Use of glucosylglycerate phosphorylases with high activity on sucrose

Abstract

Osmolytes are produced by various microorganisms as a defense mechanism to protect cells and macromolecules from damage caused by external stresses in harsh environments. Due to their useful stabilizing properties, these molecules can also be applied as active ingredients in a wide range of cosmetics and healthcare products. The present invention relates to the usage of glucosylglycerate phosphorylases that are also active on sucrose, which contrasts the strict specificity of known glucosylglycerate phosphorylases that can only use α-D-glucose 1-phosphate as glycosyl donor. The promiscuity of the sucrose-active glucosylglycerate phosphorylase can be exploited for the high-yielding and rapid synthesis of 2-O-α-D- glucosylglycerate from sucrose and D-glycerate.

Description

[0001] Use of glucosylglycerate phosphorylases with high activity on sucrose

[0002] Technical field of the invention

[0003] The invention relates to glycoside phosphorylases which catalyze, among other things, the synthesis of 2-O-a-glucosylglycerate from a suitable glycosyl donor and glycerate; where said phosphorylase shows higher affinity for D-glycerate than for fructose as the glycosyl acceptor; and where said phosphorylase is distinguished by significant activity on sucrose as a suitable glycosyl donor. Therefore, said enzymes are especially useful for the efficient synthesis of 2- O-a-glucosylglycerate from sucrose and glycerate.

[0004] Background art

[0005] The discovery of novel carbohydrate-active enzymes (CAZymes) is an area of broad interest due to the vital role that carbohydrates play in nature and in industry. A group of CAZymes that are particularly appealing for biotechnological applications are glycoside phosphorylases (GPs) (Li et al. 2022). These enzymes catalyze the breakdown of glycosidic bonds using inorganic phosphate to produce sugar 1-phosphates (i.e. phosphorolysis), but they can also catalyze the reverse reaction where such sugar 1-phosphates are used as glycosyl donor to form a new glycosidic bond (i.e. reverse phosphorolysis). Therefore, GPs are versatile synthetic tools that can be used to produce valuable carbohydrates, glycoconjugates or glycosyl phosphates.

[0006] Subfamily 18 of glycoside hydrolase family 13 (GH13_18) in the carbohydrate-active enzyme database classification (www.cazy.org) is a rich source of GP specificities. The most famous member of this subfamily is sucrose phosphorylase, of which the discovery dates back to the 1940s (Kagan and Latker 1942). This enzyme is quite promiscuous, allowing it to transfer the glucosyl moiety of sucrose not only to phosphate, but also to a variety of different acceptor substrates. Hence, the transglycosylation activity of sucrose phosphorylase has been successfully exploited for the biocatalytic production of numerous glycosides and rare sugars (Franceus and Desmet 2020). More recently, it was discovered that several GPs from GH13_18 are not sucrose phosphorylases. A promiscuous GP from Thermoanaerobacterium thermosaccharolyticum was found to prefer sucrose 6F-phosphate over sucrose as glycosyl donor substrate (Verhaeghe et al. 2014). Furthermore, some phosphorylases from GH13_18 are not active on sucrose at all, but they are specialized in the reversible phosphorolysis of 2- O-a-D-glucosylglycerate or 2-O-a-glucosylglycerol (Franceus et al. 2017; Franceus et al. 2018; Franceus and Desmet 2019).

[0007] Known glycoside phosphorylases that can be used for the synthesis of glucosylglycerate are either (a) sucrose phosphorylases, which show higher affinity for fructose than for D-glycerate as the glycosyl acceptor, or (b) strict glucosylglycerate phosphorylases, which show higher affinity for D-glycerate as glycosyl acceptor but are totally inactive on sucrose as glycosyl donor (Sawangwan et al. 2009, W02010055123, Franceus et al. 2017).

[0008] However, there is still a need to develop more efficient methods to produce 2-O-a- glucosylglycerate.

[0009] Brief description of figures

[0010] Figure 1 (a) Simplified representation of the phylogeny of GH13_18. The clade containing the X. protaetiae GP is indicated by a box. The UniProt identifier is given for the sequence of one representative of each clade, (b) Phylogeny of GH13_18. The clade of the present inventioncontaining the X. protaetiae GP is indicated by an arrow.

[0011] Figure 2 The effect of (a) pH and (b) temperature on the initial activity of XpGP. The pH profile was determined at 25°C, the temperature profile was determined at pH 7. All reactions were performed using 30 mM a-glucose 1-phosphate and 50 mM glycosyl acceptor.

[0012] Figure 3 Reactions catalyzed by the X. protaetiae glycoside phosphorylase from subfamily GH13_18. (a) Reversible phosphorolysis of 2-O-glucosylglycerate, (b) reversible phosphorolysis of sucrose, (c) transglycosylation with sucrose and glycerate to form glucosylglycerate.

[0013] Figure 4 Conversion of 0.3 M sucrose and 0.3 M D-glycerate by 5 pM XpGP at 30°C and pH 7.0 (V, sucrose; •, glucosylglycerate; o, glucose). Description of the invention

[0014] The present invention relates in first instance to the usage of a sucrose-active glucosylglycerate phosphorylase to efficiently produce 2-O-a-glucosylglycerate wherein said sucrose-active glucosylglycerate phosphorylase shows higher affinity for D-glycerate than for fructose and wherein said enzyme can use sucrose as glycosyl donor.

[0015] The present invention thus relates to the usage of a sucrose-active glucosylglycerate phosphorylase as described above wherein said sucrose-active glucosylglycerate phosphorylase is a member of a clade of the subfamily 18 of glycoside hydrolase family 13 (GH13_18) according to the carbohydrate-active enzyme database classification. The present invention indeed discloses a clade of the GH13_18 subfamily wherein the members of said clade show a higher affinity for D-glycerate than for fructose as is further shown by table 2, and wherein said enzyme can use sucrose as glycosyl donor as is further shown by table 2, table 3 and figure 4. In other words, the term 'sucrose-active glucosylglycerate phosphorylase' relates to a glucosylglycerate phosphorylase from the GH13_18 subfamily that is capable of using sucrose as glycosyl donor.

[0016] More specifically, the present invention relates to the usage of a sucrose-active glucosylglycerate phosphorylase as described above wherein said sucrose-active glucosylglycerate 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 or 15.

