WO2016116472A1 - Production of specific glucosides with cellobiose phosphorylase - Google Patents

Abstract

The present invention relates to enzymatic production methods of b-glucosides. More specifically, the present invention discloses the usage of cellobiose phosphorylases to glycosylate specific acceptors in a cosolvent- or biphasic system. The b-glucosides obtained via the latter methods can be further elongated using cellodextrin phosphorylases and a-glucose-1-phosphate as donor.

Description

Production of specific glucosides with cellobiose phosphorylase

Technical field of invention

The present invention relates to enzymatic production methods of β-glucosides. More specifically, the present invention discloses the usage of cellobiose phosphorylases to glycosylate specific acceptors in a cosolvent- or biphasic system. The β-glucosides obtained via the latter methods can be further elongated using cellodextrin phosphorylases and cc- glucose-l-phosphate as donor.

Background art

Despite the continuous development of new procedures, the synthesis of glycosides still remains a challenge to date.'¹¹ Numerous chemical routes have been described for the production of these molecules, but their application is limited by poor yields, the use of toxic catalysts and the generation of waste.'²¹ Consequently, biocata lytic approaches have received increasing attention in recent years as promising alternative for glycoside synthesis.'³¹ Indeed, enzymatic glycosylation reactions enable one-step conversions with high regio- and stereoselectivity; thereby generating 5-fold less waste.'⁴¹

Over the past decade, disaccharide phosphorylases have been identified as potent enzymes for the synthesis of glycosides. While glycosyltransferases require expensive nucleotide- activated sugars'⁵¹ and glycoside hydrolases suffer from unfavorable equilibrium constants,'³¹ these enzymes use cheap glycosyl phosphate donors and can be efficiently used in the synthesis direction.'⁶¹ Examples include the a-glucosylation of alcohols with maltose phosphorylase'⁷¹ and the synthesis of 2-O-a-D-glucopyranosyl glycerol,'⁸¹ 1-O-a-D- glucopyranosyl hydroquinone,'⁹¹ 2-O-a-D-glucopyranosyl L-ascorbic acid,'¹⁰¹ and geranyl a-D- glucopyranoside'¹¹¹ using sucrose phosphorylase (SP). Recently, it has shown that the efficiency of these glucosylation reactions can be vastly improved by the addition of ionic liquids'¹²¹ (ILs) or the use of biphasic catalysis.'¹¹¹ Moreover, various immobilization techniques have been successfully applied resulting in biocatalysts with enhanced activity and stability, while allowing their recycling in repetitive batch conversions.'¹³¹

In contrast to the extended work on a-glucosides, only a single report describing the synthesis of β-glucosides with disaccharide phosphorylases is known to date, and that involves the use of simple, short-chain alcohols.'¹⁴¹ It is thus not known and unpredictable whether longer and/or more complex acceptors could be glycosylated using disaccharide phosphorylases. Nevertheless, numerous biologically active molecules exist as β-glucosides in nature. For example arbutin, the β-glucoside of hydroquinone, can be found in wheat, pear skins and Bergenia crassifolia. ^15] This skin whitening compound is used in cosmetics to reduce irritation caused by hydroquinone'¹⁶¹ and treat urogenital tract infections.'¹⁷¹ Alkyl β-glucosides, on the other hand, are potent non-ionic surfactants with good emulsifying and antimicrobial properties.'¹⁸¹ These compounds are widely used in pharmaceuticals, detergents, and food ingredients ¹¹⁹¹ so that improved production methods for said β-glucosides are urgently needed.

Cellobiose phosphorylase (CP) catalyzes the reversible phosphorolysis of cellobiose and inorganic phosphate to a-D-glucose 1-phosphate (aGlP) and D-glucose.'²⁰¹ It is an inverting phosphorylase following a single displacement reaction, thereby forming glycosides with opposite anomeric configuration compared to the donor substrate.'²¹¹ The enzyme has been successfully used for the synthesis of various di-'²²¹ and trisaccharides'²³¹, and mutant CPs with enhanced activity towards alkyl'²⁴¹ and aryl β-glucosides'²⁵¹ have also been reported. However, all of these acceptors contain a glucose moiety as point of attachment whereas the direct glycosylation of non-carbohydrate acceptors such as long-chain alcohols, flavonoids, alkaloids, phenolics and terpenes has not been achieved. Brief description of figures

Figure 1. Kinetic thermostability of the CP from C. thermocellum. The enzyme was dissolved in MES buffer (50 mM, pH 6.5) an incubated at 37 (■), 45 (Δ), 50 ( T ), 55 (o) and 60 °C (·).

Figure 2. Half-life of CP and CP CLEAs at 50 °C. The biocatalysts were incubated in MES buffer (50 mM, pH 6.5) containing 20% AMMOENG™ 101 or 37.5% EtOAc in MES buffer (50 mM, pH 6.5) an incubated at 37 (■), 45 (Δ), 50 ( T ), 55 (o) and 60 "C (·).

Figure 3. Synthesis of octyl β-D-glucopyranoside at 50 °C. Biphasic catalysis was performed with CP CLEAs (·), octyl β-D-glucopyranoside iCLEAs ( T ) or octanol iCLEAs (o). Figure 4. (A) TLC analysis of the chain elongation of octyl β -glucoside, and (B) HPLC-analysis of the chain elongation of hydroquinone β -glucoside. Reactions were analyzed after 24h incubation while shaking at 1,400 rpm, using 100 mM of donor and acceptor, and 1 mg/mL CDP in 50 mM MES buffer at pH 6.5 and 37 °C. Description of invention

The present invention discloses methods to glycosylate various small organic compounds with cellobiose phosphorylases (CPs) in cosolvent and biphasic systems. The enzymes can be further immobilized and/or imprinted to increase both the stability and activity of the biocatalysts. More in particular, the present invention describes the stability of CPs at elevated temperatures in the presence of various solvents. CPs were found to be compatible with the ionic liquid (IL) AMMOENG™ 101, DMSO and ethylacetate (EtOAc), allowing the glycosylation of aliphatic alcohols, monoterpenoids, aromatic alcohols and phenolics. The stability of the biocatalysts could be significantly improved by cross-linking the enzymes, resulting in impressive half-lives at 50 °C. Moreover, the efficiency of the cross-linked enzyme aggregates (CLEAs) for the synthesis of octyl β-D-glucopyranoside -as a non-limiting example- could be roughly doubled by molecular imprinting with octanol. As proof of concept, three consecutive batch productions of octyl β-D-glucopyranoside were performed, revealing the excellent operational stability and recyclability of the CP octanol imprinted CLEAs (iCLEAs). Consequently, CPs was identified to be valuable and surprising alternatives for the synthesis of specific β-glucosides.

Hence, the present invention relates to an in vitro method to produce a β-glucoside comprising:

-contacting in vitro a cellobiose phosphorylase with alpha-glucose- 1 -phosphate and an acceptor, and

-glycosylating said acceptor, wherein said acceptor is a linear alcohol with a chain length of at least 8 carbon atoms, 2- phenylethanol, β-citronellol, vannillyl alcohol or hydroquinone, and, wherein said glycosylation is carried out in a cosolvent system or a biphasic system.

With the term 'β-glucoside' is particularly meant octyl β-D-glucopyranoside, nonyl β-D- glucopyranoside, decyb β-D-glucopyranoside, dodecyl β-D-glucopyranoside, vanillyl 4-0 β-D- glucopyranoside, β-citronellyl β-D-glucopyranoside, 2-phenylethyl β-D-glucopyranoside or 1- 0- β— D-glucopyranosyl hydroquinone.

The terms 'a cellobiose phosphorylase' means an enzyme that catalyzes the reversible phosphorolysis of cellobiose and inorganic phosphate to a-D-glucose 1-phosphate (aGlP) and D-glucose. Hence, the latter enzyme can glycosylate D-glucose via attaching a-D-glucose to said D-glucose in order to form the β- glucoside 'cellobiose' and to release inorganic phosphate. Examples of such enzymes -and the nucleic acid sequences encoding for such enzymes- can be derived from the following organisms: Clostridium thermocellum (Genbank AAL67138), Cellulomonas uda (AAQ20920), Cellvibrio gilvus (Genbank BAA28631), Clostridium stercorarium (UniProt KB accession 0.59316), Saccharophagus degradans (UniProt KB accession Q.21L49), Thermotoga maritima (UniProt KB accession Q.9X2G3) or Thermotoga neapolitana (UniProt KB accession 087964) . The sequence identity (in %) on the protein level among the cellobiose phosphorylases of the latter organisms is the following: gilvus 100%

uda 88.90% 100%

stercor. 62.63% 63.25% 100%

thermoc. 63.99% 62.51% 72.00% 100%

degrad. 63.50% 62.02% 66.33% 70.65% 100%

maritima 61.99% 61.50% 72.37% 74.22% 62.21% 100%

neapol. 61.62% 61.13% 70.77% 73.73% 65.96% 91.88% 100%

gilvus uda stercor. thermoc. degrad. maritima neapol

Hence, the term 'a cellobiose phosphorylase' means, more in particular, an enzyme comprising the amino acid sequence having at least 60 sequence identity with SEQ ID N° 1 (= the C. thermocellum cellobiose phosphorylase).

