Candidatus Eisenbergiella merdigallinarum. Candidatus Anaerofilum excrementigallinarum, Blautia sp. OF03-15BH or Thermoanaerobacterium thermosaccharolyticum .

[0153] The term ‘modified’ indicates that there is at least one amino acid which is absent compared to SEQ ID N° 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 or 35 and / or there is at least one amino acid which is different compared to SEQ ID N01, 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 or 35 and / or there is at least one amino acid which is additional compared to SEQ ID N° 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 or 35. Multiple modifications or combinations of several different modifications are also part of the present invention. Said modifications occur within the entire length of the protein. The term ‘mutated’ or ‘mutation’ refers to spontaneous mutation and / or to an induced mutation in the nucleic acids encoded for the enzymes of the present invention. Said mutation can be a point mutation, deletion, insertion or any other type of mutation. A mutation as used here can actually be obtained by any method known to the person skilled in the art. Specific, but non-limiting, methods that can be used for mutagenesis are site-saturation mutagenesis with degenerate primers (NNK) as described by Sanchis et al. (2008) and random mutagenesis using an error-prone polymerase or a commercial kit containing an error-prone polymerase, such as the GeneMorph II EZClone Domain Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.23Examples of additions that do not influence the enzyme’s activity that are further embodiments of the present invention are N-terminal fusion or C-terminal fusion affinity peptides with the sequence Gly- Gly-Ser-His6-Gly-Met-Ala-Ser (= a His-tag) or related peptides for purification purposes, or deletions or substitutions, preferably conservative substitutions, or any combination thereof that do not diminish activity of isomelezitose synthesis at most 5%, 10% or 20%, preferably by at most 30%, more preferably by at most 40 % and most preferably by at most 50%.

[0154] The term “homologue” relates to a sucrose 6(F)-phosphate phosphorylase, having at least a sequence identity of 90% with any of the sequences corresponding to SEQ ID N° 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 or 35. Hence, said sequence identity can be 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. A skilled person can easily determine the identity of a particular sequence with a wild-type sucrose 6(F)-phosphate phosphorylase by aligning both sequences. In analogy, a skilled person can easily define corresponding amino acids or amino acid regions within both sequences as defined by the present invention by aligning both sequences.

[0155] The term “ functional fragment” relates to the usage of a sucrose 6(F)-phosphate phosphorylase as described above wherein said enzyme contains fewer amino acids than any one of the sequences corresponding to SEQ ID NO1, 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 or 35. In other words, a skilled person can thus clearly test whether ‘a fragment of an enzyme’ is a ‘functional fragment’ via determining whether ‘a fragment of an enzyme’ (i.e., contains less amino acids than any one of the sequences corresponding to SEQ ID N° 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 or 35) catalyzes, among other things, the reversible phosphorolysis of sucrose 6(F)-phosphate.

[0156] The present invention further relates to a method to efficiently produce isomelezitose comprising: providing sucrose or a-D-glucose 1 -phosphate as glycosyl donor, providing isomaltulose as a glycosyl acceptor, providing a sucrose 6(F)-phosphate phosphorylase as described above, and contacting said glycosyl donor, said glycosyl acceptor and said sucrose 6(F)- phosphate phosphorylase in order to allow the synthesis of said isomelezitose.

[0157] It is known that sucrose can be a suitable glycosyl donor in reactions catalyzed by sucrose 6(F)- phosphate phosphorylases.19Sucrose is also a suitable glycosyl donor in the method for producing isomelezitose using a sucrose 6(F)-phosphate phosphorylase as described above.

[0158] More specifically, the present invention relates to a method as described above wherein said isomaltulose is not provided as such but is produced via another catalytic process. The term ‘another catalytic process’ here relates to a process that comprises providing a substrate, providing an enzyme or microorganism as catalyst, and contacting said substrate and catalyst in order to allow the synthesis of isomaltulose. Subsequently, said isomaltulose is used by a sucrose 6(F)-phosphate phosphorylase using the method described above. For example, the term ‘another catalytic process’ may more specifically relate to a process where sucrose is provided as substrate, a sucrose isomerase is provided as catalyst, and said sucrose and sucrose isomerase are contacted in order to allow the synthesis of isomaltulose.

