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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
  • C12N9/1066—Sucrose phosphate synthase (2.4.1.14)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
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  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01007—Sucrose phosphorylase (2.4.1.7)
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  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01014—Sucrose-phosphate synthase (2.4.1.14)

Definitions

• thermostable sucrose and sucrose-6'-phosphate phosphorylase A thermostable sucrose and sucrose-6'-phosphate phosphorylase
• Sucrose phosphorylase (SP, E.C. 2.4.1.7) is classified in the a-amylase family (GH-13) and catalyzes the reversible phosphorolysis of sucrose into a-D-glucose-l-phosphate (a-Glc-l-P) and D-fructose 1_ 4 . Thanks to its broad acceptor specificity, SP can be employed for the transfer of a glucosyl moiety to a variety of monosaccharides, sugar alcohols and even phenolic compounds. Therefore, SP is a very interesting biocatalyst for the production of a-D-glucosides as industrial fine chemicals 5 .
• SP have so far only been isolated from mesophilic sources 5 ' 11 with the enzymes originating from Leuconostoc mesenteroides, Bifidobacterium adolescentis and Pelomonas saccharophyla the most extensively described in literature 5 ' 6 .
• the SP from B. adolescentis is the most thermostable isolate today with a half-life time of 12 hours at 60°C 12 . This limited thermostability of mesophilic isolates could hamper the commercial exploitation of SP.
• sucrose-6'-phosphate Although progress has been made in stabilizing industrially important biocatalysts, there is still a need to identify a sucrose phosphorylase that is more stable than the best enzymes available today or to identify other, specific substrates of said phosphorylases such as sucrose-6'-phosphate.
• FIG. 5 Homology model of the Thermobacterium thermosaccharolyticum SP showing the main enzyme-substrate interactions
• the present invention further relates to the use as described above wherein said amino acid sequence is encoded by the nucleic acid sequence as depicted by SEQ ID N°2 or SEQ ID N°3.
• nucleic acid sequence as depicted by SEQ I D N° 2 is the following Thermoanaerobacterium thermosaccharolyticum sucrose phosphorylase or sucrose-6'-phosphate phosphorylase encoding nucleic acid sequence:
• the nucleic acid sequence as depicted by SEQ I D N° 3 is the following Thermoanaerobacterium thermosaccharolyticum sucrose phosphorylase or sucrose-6'-phosphate phosphorylase encoding nucleic acid sequence which, compared to SEQ ID N°2, has been codon optimized for expression in E.
• the invention further relates to the use as described above wherein said amino acid sequence is depicted by SEQ ID N°4.
• the amino acid sequence as depicted by SEQ I D N° 4 is the following Thermoanaerobacterium thermosaccharolyticum sucrose phosphorylase or sucrose-6'-phosphate phosphorylase amino acid sequence which, compared to SEQ ID N° 1, comprises a N-terminal His 6 -tag (underlined): MGGSHHHHHHGMASMALKNKVQLITYPDSLGGNLKTLNDVLEKYFSDVFGGVHILPPFPSSGD GFAPITYSEIE PKFGTWYDIKKMAENFDILLDLMVNHVSRRSIYFQDFLKKGRKSEYADMFITLDKLWKDGKPVKGDIEKMFLRRT LPYSTFKIEETGEEEKVWTTFGKTDPSEQIDLDVNSHLVREFLLEVFKTFSNFGVKIVRLDAVGYVIKKIGTSCFFVEP EIYEFLDWAKGQAASYGIELLLEVHSQFEVQYKLAERGFLIYDFILPFTVLYTLINK
• the term 'thermostability' thus means that the sucrose phosphorylase or sucrose-6'-phosphate phosphorylase of the present invention has, during a phosphorolysis or synthesis reaction as described elsewhere in the present disclosure, at a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or, 7.5, and, at a temperature of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 °C, a half-life of 20, 21, 22, 25, 30, 35, 40,..., 45,..., 50, 55,..., 60, 61, 62, 63,..., 70,...75,...80, 81, 82,
• thermostability means that said sucrose phosphorylase or sucrose-6'-phosphate phosphorylase has, during a phosphorolysis or synthesis reaction at an enzyme concentration of about 8.5 ⁇ g/ml, at a pH between 6.0 and 6.5 and at a temperature of about 60°C, a half-life of at least 60 hours.