With the cellobiose phosphorylase of Clostridium stercorarium corresponding with the amino acid sequence SEQ ID N°6 is meant the following amino acid sequence:

MKFGYFDDVNREYVITTPATPYPWINYLGCQDFFSLISNTSGGY CFYRDARLRRITRYRYNNVPIDSGGRYFYIYDSGDYWTPGWMPVKRELDRYECRHGLG YTRITGERNGVEVSQLAFVPLNYNGEVNQVVITNKSGSEKEIALFSFVEFCLWNAMDD MTNFQRNFSTGEVEVEGSAIYHKTEYRERRNHYAFFWVNSPIDGFDTDRESFLGLYNG FDSPKNVAAGKPTNSIASGWSPIASHYIKMSLKPGEKRSYIFVLGYVENPPEEKWERK GVINKKRAREMQQKFIDDTCVEKAFQELKDYWADLCSKFALESHDEKLNRMVNIWNPY QCMVTFNMSRSASYFESGISRGMGFRDSAQDLLGFVHQVPERARQRILDLASTQFEDG SAYHQYQPLTKKGNSDIGSGFNDDPLWLILATAQYIKETGDFGILDEMVPFDCDENKK DTLFEHLKRSFYHVVNNLGPHGLPLIGRADWNDCLNLNCFSTQPMNPFTPADKFEGRV AESVFIAGMFVLIGPEYVELCKRRGLSEEAAEAEKHIQNMVNAVLTHGYDGEWFLRAY DHFGNKIGSKECSEGQIFIEPQGICVMAGIGVKEGLAQKALDSVMKRLDTKYGIVLHT PAYTEYYLNLGEISSYPPGYKENAGIFCHNNPWIAIAETVIGRGDRAFEVYSKIAPAY IEDISDIHRTEPYVYSQMIAGRTRWSFGEAKNSWLTGTAAWNFVAITQYILGVRPVYD GLMVDPCIPASWDGFTVTREFRGSKYRIRVENPEHICKGVNKVIMDGKEIEGQVLPVS PKESEHEVIVIMG

With the cellobiose phosphorylase of Saccharophagus degradans corresponding with the amino acid sequence SEQ. ID N°7 is meant the following amino acid sequence: MKFGHFDDKAREYVITDPKTPYPWINYLGNEDFFSLVSNTGGGYSFYKDAKFRRLTRYRYNNVPVDNGG KYFYINDSGDVWSPGWKPVKAELDAYSCAHGLSYTRITGERNGIQAEVLSFIPLGTWAEIQKVSLKNTSGA TKKFKLFSFAEWCLWNAEDDMTNFQRNFSTGEVEVEDSVIYHKTEFKERRNHYAFYSVNAPIQGFDTDR DKWKGLYNDFDKPDAVFEGEPRNSEAHGWSPIASHYLEVELAPGESKDLIFVLGYIEVAPENKWESKGVI NKSPAKELIARFDSVEKVDAELTKLADYWANLLSTYSVESGDEKLDRMVNIWNQYQCMVTFNMSRSASF FESGIGRGMGFRDSNQDLIGFVHQVPERARERIIDIASTQFEDGSAYHQYQPLTKRGNNAIGGNFNDDPL WLILSTTDYIKETGDFSILEEQVPYDNDASKATSHFEHLKRSFYHTVNNLGPHGLPLIGRADWNDCLNLNC FSEDPNESFQTTGNKTGRTAESLMIAGLFVLYGNEFVKLCREIGQDGEAAEAQAHIDQMVEAVKKHGW DGEWFLRAYDYYGKKVGSKENEEGKIFIESQGFCGMAGIGLEDGLVEKSMDSVKEWLDCDYGIVLQQPA FTKYYIEYGEISTYPAGYKENAGIFCHNNPWIMITETLLGRGDKAFEYYRKIAPAYLEEISDLHKVEPYAYCQ MIAGKDAYLPGEGKNSWLTGTASWNFAAITQYILGVKPDYSGLAINPCIPSSWDGFKVTRKYRGATYNIIV TNPTHVSKGVKSLTLNGNAIDGYIVPPQQAGTVCNVEVTLG

With the cellobiose phosphorylase of Thermotoga maritimans corresponding with the amino acid sequence SEQ. ID N°8 is meant the following amino acid sequence:

MRFGYFDDVNREYVITTPQTPYPWINYLGTEDFFSIISHMAGGYCFYKDARLRRITRFRYNNVPTDAGGR YFYIREENGDFWTPTWMPVRKDLSFFEARHGLGYTKITGERNGLRATITYFVPRHFTGEVHYLVLENKAEK PRKIKLFSFIEFCLWNALDDMTNFQRNYSTGEVEIEGSVIYHKTEYRERRNHYAFYSVNQPIDGFDTDRESF IGLYSGFEAPQAVVEGKPRNSVASGWAPIASHYLEIELAPSEKKELIFILGYVENPEEEKWEKPGVINKKRAK EMIEKFKTGEDVEHALKELREYWDDLLGRIQVETHDEKLNRMVNIWNQYQCMVTFNISRSASYFESGISR GIGFRDSNQDILGFVHMIPEKARQRILDLASIQFEDGSTYHQFQPLTKKGNNEIGGGFNDDPLWLILSTSA YIKETGDWSILGEEVPFDNDPNKKASLFEHLKRSFYFTVNNLGPHGLPLIGRADWNDCLNLNCFSKNPDES FQTTVNALDGRVAESVFIAGLFVLAGKEFVEICKRRGLEEEAREAEKHVNKMIETTLKYGWDGEWFLRAY DAFGRKVGSKECEEGKIFIEPQGMCVMAGIGVDNGYAEKALDSVKKYLDTPYGLVLQQPAYSRYYIELGEI SSYPPGYKENAGIFCHNNPWVAIAETVIGRGDRAFEIYRKITPAYLEDISEIHRTEPYVYAQMVAGKDAPR HGEAKNSWLTGTAAWSFVAITQHILGIRPTYDSLVVDPCI PKEWEGFRITRKFRGSIYDITVKN PSHVSKGV KEIIVDGKKIEGQVLPVFEDGKVHRVEVVMG

With the cellobiose phosphorylase of Thermotoga neapolitana corresponding with the amino acid sequence SEQ. ID N°9 is meant the following amino acid sequence: MKFGYFDDKNREYVIVTPRTPYPWINYLGTEDFFSI ISHMAGGY

CFYKDARLRRITRFRYNNVPTDAGGRYFYI REEDGDFWSPTWMPVRRDLSFFEARHGL GYTKIAGDINGLRATITFFVPRHFTGEVHHLVLQNRTERPRRI KLFSFIEFCLWNALD DMTN FQRNYSTGEVEI EGSVIYHKTEYRERRNHYAFYSVN HSIDGFDTDRESFMGLYN GFEAPQAVVEGNPRNSVASGWAPIASHYLELEI PPLGEKELIFI LGYVENPEEEKWER PGVI NKKRAKEM IERFKTGEDVERALKELKEYWDELLGRIQVETHDEKLNRMVN IWNQ YQCMVTFNIARTSSYFESGISRGIGFRDSNQDILGFVHM I PEKARQRILDLASIQFED GSTYHQFQPLTKKGNN EIGGGFNDDPLWLI LSTSAYIKETGDWSILN EEVPFDNDPDK KATLFEHLKRSFYFTVNNLGPHGLPLIGRADWN DCLNLNCFSKN PDESFQTTVNALDG RVAESVFIAGLFVLAGKEFVEICRRLGLEDEAKEAEKHVKKM IETTLEYGWDGEWFLR AYDAFGRKVGSKECEEGKIFIEPQGMCVMAGIGVENGYAKKALDSVKEHLDTHYGLVL QQPAYSRYYIELGEISSYPPGYKENAGIFCHNN PWVAIAESVIGRGDRAFEIYRKITP AYLEDISEI HRTEPYVYAQMVAGKDAPRHGEAKNSWLTGTAAWSFVAITQYILGVRPT YDGLMVDPCIPEDWDGFKITRRFRGATYEITVKN PHHVSKGVKEIIVDGKKIEGQVLP VFNDGKVHRVEVLMG The terms 'contacting in vitro a cellobiose phosphorylase with alpha-glucose- 1 -phosphate and an acceptor' means adding the acceptors 2-phenylethanol, β-citronellol, vannillyl alcohol, hydroquinone or a linear alcohol with a chain length of at least 8 carbon atoms to a buffer containing the donor aGlP in the presence of a CP enzyme.

The terms ' a linear alcohol with a chain length of at least 8 carbon atoms' means an octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol... and/but particularly relates to an octanol, nonanol, decanol or dodecanol.

The term 'glycosylating' means the reaction in which aGlP (the donor) is attached to a functional group of one of the following acceptors: 2-phenylethanol, β-citronellol, vannillyl alcohol, hydroquinone or a linear alcohol with a chain length of at least 8 carbon atoms. The terms 'a cosolvent system' means that the above-cited glycosylation reaction is undertaken by adding to said buffer -which contains the donor aGlP, the indicated acceptors and the enzyme CP- a cosolvent such as -but not limited to- dimethylsulfoxide (DMSO) or the quaternary ammonium salt AM MOENG™ 101. The latter cosolvent will form 1 phase with said buffer, will increase the solubility of the acceptors within said single phase and will thus improve the glycosylation reaction.