[0159] The present invention further relates to a method as described above wherein said glycosyl donor is not provided as such but is produced via another catalytic process. The term ‘another catalytic process’ here relates to a process that comprises providing a substrate, providing an enzyme or microorganism as catalyst, and contacting said substrate and catalyst in order to allow the synthesis of a-D-glucose 1-phosphate. Subsequently, said a-D-glucose 1-phosphate is used as glycosyl donor by a sucrose 6(F)-phosphate phosphorylase using the method described above. For example, the term ‘another catalytic process’ here may more specifically relate to a process where sucrose and phosphate are provided as substrates, a sucrose phosphorylase is provided as catalyst, and said sucrose, phosphate and sucrose phosphorylase are contacted in order to allow the synthesis of a-D-glucose 1-phosphate.

[0160] EXAMPLES

[0161] MATERIALS AND METHODS

[0162] Materials

[0163] All chemicals were obtained from Merck, unless stated otherwise. The carbohydrates were purchased from Biosynth.

[0164] Sequence analysis

[0165] Protein identifiers classified within CAZy family GH13 18 were extracted from the CAZy database (http: / / www.cazy.org) and their corresponding amino acid sequences were obtained from GenBank. Duplicate sequences were removed, leaving a total of 2804 sequences. Next, a multiple sequence alignment was performed with Clustal Omega using default parameters.24A phylogenetic tree was constructed using RAxML v8.2.12 with the LG+I+G substitution model and the tree was visualized using iTOL v5.25,26The selected sequence of the sucrose 6(F)- phosphate phosphorylases originates from Sphaerochaeta globosa str. Buddy (A / rSPP). Cloning, expression and purification

[0166] The full-length amino acid sequence was codon-optimized for E. coli and then synthesized and subcloned into a pET21a vector at the Nhel and Xhol sites by Life Technologies (Belgium), including a C-terminal Hise tag. The plasmid was transformed in E. coli BL21(DE3) electrocompetent cells. Precultures of 5 mL were grown overnight at 37 °C and 200 rpm in LB medium (500 mL, 5 g-L'1NaCl, 5 g-L’1yeast extract, 10 g-L’1tryptone) supplemented with 100 pg mL'1ampicillin. Next, 500 mL LB, containing 100 pg mL'1ampicillin was inoculated with the preculture and incubated at the same conditions until an ODeoo of 0.6 was reached. The temperature was lowered to 18 °C and expression was induced by adding isopropyl P-D-1- thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Cells were harvested by centrifugation after 16h, the supernatant was discarded, and the pellet was frozen at -20 °C.

[0167] For enzyme extraction and purification, the pellet was thawed and resuspended and incubated for 30 min in 8 mL lysis buffer (10 mM imidazole, 0.1 mM phenylmethyl sulfonyl fluoride, 1 mg mL-1 lysozyme, 50 mM phosphate-buffered saline; pH 7.4). The lysate was sonicated three times for 2 min (Branson sonifier 450, level 3, 50% duty cycle) after which the soluble fraction was obtained by centrifugation (20,000 g for 30 min). The clarified lysate was His-tag purified by nickel-nitrilotriacetic acid chromatography following the instructions of the supplier (HisPur Ni-NTA; Thermo Fisher Scientific). After elution the purified enzyme was exchanged to o 50 mM 3-(N-morpholino) propanesulfonic acid (MES) at pH 5.5 using an Amicon Ultra- 15 centrifugal filter unit with a 30-kDa cut-off (Merck). The protein concentration was measured using a NanoDrop ND- 1000 (Thermo Fisher Scientific) with the extinction coefficient and molecular weight as determined by the ProtParam tool (https: / / web.expasy.org / cgi- bin / protparam / protparam).

[0168] Enzyme characterization

[0169] Reaction products were detected as follows. In the phosphorolysis direction of the reversible reaction, fructose release was measured in the phosphorolysis direction of the reversible reaction was measured using the colorimetric bicinchoninic acid (BCA) reducing sugars assay.15In the synthesis direction of the reversible reaction, inorganic phosphate released from a-glucose 1 -phosphate was detected using the colorimetric phosphomolybdate assay.22Samples could also be analyzed by high-performance anion exchange chromatography (HPAEC; Dionex ICS-6000; Thermo Fisher Scientific) with a CarboPac PA20 pH-stable column and pulsed amperometric detection (PAD).