[0017] The latter SEQ ID N° correspond to the following amino acid sequences:

[0018] 1) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Xylanimonas protaetiae and having the following amino acid sequence (SEQ ID N° 1):

[0019] MTGVDILVYADRIGGDLRALRELLTTELVGFAGVHILPFFTPFDGDDAGFDPTDHTVVDSRLGTWADVR ALADDGFEVTADLIVNHVSADSPEFRDWLAQGAHSEHEGMFLTFDRVFGGHATEAGITAFYRPRPGLPF TAYQQADGTRRLVWTTFMPSQVDLDVQHPAGEAYLRRVIRTLADGGVRTVRIDAVGYAIKTVGTDSFM TDETLEYVRHLAALCHEAGMRVLVEVHAHYTQQQRIAPLVDLVYDFALPALLLHSLVARTSDRLARWLDI RPANAITVLDTHDGIGIIDAGPVTTGGRELPGLIDHAEMAAVFAAASAATDGESDRASVIPAWCDLPHQI NATFYSALGRDDDAYLLCRAVQLFVPGEPQIYYVGLLAGVDDVDLWRSTGQGRDINRHRYTHAEVVEAL DRPVVRALRGLVRLRSRHPAFGGEFSWRQTGTAGLELDWVNGDAGAVLTVDLAERHVEIVLRDADGQ

[0020] RRLTGVEEIIDA

[0021] 2) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Diaminobutyricimonas aerilata and having the following amino acid sequence (SEQ ID N° 2, SEQ ID N° 2 is 68% identical to SEQ ID N°l):

[0022] MTTRGVSLLAYADRFGGGMTGLRDLLDGPLAEFAGVHILPFFVPFDGADAGFDPIDHATVDPRLGDWD DVRAIAARREVTADLIVNHVSASSGEFRDWLADGEASEHHGMFLTFDTVFPNGGTEREITAFYRPRPGLP FTSYQSPDGTRRLVWTTFMPSQVDLDVAHPAAHAYLRRVLAALADGGVTTVRLDAVGYAVKTPGSDSF

[0023] MTEHTLGFVREITAMVREYGMRVLVEVHAHYTQQQAIAPLVDLVYDFALPPLLLHSLGTGTIDRLIRWLEI RPRNAVTVLDTHDGIGVIDAGPSGDRPGLLSTEEMAAIFDRAAAATDGHSSIASVIPAWAPMPHQINAT FFSVLEADAGSYLLARAVQFFVPGEPQVYYVGLLAGLDDVELFRRTGQGRDVNRHVYTRDEIDAALAGE VPRALLDLVRLRSAHPAFEGAFSVRAVSATELELAWTAGEHHAVLHADLAARTHRIDATPIPRVE

[0024] 3) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Aquiluna borgnonia and having the following amino acid sequence (SEQ ID N° 3, SEQ ID N° 3 is 56 % identical to SEQ ID N°l):

[0025] MSGASILVYAERVGGSLGEIEKLLKGPLADFSGIHVLPFFHPYDGDDAGFDPIDHTIVDPRLGNWSDFKRI

[0026] SETHELTADLIVNHASYLSPEFIDWQEKGDASEFAGMFLTFDTVFPNGGNEEGITSFYRPRPGFPFTAYNV AGKRRLVWTTFMPSQVDIDIEHPAGQAYLDRILVALKSGGVKVVRLDAVGYAVKTPGTDSFMTKETLEF VKEITEKIHSYGMRVLVEVHAHYTQQLDIAPLVDLIYDFQTAPLLLHSFFTGTVDRLDNWFKIRPDNCLNV

[0027] LDTHDGYGVIDGGPIGGRPGLVTQEEMAHIFAVAEKNTGGHSKLASVIPQWFELPHQINATLPNILADDT AYVISRMVQFLLPGEPQVYYVGLFNGMDDQKLFAESGQGRDVNRHHYTPAEISKALESEVTQAIIAMAR IRRSSAFEGTFSWRVTGSDSMNLSWSGAAEELSFDFSTKMGSPHFKLEVRGDGGIRNFSSVAELASY

[0028] 4) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Aquiluna sp. KACHI24 and having the following amino acid sequence (SEQ ID N° 4, SEQ ID N° 4 is 56 % identical to SEQ ID N°l): MAGTSILVYAERVGGNLSEIEKLLQGPLKDFDGIHVLPFFHPYDGDDAGFDPIDHTIVDPRLGTWSDFNRI SQTHELTADLIVNHASNLSPEFLDWQEKGDASEYNGLFLTFDTVFPNGGTEEMITSFYRPRPGFPFTAYEV AGKRRLVWTTFMPSQVDVDIKHEQGMAYLKRILEALKSGGVKVVRLDAVGYAVKTPGTDSFMTKETLEF VKEITDLIHSYDMRVLVEVHAHYTQQLDIAPLVDLIYDFQTAPLLLHSFFTGTVDRLDNWFKLRPNNCLNV LDTHDGYGVIDGGPIGGRPGLITQDEMAEIFKVAERNTGGHSAIASVIPQWFSLPHQINATLPNIVANDT AYVIMRAVQFFLPGEPQVYYVGLFNGMDDQELFRKTGQGRDVNRHNYTPDEIAQALEQPVTKAIIALAR VRKHPAFEGQFGWSVTGEDSMELSWTKGEDKLTLSFAFKNPKFRITSVINGESTVYEGVEGLASF

[0029] 5) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Cellulomonas sp. C5510 and having the following amino acid sequence (SEQ ID N° 5, SEQ ID N° 5 is 62% identical to SEQ ID N°l):

[0030] MDDHGVHLLAYTERLGGPTPDLAGVRALLADGPLAAFSGVHLLPFFVPFDGDDAGFDPVDHGTVDPRL GTWDDVRALAAAGVHVTADLIVNHVSADSAEFRDWLARGPQSPSDGMFLTFDTVFPDGADEAAITAF YRPRLGLPFTPYQRADGTRRLVWTTFMPTQVDLDVHHPAARDYLRRILRTLREGGVSTVRLDAVGYAVK

[0031] TPGTDSFMTEHTLAFVEEITALAHAEGLQVLVEVHAHHTQQAAIAPLVDLVYDFALPPLLLHALATGATD RLAHWLEIRPANAVTVLDTHDGIGVIDAGPSGDRPGILSTEEMAGVFAEAERRTGGESAQASARVAWA ALPHQVNSTFWSVLGADATAYLLARCVQLFVPGRPQIYYVGLLAGANDMPRYAATGQGREVNRHVYTP