The terms a 'biphasic system' means that the above-cited glycosylation reaction is undertaken by adding to said buffer -which contains the donor aGlP, the indicated acceptors and the enzyme CP- an organic solvent such as ethylacetate. The latter addition will result in 2 phases: an organic phase in which the acceptors are dissolved and a water (buffer) phase in which the enzyme and the donor are present. The latter system will thus improve the glycosylation reaction.

Hence, the present invention particularly relates to a method as described above wherein said alcohol with a chain length of at least 8 carbon atoms is octanol, nonanol, decanol or dodecanol.

The invention further relates to a method as described above wherein the solvents used in said cosolvent system are dimethylsulfoxide (DMSO) or the ionic liquid AMMOENG™ 101, or, wherein the organic solvent in said biphasic system is ethylacetate (EtOAc). The ionic liquid AMMOENG™ 101 (also known as TEGO K5) is a mixture of quaternary ammonium compounds, coco alkylbis (hydroxyethyl)methyl, ethoxylated, chlorides, methyl chloride.

The present invention further relates to a method as described above wherein said cellobiose phosphorylase is a cellobiose phosphorylase comprising the amino acid sequence SEQ ID N° 1 (Clostridium thermocellum), SEQ ID N° 2 (Cellvibrio gilvus) or SEQ ID N° 3 (Cellulomonas uda), or, a variant of each of said sequences having at least 95% sequence identity with each of said sequences.

With the cellobiose phosphorylase of Clostridium thermocellum corresponding with the amino acid sequence SEQ ID N°l is meant the following amino acid sequence: Genbank accession number: AAL67138

MKFGFFDDAN KEYVITVPRTPYPWINYLGTEN FFSLISNTAGGYCFYRDARLRRITRYRYN NVPI DMGGRY

FYIYDNGDFWSPGWSPVKRELESYECRHGLGYTKIAGKRNGIKAEVTFFVPLNYNGEVQ.KLI LKNEGQ.DK

KKITLFSFI EFCLWNAYDDMTNFQRNFSTGEVEI EGSVIYHKTEYRERRNHYAFYSVNAKISGFDSDRDSFI GLYNGFDAPQAVVNGKSNNSVADGWAPIASHSI EI ELNPGEQKEYVFI IGYVENKDEEKWESKGVINKKK AYEM I EQFNTVEKVDKAFEELKSYWNALLSKYFLESHDEKLNRMVN IWNQYQCMVTFNMSRSASYFES GIGRGMGFRDSNQDLLGFVHQI PERARERLLDLAATQLEDGSAYHQYQPLTKKGNNEIGSNFNDDPLWL ILATAAYIKETGDYSILKEQVPFNNDPSKADTM FEHLTRSFYHVVNN LGPHGLPLIGRADWNDCLNLNCFS TVPDESFQTTTSKDGKVAESVM IAGM FVFIGKDYVKLCEYMGLEEEARKAQQHI DAM KEAILKYGYDGE WFLRAYDDFGRKVGSKENEEGKI FIESQGFCVMAEIGLEDGKALKALDSVKKYLDTPYGLVLQN PAFTRYY IEYGEISTYPPGYKENAGI FCHNNAWI ICAETVVGRGDMAFDYYRKIAPAYIEDVSDIHKLEPYVYAQMVA GKDAKRHGEAKNSWLTGTAAWN FVAISQWILGVKPDYDGLKIDPCI PKAWDGYKVTRYFRGSTYEITVK NPNHVSKGVAKITVDGNEISGN ILPVFNDGKTHKVEVIMG

With the cellobiose phosphorylase of Cellvibrio gilvus corresponding with the amino acid sequence SEQ ID N°2 is meant the following amino acid sequence:

Genbank accession number: BAA28631

MRYGHFDDAAREYVITTPHTPYPWINYLGSEQFFSLLSHQAGGYSFYRDAKM RRLTRYRYN NI PADAGG RYLYVNDGGDVWTPSWLPVKADLDHFEARHGLGYSRITGERNGLKVETLFFVPLGENAEVQKVTVTNTS DAPKTATLFSFVEFCLWNAQDDQTNYQRNLSIGEVEVEQDGPHGSAIYHKTEYRERRDHYAVFGVNTRA DGFDTDRDTFVGAYNSLGEASVPRAGKSADSVASGWYPIGSHSVAVTLQPGESRDLVYVLGYLENPDEE KWADDAHQVVNKAPAHALLGRFATSEQVDAALEALNSYWTN LLSTYSVSSTDEKLDRMVN IWNQYQC MVTFNMSRSASFFETGIGRGMGFRDSNQDLLGFVHLIPERARERI IDIASTQFADGSAYHQYQPLTKRGN NDIGSGFNDDPLWLIAGVAAYI KESGDWGILDEPVPFDN EPGSEVPLFEHLTRSFQFTVQNRGPHGLPLIG RADWNDCLNLNCFSTTPGESFQTTENQAGGVAESVFIAAQFVLYGAEYATLAERRGLADVATEARKYVD EVRAAVLEHGWDGQWFLRAYDYYGNPVGTDAKPEGKIWIEPQGFAVMAGIGVGEGPDDADAPAVKA LDSVNEM LGTPHGLVLQYPAYTTYQIELGEVSTYPPGYKENGGI FCHN NPWVI IAETVVGRGAQAFDYYK RITPAYREDISDTHKLEPYVYAQM IAGKEAVRAGEAKNSWLTGTAAWNFVAVSQYLLGVRPDYDGLVVD PQIGPDVPSYTVTRVARGATYEITVTNSGAPGARASLTVDGAPVDGRTVPYAPAGSTVRVEVTV

With the cellobiose phosphorylase of Cellulomonas uda corresponding with the amino acid sequence SEQ ID N°3 is meant the following amino acid sequence: Genbank accession number: AAQ20920

MRYGHFDDEAREYVITTPHTPYPWINYLGSEQFFSLLSHQAGGYSFYRDAKMRRLTRYRYNNIPADAGG RYLYVNDGGDVWTPSWLPVKADLDHFEARHGLGYSTITGERNGVRVETLFFVPVGENAEVQKVTVTNTS DSYKSLTLFSFVEFCLWNAQDDQTNYQRNLSIGEVEVEQESPHGSAIYHRTEYRERRDHYAVFAVNTQAE GFDTDRDTFVGAYNSLGEAAVPLKGESANSVASGWYPIGSHSVAVSLAPGESRELVYVLGYVENPDEEK WADDAKQVVNKERAHALLSRFATSEQTDAAFAALKDYWTDLLSTYSVSSNDEKLDRMVNIWNQYQCM VTFNMSRSASFFETGIGRGMGFRDSNQDLLGFVHLIPERARERIIDIASTQFADGSAYHQYQPLTKRGNN DIGSGFNDDPLWLIAGTAAYIKETGDFSILDEPVPFDNEPGSEVPLFEHLTRSFEFTVTHRGPHGLPLIGRA DWNDCLNLNCFSTTPGESFQTTENQAGGVAESTFIAAQFVLYGEQYAELAARRGLADVADRARGHVAE MRDALLTDGWDGSWFLRAYDYYGNPIGTDAHDEGKIWIEPQGFAVMAGVGVGEGPQDTDAPAIKAL DSVNEMLATDHGMVLQYPAYTTYQVHMGEVSTYPPGYKENGGIFCHNNPWVIIAETVVGRGGRAFDY YKRITPAYREDISDVHRLEPYVYAQMIAGKEAVRHGEAKNSWLTGTAAWNFVTVSQYLLGVRPEYDGLV VDPQIGPDVPSFTVTRVARGATYEITVTNSGTDGSRGRLVVDGTPVEGNLVPYAPAGSTVRVDVTL

The terms 'a variant of each of said sequences having at least 95% sequence identity with each of said sequences' refers to proteins having at least 95 % sequence identity (i.e. having at least 95, 96, 97, 98 or 99% sequence identity) with SEQ ID N° 1, 2 or 3 and that retain said cellobiose phosphorylase activity. The percentage of amino acid sequence identity is determined by alignment of the two sequences and identification of the number of positions with identical amino acids divided by the number of amino acids in the shorter of the sequences x 100. Typically the latter 'variant' may differ from the protein as depicted by SEQ. ID N° 1, 2 or 3 only in conservative substitutions and/or modifications, such that the ability of the protein to have cellobiose phosphorylase activity is retained. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of protein chemistry would expect the nature of the protein to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

Variants may also (or alternatively) be proteins as described herein modified by, for example, the deletion or addition of amino acids that have minimal influence on the cellobiose phosphorylase activity (as defined above), secondary structure and hydropathic nature of the enzyme. Hence the term variants also refers to fragments of SEQ N° 1, 2 or 3 containing fewer amino acids than the amino acid sequence as depicted by SEQ. ID N° 1, 2 or 3 and that retain said cellobiose phosphorylase activity.

The present invention further relates to a method as described above wherein said cellobiose phosphorylase is immobilized. The term 'immobilized' refers to any method known to a skilled person to rigidify and/or stabilize enzymes. Non-limiting examples of such methods are the binding of enzymes -preferably covalent binding- to carriers or the production of cross-linked enzyme aggregates (CLEA) which do not contain carriers as is for example described in more detail in WO 2011/124538.