[0170] Enzyme activity on various candidate glycosyl acceptors was evaluated by incubating 0.35 pM purified enzyme, 25 mM a-glucose 1 -phosphate and 50 mM putative substrate in 50 mM MOPS buffer at pH 7.0 and 30°C for 30 min. A 50 pL-sample was analyzed using the phosphomolybdate assay, and a sample from a reaction without glycosyl acceptor was used as negative control. The influence of pH on the activity of A / rSPP was determined by measuring the specific activity in reactions with 100 mM a-glucose 1 -phosphate and 50 mM glycosyl acceptor at 37°C in 50 mM acetate (pH 3.5 - 4.5), 2-morpholinoethanesulfonic acid (MES) (pH 5.0 - 6.5) or MOPS (pH 7.0 - 7.5). The optimal temperature was determined using the same substrate concentrations in 50 mM MES at pH 5.5. Reaction samples were analyzed using the phosphomolybdate assay.

[0171] The apparent kinetic parameters for A / rSPP were determined at optimal temperature (37°C) and pH (5.5). For each of the kinetic measurements, the concentration of the respective substrate was varied, whereas a constant concentration of 100 mM was used for the remaining cosubstrate. The reaction was monitored for 5 min with regular sampling after which the specific activity was calculated. All reactions were performed in triplicate and the final kinetic parameters were obtained by nonlinear regression of the Michaelis-Menten equation using Sigma Plot 15. The molecular weight (55.3 kDa) was used to calculate the turnover number kcat.

[0172] The enzyme’s kinetic stability was evaluated by incubating the purified protein (77 pg.mL-1) at 37°C. At regular time intervals, the residual activity was determined at 30°C in 50 mM MES, pH 5.5 by measuring the phosphate release with the phosphomolybdate assay. Assuming first- order inactivation kinetics, the experimental data were fitted to the following equation: f=a.exp(-b.x), from which the t50 value was then calculated.

[0173] Optimization of reaction conditions

[0174] Isomelezitose synthesis was conducted in Eppendorf tubes (500 pL) at optimal reaction conditions (50 mM MES pH 5.5; 37°C). Purified SphSPP (0.02 mg.mL'1) was incubated with varying substrate concentrations and monitored for 24 h. Samples were taken regularly, and subsequent analysis was carried out using high-performance anion exchange chromatography (HPAEC; Dionex ICS-6000; Thermo Fisher Scientific) with a CarboPac PA20 pH-stable column and pulsed amperometric detection. Isomelezitose production was quantified by reference to the fructose release that occurs when sucrose is converted, taking into account the glucose release caused by the competing hydrolysis reaction.

[0175] Isomelezitose production

[0176] A proof-of-concept production was performed on 1 L scale with 400 mM sucrose and 200 mM isomaltulose in 50 mM MES buffer (pH 5.5, 37°C). His-tag purified SphSPP was added to the mixture at a final concentration of 0.02 mg.mL-1. Isomelezitose synthesis was then monitored for 9 h with sampling at regular time points, samples were subsequently inactivated at 95°C for 10 min. The obtained solution was cooled to 45°C, then invertase was added to remove the remaining substrates. After 4 h, invertase was inactivated by heating the solution again to 95°C for lOmin. For removal of all ions, the reaction mixture was treated with a mixed bed ion exchange resin (AG501- X8 (D), Bio-Rad), after which all suspended solids were removed by vacuum filtration with a 0.2 pm PES filter. Isomelezitose was finally isolated from the monosaccharides by further purification through preparative liquid chromatography (prepLC; Sugar Purification System, Knauer, Eurokat Ca-resin, mesh 25-56 pm), after which the different fractions were concentrated with a rotavapor (Rotavapor R-200, Biichi) at 40°C to a final Brix value of 20. Crystallization of the trisaccharide was finally achieved by lyophilization (VaCo5 lyophilizer, Zirbus technology), resulting in a powdered product. NMR analysis

[0177] The structure of isomelezitose was elucidated by NMR analysis of highly pure isomelezitose (> 99%) derived from the proof-of-concept production. 'H NMR and13C NMR spectra were recorded at 700.13 and 176.05 MHz, respectively, on a Bruker Avance III 700MHz spectrometer in D2O at 30°C. In case of the 'H NMR spectra, protons were referenced to the residual signal of D2O (5H 4.732 ppm), for13C NMR spectra carbons were referenced to the signal of acetone (5C 30.50 ppm). Spin systems of individual monosaccharide units (determined by COSY, HSQC, 1D-TOCSY, and HSQCTOCSY) were linked using diagnostic correlations extracted from the HMBC experiment. All experiments (XH NMR,13C NMR, gCOSY,JH-13C gHSQC,JH-13C gHMBC, HSQC-TOCSY, 1DTOCSY) were performed using Bruker TopSpin 3.6 software. The final NMR data were compared with spectra previously reported by Cote et al. (2008).11