• thermostability thus specifically means that that the sucrose phosphorylase or sucrose-6'-phosphate phosphorylase of the present invention has, during a phosphorolysis or synthesis reaction as described elsewhere in the present disclosure, and at an enzyme concentration of 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0 ⁇ g/ml, at a pH of 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5 and at a temperature of 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 °C, a half-life of 60, 61, 62, 63, 64, 65,..., 70,...75,...80, 81, 82, 83, 84, 85, or more hours.
• the 'thermostability' of the enzyme of the present invention can be determined by any method known in the art. More specific enzyme assays, such as enzyme activity assays, determinations of the temperature optimum of the enzymes, the influence of pH on enzyme activity, determination of the kinetic temperature stability and the determination of the thermodynamic stability of the enzyme of the present invention are described elsewhere in the present disclosure.
• the present invention further relates to the use as described above wherein said polypeptide has an amino acid sequence which is at least 90% identical to the amino acid sequence as depicted by SEQ ID N°l or wherein said polypeptide is a fragment of the amino acid sequence as depicted by SEQ ID N°l. More specifically, the present invention relates to the use as described above wherein said variant or fragment comprises the amino acid regions 45-56, 131-207, 236-310 and 340-358 of SEQ ID N°l.
• Such fragment can -for example- be a protein with a deletion of 10% or less of the total number of amino acids at the C- and/or N-terminus. More specifically, said fragment comprises the amino acid regions 45-56, 131- 207, 236-310 and 340-358 of SEQ ID N°l or comprises the amino acid histidine at amino acid position 344 of SEQ ID N° 1.
• the term "variant" refers to a protein having at least 90 % sequence identity (i.e. having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity) with SEQ ID N° 1 and that retains said sucrose phosphorylase- or sucrose-6'-phosphate phosphorylase activity. More specifically, said variant comprises the amino acid regions 45-56, 131-207, 236-310 and 340-358 of SEQ ID N°l or comprises the amino acid histidine at amino acid position 344 of SEQ ID N° 1.
• 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.
• the latter 'variant' may differ from the protein as depicted by SEQ ID N° 1 only in conservative substitutions and/or modifications, such that the ability of the protein to have sucrose phosphorylase- or sucrose-6'-phosphate 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.
• 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 sucrose phosphorylase- or sucrose-6'-phosphate phosphorylase activity as defined above, secondary structure and hydropathic nature of the enzyme.
• variants also refers to any glycosylated protein or fragments thereof as described above.
• the present invention further relates to a method to produce D-fructose, D-fructose-6-phosphate and/or alpha-D-glucose -1-phosphate at a temperature above 50°C (i.e. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C) comprising: i) contacting a polypeptide as defined above with sucrose or sucrose-6'-phosphate and inorganic phosphate, ii) phosphorolyse sucrose or sucrose-6'-phosphate to obtain D-fructose, D-fructose-6-phosphate and/or alpha-D- glucose -1-phosphate, and iii) purifying said D-fructose, D-fructose-6-phosphate and/or alpha-D- glucose -1-phosphate.
• the present invention also relates to a method to produce an alpha-D-glucoside at a temperature above 50°C (i.e. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C)comprising: i) contacting a polypeptide as defined above with either alpha-D-glucose -1- phosphate, sucrose or sucrose-6'-phosphate as donor and an appropriate acceptor such as a monosaccharide, a phosphorylated monosaccharide, an aliphatic or aromatic alcohol, a furanone, a flavanoid or a phenolic compound, ii) glycosylating said monosaccharide, phosphorylated monosaccharide, aliphatic or aromatic alcohol, furanone, flavanoi
• the present invention also relates to an isolated amino acid sequence as depicted by SEQ ID N°4 and an isolated nucleic acid sequence as depicted by SEQ ID N°3.
• the term 'nucleic acid' as used herein corresponds for example to DNA, cDNA, NA, sense and anti- sense nucleic acids and the like.
• Said nucleic acids can be incorporated in appropriate vectors such as plasmids and appropriate host cells such as Escherichia can be transfected with said vectors.
• the present invention further relates to a method to produce an alpha-D-glucoside comprising: i) contacting a polypeptide as defined above with either alpha-D-glucose -1-phosphate, sucrose or sucrose-6'-phosphate as donor and an appropriate acceptor, ii) glycosylating said acceptor to obtain an alpha-D-glucoside, and iii) purifying said alpha-D-glucoside, wherein said glycosylation is carried out in a two-phase system with an organic solvent phase containing the acceptor and an aqueous buffer phase containing said polypeptide as defined above and said donor.