Hence, the present invention more specifically relates to a method as described above wherein said immobilized cellobiose phosphorylase is a cross-linked enzyme aggregate or CLEA.

The present invention further relates to a method as described above, wherein said cross- linked cellobiose phosphorylase aggregate is imprinted with the acceptor.

The term 'imprinting' refers to increasing the activity of the cellobiose phosphorylases or immobilized enzymes of the present invention through pretreating the enzymes with the acceptors 2-phenylethanol, β-citronellol, vannillyl alcohol, hydroquinone or a linear alcohol with a chain length of at least 8 carbon atoms.

More specifically, the present invention relates to a method as described above wherein said acceptor is octanol.

In addition, the present invention relates to a method as described above wherein said β-glucoside is further elongated by:

-contacting in vitro a cellodextrin phosphorylase with alpha-glucose- 1 -phosphate as donor and one of the following glucosides as acceptor : octyl β-D-glucopyranoside, nonyl β-D- glucopyranoside, decyl β-D-glucopyranoside, dodecy^-D-glucopyranoside, vanillyl 4-0 β-D- glucopyranoside, β-citronellyl β-D-glucopyranoside, 2-phenylethyl β-D-glucopyranoside or 1- Ο-β-D-glucopyranosyl hydroquinone, and

-glycosylating said glucoside as acceptor. The term 'a cellodextrin phosphorylase' (CDP) refers to glycoside phosphorylases that catalyze the reversible phosphorolysis of cellooligosaccharides into a-glucose-l-phosphate (G1P) and cellodextrins with reduced chain length (Kitaoka et al., 1992 and Kitaoka et al., 2002). They are involved in the degradation of cellulosic biomass in vivo. More specifically, the present invention relates to the CDPs derived from Clostridium stercorarium (Reichenberger et al., 1997) and Clostridium thermocellum (Sheth 1969, Arai et al., 1994, Samain et al., 1995, Kawaguchi et al., and Sheth & Alexander, 1998) or, a variant of each of said sequences having at least 95% sequence identity with each of said sequences in a similar manner as defined above for the CPs of the present invention. More specifically, the present invention relates to the CDP encoded by the gene with Genbank number U60580 derived from C. stercorarium strain DSM8532 and its activity as described in WO 2011/144706. The latter enzyme has a the following amino acid sequence (SEQ. ID N° 4):

Genbank accession number: AAC45511

MRYGYFDEKAREYVITRPDTPTPWI NYIGNGKYGGIVTNTGGGYSFHKDPQN RRITRYRYN NLPTDRPGR YIYVRDRLTGEYWNPGYQPVQRKLDSYRCRHGMGYTVLEGEYKGIAADVTYFVPDDRDFEIWLVQI RNL CHVERNLQVFSYAEFCFWDAI M DQQNVDWVQQI NQGRYEDRLITWHPHHFKDACAFFATNAEI NSFD TNLEAFIGRYRCESN PIAVETGACSNSVSYRMNGVGAFCI DVN LKPGEEREII FILGFTEN KSTI RDEI RDYLN VEYAKEALKRLKDSWEEYLDKLQIETPDRETNLFVNTWNQYQCKITFNWSRFVSMYSWGLGRGIGI RDS AQDTLGVM HSI PELAGGLIKRLI HCQYTDGRVYHLFFPLTGEGGIGDAPVVKFDWYSDDHLWLPIAANAY LKETAN FDFFQSVVPYNDNKTEGTVWEHLN RAM EFTYNHRGPHALPYSRADWN DTLNLDMGNGIAET LFTSM LFSEPPLKRFRCRISDKRIATKYRYWYDEM KQAI NEWCWDGEWYI RAFDDEGNVLGSGKNRYGK IFINSQSWAVLSMVAPEEYAKKCLESVYRHLNTKYGIVKVYPAYPEYN PKIGGMTTYPPGAKENGGI FAHT NPWVM IAECM MGNGRRAYQYYRQI LPLTRN DDADLLEVEPYVYCQN ILGKEHPQFGIGRNSWLTGTAA WNMVAVSQYILGIRPEYDGLTVDPCIPPDWKGFKVRRIFRGCVYN IEVRN PEGVRRCEKNCRRGVETDKI PVKPAGTVCECVVI MG

The activity of 'further elongating a glucoside of the present invention' is also described in detail in WO 2011/144706.

The present invention will now be illustrated by the following non-limiting examples. Examples

Materials and Methods

Materials

The IL AM MOENG 101 was kindly provided by Evonik Industries AG, and EtOAc was bought from Fiers NV (Kuurne. Belgium). The other I Ls were purchased from loLiTec Ionic Liquids Technologies GmbH, and had a purity of at least 99 %. All other chemicals were analytical grade and purchased from Sigma-Aldrich.

Production and purification of cellobiose phosphorylase

Overexpression of CP was carried out using E. coli JW0987 cells transformed with the expression vector pTrc99a harbouring the following codon optimized CP gene from Clostridium thermocellum YM4 (SEQ. ID N°5):

ATGAAATTTGGCTTTTTTGATGATGCCAATAAAGAATATGTGATTACCGTTCCGCGTACCCCGTATCC GTGGATTAATTATCTGGGCACCGAAAATTTTTTTAGCCTGATTAGCAATACCGCAGGCGGTTATTGTT TTTATCGTGATGCACGTCTGCGTCGTATTACCCGTTATCGTTATAATAATGTGCCGATTGATATGGGT GGTCGCTATTTTTATATTTATGATAATGGCGATTTTTGGAGTCCGGGTTGGAGTCCGGTTAAACGTGA ACTGGAAAGCTATGAATGTCGTCATGGTCTGGGCTATACCAAAATTGCAGGTAAACGCAATGGCATT AAAGCCGAAGTTACCTTTTTTGTGCCGCTGAATTATAACGGCGAAGTGCAGAAACTGATTCTGAAAA ATG AAG G CCAG G ATAAAAAAAAAATTACCCTGTTTAG CTTTATTG AATTTTG CCTGTG G AATG CCTAT GATGATATGACCAATTTTCAGCGCAATTTTAGCACCGGTGAAGTTGAAATTGAAGGCAGCGTGATTT ATCATAAAACCGAATATCGCGAACGTCGCAATCATTATGCCTTTTATAGCGTGAATGCCAAAATTTCA GG CTTTG ATAG CG ATCG CG ATAG CTTTATTG GTCTGTATAATG GTTTTG ATG CACCG CAG G CAGTTGT TAATGGCAAAAGCAATAATAGCGTTGCAGATGGTTGGGCACCGATTGCAAGCCATAGCATTGAAATT GAACTGAATCCGGGTGAACAGAAAGAATATGTTTTTATTATTGGCTATGTGGAAAATAAAGATGAAG AAAAATGGG AAAG CAAAGG CGTG ATTAATAAAAAAAAAG CCTATG AAATG ATTG AACAGTTTAATA CCGTG G AAAAAGTG G ATAAAG CCTTTG AAG AACTG AAAAG CTATTG G AATG CCCTG CTG AG CAAAT ATTTTCTGGAAAGCCATGATGAAAAACTGAATCGCATGGTGAATATTTGGAATCAGTATCAGTGCAT GGTGACCTTTAATATGAGCCGTAGCGCAAGCTATTTTGAAAGCGGTATTGGTCGTGGTATGGGTTTT CGTGATAGCAATCAGGATCTGCTGGGTTTTGTTCATCAGATTCCGGAACGTGCACGTGAACGTCTGC TGGATCTGGCAGCAACCCAGCTGGAAGATGGTAGCGCATATCATCAGTATCAGCCGCTGACCAAAAA AG G C AATAATG AAATTGG CAG CAATTTTAATG ATG ATCCG CTGTGG CTG ATTCTG G C AACCG C AG C A TATATTAAAGAAACCGGTGATTATAGCATTCTGAAAGAACAGGTGCCGTTTAATAATGATCCGAGCA AAGCAGATACCATGTTTGAACATCTGACCCGTAGCTTTTATCATGTGGTTAATAATCTGGGTCCGCAT GGTCTGCCGCTGATTGGTCGTGCAGATTGGAATGATTGTCTGAATCTGAATTGCTTTAGCACCGTTCC GGATGAAAGCTTTCAGACCACCACCAGTAAAGATGGTAAAGTTGCAGAAAGCGTTATGATTGCCGG TATGTTTGTGTTTATTGGCAAAGATTATGTGAAACTGTGCGAATATATGGGCCTGGAAGAAGAAGCA CGTAAAGCACAGCAGCATATTGATGCAATGAAAGAAGCCATTCTGAAATACGGCTATGATGGTGAAT GGTTTCTG CGTG C ATATG ATG ATTTTG GTCGTAAAGTGG G CAG C AAAG AAAATG AAG AAG G CAAAA TTTTTATTGAAAGCCAGGGCTTTTGCGTGATGGCAGAAATTGGTCTGGAAGATGGCAAAGCACTGAA AGCCCTGGATAGCGTTAAAAAATATCTGGATACCCCGTATGGTCTGGTTCTGCAGAATCCGGCATTTA CCCGTTATTATATTGAATATGGCGAAATTTCCACCTATCCGCCGGGTTATAAAGAAAATGCCGGTATT TTTTGCCATAATAATGCCTGGATTATTTGTGCCGAAACCGTTGTTGGTCGTGGTGATATGGCCTTTGA TTATTATCGTAAAATTGCACCGGCATATATTGAAGATGTGAGCGATATTCATAAACTGGAACCGTATG TTTATGCACAGATGGTTGCAGGTAAAGATGCAAAACGTCATGGTGAAGCAAAAAATAGCTGGCTGA CCGGCACCGCAGCATGGAATTTTGTTGCAATTAGCCAGTGGATTCTGGGTGTTAAACCGGATTATGA TGGCCTGAAAATTGATCCGTGTATTCCGAAAGCATGGGATGGTTATAAAGTGACCCGTTATTTTCGTG GTAGCACCTATGAAATCACCGTGAAAAATCCGAATCATGTGAGCAAAGGTGTGGCAAAAATTACCGT TGATGGCAATGAAATTAGCGGTAATATTCTGCCGGTGTTTAATGATGGCAAAACCCATAAAGTGGAA GTGATTATGGGTTAA

