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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.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
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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
• the present invention relates to the identification, recombinant expression and characterization of a sucrose and sucrose-6'-phosphate phosphorylase of the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum.
• the enzyme has an optimal temperature of about 55°C and has a high specificity for its substrates. More importantly, the sucrose and sucrose- 6'-phosphate phosphorylase of the present invention has a half-life of at least 60 hours at the industrially relevant temperature of about 60°C. This enzyme is thus -for example- useful for the industrial production of D-fructose, D-fructose-6-phosphate, alpha-D-glucose -1-phosphate or alpha-D-glucosides.
• 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 .
• sucrose-6'-phosphate phosphorylase has not been described before.
• thermostable enzymes are also desired for enzyme engineering. Compared to its mesophilic homologues, thermostable enzymes have a higher capacity to evolve because of a higher mutational robustness conferred by the extra stability 9 ' 10 .
• 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.
• mesophilic isolates can be stabilized by immobilization or by mutagenesis 13 .
• the SP enzyme from B. adolescentis for example, has been covalently attached to Sepabeads and prepared as cross-linked enzyme aggregate (CLEAS), resulting in a drastically improved thermostability 12 14 .
• increasing the stability by mutagenesis is a good alternative, of which two examples are available today. The first one is the SP from Streptococcus mutans, which has been stabilized by the introduction of 8 mutations 1S . Unfortunately, the resulting enzyme variant retains only 60% of its activity after 20 minutes incubation at 60 °C, which is not enough for industrial applications.
• the second example is a variant of the SP from B. adolescentis containing 6 mutations and whose half-life at the industrially relevant temperature of 60 °C has more than doubled compared to the wild-type enzyme 16 .
• 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. 1 Temperature optimum (left) and pH optimum (right) for T. thermosaccharolyticum SP. The activity was assayed using 350 mM substrate in the phosphorolysis reaction and 200 mM substrate in the synthetic reaction.
• Figure 2 The kinetic stability of the SP enzymes of Thermoanaerobacterium thermosaccharolyticum and Bifidobacterium adolescentis. The enzymes were incubated at 60°C for various times at a protein concentration of 8.5 ⁇ g/mL, after which their residual activity was compared with that of the untreated enzymes. All assays were performed in triplicate and had a coefficient of variation (CV) of ⁇ 10%.
• CV coefficient of variation
• FIG 3 Temperature of inactivation for 1 hour incubation for the SP enzymes of Thermoanaerobacterium thermosaccharolyticum (TtSP) and Bifidobacterium adolescentis (BaSP). Inactivation was carried out at a protein concentration of 35 ⁇ g/mL for both enzymes.
• Figure 4 Differential scanning fluorometry for Bifidobacterium adolescentis SP (BaSP), Thermoanaerobacterium. thermosaccharolyticum SP (TtSP). The spectrum was recorded at a protein concentration of 400 ⁇ g/mL.
• FIG. 5 Homology model of the Thermobacterium thermosaccharolyticum SP showing the main enzyme-substrate interactions
• thermosaccharolyticum DSM 571 three putative sucrose phosphorylases isolated from thermophiles and classified among the a-amylase subfamily 18 were recombinantly expressed in Escherichia coli.
• Thermoanaerobacterium thermosaccharolyticum DSM 571 catalyzes the phosphorolysis of sucrose with an optimal temperature of 55°C.
• This new enzyme was found to be the most stable SP enzyme so far, with a half-life time of 60 hours at the industrially relevant temperature of 60°C and a half temperature of inactivation ( ⁇ 50 ) of 69°C after 1 hour incubation.
• this new SP had a melting temperature of 78.5°C.
• the putative SPs from Meiothermus silvanus DSM 9946 and Spirochaeta thermophila DSM 6192 did not catalyze the phosphorolysis of sucrose.
• the present invention discloses that the above-described SP enzyme from Thermoanaerobacterium thermosaccharolyticum DSM 571 is much more active on fructose-6- phosphate as acceptor than on free fructose.
• the latter enzyme has thus a unique specificity that has never been described before and could be designated as a sucrose-6'-phosphate phosphorylase (S6'PP).
• the present invention relates in first instance to the use of a polypeptide having the amino acid sequence as depicted by SEQ ID N°l as a thermostable sucrose phosphorylase and/or as a thermostable sucrose-6'-phosphate phosphorylase.
• 'sucrose-6'-phosphate phosphorylase' refers to an enzyme which catalyzes the reversible phosphorolysis of sucrose-6'-phosphate into a-D-glucose-l-phosphate (a-Glc-l-P) and D-fructose-6- phosphate.
• 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.
• the present invention relates to the use as described above wherein said variant or fragment phosphorolyzes sucrose-6'-phosphate and comprises the amino acid histidine at amino acid position 344.