[0178] RESULTS

[0179] Identification and expression of a glycoside phosphorylase

[0180] All sequences of subfamily 18 of glycoside hydrolase family 13 were extracted from the carbohydrate-active enzyme database. This subfamily is known to contain various glycoside phosphorylases. After analysis of the phylogenetic tree, a sequence encoding a putative glycoside phosphorylase (GP) from Sphaerochaeta globosa strain Buddy was selected from an unexplored clade. The putative glycoside phosphorylase from S. globosa str. Buddy (A / vSPP), containing a C-terminal Hise-tag, was recombinantly expressed in Escherichia coli and purified to apparent homogeneity by affinity chromatography. Approximately 5 mg of purified enzyme could be obtained from a 500-mL culture.

[0181] Characterization of .S / i / z.S P

[0182] A hSPP was screened on an extensive panel of candidate glycosyl acceptors in reactions with a-glucose 1-phosphate as glycosyl donor (Table 1). The panel of candidate glycosyl acceptors included mono- and disaccharides as well as phosphorylated sugars. The activity on these glycosyl acceptors was quantified by monitoring the release of phosphate from a-glucose 1- phosphate using the colorimetric phosphomolybdate assay. Very high activity was observed in reactions with fructose 6-phosphate, indicating that A / rSPP is a sucrose 6(F)-phosphate phosphorylase, which is a known activity of some glycoside phosphorylases in family GH13 18. High activity was also observed in reactions with fructose, indicating the synthesis of sucrose. This observation matches the finding that A / rSPP is a sucrose 6(F)-phosphate phosphorylase. Indeed, certain sucrose 6(F)-phosphate phosphorylases are known to show significant activity on fructose as glycosyl acceptor, and on sucrose as glycosyl donor.19Significant activity was also observed in reactions with L-sorbose, which is a common promiscuous activity of glycoside phosphorylases from this family.20

[0183] Unexpectedly, A / rSPP also showed very high activity in reactions with isomaltulose as glycosyl acceptor. Isomaltulose is a disaccharide (6-O-a-D-glucopyranosyl-D-fructofuranose). In this case, A / rSPP binds the glucosyl moiety of the glucosyl donor a-glucose 1 -phosphate to form a covalent glucosyl-enzyme intermediate, which is then intercepted by isomaltulose to selectively form a 1,2-glycosidic bond, resulting in a non-reducing trisaccharide known as isomelezitose (a-D-glucopyranosyl-(l,6)-P-D-fructofuranosyl-(2, l)-a-D-glucopyranoside). The obtained product was isolated and structurally elucidated by NMR spectroscopy, chemical shifts were in accordance with the rounded values previously reported by Cote et al. (2008).11

[0184] Table 1 Screening of a panel of possible glycosyl acceptor substrates. The substrates were ranked by the ratio of total activity in reactions with a candidate acceptor substrate, to the hydrolytic activity in control reactions without acceptor substrate (Ratio > 3.5: +++; ratio 2 - 3.5: ++; ratio 1.2 - 2: +; ratio < 1.2: -).

[0185] Substrate Activity

[0186] Fructose ++

[0187] Fructose 6-phosphate +++

[0188] Glycerol +

[0189] D-Glycerate +

[0190] L-Glucose

[0191] D-Glucose

[0192] D-Galactose

[0193] L-Mannose + D-Mannose

[0194] L-Tagatose

[0195] D-Tagatose

[0196] Psicose

[0197] L-Xylose

[0198] D-Xylose

[0199] L-Arabinose +

[0200] D-Arabinose

[0201] L-Gulose

[0202] D-Gulose +

[0203] L-Allose

[0204] D-Allose +

[0205] L-Talose

[0206] D-Talose

[0207] L-Idose

[0208] D-Idose

[0209] L-Altrose +

[0210] D-Altrose +

[0211] L-Sorbose +++

[0212] L-Ribose

[0213] L-Rhamnose

[0214] L-Fucose

[0215] D-Fucose

[0216] D-Lyxose

[0217] D-Allulose +

[0218] D-Ribulose

[0219] A-Acetylglucosamine

[0220] Glucosamine

[0221] A-Acetylmannosamine

[0222] Sorbitol ++

[0223] Xylitol +

[0224] Mannitol +

[0225] Ribitol + Arabitol +

[0226] Erythritol +

[0227] Trehalose

[0228] Kojibiose

[0229] Maltose +

[0230] Isomaltose

[0231] Sophorose

[0232] Cellobiose

[0233] Gentiobiose

[0234] Melibiose

[0235] Lactulose

[0236] Sucrose

[0237] Lactose

[0238] Isomaltulose +++

[0239] Glucose 6-phosphate +

[0240] Sucrose 6-phosphate

[0241] The optimal pH of A / rSPP was 5.5 in the synthesis direction of the reversible reaction (Figure 1A). Its optimal temperature for activity was found to be 37°C (Figure IB). In addition, the kinetic stability was evaluated at 37°C, at which the enzyme had a half-life time of 1.6 h.