[0032] GELEAALAGEVTRAQLGLVSLRTGHPAFAGTFAARAAGDGRLELAWRDDAGASAVLTVDVTPGRQGFL VEVAGGPEGERRYASVADLAELAG

[0033] 6) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Cellulomonas sp. ES6 and having the following amino acid sequence (SEQ ID N° 6, SEQ ID N° 6 is 63% identical to SEQ ID N°l):

[0034] MDDHGVHLLAYTERLGGPTPDLAGVRALLAGGPLAVFSGVHLLPFFVPFDGDDAGFDPVDHGTVDPRL GTWDDVRALADAGVNVTADLIVNHVSADSAEFRDWLARGPRSPSDGMFLTFDTVFPHGADEAAITAFY RPRLGLPFTPYQRADGTRRLVWTTFMPTQVDLDVHHPAARDYLRRILRTLREGGVSTVRLDAVGYAVKT

[0035] PGTDSFMTEHTLGFVEEITALAHAEGVQVLVEVHAHHTQQAAIAPLVDLVYDFALPPLLLHALATGATDR LAHWFAIRPANAVTVLDTHDGIGVIDAGPSGERPGILSAEEMAAVFAEAERRTGGESAQASARVAWAA LPHQVNSTFWSVLGEDATAYLLARCVQLFVPGRPQIYYVGLLAGTNDMPRYAATGQGREVNRHVYTPG ELAEALTGEVTRAQLGLVALRTAHPAFAGEFTARELGEGRLELAWRGDAGAAAVLTVDVTPGRQGFVV DVAGGPEGERRFASVADLADLAALAG

[0036] 7) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Herbiconiux sp. L3i23 and having the following amino acid sequence (SEQ ID N° 7, SEQ ID N°

[0037] 7 is 66 % identical to SEQ ID N°l)

[0038] MTDTRPVHLLTYAERLGGDLAGVARLLDGPLSDFGGVHLLPFFVPFDGDDAGFDPVDHASVDPRLGTW DDVRTIAADREVTADLIVNHVSSGSAEFADWLERQELSPHDGMFLTFDTVFPDGASEAAITAFYRPRPGL PFTAYRAADGRRRLVWTTFMPSQVDLDVRHPAAVAYLERLLRTLADGGVTTVRLDAVGYAIKTPGSDSF

[0039] MTDETLAFVRDITALARRHGLRVLVEVHAHYSQQLAIAPLVDLVYDFALPALLLHSLGTGTVDRLTEWFAI RPVNAITVLDTHDGIGIIDAGPSAESPGLISHDEMAAIFERASVATVGHSDIASVIPAWMTLPHQINATFFS ALGADPTAYLLCRAVQMFVPGVPQIYYVGLLGGEDDTALFEQTGQGRDVNRHTYSPDEFERALGEPVA

[0040] QAILGLVRLRSRHPAFDGEFDYETASDGALSMRWIHDASTARLDIVTGPTPSFVVTLTGDEGERRFDSVE ALALL

[0041] 8) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Leifsonia sp. 157MF and having the following amino acid sequence (SEQ ID N° 8, SEQ ID N° 8 is 67 % identical to SEQ ID N°l):

[0042] MTGVDLLAYADRLGGDLPRLRALLEGPLRDFAGVHILPFFVPFDGADAGFDPIDHGAVDPRLGTWDDV RALAAEREVTADLIVNHVSSDSAEFVDWLANGEQSAHDGMFLTFDTVFPDGATEDGITAFYRPRPGLPF TAYRMADGRRRLVWTTFMPTQVDLDVANDRARVYLRRILATLRSGGVTTVRLDAVGYAVKTPGTDSF

[0043] MTPETLEFVREIVAMARDEGLRVLVEVHAHYTQQLAIAPLVDYVYDFALAPLLLHSLGTGTSDRLVEWM RIRPQNAITVLDTHDGIGIIDAGPSGDRPGLIDQREMAAIFERAAVATGGHSTVASVIPAWMSLPHQINA TFFSALGADARSYLLARAVQLWLPGKPQLYYVGLFGGLDDVALFQRTGQGRDVNRHVYTPDEVEAALAS

[0044] EVTRAQLALVRLRSAHPAFQGEFRYRVEADGSLDLEWVLGDERATLTVSFAPEPEFRIRLAGAGGQLTAD SVPALAAL 9) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Leifsonia xyli and having the following amino acid sequence (SEQ ID N° 9, SEQ ID N° 9 is 66 % identical to SEQ ID N°l):

[0045] MTGVDLLAYADRLGGDLPRLRALLDGPLRDFAGVHTLPFFVPFDGADAGFDPIDHGAVDPRLGTWDDV RAISANREVTADLIVNHVSSESAEFVDWLALGEESAHDGMFLTFDTVFPDRASEDDITAFYRPRPGLPFTA YRMADGRRRLVWTTFMPSQVDLDVANDRARAYLRRILAALHDGGVTTVRLDAVGYAVKTPGSDSFMT

[0046] PETLEFVREIVALAREEGLRVLVEVHAHYTQQLAIAPLVDLVYDFALAPLLLHSLGTGTSDRLEEWLRIRPQ NAITVLDTHDGIGIIDAGPSGDRPGLISQDDMAAIFARAAEATHGHSTIASVIPAWMSLPHQINATFFSAL GADADAYLLCRAVQLWLPGRPQLYYVGLLGGLDDVALFQRTGQGRDVNRHVYTPQEFDAAMASDVTR

[0047] AQLALVRLRSAHPAFSGEFGYRVEADALVLEWTLGDERATLEVAFAPEPRFRIHLEGVDGPLTADSVAAL AAL

[0048] 10) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Naasia lichenicola and having the following amino acid sequence (SEQ ID N° 10, SEQ ID N° 10 is 59% identical to SEQ ID N° 1):

[0049] MSTRGVSLLAYADRLGGDLRRLQAVLDGPLADLTGVHILPFYTPFDGDDAGFDPVDHTAVDPRLGRWS DLAAIGGGRELTADIIVNHSSALSPEFLDWTAHGTESRFDGMFLRYDAVFPSGASEADITAFYRPRPGLPF TPYAVAGERRLIWTTFMPSQVDLDLRHPAALAYLDRIVGALAQAGVDVVRLDAVGYAVKTPGTSSFMTE