The strain was routinely grown at 37 °C on 500 mL LB medium (10 g L ¹ tryptone, 5 g L ¹ yeast extract, 5 g L ¹ NaCI) supplemented with ampicillin (100 mg L ¹). After overnight growth, the culture was inoculated into 15 L of double LB medium (20 g L ¹ tryptone, 10 g L ¹ yeast extract, 5 g L ¹ NaCI) supplemented with glucose (30 g L ¹) and ampicillin (100 mg L ¹) in a 30 L Biostat C reactor (B. Braun Biotech I nc., Pennsylvania, USA). The temperature, pH and stirrer speed were set at 37 °C, 7 and 800 rpm respectively. Adequate aeration was achieved by passing 1.1 vvm air through the reactor, and foaming was prevented by manually adding anti-foam (10% (v/v) antifoam silicone Snapsil RE 20, VWR BDH Prolabo, BE) when required. Induction was performed by adding I PTG to a final concentration of 0.1 mM as soon as the OD o reached 0.6. After 8 h of growth (OD₆oo ~ 34), the cells were harvested by centrifugation (10000 g, 4 °C, 20 min), and frozen at -20 °C. Next, the obtained pellets were lysed as described earlier,'¹³¹ and the /V-terminal His6-tagged protein was purified by nickel-nitrilotriacetic acid metal affinity chromatography. The protocol as described by the supplier (Qjagen, USA) was used, except for the imidazole concentration of the elution buffer, which was reduced to 175 mM. The obtained enzyme solution was washed with MES buffer (50 mM, pH 6.5) and concentrated using centricons (Amicon Ultra 30K, Millipore, DE).

Preparation of CLEAs and iCLEAs

Cross-linked enzyme aggregates of CP were prepared by adding 100 μί ieri-butanol to 100 μί His6-tagged purified protein (2.4 mg mL^-1) under agitation in a thermoshaker (Eppendorf, DE) (1000 rpm). After 30 min incubation at 4 °C, varying amounts of glutaraldehyde (25% (v/v)) were added and the mixture was kept under stirring for 15, 30, 60, 75, 90, 120, 150 or 180 min. Reduction of the formed imine bonds was achieved by adding 500 μί sodium bicarbonate buffer (100 mM, pH 10) supplemented with sodium borohydrate (1 mg mL^-1). After 15 min incubation at 4°C, another 500 μί was added and allowed to react during 15 min. Next, the resulting CLEAs were harvested by centrifugation (17000 g, 4 °C, 15 min), and subsequently washed 5 times with 1 mL MES buffer (50 mM, pH 6.5). iCLEAs were prepared by incubating CP with 250 mM octanol (3.25 mg) or octyl β-D-glucopyranoside (7.31 mg) during 30 min at 37 °C prior to the addition of ieri-butanol.

Large-scale production of CLEAs and iCLEAs was performed by adding 4 mL ieri-butanol to 4 mL His6-tagged purified protein (2.4 mg mL^-1) in 50 mL falcons. The glutaraldehyde:protein ratio was set at 0.6 (5.76 mg), and the reaction mixture was incubated during 90 min at 4 °C. Next, two times 10 mL sodium bicarbonate buffer (100 mM, pH 10) supplemented with sodium borohydrate (1 mg mL^-1) was added. The CLEAs were then harvested by centrifugation (10000 g, 4 °C, 20 min), washed 5 times with 20 mL MES buffer (50 mM, pH 6.5) and freeze- dried (Alpha 1-4, Christ, DE). Activity assays

The activity of CP and CP CLEAs was determined in the synthesis direction by measuring the release of phosphate from aGlP with the method of Gawronski and Benson.'³³¹ One unit of CP activity corresponds to the release of 1 μιηοΙ phosphate from 50 mM aGlP and 50 mM glucose in a 50 mM MES buffer at pH 6.5 and 37 °C. The activity of the CLEAs was determined by adding 1 mL substrate buffer (50 mM aGlP and 50 mM glucose in 50 mM MES buffer pH 6.5) to the obtained biocatalyst. The reactions were performed in a thermoshaker (Eppendorf, DE) at 1000 rpm. Alternatively, a citrate-phosphate buffer (pH 4-5.4), MES buffer (pH 5.5-6.5), MOPS buffer (pH 6.6-7.5) and tricine buffer (pH 7.6-9) were used. Unless stated otherwise, the temperature was set at 37 °C. The immobilization yield is defined as the ratio of the activity detected in the CLEA preparation to that present in the original enzyme solution. Protein concentrations were measured according to the Lowry method, using bovine serum albumin as standard.'³⁴¹ All assays were performed in triplicate and had a CV of less than 10 %.

Stability assays The kinetic thermostability was determined by diluting 20 U mL ¹ CP in a 50 mM MES (pH 6.5). If required, solvents were added and the mixtures were incubated in a water bath at various temperatures. At regular intervals, samples were taken and diluted 200 times in MES buffer (50 mM, pH 6.5). The diluted samples were stored at 4 °C, and their activity was determined at 37 °C using the Gawronski method. The stability of the CLEAs was evaluated by incubating 6 mg CP CLEA in 1.5 mL MES buffer (50 mM, pH 6.5) in a thermoshaker (Eppendorf, DE) at 1000 rpm. Alternatively, the MES buffer contained 20% AMMOENG™ 101 or 37.5% EtOAc. At regular intervals, homogeneous samples (100 μί) were taken after intensive mixing, centrifuged (17000 g, 4 °C, 15 min) and washed three times with 1 mL MES buffer (50 mM, pH 6.5). The samples were stored at 4 °C, and the activity was determined at 37 °C using the Gawronski assay. The tso-values were calculated from the equations obtained by fitting the linear part of the stability curves. Analytical methods

The formation of glucosides was assessed by TLC or HPLC. Separation was performed on Merck Silica gel 60 F254 precoated plates. The eluens was a mixture of EtOAc:MeOH :water (30:5:4), and spots were visualized by UV detection at 254 nm, or charring with 10% (v/v) H2SO4. The concentration of glucosides was determined by HPLC analysis. Adequate separation and detection was achieved using an X-bridge amide column (250 mm χ 4.6 mm, 3.5 μιη, Waters, USA) coupled with an Alltech 2000ES evaporative light scattering detector, as previously described.'¹¹¹ When using biphasic catalysis, homogeneous samples, obtained after intensive mixing, were diluted in DMSO. The resulting concentrations thus refer to the amount of product present in the total reaction volume. All calibration curves were prepared with authentic samples.

Glycosylation reactions with CP Biphasic catalysis was performed by adding water immiscible acceptors (300 μί) to 500 μί MES buffer (50 mM, pH 6.5) containing 200 mM aGlP. Alternatively, 300 μΙ_ EtOAc supplemented with various acceptors (30 mg) was used as organic phase. Cosolvent catalysis was achieved by adding AM MOENG™ 101 (200 μί) and various acceptors (37.5 mg) to 800 μί M ES buffer (50 mM, pH 6.5) containing 200 mM aGlP. All reactions were performed in a thermoshaker (Eppendorf, DE) at 1000 rpm and 50 °C in the presence of 5 U mL ¹ CP. After 24 h, samples were inactivated (10 min at 95 °C) and subjected to TLC or HPLC analysis.

Production and purification of glucosides The glycosylation of hexanol, heptanol, octanol, nonanol, decanol, dodecanol, β-citronellol and 2-phenylethanol was carried out by adding the acceptors (18.75 mL) to 31.25 mL M ES buffer (50 mM, pH 6.5) containing 200 mM aGlP. Glycosylation of vannillyl alcohol and hydroquinone was achieved by using EtOAc (18.75 mL) containing the acceptors (1.875 g) as organic phase. All reactions were performed in magnetically stirred flasks at 50 °C in the presence of 5 U mL ¹ CP. After 48 h, the reaction mixtures were heated (10 min, 95 °C) and centrifuged (10000 g, 4 °C, 20 min) to remove debris. The samples were then evaporated in vacuo and the residue was purified by column chromatography (silicagel, EtOAc:MeOH:water

(30:5:4)).