[0242] The kinetic parameters of A / rSPP were determined at the optimal pH and temperature conditions in both the synthesis and phosphorolysis direction. Michaelis-Menten kinetics were observed for the for the glycosyl donors a-glucose 1 -phosphate and sucrose, and also for the glycosyl acceptors fructose, fructose 6-phosphate, phosphate, and isomaltulose (Table 2). The kinetic parameters for phosphate, a-glucose 1 -phosphate, sucrose, fructose 6-phosphate and fructose confirm that A / rSPP is indeed a sucrose 6(F)-phosphorylase, as described in the past by Verhaeghe and co-workers co-workers.19Indeed, the affinity for fructose 6-phosphate (KM = 6.8 mM) is higher than the affinity for fructose (KM = 39.3 mM). Further, the kinetic parameters confirm that the SPP shows very high affinity for isomaltulose as glycosyl acceptor (KM= 13 mM). Table 2 Apparent kinetic parameters of SphSPP at pH 5.5 and 37 °C.

[0243] Reaction Substrate A„, (mM) Acoz (s1) vv’

[0244] Phosphorolysis Sucrose" 53.5 + 8.8 95.1 + 6.4 1.8

[0245] Phosphate62.2 + 0.3 62.7 + 3.2 28.6

[0246] Synthesis D-Fructosec39.3 + 4.4 22.9 + 1.2 0.58

[0247] D-Fructose 6-phosphate" 6.8 + 0.9 20.6 + 0.9 3.0 a-D-Glucose 1 -phosphate" 25.2 + 4.2 16.9 + 1.2 0.67

[0248] Isomaltulose" 13.0 + 1.7 16.8 + 0.8 1.29aInitial rates were measured with BCA reducing sugars assay, at 37 °C in 100 mM phosphate buffer pH 5.5.bInitial rates were measured with BCA reducing sugars assay, at 37°C in 50 mM MES buffer pH 5.5 in the presence of 100 mM sucrose.cInitial rates were measured with phosphomolybdate assay, at 37°C in 50 mM MES buffer pH 5.5 in the presence of 100 mM a-D-glucose 1 -phosphate.

[0249] Process optimization for isomelezitose production

[0250] SphSPP can be used for the synthesis of isomelezitose from the low-cost bulk sugars sucrose and isomaltulose (Figure 2). SphSPP (0.02 mg.mL-1) was incubated with varying concentrations of sucrose as glycosyl donor and isomaltulose as glycosyl acceptor at 37°C and pH 5.5 (Table 3). Notably, a 2-fold excess and 3-fold excess of sucrose yielded conversions of 96% and 57%, respectively, as calculated from the limiting substrate after a 6-hour reaction period. Conversely, under analogous conditions with a 2-fold excess of isomaltulose, 28% conversion was observed after 6 h. Further, it is worth noting that the double displacement mechanism employed by glycoside phosphorylases from family GH13 18 introduces the risk of water acting as a glycosyl acceptor and intercepting the covalent reaction intermediate, leading to the undesirable irreversible hydrolysis of the glycosyl donor. Therefore, it is interesting that only a minimal amount of glucose (<1%) was detected for each reaction condition, indicating that the undesirable hydrolytic activity is very low under these reaction conditions.

[0251] Table 3 The effect of varying concentrations of sucrose and isomaltulose on isomelezitose production by A / rSPP. All reactions were performed using 0.02 mg.mL'1A / rSPP and incubated at 37 °C and pH 5.5.

[0252] Sucrose (mM) Isomaltulose (mM) Yield (%)aHydrolysis (%)b200 400 28 0.0 200 200 37 0.4

[0253] 400 200 96 0.6

[0254] 600 200 57 0.5aYields relative to the concentration of limiting substrate added.bFraction of converted sucrose that was hydrolyzed to glucose and fructose.