[0050] HTLAFVEELAARLRGAGLRVLVEVHAHHSQQIAIAPLVDLVYDFASAPLMLHALGDGDIAPLLRWLELRP ANAVTVLDTHDGIGIIDAGPAEGLPGLIDEDQMRAIFERAADATAGHSDLASVVPQWASMPHQINATF PSVVADDEAYVLCRAVQVMLPGEPQFYYVGLLDGRDDEQRFLETGQGREVNRHVYDDAELSAAVARPV

[0051] PRALLGLARLRRDHPAFDGDFGWMQTGPTSLRLQWQSADELLVLDADFAPGARQAVLTSERDGERRIL HGAAGLAAI

[0052] 11) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Rhodoluna lacicola and having the following amino acid sequence (SEQ ID N° 11, SEQ ID N° 11 is 57% identical to SEQ ID N°l): MSGTSILVYAERVGGNLGEIEKLLAGPLADFDGIHVLPFFHPYDGDDAGFDPIDHTIVDPRLGDWSDFKRI SQTHELTADLIVNHASYLSPEFKDWQEKGEQSEYDGMFLTFDVVFPNGGTEEGITSFYRPRPGMPFTAYE VGGKRRLVWTTFMPSQVDIDIKHDAGKAYLVRILEALKSGGVKVVRLDAVGYAVKTPGTDSFMTKETLD FVKEISELIHSYDMRVLVEVHAHYTQQLEIAPLVDLIYDFQTAPLLLHSLFTGTVDRLGDWFKIRPNNCLNV

[0053] LDTHDGYGVIDGGPIGERPGLITQDEMANIFKVAEKNTDGHSAIASVIPQWFTLPHQINATLPNIVKNDT GYVIMRAVQFFLPGEPQVYYVGLFNGMDDRDLFARSGQGRDTNRHNYTPAEIQEALQQPVTQAIIALA RVRKHGAFNGEFSWGIIDDQTMRLEWKNGNDVLSLEFKTTVDAPSFVIEATTDGGVRRFSSVEELAKY

[0054] 12) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Rhodoluna lacicola and having the following amino acid sequence (SEQ ID N° 12, SEQ ID N° 12 is 57% identical to SEQ ID N°l):

[0055] MSGTSILVYAERVGGNLGEIEKLLAGPLADFDGIHVLPFFHPYDGDDAGFDPIDHTIVDPRLGDWSDFKRI SQTHELTADLIVNHASYLSPEFKDWQEKGEQSEYDGMFLTFDVVFPNGGTEEGITSFYRPRPGMPFTAYE VGGKRRLVWTTFMPSQVDIDIKHDAGNAYLVRILEALKSGGVKVVRLDAVGYAVKTPGTDSFMTKETLD FVKEISELIHSYDMRVLVEVHAHYTQQLEIAPLVDLIYDFQTAPLLLHSLFTGTVDRLGDWFKIRPNNCLNV LDTHDGYGVIDGGPIGERPGLITQDEMANIFKVAEKNTDGHSAIASVVPQWFTLPHQINATLPNIVKNDT GYVIMRAVQFFLPGEPQVYYVGLFNGMDDRDLFARSGQGRDTNRHNYTPAEIQEALQQPVTQAIIALA RVRKHAAFNGEFSWGIIDDETMRLEWKNGDAILSLEFKTTVDAPSFVVEATTDGGVRRFSSVEELAKY

[0056] 13) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Rhodoluna sp. KAS3 and having the following amino acid sequence (SEQ ID N° 13, SEQ ID N° 13 is 57% identical to SEQ ID N°l):

[0057] MTGASILVYAERVGGNLGEIEKLLKGPLKDFSGIHVLPFFHPYDGDDAGFDPIDHKIVDPRLGNWADFKRI SETHELTADLIVNHASALSPEFIDWQEKGDASEYAGLFLTFDTVFPNGGTEEGITSFYRPRPGMPFTAYEV AGKRRLVWTTFMPSQVDIDIKHEKGQNYLKSVLEALQSGGVKVVRLDAVGYAVKTPGTDSFMTPETLEF

[0058] VEEITELIQSYGMRVLVEVHAHYTQQLDIAPLVDLIYDFQTAPLLLHSFFTGTVDRLDDWFKIRPNNCLNV LDTHDGYGVIDGGPINGRPGLITQDEMAFIFKKAEENTNGHSAIASVVPQWFTLPHQINATLPNIVANDT AYTIMRAVQFFLPGEPQVYYVGLFNGMDDVELFNKTEQGRDVNRHNYTPEEIAVALEQPVTKAIIALARL

[0059] RNLPVFDGEFSWNVTGESSLRMGWVNGDDSISLEFNTSVDAPEFVIEVKDAAGTRSYNSVEALGSAE 14) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Rhodoluna planktonica and having the following amino acid sequence (SEQ ID N° 14, SEQ ID N° 14 is 58% identical to SEQ ID N°l):

[0060] MGSSILVYAERVGGNLGEIEKLLHGPLSDFDGIHVLPFFHPYDGDDAGFDPIDHKIVDPRLGTWADFKRIS ETHELTADLIVNHASNLSPEFLDWQAKGAASDYDGLFLTFDVVFPNGATEEGITSFYRPRPGMPFTAYEV DGKRRLVWTTFMPSQVDIDIKHPQGQAYLRSVLDALASGGVKVVRLDAVGYAVKTPGTDSFMTAETLK

[0061] FVEEITELIHSYGMRVLVEVHAHYTQQLDIAPLVDLIYDFQTAPLLLHSYFTGSVDRLDKWFAIRPNNCLNV LDTHDGYGVIDGGPIGERPGLITQDEMANIFRVAEKNTNGHSAVASVIPQWFSLPHQINATLPNIVAND TAYVGMRAVQFFLPGEPQVYYVGLFNGMDDQELFRQTGQGRDVNRHNYTPAEISAALQQPVTQAIIAL

[0062] ARVRKAKAFGGSFDWQVTGDSSIMLKWTNGSDTASLEINTAVDAPSLCIKVTEDSVAREFCSVEELAKF

[0063] 15) Sucrose-active glucosylglycerate phosphorylase originating from the bacterium Cryobacterium fucosi and having the following amino acid sequence (SEQ ID N° 15, SEQ ID N° 15 is 62% identical to SEQ ID N° 1):

[0064] MSDVELLVYADRLGGTIPELQAVLDGPLRAFSSVHVLPFYIPFDGADAGFDPVDHAAVDPRLGTWDDLR SLSGTRGLTADLIVNHVSSSSAEFVDWLARGDASEFAGMFLTFDTVFPNGGTEHDITAFYRPRPGLPFTA