Synthesis and purification of octyl β-D-glucopyranoside

Synthesis of octyl β-D-glucopyranoside was carried out at 50 mL scale in a biphasic system consisting of octanol (18.75 mL) and 31.25 mL MES buffer (50 mM, pH 6.5) containing 200 mM aGlP. The reactions were performed in magnetically stirred flasks at 50 °C in the presence of 40 mg CP CLEA, octanol iCLEA or octyl β-D-glucopyranoside iCLEA respectively. At regular intervals, samples were inactivated (10 min at 95 °C) and subjected to HPLC analysis. Alternatively, the reaction was stopped after 24 h incubation at 50 °C. The octanol iCLEAs were recuperated by centrifugation (10000 g, 4 °C, 20 min), and the reaction mixture was passed through a pretreated hydrophobic membrane (Accurel PP1E, Membrane, Germany). Prior to its application, the membrane was consecutively treated with 50 mL of the following solutions: hexadecane, a hexadecane:water (50:50), octanol and finally an octanol:water (50:50). The resulting octanol phase was then evaporated in vacuo and the residue was weighed. The iCLEAs were recycled for the next batch conversion.

Structure elucidation of glucosides

The structures of the newly formed glucosides were determined by a combination of ID NMR (1H NMR and 13C NMR) and 2D NMR (gCOSY, gHSQC and gHMBC) spectroscopy. Residual signals of solvent were used as internal standard (<¾ 3.330 ppm, <¾ 49.30 ppm), and digital resolution enabled us to report <¾ to three and <¾ to two decimal places. The proton spin systems were assigned by COSY, and then the assignment was transferred to carbons by HSQ.C. HMBC experiments enabled to assign quaternary carbons and to join individual spin systems together. Chemical shifts are given in £scale [ppm], and coupling constants in Hz. Hexyl β-D-glucopyranoside

^XH NMR (CD3OD): 50.926 (3H, t, J = 7.1 Hz, H-6'), 1.33 (2H, m, H-4'), 1.35 (2H, m, H-5'), 1.404 (2H, m, H-3'), 1.638 (2H, m, H-2'), 3.200 (1H, dd, J = 9.2, 7.8 Hz, H-2), 3.281 (1H, ddd, J = 9.6, 5.6, 2.3 Hz, H-5), 3.320 (1H, dd, J = 9.6, 8.7 Hz, H-4), 3.381 (1H, dd, J = 9.2, 8.7 Hz, H-3), 3.554 (1H, dt, J = 9.6, 6.8 Hz, H-l'u), 3.696 (1H, dd, J = 11.9, 5.6 Hz, H-6u), 3.880 (1H, dd, J = 11.9, 2.3 Hz, H-6d), 3.916 (1H, dt, J = 9.6, 6.9 Hz, H-l'd), 4.273 (1H, d, J = 7.8 Hz, H-l); ¹³C NMR (CD3OD): δ 14.68 (C-6'), 23.91 (C-5'), 27.01 (C-3'), 30.98 (C-2'), 33.10 (C-4'), 62.97 (C-6), 71.14 (C-1'), 71.83 (C-4), 75.28 (C-2), 78.05 (C-5), 78.29 (C-3), 104.54 (C-1)

Heptyl β-D-glucopyranoside

^XH NMR (CD3OD): δ 0.921 (3H, t, J = 7.1 Hz, H-7'), 1.33 (2H, m, H-5'), 1.34 (2H, m, H-6'), 1.36 (2H, m, H-4'), 1.398 (2H, m, H-3'), 1.640 (2H, m, H-2'), 3.197 (1H, dd, J = 9.2, 7.8 Hz, H-2), 3.279 (1H, ddd, J = 9.6, 5.6, 2.3 Hz, H-5), 3.317 (1H, dd, J = 9.6, 8.7 Hz, H-4), 3.379 (1H, dd, J = 9.2, 8.7 Hz, H-3), 3.553 (1H, dt, J = 9.6, 6.8 Hz, H-l'u), 3.695 (1H, dd, J = 11.9, 5.6 Hz, H-6u), 3.880 (1H, dd, J = 11.9, 2.3 Hz, H-6d), 3.915 (1H, dt, J = 9.6, 6.9 Hz, H-l'd), 4.271 (1H, d, J = 7.8 Hz, H-l); ¹³C NMR (CD3OD): δ 14.72 (C-7'), 23.92 (C-6'), 27.31 (C-3'), 30.54 (C-4'), 31.04 (C-2'), 33.25 (C- 5'), 62.99 (C-6), 71.14 (C-1'), 71.86 (C-4), 75.31 (C-2), 78.08 (C-5), 78.31 (C-3), 104.56 (C-1)

Octyl β-D-glucopyranoside

^XH NMR (CD3OD): δ 0.921 (3H, t, J = 7.1 Hz, H-8'), 1.31 (2H, m, H-6'), 1.33 (4H, m, H-4', H-7'), 1.34 (2H, m, H-5'), 1.402 (2H, m, H-3'), 1.640 (2H, m, H-2'), 3.191 (1H, dd, J = 9.2, 7.8 Hz, H-2), 3.274 (1H, ddd, J = 9.6, 5.5, 2.2 Hz, H-5), 3.309 (1H, dd, J = 9.6, 8.6 Hz, H-4), 3.370 (1H, dd, J = 9.2, 8.6 Hz, H-3), 3.553 (1H, dt, J = 9.6, 6.8 Hz, H-l'u), 3.690 (1H, dd, J = 11.9, 5.5 Hz, H-6u), 3.881 (1H, dd, J = 11.9, 2.2 Hz, H-6d), 3.917 (1H, dt, J = 9.6, 6.9 Hz, H-l'd), 4.267 (1H, d, J = 7.8 Hz, H-l); ¹³C NMR (CD3OD): δ 14.72 (C-8'), 24.00 (C-7'), 27.39 (C-3'), 30.70^a (C-4'), 30.87^a (C- 5'), 31.08 (C-2'), 33.29 (C-6'), 63.04 (C-6), 71.18 (C-1'), 71.92 (C-4), 75.38 (C-2), 78.16 (C-5), 78.38 (C-3), 104.62 (C-1) Nonyl β-D-glucopyranoside

^XH NMR (CD3OD): δ 0.921 (3H, t, J = 7.1 Hz, H-9'), 1.31 (2H, m, H-7'), 1.32 (2H, m, H-8'), 1.33 (6H, m, H-4', H-5', H-6'), 1.403 (2H, m, H-3'), 1.640 (2H, m, H-2'), 3.187 (1H, dd, J = 9.2, 7.8 Hz, H-2), 3.271 (1H, ddd, J = 9.6, 5.5, 2.2 Hz, H-5), 3.303 (1H, dd, J = 9.6, 8.5 Hz, H-4), 3.363 (1H, dd, J = 9.2, 8.5 Hz, H-3), 3.553 (1H, dt, J = 9.6, 6.8 Hz, H-l'u), 3.686 (1H, dd, J = 11.9, 5.5 Hz, H- 6u), 3.881 (1H, dd, J = 11.9, 2.2 Hz, H-6d), 3.918 (1H, dt, J = 9.6, 6.9 Hz, H-l'd), 4.265 (1H, d, J = 7.8 Hz, H-l); ¹³C NMR (CD3OD): δ 14.73 (C-9'), 24.03 (C-8'), 27.42 (C-3'), 30.73^a (C-6'), 30.94^a (C-5'), 31.02^a (C-4'), 31.11 (C-2'), 33.36 (C-7'), 63.08 (C-6), 71.20 (C-1'), 71.97 (C-4), 75.43 (C- 2), 78.21 (C-5), 78.44 (C-3), 104.67 (C-1)

Decyl β-D-glucopyranoside ^XH NMR (CD3OD): δ 0.920 (3H, t, J = 7.1 Hz, H-10'), 1.30 (2H, m, H-8'), 1.32 (2H, m, H-7'), 1.33 (8H, m, H-4', H-5', H-6', H-9'), 1.403 (2H, m, H-3'), 1.640 (2H, m, H-2'), 3.189 (1H, dd, J = 9.2, 7.8 Hz, H-2), 3.273 (1H, ddd, J = 9.6, 5.5, 2.2 Hz, H-5), 3.305 (1H, dd, J = 9.6, 8.5 Hz, H-4), 3.365 (1H, dd, J = 9.2, 8.5 Hz, H-3), 3.553 (1H, dt, J = 9.6, 6.8 Hz, H-l'u), 3.688 (1H, dd, J = 11.9, 5.5 Hz, H-6u), 3.882 (1H, dd, J = 11.9, 2.2 Hz, H-6d), 3.918 (1H, dt, J = 9.6, 6.9 Hz, H-l'd), 4.266 (1H, d, J = 7.8 Hz, H-l); ¹³C NMR (CD3OD): δ 14.74 (C-10'), 24.03 (C-9'), 27.41 (C-3'), 30.77^a (C-7'), 30.93^a (C-6'), 31.0 (C-5'), 31.05^a (C-4'), 31.10 (C-2'), 33.36 (C-8'), 63.07 (C-6), 71.20 (C-1'), 71.95 (C-4), 75.41 (C-2), 78.20 (C-5), 78.42 (C-3), 104.66 (C-1)

Dodecyl β-D-glucopyranoside

^XH NMR (CD3OD): δ 0.920 (3H, t, J = 7.1 Hz, H-ll'), 1.31 (4H, m, H-8', H-9'), 1.32 (6H, m, H-4',

H-5', H-6'), 1.33 (4H, m, H-7', H-10'), 1.402 (2H, m, H-3'), 1.640 (2H, m, H-2'), 3.189 (1H, dd,