• the term 'a two-phase system' relates to any two-phase system described in the art such as for example described in the work of Vulfson 17 and Carrea 18 .
• the term 'organic phase' relates to a phase wherein for example ethyl acetate, n-butyl acetate, methyl ieri-butyl ether, diethyl ether, pentane, hexane or octane is present as solvent and further contains the acceptor molecule (for example at a concentration of 1-500 g/L).
• aqueous buffer phase' contains the enzyme of the present invention (for example at a concentration of 0.1-500 U mL "1 ) and donor substrate (for example at a concentration of 0.05-3 M).
• the latter method can take place at temperature between 30 and 70 °C, at a pH between 6 and 9, and the ratio between the aqueous and organic (solvent) phase can be between 0.01 and 100.
• the present invention specifically relates to a method as described above wherein said acceptor is a non-carbohydrate acceptor (i.e. any appropriate acceptor which is not a carbohydrate), and more specifically to a method as describe above wherein said non-carbohydrate acceptor is cinnamyl alcohol, geraniol, propyl gallate, ethyl gallate, resorcinol, pyrogallol, saligenin or methyl gallate.
• a non-carbohydrate acceptor i.e. any appropriate acceptor which is not a carbohydrate
• the present finally relates to the use as described above wherein said amino acid sequence is encoded by the nucleic acid sequence as depicted by SEQ ID N°2 or SEQ ID N°3.
• the present invention will further be illustrated by the following non-limiting examples.
• the synthetic genes were cloned into the constitutive expression vector pCXP34h 11 using the respective restriction endonucleases.
• the resulting expression plasmids were transformed in E. coli CGSC 8974.
• for the SP from B for the SP from B.
• the expression plasmid constructed in 11 was used.
• 2 % of an overnight culture was inoculated in 500 mL LB medium containing 100 ⁇ g/mL ampicillin in a 2 L shake flask and incubated at 37 °C with continuous shaking at 200 rpm for 6 hours.
• the produced biomass was harvested by centrifugation for 15 minutes at 12000 x g and 4 °C, washed with 50 mL PBS buffer (300 mM NaCI and 50 mM NaH 2 P0 4 at pH 8) and the obtained cell pellets were stored at -20 °C.
• the cell pellets were then thawed and dissolved in 20 mL lysis buffer (300 mM NaCI, 10 mM imidazole , 0.1 mM PMSF and 50 mM NaH 2 P0 4 at pH 8) supplemented with lysozyme and DNasel in a final concentration of 1 mg/mL and 6 mU/mL, respectively.
• This cell suspension was incubated on ice for 30 minutes and sonicated 3 times for 2.5 minutes (Branson sonifier 250, level 3, 50 % duty cycle).
• the His 6 -tagged proteins were purified by Ni-NTA chromatography as described by the supplier (Qiagen), after which the buffer was exchanged to 50 mM MOPS pH 7 in a Centricon YM-30 (Millipore). The protein content was analyzed measuring the absorbance at 280 nm. The extinction coefficients for the His 6 -tagged proteins were calculated using the Protparam tool on the expasy server (URL: http://web.expasy.org/protparam/).
• Enzyme assays and enzyme characterization The enzyme activity has been measured in both directions of the equilibrium reaction using a discontinuous assay.
• Initial reaction rates for the phosphorolysis of sucrose were measured by quantifying the release of fructose using the bicinchoninic acid (BCA) assay and the release of inorganic phosphate from a-glucose-l-phosphate was monitored in the synthetic direction using the phosphomolybdate assay like described before 6 .
• First the temperature optimum was determined in the phosphorolytic direction using 350 mM sucrose and 350 mM sodium phosphate buffer at pH 6.5 in a range from 40 to 70 °C. Reactions were monitored for 15 min in a heating block with sampling at regular intervals. Inactivation of the samples occurred by the alkaline environment of the assay solution.
• the influence of pH on enzyme activity was measured in the range of pH 4.5 to 8 using acetate (pH 4.5), MES (pH 5.0 - 6.5) and MOPS (pH 7 - 8)) buffers with a concentration of 50 mM.
• the pH of the substrate solutions was set at the temperature of measurement with NaOH or HCI.
• the apparent kinetic parameters for sucrose as well as a-Glc-l-P as donor and for inorganic phosphate and fructose as acceptor were determined at optimal pH and temperature. The parameters were calculated by non-linear regression of the Michaelis-Menten equation using Sigma Plot 11.0.