[0065] YQGADGSRHLVWTTFMPSQVDLDVNHPAALRYLRRVLRALADGGVSTVRLDAVGYAVKTPGTDSFMT PETLEFAEVVVALAHEAQVEVLVEVHAHYSQQLAIAPLVDWVYDFALAPLLLHSLGTGTVDRLARWFEIR PENAITVLDTHDGIGIIDAGPSGDRPGLISQDEMASIFARADVATGGHSSIASVVPAWFTLPHQINATFFS

[0066] TLAGETAPYLLARAVQHFLPGRPQVYYVGLLGGLDDTTLFAETGNGRDVNRHRYPAGEIDLALGTEVTQ AQLGLARLRTTHPAFDGEFSWETSGDDEARLLWSNGEARAELTVRTTLGAPSFRIELADAGRERVLATPA DLASWERFGR

[0067] Furthermore, the present invention relates to the usage of a sucrose-active glucosylglycerate phosphorylase as described above wherein said enzyme has an amino acid sequence which is at least 90 % identical to SEQ ID N°l, or, which is at least 90 % identical to SEQ ID N° 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0068] In other words, the latter terms relate to sucrose-active glucosylglycerate phosphorylases having an amino acid sequence which is 90, 95, 96, 97, 98 or 99% identical to the sequence as depicted by SEQ ID N°1 and, which show a higher affinity for D-glycerate than for fructose and wherein said enzyme can use sucrose as glycosyl donor, or, to sucrose-active glucosylglycerate phosphorylases having an amino acid sequence which is 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the sequence as depicted by SEQ ID N° 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 and, which show a higher affinity for D-glycerate than for fructose and wherein said enzyme can use sucrose as glycosyl donor

[0069] Moreover, the present invention relates to the usage of a sucrose-active glucosylglycerate phosphorylase as described above wherein said enzyme is a functional fragment of a sucroseactive glucosylglycerate phosphorylase having an amino acid sequence corresponding to SEQ ID N° 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The term 'functional fragment' relates to an enzyme having an amino acid sequence which 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 or 15 and, which shows a higher affinity for D-glycerate than for fructose and wherein said enzyme can use sucrose as glycosyl donor. 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 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 or 15) shows a higher affinity for D-glycerate than for fructose and can use sucrose as glycosyl donor.

[0070] The present invention further relates to a method to efficiently produce 2-O-a- glucosylglycerate comprising:

[0071] -providing sucrose as a suitable glycosyl donor,

[0072] -providing glycerate as a glycosyl acceptor;

[0073] -providing a sucrose-active glucosylglycerate phosphorylase as describe above, and -contacting said glycosyl donor, said glycosyl acceptor and said sucrose-active glucosylglycerate phosphorylase in order to allow the synthesis of said 2-O-a- glucosylglycerate.

[0074] More specifically, the present invention relates to a method as described above wherein said glycerate is glyceric acid, D-glycerate, L-glycerate, or a mixture thereof.

[0075] The present invention further relates to a method as described above wherein said glycerate is not provided as such but is first produced via another catalytic process. The term 'another catalytic process' 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 glycerate. Subsequently, said glycerate is used by a sucrose-active glucosylglycerate phosphorylase using the method described above. For example, the term 'another catalytic process' may more specifically relate to a process where glycerol is provided as substrate, an alditol oxidase is provided as catalyst, and said glycerol and alditol oxidase are contacted in order to allow the synthesis of glycerate.

[0076] Examples

[0077] MATERIALS AND METHODS

[0078] Materials

[0079] All chemicals were obtained from Merck, unless noted otherwise. D-Glycerate was supplied as D-glyceric acid calcium salt dihydrate. The carbohydrates for the putative substrate screening were obtained from Biosynth.

[0080] Sequence analysis

[0081] The identifiers of proteins classified in GH13_18 were extracted from the CAZy database (http: / / www.cazy.org) and their amino acid sequences were obtained from GenBank. Duplicates were removed, leaving a total of ~l,900 unique sequences. Clustal Omega was used to perform a multiple sequence alignment with default parameters (Sievers et al. 2011). A phylogenetic tree was constructed using RAxMLv8.2.12 with the LG+I+G substitution model and the tree was visualized using iTOL v5 (Stamatakis 2014; Letunic and Bork 2021). The selected sequences of the glycoside phosphorylases of interest originated from Xylanimonas protaetiae (XpGP; SEQ ID N°l), Diaminobutyricimonas aerilata (DaGP; SEQ ID N°2), Aquiluna borgnonia (AbGP; SEQ ID N°3), Aquiluna sp. KACHI24 (A GP; SEQ ID N°4), Cellulomonas sp. C5510 (CelGP; SEQ ID N°5), Cellulomonas sp. ES6 (Ce2GP; SEQ ID N°6), Herbiconiux sp. L3i23 (HeGP; SEQ ID N°7), Leifsonia sp. 157MF (LelGP; SEQ ID N°8), Leifsonia xyli (Le2GP; SEQ ID N°9), Naasia lichenicola (NIGP; SEQ ID N°10), Rhodoluna lacicola (RhlGP; SEQ ID N°ll), Rhodoluna lacicola (Rh2GP; SEQ ID N°12), Rhodoluna sp. KAS3 ( / ? / )3GP; SEQ ID N°13), Rhodoluna planktonica (Rh4GP; SEQ ID N°14), Cryobacterium fucosi (CfGP; SEQ ID N°15).

[0082] Cloning, expression and purification

[0083] The full-length amino acid sequences of XpGP, DaGP, AbGP, AkGP, CelGP, Ce2GP, HeGP, LelGP, Le2GP, NIGP, RhlGP, Rh2GP, Rh3GP, Rh4GP and CfGP were codon-optimized for E. coli, synthesized and subcloned into a pET21a vector at the Nhe\ and Xho\ sites by Life Technologies (Belgium), introducing a C-terminal short linker and Hisetag. The enzymes were recombinantly expressed as follows. The plasmid was used for transformation of E. coli BL21(DE3) electrocompetent cells. LB medium (500 mL; 10 g-L1tryptone, 5 g-L1NaCI, 5 g-L1yeast extract) supplemented with 100 pg-mL1ampicillin was inoculated with an overnight culture (5 mL) containing these cells and incubated at 37°C and 200 rpm until ODeoo reached 0.6. The temperature was then lowered to 18°C and expression was induced by adding isopropyl p-D-l-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. After incubation for 16 h, cells were harvested by centrifugation, the supernatant was discarded, and the pellet was frozen at -20°C.