= 9.2, 7.8 Hz, H-2), 3.272 (1H, ddd, J = 9.6, 5.5, 2.2 Hz, H-5), 3.305 (1H, dd, J = 9.6, 8.5 Hz, H-4), 3.365 (1H, dd, J = 9.2, 8.5 Hz, H-3), 3.553 (1H, dt, J = 9.6, 6.8 Hz, H-l'u), 3.688 (1H, dd, J = 11.9,

5.5 Hz, H-6u), 3.881 (1H, dd, J = 11.9, 2.2 Hz, H-6d), 3.918 (1H, dt, J = 9.6, 6.9 Hz, H-l'd), 4.266

(1H, d, J = 7.8 Hz, H-l); ¹³C NMR (CD3OD): δ 14.74 (C-ll'), 24.03 (C-10'), 27.41 (C-3'), 30.77^a (C- 8'), 30.93^a (C-7'), 31.05^a (C-5', C-6'), 31.10³ (C-4', C-2'), 33.37 (C-9'), 63.07 (C-6), 71.20 (C-1'), 71.95 (C-4), 75.41 (C-2), 78.20 (C-5), 78.42 (C-3), 104.66 (C-1)

2-Phenylethyl β-D-glucopyranoside

^XH NM R (CD3OD): δ 2.941 (IH, m,∑J = 25.0 Hz, H-2'u), 2.973 (IH, m,∑l = 25.0 Hz, H-2'd), 3.205 (IH, dd, J = 9.2, 7.8 Hz, H-2), 3.286 (IH, m, H-5), 3.305 (IH, m, H-4), 3.369 (IH, dd, J = 9.2, 8.6 Hz, H-3), 3.684 (IH, dd, J = 11.9, 5.5 Hz, H-6u), 3.779 (IH, ddd, J = 9.7, 8.2, 6.7 Hz, H-l'u), 3.882 (IH, dd, J = 11.9, 2.1 Hz, H-6d), 4.111 (IH, ddd, J = 9.7, 8.2, 6.8 Hz, H-l'd), 4.322 (IH, d, J = 7.8 Hz, H-l), 7.197 (IH, m, H-para), 7.267 (4H, m, H-ortho, H-meta); ¹³C N MR (CD₃OD): δ 37.53 (C-2'), 63.05 (C-6), 71.94 (C-4), 72.01 (C-1'), 75.40 (C-2), 78.26 (C-5), 78.40 (C-3), 104.68 (C-1), 127.50 (C-para), 129.64 (C-meta), 130.32 (C-ortho), 140.36 (C-ipso)

β-Citronellyl β-D-glucopyranoside

^XH NM R (CD3OD): δ 0.935 (3H, d, J = 6.6 Hz, 3'-Me), 1.18 (IH, m, H-4'u), 1.37 (IH, m, H-4'd), 1.44 (IH, m, H-2'u), 1.610 (IH, m, H-3'), 1.627 (3H, m, 7'-Me), 1.68 (IH, m, H-2'd), 1.692 (IH, q, J = 1.3 Hz, H-8'), 1.999 (IH, m, H-5'u), 2.040 (IH, m, H-5'd), 3.184 (IH, dd, J = 9.2, 7.8 Hz, H- 2), 3.275 (IH, ddd, J = 9.6, 5.4, 2.2 Hz, H-5), 3.305 (IH, dd, J = 9.6, 8.5 Hz, H-4), 3.365 (IH, dd, J = 9.2, 8.5Hz, H-3), 3.606 (IH, ddd, J = 9.6, 8.0, 5.8 Hz, H-l'), 3.691 (IH, dd, = 11.9, 5.4 Hz, H- 6u), 3.885 (IH, dd, J = 11.9, 2.2 Hz, H-6d), 3.956 (IH, ddd, J = 9.6, 7.8, 6.8 Hz, H-l'd), 4.267 (IH, d, J = 7.8 Hz, H-l), 5.127 (IH, m, H-6'); ¹³C NM R (CD3OD): δ 18.02 (7'-Me), 20.21 (3'-Me), 26.19 (C-8'), 26.77 (C-5'), 30.88 (C-3'), 38.04 (C-2'), 38.73 (C-4'), 63.07 (C-6), 69.43 (C-1'), 71.97 (C- 4), 75.41 (C-2), 78.21 (C-5), 78.45 (C-3), 104.64 (C-1), 126.19 (C-6'), 132.25 (C-7') Vanillyl 4-0- -D-glucopyranoside

^XH NM R (CD3OD): δ 3.253 (1H, dd, J = 9.1, 7.8 Hz, H-2), 3.272 (1H, ddd, J = 9.5, 5.8, 2.3 Hz, H- 5), 3.311 (1H, dd, J = 9.5, 8.7 Hz, H-4), 3.354 (1H, dd, J = 9.1, 8.7 Hz, H-3), 3.710 (1H, dd, J = 11.9, 5.8 Hz, H-6u), 3.878 (3H, s, 3'-0Me), 3.918 (1H, dd, J = 11.9, 2.3 Hz, H-6d), 4.336 (1H, d, J = 7.8 Hz, H-l), 4.609 (1H, d, J = 11.4 Hz, CH₂ u), 4.838 (1H, d, J = 11.4 Hz, CH₂ d), 6.769 (1H, d, J = 8.0 Hz, H-5'), 6.845 (1H, dd, J = 8.0, 2.0 Hz, H-6'), 7.071 (1H, d, J = 2.0 Hz, H-2'); ¹³C NM R (CD3OD): δ 56.67 (3'-0Me), 63.14 (C-6), 72.04^a (CH₂), 72.05^a (C-4), 75.42 (C-2), 78.32 (C-5), 78.40 (C-3), 103.04 (C-l), 113.62 (C-2'), 116.09 (C-5'), 122.76 (C-6'), 130.60 (C-l'), 147.65 (C- 4'), 149.23 (C-3')

Ι-Ο-β-D-Glucopyranosyl hydroquinone ^XH NM R (CD3OD): δ 3.395 (2H, m, H-4, H-5), 3.428 (1H, dd, J = 9.2, 7.5 Hz, H-2), 3.460 (1H, m, H-3), 3.718 (1H, m, H-6u), 3.902 (1H, m, H-6d), 4.755 (1H, d, J = 7.5 Hz, H-l), 6.718 (2H, m,∑l = 9.1 Hz, H-meta), 6.985 (2H, m, U = 9.1 Hz, H-ortho); ¹³C NM R (CD₃OD): δ 62.84 (C-6), 71.72 (C-4), 75.27 (C-2), 78.27 (C-3), 78.32 (C-5), 103.92 (C-l), 116.90 (C-meta), 119.67 (C-ortho), 152.72 (C-ipso), 154.08 (C-para)

Results

Development of a suitable glycosylation system To date, C. thermocellum is the only known thermophilic source of CP, which makes the corresponding enzyme the most promising representative for practical applications. Indeed, carbohydrate conversions are preferably performed at elevated temperatures, mainly to avoid microbial contamination ^26]. However, the exact half-life of the protein was not yet known, and was therefore, measured here at different temperatures (Figure 1).

The CP was found to be rapidly inactivated at 60 °C, CP was found to be remarkably stable at 37 °C. However, applying enzymes much below their optimal temperature typically comes at the expense of a lower turnover. A balance between stability and activity was found for CP at 50 °C, retaining over 58% of its initial activity after 24 h, while operating at roughly 80% of its maximal velocity.'²⁷¹

In addition, the typically very low affinity of disaccharide phosphorylases for non- carbohydrate acceptors urges the addition of solvents.'²⁸¹ Therefore, the half-life (tso) of CP was also determined in the presence of some commonly used organic cosolvents, as well as in some ILs (Table 1).

Table 1. Stability of the CP from C. thermocellum at pH 6.5 and 50 °C, in the presence of various organic solvents and ILs. tso (min)

Solvent 20% 37.5%

MeOH 229 ± 12 11.2 ± 0.9

EtOH 42.1 ± 3.3 3.5 ± 0.3

DMSO 736 ± 49 256 ± 19

[BMIM][dca] 24.2 ± 2.2 3.5 ± 0.2

[BMIM][I] <1 <1

[BMIM][BF₄] 3.2 ± 0.1 <1

[MMIM] ][MeS0₄] 82.6 ± 6.2 10.3 ± 0.6

AMMOENG™ 101 1092 ± 96 654 ± 48

EtOAc 966 ± 78 902 ± 64

Both methanol and ethanol were found to destabilize CP, reducing the tso at 50 °C from over 32 h to less than 4 and 1 h, respectively. Moreover, these short chain alcohols can be glycosylated by CP,'¹⁴¹ impairing their application as cosolvent. Also the ILs [BMIM][dca], [BMIM] [I] and [BMIM][BF₄] were found to destabilize CP, while DMSO and the quaternary ammonium salt AMMOENG™ 101 were less deleterious. Interestingly, the enzyme was also found to be compatible with EtOAc, allowing the use of biphasic catalysis. In contrast to the application of cosolvents, increasing the EtOAc concentration from 20 to 37.5% did not significantly influence the stability of CP (Table 1). Next, the activity of the enzyme was assessed in the most promising solvent systems. CP displayed a relative activity of 94 and 92% in 20% IL and 37.5% EtOAc respectively, thus allowing its use in the latter solvent systems. Exploring the glycosylation potential of CP

Having established adequate cosolvent and biphasic catalysis conditions, the glycosylation potential of CP towards various small organic compounds was evaluated (Table 2).