• the kinetic temperature stability was examined by incubating purified enzyme (35 ⁇ g/mL) for 1 h in a gradient thermocycler (Biometra, Goettingen, Germany) set to a temperature range of 57 - 73°C, followed by 15 min cooling to 16°C.
• the residual activity of the enzyme was determined in the phophorolytic direction using the standard conditions described above.
• the half life time (tso) was evaluated by incubating purified enzyme (8.5 ⁇ g/mL) in a water bath at 60 °C with sampling at regular time intervals. The residual activity was then measured and compared to the activity of the untreated enzyme.
• thermodynamic stability was measured using differential scanning fluorimetry (DSF) 21 in a Rotor-Gene Q cycler with HRM channel (Qiagen).
• DSF differential scanning fluorimetry
• 10 ⁇ g purified protein was used with 1.25 ⁇ SYPRO Orange (400x diluted) (Sigma-Aldrich) in 25 ⁇ .
• the gain was optimized before the temperature increase was started. The temperature increases from 35°C to 95°C, rising 1°C each step in 5 s steps.
• the fluorescent signal is detected at 510 nm with the green detection filter and the excitation occurs at 460 nm with a HRM lamp.
• the melting temperature (T m ) is determined by calculating the maximum of the first derivative of the melt curve using the Rotor-Gene Q software (Qiagen).
• Enzymatic glucosylation of non-carbohydrate acceptors The glucosylation of various non-carbohydrate acceptors was carried out at 100 mL scale in magnetically stirred reaction vessels. To that end, a two-phase system was used with either ethyl acetate, n-butyl acetate, methyl ieri-butyl ether, diethyl ether, pentane, hexane or octane as organic phase containing the acceptor molecule (1-500 g L 1 ), and an aqueous buffer system containing the enzyme (0.1-500 U mL "1 ) and donor substrate (0.05-3 M).
• the temperature was varied between 30 and 70 °C, the pH between 6 and 9, and the ratio between the aqueous and solvent phase between 0.01 and 100.
• samples were taken and subjected to HPLC analysis. Separation was achieved using a Waters X-bridge amide column (250 x 4.6 mm, 3.5 ⁇ ) with milliQ water (solvent A) and acetonitrile (solvent B), both containing 0.2 % triethylamine, as the mobile phase.
• the flow rate and temperature were set at 1.0 mL min "1 and 30 °C, respectively.
• the gradient elution was as follows: 95 % of solvent A (0 - 12 min), 5 to 25 % solvent B (12 - 15 min), 25 % solvent B (15 - 40 min), 25 to 5 % solvent B (40 - 41 min) and 95 % solvent A (41 - 50 min). Adequate detection was obtained with an Alltech 2000ES evaporative light scattering detector (ELSD).
• ELSD evaporative light scattering detector
• thermostable sequences To obtain a better understanding in the genetic diversity of SP, a phylogenetic tree was constructed from all the putative SP genes classified in the a-amylase family subfamily 18 (GH13-18). So far, there is only one specificity demonstrated for this subfamily, i.e. the phosphorolysis of sucrose 4 .
• a-amylase family subfamily 18 GH13-18. So far, there is only one specificity demonstrated for this subfamily, i.e. the phosphorolysis of sucrose 4 .
• Thermaceae From the 115 different species harboring a (putative) SP gene, 8 of them grow with optimal temperatures between 55 and 67.5 °C (Table 1). From the family Thermaceae, putative SP sequences from 4 different species are available and they are 555 to 588 AA in length and phylogenetically related. Besides members from the Thermaceae family, two sequences originating from Thermoanaerobacterium are classified in GH13-18 (Table 1) and they are for 91 % identical. The sequences are 488 AA long and they are most related to SPs from lactic acid bacteria which are extensively described in literature. Furthermore a putative SP can be found in Spicrochaeta thermophila and Geobacillus thermodenitrificans.
• fructose-6-phosphate was found to generate an activity that is almost twice as high as that on fructose (Table 3). Furthermore, detailed kinetic analysis revealed that the affinity for the phosphorylated acceptor is also much higher, corresponding to a Km of 15 mM instead of 42 mM.
• the enzyme also prefers sucrose-6'-phosphate over sucrose as substrate in the phosphorolysis reaction. It represents a unique specificity that has never before been described and can be designated as sucrose-6'-phosphate phosphorylase. It is important to note that the phosphate group can only be present on the fructose moiety and not on the glucose moiety (Fig. 6). Indeed, absolutely no activity could be detected with fructose as acceptor and glucose-l,6-bisphosphate as donor.

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