[0084] For enzyme extraction and purification, the obtained pellet was thawed, resuspended in 8 mL lysis buffer (10 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg-mL1lysozyme, 50 mM phosphate-buffered saline; pH 7.4) and incubated on ice for 30 min. The lysate was sonicated three times for 2.5 min (Branson sonifier 450, level 3, 50% duty cycle) and the soluble fraction of the lysate was obtained by centrifugation (20,000 g for 30 min). The clarified lysate containing recombinantly expressed enzyme with a C-terminal Hise-tag was purified by nickel-nitrilotriacetic acid chromatography following the instructions of the supplier (HisPur Ni-NTA; Thermo Fisher Scientific). The purified enzyme solution was concentrated and the buffer was exchanged to 50 mM 3-( / V-morpholino)propanesulfonic acid (MOPS) at pH 7.0 using an Amicon Ultra-15 centrifugal filter unit with a 30-kDa cutoff (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).

[0085] Detection of reaction products

[0086] The release of inorganic phosphate from a-glucose 1-phosphate was monitored using the colorimetric phosphomolybdate assay (Gawronski and Benson 2004). The release of fructose from sucrose was monitored using the colorimetric bicinchoninic acid reducing sugars assay (Aerts et al. 2011). When using these assays to determine specific activities, one unit of activity was defined as the amount of enzyme that releases one pmol of the measured product (i.e. inorganic phosphate or fructose) per minute under the specified reaction conditions. All measurements of specific activities were performed in triplicate. Transglycosylation reactions starting from sucrose were monitored by high-performance anion exchange chromatography (HPAEC; Dionex ICS-600; Thermo Fisher Scientific) with a CarboPac PA20 pH-stable column and pulsed amperometric detection. The column was kept at 30°C with a flow rate was 0.5 mL-min-1. The eluent composition was 30 mM NaOH for 8.5 min after which the NaOH concentration increased to 100 mM for 1.5 min. Next, the NaOAc concentration was gradually increased from 0 mM to 300 mM over 4 min. The eluent composition was kept constant for 0.5 min, gradually changed back to 30 mM NaOH and 0 mM NaOAc over 0.5 min, and kept constant for another 5 min. Before analysis, all reactions were inactivated by the low pH of the assay solution (phosphomolybdate assay) or by heating the sample at 95°C for 5 min (HPAEC).

[0087] Enzyme characterization

[0088] The activity of GPs 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.

[0089] The influence of pH on the activity of XpGP was determined my measuring the specific activity in reactions with 30 mM a-glucose 1-phosphate and 50 mM glycosyl acceptor at 30°C in 50 mM acetate (pH 4.5 - 5.5), 2-morpholinoethanesulfonic acid (pH 5.5 - 6.5), MOPS (pH 7.0- 7.5) or Tris (pH 8.0). The optimal temperature was determined using the same substrate concentrations in 50 mM MOPS at pH 7.0. Reaction samples were analyzed using the phosphomolybdate assay.

[0090] The thermodynamic stability of XpGP was evaluated by differential scanning fluorimetry in a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) using the FRET channel. Purified protein (22.5 pL of a 0.5 mg-mL1solution) was mixed with SYPRO Orange Protein Stain (2.5 pL of a 50X Stain solution). The temperature was increased from 20°C to 99°C at a rate of 0.5°C-min1and fluorescence emissions were plotted as a function of temperature.

[0091] The kinetic stability of XpGP was evaluated by monitoring its residual specific activity at various time points during the incubation of lpM purified enzyme at 30°C and at 45°C in 50 mM MOPS, pH 7.0. Specific activities were determined with 30 mM a-glucose 1-phosphate and 50 mM fructose at 30°C and pH 7.0 and reaction samples were analyzed using the phosphomolybdate assay.

[0092] Kinetic parameters were determined at the optimal pH (7.0) and temperature (30°C) in 50 mM MOPS buffer. When varying the acceptor substrate concentration (fructose, D-glycerate and L-glycerate), 30 mM a-glucose 1-phosphate was added as the cosubstrate and reaction samples were analyzed using the phosphomolybdate assay. When varying the donor substrate concentration (sucrose), 100 mM phosphate was added as the cosubstrate and reaction samples were analyzed using the bicinchoninic acid reducing sugars assay. The kinetic parameters were calculated by nonlinear regression of the Michaelis-Menten equation using SigmaPlot 14.5.

[0093] Optimization of reaction conditions

[0094] Reactions for the synthesis of glucosylglycerate from sucrose and glycerate were performed in Eppendorf tubes with a total reaction volume of 500 pM, without agitation, in 100 mM MOPS buffer at pH 7.0 and 30°C. When monitoring the reaction over time, 5 pM purified enzyme was incubated with 300 mM sucrose and 300 mM D-glycerate. Samples were taken at various time points for 4 h and subsequently analyzed by HPAEC. When optimizing the substrate concentrations, the purified enzyme concentration was 3.7 pM, reactions were stopped after 4 h of incubation and yields were determined by HPAEC. The conversion of sucrose to glucosylglycerate was monitored and quantified by determining the difference between the overall conversion of sucrose (i.e. release of fructose) and the contaminating hydrolytic conversion if sucrose (i.e. release of glucose).

[0095] NMR analysis

[0096] Purified XpGP (5 p.M ) was incubated with 0.3 M sucrose and 0.3 M D-glycerate for 4 h at 30°C. The reaction mixture was inactivated (5 min, 95°C), centrifuged and freeze-dried.1H NMR and13C NMR spectra were recorded at 400 and 100.6 MHz, respectively, on a Bruker Avance III HD Nanobay III instrument. Deuterated water was used as a solvent, and acetone was used as an internal chemical shift standard (6H 2.23 ppm and 6c 31.5 ppm). All spectra were processed using TOPSPIN 3.6.3 software and compared to spectral data previously reported for ( / ?)-2-O-a-D-glucopyranosylglycerate (Sawangwan et al. 2009).