Table 2. Glycosylation potential of the CP from C. thermocellum. TLC or HPLC analysis was performed after 48 h incubation at 50 °C.

Acceptor Product Acceptor Product

(mM) (mM)

Pentanol + a) l ?-phenylethanol . a)

Hexanol 71 ^a> lS-phenylethanol - a)

heptanol 72 ^a> 2-phenylethanol 59 ^a>

octanol 68 ^a> cinnamyl alcohol + a)

nonanol 55 ^a> menthol _ )_; _ c)

decanol 48 ^a> vannillyl alcohol 25 ^b>, 26 ^c>

dodecanol 30 ^a) phenol _ )_; _ c)

cyclohexanol + a) p-nitrophenol - b)_r - c)

2-hexanol + a) hydroquinone 2 b) 2 ^c)

2-octanol + a) catechol - _ c)

linalool - a) resorcinol - b)_r - c)

eugenol . a) pyrogallol + ^b + ^c)

β-citronellol 20 ^a> curcumin - b)_r - c)

geraniol + a) quercetin - _ c)

nerolidol - a) resveratrol - b)_r - c)

anisyl alcohol + a) vanillin + ^b + ^c)

a⁾ Glycosylation was performed using 37.5% acceptor as organic phase. ^b) Glycosylation was performed using 37.5% EtOAc as organic phase. ^c) Glycosylation was performed in a cosolvent system containing 20% IL.

The glycosylation of short-chain alcohols has already been reported for CP^[14] but surprisingly, we have found that longer chains can also be accepted, although they are more challenging. For example, after 48 h incubation under optimal reaction conditions, about 70mM could be obtained of the hexyl β-D-glucopyranoside but only about 50 mM of the decyl β-D- glucopyranoside. Secondary alcohols were found to be even more challenging, with very little product being formed when using 2-hexanol or 2-octanol as acceptor.

Interestingly, the glycosylation potential of CP is not limited to linear aliphatic alcohols. Next to cyclohexanol, some substituted alcohols with olfactory properties were successfully glucosylated. Despite being produced in lower concentrations, we were able to confirm the formation of vanillyl 4-0^-D-glucopyranoside, β-citronellyl β-D-glucopyranoside and 2- phenylethyl β-D-glucopyranoside. Glycosylation of both R and S-l-phenylethanol failed, confirming the difficult glycosylation of secondary alcohols.

CP was also able to couple a glucose moiety to the phenolic hydroxyl groups of hydroquinone, pyrogallol and vanillin. Although these reactions were rather inefficient compared to SP, 1-0- β-D-glucopyranosyl hydroquinone could be isolated, and its structure was confirmed by NMR spectroscopy. Remarkably, no significant differences were observed between the IL based cosolvent and biphasic system.

Based on these results, we have also evaluated the glycosylation potential of the CP from C. uda and C. gilvus. To that end, the temperature was lowered to 37 °C because these enzymes do not originate from thermophilic sources. Nevertheless, a similar pattern as that with the enzyme from C. thermocellum was observed. For example, the glycosylation of long-chain alcohols could be achieved, which confirms cellobiose phosphorylase as a new biocatalyst for the synthesis of industrially relevant products such as octyl β-glucoside.

Cross-linked enzyme aggregates of CP Over the past decade, immobilization has proven to be a valuable technique to enhance the operational stability of enzymes.'³⁰¹ Unlike other immobilization techniques, the formation of cross-linked enzyme aggregates (CLEAs) doesn't require expensive carriers, and avoids dilution of the enzyme's activity.'³¹¹ First, the precipitation of CP was evaluated in the presence of ieri-butanol (Figure Al). While 80% of the enzyme remained soluble after the addition of 20% ieri-butanol, all CP was precipitated after 30 min incubation at a solvent concentration of 50%. Lower concentrations or shorter incubation failed to yield complete precipitation (Figure Al).

Next, the glutaraldehyde (GA) based cross-linking step was optimized by varying the amount of cross-linker and the incubation time (Figure A2). A maximal yield of 67% was reached when incubating the enzyme at a GA:protein ratio of 0.6 for 90 min. Lower ratios or shorter incubation resulted in less CLEA, while further increasing the amount of GA or the incubation time significantly reduced the activity of the immobilized biocatalyst (Figure A2).

In parallel with previous studies on SP, the optimal temperature of the obtained CP CLEAs shifted from 70 °C for the soluble enzyme, to an impressive 90 °C (Figure A3).^[32] Moreover, the pH range was found to have broadened (Figure A4), indicating enhanced operational stability. The latter was further investigated by determining the stability of the CP CLEAs at 50 °C (Figure 2).

Immobilization boosted the half-life at 50 °C from 34 h to almost 11 days. A similar pattern was observed upon addition of the IL AMMOENG™ 101 or EtOAc, revealing close correlation between thermal and solvent stability.

Imprinted cross-linked enzyme aggregates of CP

It has been recently shown that the activity of CLEAs can be improved by molecular imprinting.¹¹³¹ The applicability of the latter technique was evaluated using the glycosylation of octanol as case study. To that end, CP was incubated with 250 mM octanol or octyl β-D- glucopyranoside (OG) prior to the formation of CLEAs. The effectiveness of the obtained immobilizates for the synthesis of OG was then evaluated (Figure 3).

Imprinting with OG did not significantly increase the transglycosylation activity of the enzyme, but the addition of octanol roughly doubled the initial rate of OG synthesis (Figure 3). Interestingly, the activity of both iCLEAs towards glucose was decreased by 80 and 45%, respectively, further illustrating their altered specificity. In contrast to SP,^[13] imprinting with the acceptor molecule rather than the glucoside was found to enhance the desired glycosylation activity.

Finally, the applicability and reusability of the octanol iCLEAs was assessed at 50 mL scale. After 24 h incubation at 50 °C, the iCLEAs were recuperated by centrifugation and the reaction mixture was subjected to hydrophobic membrane filtration. Next, the octanol phase was evaporated in vacuo, yielding 674 mg octyl β-D-glucopyranoside. This procedure was repeated three times without loss of productivity, revealing excellent mechanical stability and recyclability of the biocatalyst.

Chain elongation by cellodextrin phosphorylase

Although cellodextrin phosphorylase (CDP) is not able to glycosylate non-carbohydrate acceptors directly, it can elongate the carbohydrate chain of glucosylated products.¹³⁵¹ Indeed, we show here that the β -glucosides synthesized by the CP from C. thermocellum can be converted to the corresponding β -cellooligosaccharides by the CDP from the same organism. For example, incubating CDP with 100 mM octyl β -glucoside and 100 mM G1P results in the formation of 14 mM octyl β -cellobioside, 6 mM octyl β -cellotrioside and trace amounts of products with a higher degree of polymerization (Fig. 4A). Similarly, 100 mM of the s glucoside of hydroquinone is converted to 24 mM and 10 mM of the corresponding β - cellobioside and -trioside, respectively (Fig. 4B). All products could be efficiently purified by flash chromatography and their structure was confirmed by NMR analysis. The production of cellooligosaccharide derivatives of long-chain alcohols is of particular relevance because so- called alkyl polyglycosides (APG) are widely used as non-ionic surfactants in household and industrial applications.

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Claims

Claims

1. An in vitro method to produce a β-glucoside comprising:

-contacting in vitro a cellobiose phosphorylase with alpha-glucose-l-phosphate and an acceptor, and -glycosylating said acceptor,

wherein said acceptor is a linear alcohol with a chain length of at least 8 carbon atoms, 2- phenylethanol, β-citronellol, vannillyl alcohol or hydroquinone, and,

wherein said glycosylation is carried out in a cosolvent system or a biphasic system.

2. A method according to claim 1 wherein said cellobiose phosphorylase comprises an amino acid sequence having at least 60% sequence identity with SEQ ID N° 1.

3. A method according to claims 1-2 wherein said alcohol with a chain length of at least 8 carbon atoms is octanol, nonanol, decanol or dodecanol.

4. A method according to claims 1-3 wherein said cellobiose phosphorylase is a cellobiose phosphorylase comprising the amino acid sequence SEQ ID N° 1, SEQ ID N° 2 or SEQ ID N° 3, or, a variant of each of said sequences having at least 95% sequence identity with each of said sequences.

5. A method according to claims 1-4 wherein the solvents used in said cosolvent system are dimethylsulfoxide or the ionic liquid AMMOENG™ 101, or, wherein the organic solvent in said biphasic system is ethylacetate.

6. A method according to claims 1-5 wherein said cellobiose phosphorylase is immobilized.

7. A method according to claim 6 wherein said immobilized cellobiose phosphorylase is a cross-linked enzyme aggregate.

8. A method according to claim 7, wherein said cross-linked cellobiose phosphorylase aggregate is imprinted with the acceptor.

9. A method according to claim 8, wherein said acceptor is octanol.

10. A method according to claims 1-9 wherein said glucoside is further elongated by: -contacting in vitro a cellodextrin phosphorylase with alpha-glucose- 1 -phosphate as donor and glucoside obtainable by claims 1-8 as acceptor, and

-glycosylating said glucoside as acceptor.