• the term 'fragment' refers to a protein (or peptide or polypeptide) containing fewer amino acids than the amino acid sequence as depicted by SEQ ID N° 1 and that retains said sucrose phosphorylase- or sucrose-6'-phosphate phosphorylase activity.
• 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 the use of a microorganism expressing a polypeptide having the amino acid sequence as depicted by SEQ ID N°l or SEQ ID N°4 to provide a thermostable sucrose phosphorylase or a thermostable sucrose-6'-phosphate phosphorylase.
• the latter microorganism can be any microorganism known in the art but specifically relates to the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum or to the bacterium Escherichia coli.
• 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.
• thermophiles Meiothermus silvanus DSM 9946 MsSP
• Spirochaeta thermophila DSM 6192 StSP
• Thermoanaerobacterium thermosaccharolyticum DSM 571 TtSP
• their genes were chemically synthesized with a codon usage that is optimal for the host strain E. coli.
• a N-terminal His-tag was also added, which allowed their purification in a single- step by means of affinity chromatography.
• the purified enzymes were assayed for phosphorolytic activity on different a-linked disaccharides existing of a glucose moiety connected to either a fructose or glucose moiety.
• T. saccharolyticum could catalyze the phosphorolysis of sucrose.
• the isolates from S. thermophila and M. silvanus did not phosphorolyze any of the selected dissacharides with a significant activity.
• Changing the reaction pH in the range of 4.5 to 8 or adding CaCI 2 or MgCI 2 as cofactors (0.1-10 mM) did not help to phosphorolyze sucrose.
• TtSP has a somewhat higher K m of 76.5 ⁇ 10.3 mM for sucrose than what is typically expected for a SP, i.e. between 1 and 15 mM for mesophilic isolates 6 .
• K m for a-Glc-l-P and inorganic phosphate is in the same range as previously reported for other SPs 31 .
• TtSP has a k cat of around 60 s "1 and this is in the same range as the most efficient SP enzymes like those from Leuconostoc mesenteroides ATTC 12291 and B. adolescentis 6 .
• thermosaccharolyticum SP Thermoanaerobacterium thermosaccharolyticum SP (TtSP). Michaelis Menten curves were obtained using 11 levels of varying substrate concentration together with 350 mM of co-substrate in the phophorolytic direction and 200 mM of co-substrate in the synthetic direction at 55°C and pH 6.5.
• 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.
• SP is a promiscuous enzyme that is able to transfer a glucose moiety from either sucrose or a-Glc-l-P to various alternative acceptors, al beit a reduced rate 6 .
• the acceptor specificity of TtSP was, therefore, tested with different monosaccharides, sugars alcohols and non- carbohydrate molecules.
• the monosaccharide /.-sorbose is the best alternative acceptor for TtSP and generates an activity that is almost half of that of the wild-type reaction.
• D-psicose and D-tagatose generate a 10-fold lower transglucosylation rate, while D-galactose and D-mannose are almost not accepted.
• the transglucosylation efficiency is also pretty high with glycerol (a sugar alcohol), whereas activity towards non-carbohydrate acceptors is low but in the same order as with other SP enzymes (2).
• the concentration of the produced glycosides is well within the industrial relevant range (35-85 g L "1 ). Although the yields with cinnamyl alcohol and geraniol are rather low due to the use of the pure acceptor as solvent phase, more than reasonable product concentrations of 41 and 61 g L "1 , respectively, were achieved. Table 4. Production of glycosides with TtSP
• thermostability of the SP of T. thermosaccharolyticum The influence of the temperature on the stability was determined for the TtSP enzyme and compared to currently the most stable SP enzyme, isolated from B. adolescentis (BaSP).
• the kinetic stability i.e. the length of time a protein remains active before undergoing irreversible denaturation 3 2 , was assessed using two measures. First, the half-life (t 50 ) at the industrially relevant temperature of 60°C was evaluated and was found to have increased almost 3-fold for TtSP (60 h) compared to BaSP (21h) ( Figure 2).
• thermodynamic stability i.e. the tendency of a protein to reversibly unfold 32
• T m melting temperature
• sucrose phosphorylase is a promising biocatalyst for the production of a- glucosides.
• thermostable respresentatives are desired.
• the SP from the thermophilic organism T. thermosaccharolyticum is described. Determination of the enzymatic properties revealed that this enzyme is the most stable SP yet reported, with a half-life of 60 hours at 60°C. Inspection of its acceptor promiscuity showed a similar pattern as for other SP enzymes 6 , reflecting the industrial potential of this new biocatalyst.

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• 2013
  • 2013-10-16 EP EP13776826.3A patent/EP2909318A1/de not_active Withdrawn
  • 2013-10-16 WO PCT/EP2013/071587 patent/WO2014060452A1/en active Application Filing

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