[0097] RESULTS

[0098] Identification of sequences of interest

[0099] All sequences from family GH13_18 were extracted from sequence databases and the phylogenetic tree was constructed in search of unexplored clades that may contain GPs with novel function (Figure 1). To assist the sequence-based search, we inspected which amino acid residues are present in certain regions of the sequence that are known to be involved in substrate recognition. One example is the region that corresponds to positions 342 - 345 in the sequence of the sucrose phosphorylase from Bifidobacterium adolescentis. Another example is the region surrounding the catalytic acid / base residue. These efforts led to the discovery of a clade of atypical putative GPs. Three representatives from this clade were selected for expression and characterization. The selected sequences encoding the GPs of interest originated from Xylanimonas protaetiae (XpGP), Diaminobutyricimonas aerilata (DaGP) and Aquiluna borgnonia (AbGP). The enzymes, containing a C-terminal Hise-tag, were recombinantly expressed in Escherichia coli and purified to apparent homogeneity by affinity chromatography. Approximately 20 mg of purified enzyme could be obtained from a 250-mL culture. Characterization of XpGP

[0100] XpGP was screened on a diverse panel of candidate glucosyl acceptors in reactions with a-D- glucose 1-phosphate as the glucosyl donor (Table 1). Very high activity was observed in reactions with D-glycerate, indicating that XpGP is likely to be a glucosylglycerate phosphorylase. Several glucosylglycerate phosphorylases have already been discovered in

[0101] GH13_18, although those are located in a different phylogenetic clade and they share low homology with XpGP (~30% sequence identity). However, in contrast to all known glucosylglycerate phosphorylases from the same subfamily, which are known to have a very strict substrate specificity, XpGP also showed high activity in the presence of fructose.

[0102] 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 > 10: ++++; 5 - 10: +++, 2 - 5: ++, 1.2 - 2: +).

[0103] Substrate Activity

[0104] Fructose +++

[0105] Fructose 6- ++ phosphate

[0106] Glycerol

[0107] D-Glycerate ++++

[0108] L-Glycerate +++

[0109] Glucose

[0110] Galactose

[0111] Mannose

[0112] Tagatose

[0113] Psicose

[0114] Xylose

[0115] L-Arabinose ++

[0116] L-Rhamnose

[0117] L-Fucose N-

[0118] Acetylglucosamine

[0119] Sorbitol ++

[0120] Xylitol +

[0121] Erythritol

[0122] Trehalose

[0123] Kojibiose

[0124] Maltose

[0125] Isomaltose

[0126] Sophorose

[0127] Cellobiose

[0128] Glucose 6- + phosphate

[0129] D-Lactate +

[0130] 3-

[0131] Hydroxypropionate

[0132] The optimal pH of XpGP was 7.0 (Figure 2A). Its optimal temperature for activity was 30°C, matching the optimal growth temperature of X. protaetiae (28 - 30°C) (Figure 2B) (Heo et al. 2020). The apparent folding transition temperature (Tm) of XpGP was found to be 41 ± 1 °C by differential scanning fluorimetry. In addition, its kinetic stability was evaluated at 30°C and 45°C. At 30°C, the enzyme retained 55% of its initial activity after 24 h of incubation. At at 45°C, 40% of its activity was retained after 1 h of incubation and it was fully inactivated after 4 h. The kinetic parameters were determined in the synthesis direction of the reversible phosphorolysis reaction at the optimal pH and temperature and the enzymes were found to exhibit Michaelis-Menten kinetics on the tested substrates (Table 2). The catalytic efficiency of XpGP for D-glycerate (kcat / / M = 2.4 mM4s4) was in line with that of several other GPs for their native acceptor substrate, further confirming the results from the substrate screening. XpGP showed a clear preference for D-glycerate over its enantiomer L-glycerate and over fructose. Further, the enzyme was confirmed to regioselectively synthesize 2-O-OL-D- glucosylglycerate from sucrose and D-glycerate by NMR spectroscopy. We also determined the kinetic parameters for sucrose in the phosphorolysis direction of the reaction and found that XpGP showed low affinity for sucrose (KM = 67 mM) when compared to the values reported for sucrose phosphorylases (K = 1 - 15 mM). However, XpGP also behaves differently from the previously reported strict glucosylglycerate phosphorylases, which are not active on sucrose at all (Franceus et al. 2017). In summary, the kinetic characterization of XpGP indicates that the enzyme can be regarded as a sucrose-active glucosylglycerate phosphorylase (Figure 3). The kinetic parameters of the other GPs from the same clade of interest matched those of XpGP, preferring D-glycerate over fructose as their glycosyl acceptor. The affinity of DaGP for D-glycerate and fructose was 25 mM and 55 mM, respectively. The affinity of AbGP for D-glycerate and fructose was 20 mM and 60 mM, respectively.

[0133] Table 2 Apparent kinetic parameters of the studied phosphorylases at pH 7.0 and 30°C.

[0134] Enzyme Substrate KM kcat

[0135] (mM) (s1) (mM s4)

[0136] XpGP D-Glycerate320 48 2.4

[0137] L-Glyceratea46 42 0.9

[0138] Fructose351 77 1.5

[0139] Sucroseb67 70 1.0

[0140] DaGP D-Glyceratea15 36 2.4

[0141] Fructose395 63 0.7

[0142] AbGP D-Glyceratea18 45 2.5

[0143] Fructose360 76 1.3

[0144] 3In 50 mM MOPS buffer with 30 mM a-D-glucose 1-phosphate b In 100 mM phosphate buffer

[0145] One-step synthesis of glucosylglycerate from sucrose and glycerate

[0146] Sucrose phosphorylases have long been used for their ability to transfer the glycosyl moiety of sucrose to a diverse range of other molecules to form the corresponding glucosides in a single step. However, those alternative molecules are typically converted rather slowly, and D-glycerate is no exception (Aerts et al. 2011). Transglycosylation reactions with D-glycerate proceed at rates that are comparable to the contaminating hydrolytic reaction, where the enzyme transfers the glucosyl group of sucrose to water instead. Nevertheless, it has been reported that an attractive yield (90% of D-glycerate converted) can be obtained with the sucrose phosphorylase from Leuconostoc mesenteroides (LmSP) by adding a 2.5-fold molar excess of sucrose, albeit after long incubation times (Sawangwan et al. 2009). Strict glucosylglycerate phosphorylases cannot catalyze the synthesis of glucosylglycerate when sucrose is used as the glycosyl donor (Franceus et al. 2017). In contrast, the sucrose-active glucosylglycerate phosphorylases show high activity on both sucrose and the D-glycerate. Therefore, we hypothesized that they may be especially attractive catalyst for the transglycosylation process starting from those substrates (Figure 3C).