[0255] Production of isomelezitose on 1-L scale

[0256] The ability of the SPP to be used for efficient production of isomelezitose was demonstrated on 1-L scale. The use of a two-molar excess of sucrose resulted in the highest conversion, achieving a final isomelezitose concentration of 185 mM after 24 h (Figure 3). This approach minimizes the initial amount of isomaltulose required, hereby simplifying downstream processing and reducing overall substrate costs. To obtain a pure product, the residual sucrose should be removed from the reaction mixture, which can be easily achieved by an invertase that hydrolyzes sucrose into its building blocks glucose and fructose. These monosaccharides have very high solubilities in water, at 909 g.L’1and 3750 g.L’1at 25°C, respectively, allowing for the potential crystallization of isomelezitose out of the reaction mixture if permitted by its solubility. Here, isomelezitose was isolated from the monosaccharides by preparative liquid chromatography. In total 62.8 g of isomelezitose (purity of >95%) was obtained.

[0257] Additional sucrose-active isomaltulose active sucrose 6-phosphate phosphorylases

[0258] Next, we evaluated whether the unexpectedly high activity of A / rSPP on isomaltulose as glycosyl acceptor may be a common property of SPPs that had not yet been discovered before. We expressed and purified SPP from Thermoanaerobacterium thermosaccharolyticum ( / / SPP) (40% identical to A / rSPP) as described above and incubated the enzyme with a-D-glucose 1- phosphate and isomaltulose and confirmed the production of isomelezitose by high- performance anion exchange chromatography. Next, we determined the kinetic parameters of 7 / SPP for isomaltulose (KM = 59.0 mM; kcat = 4.4 s’1; kcat / KM = 0.075 mM’hs’1), which showed that isomaltulose can efficiently be used as glucosyl acceptor by TtSPP for the synthesis of isomelezitose.

[0259] Next, the SPPs from Lachnosporaceae bacterium, Lachnospiraceae bacterium, Clostridia bacterium, Clostridiales bacterium, Clostridium sp. chh4-2, Hungatella sp., Lacrimispora sp. 210928-DFI.3.58, Treponema socranskii. Treponema socranskii subsp. paredis ATCC 35535, Treponema sp. Marseille-Q4523, Roseburia inulinivorans, Spirochaetaceae bacterium, Spirochaetae bacterium HGW-Spirochaetae-8, Sphaerochaetaceae bacterium, Sphaerochaeta globosa str. Buddy, Candidates Choladousia intestinigallinarum, Candidates Eisenbergiella merdigallinarum. Candidates Anaerofdum excrementigallinarum and Blautia sp. OF03-15BH were confirmed to be capable of synthesizing isomelezitose in reactions containing sucrose or glucose 1 -phosphate as glycosyl donor, and isomaltulose as glycosyl acceptor

[0260] REFERENCES

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Claims

1. CLAIMS1. Use of a sucrose 6(F)-phosphate phosphorylase(EC 2.4.1.329) to produce isomelezitose from sucrose or alpha-D glucose 1 phosphate as glycosyl donor and isomaltulose as glycosyl acceptor.

2. Use of a sucrose 6(F)-phosphate phosphorylase to produce isomelezitose according to claim 1 wherein said sucrose 6(F)-phosphate phosphorylase has an amino acid sequence corresponding to SEQ ID N° 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 or 35.

3. Use of a sucrose 6(F)-phosphate phosphorylase to produce isomelezitose according to claim 1 wherein said sucrose 6(F)-phosphate phosphorylase has an amino acid sequence which is at least 90% identical to any of the sequences corresponding to SEQ ID N° 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 or 35.

4. Use of a sucrose 6(F)-phosphate phosphorylase to produce isomelezitose according to claims 2 - 3 wherein said sucrose 6(F)-phosphate phosphorylase is a functional fragment of an enzyme having an amino acid sequence corresponding to SEQ ID N° 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 or 35.

5. A method to efficiently produce isomelezitose comprising: providing sucrose or a-D-glucose 1 -phosphate as glycosyl donor, providing isomaltulose as a glycosyl acceptor, providing a sucrose 6(F)-phosphate phosphorylase as defined by claims 1 - 4 and contacting said glycosyl donor, said glycosyl acceptor and said sucrose 6(F)- phosphate phosphorylase in order to allow the synthesis of said isomelezitose.

6. A method according to claim 5 wherein said isomaltulose is not provided as such but is first produced via another catalytic process.

7. A method according to claim 5 wherein said a-D-glucose 1 -phosphate is not provided as such but is first produced via another catalytic process.34

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