[0147] Incubating XpGP with equimolar amounts of sucrose and D-glycerate (300 mM) at 30°C and pH 7.0 showed that the substrates were readily converted with a yield of 92% (Figure 4). The equivalent reactions with DaGP and AbGP resulted in yields of 87% and 90%, respectively. In contrast, LmSP can convert no more than 60% of D-glycerate at those concentrations because sucrose is gradually depleted due to hydrolysis (Sawangwan et al. 2009). Glucose was released by XpGP, DaGP and AbGP only in small amounts (< 1%), indicating that the undesired hydrolytic side reaction could effectively be suppressed by the presence of D-glycerate. Furthermore, transglycosylation reactions with these enzymes proceeded considerably faster. Reactions with 5 pM purified XpGP and equimolar substrate concentrations reached maximal conversion after approximately 2 h, whereas the equivalent reaction with purified LmSP only converted 3% of sucrose at that time.

[0148] Next, the concentrations of sucrose and D-glycerate in reactions with XpGP were varied and the yield was determined after 4 hours of incubation (Table 3). The findings reaffirmed that significant hydrolysis of sucrose takes place only at low concentrations of D- glycerate. Hydrolytic activity was largely eliminated at a concentration of 300 mM, resulting in a yield of 92%. Providing a 1.33-fold molar excess of sucrose allowed reaching a yield of up to 98%. In comparison, the optimal reaction conditions with LmSP required a 2.5-fold molar excess of sucrose (Sawangwan et al. 2009). Further, L-glycerate was utilized as glycosyl acceptor far less efficiently than the preferred enantiomer, which may be a result of the lower affinity of XpGP for this substrate.

[0149] Table 3 Optimization of enzymatic synthesis of glucosylglycerate by XpGP. Reactions were performed with 3.7 pM purified enzyme at 30°C and pH 7.0 and yields were determined after 4 h of incubation.

[0150] Sucrose D-Glycerate Hydrolysis3Yieldb

[0151] (mM) (mM) (%) (%)

[0152] 200 0 67

[0153] 3003001 92

[0154] 400 1 98

[0155] 500 1 98

[0156] 100 24 62

[0157] 300200 4 87

[0158] 350 1 83

[0159] 400 0 74

[0160] 300 300c39 15

[0161] 3Fraction of converted sucrose that was hydrolyzed to glucose and fructose. b Yields relative to the concentration of glycerate added c Using L-glycerate

[0162] Additional sucrose-active glucosylglycerate phosphorylases

[0163] Other proteins showing significant homology to XpGP (> 55% sequence identity according to EMBOSS-Needle) were recombinantly expressed in E. coli and purified by affinity chromatography using the same protocol described above. These putative glycoside phosphorylases originated from Aquiluna sp. KACHI24 (A / cGP), Cellulomonas sp. C5510 (CelGP), Cellulomonas sp. ES6 (Ce2GP), Herbiconiux sp. L3i23 (HeGP), Leifsonia sp. 157MF (LelGP), Leifsonia xyli (Le2GP), Rhodoluna lacicola (RhlGP; accession WP_051636357.1), Rhodoluna lacicola (Rh2GP; accession WP_264913852.1), Rhodoluna sp. KAS3 (Rh3GP), Rhodoluna planktonica (Rh4GP), Cryobacterium fucosi (CfGP), Naasia lichenicola (NIGP). Between 5 and 40 mg of purified protein could be obtained from a 250-mL culture. It was confirmed that these additional proteins with significant homology to XpGP were glucosylglycerate phosphorylases by determining the kinetic parameters for D-glycerate in the synthesis direction of the reversible phosphorolysis reaction, using a-glucose 1-phosphate as glycosyl donor, using the same reaction conditions described above. The enzymes were found to exhibit Michaelis-Menten kinetics and they were found to show high affinity for D- glycerate, with KM values that are in line with the values reported for known glucosylglycerate phosphorylases (Table 4). Next, it was confirmed that these additional proteins were sucroseactive glucosylglycerate phosphorylases by performing the one-step synthesis of glucosylglycerate from 300 mM sucrose and 300 mM D-glycerate, using the same reaction conditions as described above for XpGP, DaGP and AbGP (3.7 pM of purified enzyme, 30°C, pH 7.0). The substrates were readily converted with high yields, and the undesired hydrolytic side reaction was suppressed (Table 4). In conclusion, these additional phosphorylases can also be used for the efficient synthesis of glucosylglycerate from sucrose and D-glycerate, matching the beneficial properties that were observed using XpGP, DaGP or AbGP.

[0164] Table 4 Apparent affinity for D-glycerate of sucrose-active glucosylglycerate phosphorylases (pH 7.0, 30°C, 30 mM , a-D-glucose 1-phosphate, 50 mM MOPS buffer), and glucosylglycerate yields obtained in reactions with 300 mM sucrose and 300 mM D-glycerate (3.7 pM purified enzyme, 30°C, pH 7.0, 4 h).

[0165] Enzyme KM Hydrolysis Yield REFERENCES

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[0182] Letunic I, Bork P (2021) Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49:W293-W296 Li A, Benkoulouche M, Ladeveze S, Durand J, Cioci G, Laville E, Potocki-Veronese G (2022) Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases. Int J Mol Sci 23. https: / / doi.org / 10.3390 / ijms23063043

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Claims

Claims1. Use of a sucrose-active glucosylglycerate phosphorylase to efficiently produce 2-O-a- glucosylglycerate wherein said sucrose-active glucosylglycerate 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 or 15.

2. Use of a sucrose-active glucosylglycerate phosphorylase to efficiently produce 2-O-a- glucosylglycerate wherein said enzyme has an amino acid sequence which is at least 90 % identical to the sequence corresponding to SEQ ID N° 1, or, which is at least 90 % identical to the sequences corresponding to SEQ ID N° 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

3. Use of a sucrose-active glucosylglycerate phosphorylase according to claims 1 - 2 wherein said enzyme is a functional fragment of a sucrose-active glucosylglycerate phosphorylase having an amino acid sequence corresponding to SEQ ID N° 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

4. A method to efficiently produce 2-O-a-glucosylglycerate comprising:-providing sucrose as a suitable glycosyl donor,-providing glycerate as a glycosyl acceptor;-providing a sucrose-active glucosylglycerate phosphorylase according to claims 1-3 and-contacting said glycosyl donor, said glycosyl acceptor and said sucrose-active glucosylglycerate phosphorylase in order to allow the synthesis of said 2-O-a- glucosylglycerate.

5. A method according to claim 4 wherein said glycerate is glyceric acid, D-glycerate, L- glycerate, or a mixture thereof.

6. A method according to claims 4-5 wherein said glycerate is not provided as such but is first produced via another catalytic process.

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