Technical field of invention

The present invention relates to the production of the disaccharide kojibiose which is known to be a powerful prebiotic. The invention indeed discloses the generation of genetically modified sucrose phosphorylases which convert -via a transglycosylation reaction- sucrose into kojibiose in a very efficient manner. Hence, the present invention relates to a cost-effective production method of kojibiose which is useful within industry.

Background art Kojibiose (2-O-a-D-glucopyranosyl-D-glucopyranose) is a promising prebiotic for the stimulation of beneficial bacterial populations in the human gut ¹ ' ² . The a-1,2 bond can indeed be handled by specific micro-organisms such as Bifidobacteria, but it is largely resistant to the action of enzymes in the digestive tract ³ ' ⁴ . Moreover, kojibiose and derivatives have the ability to inhibit a-glucosidase I in different tissues and organisms ^{5- 8} . Accordingly, these are appealing molecules because inhibitors that act upon a- glucosidase I are suggested to be interesting candidate drugs for the treatment of human immunodeficiency virus type 1 (HIV-1) infections ⁹ . Given their a-glucosidases inhibitory action, they can also limit the digestion of dietary carbohydrates and could therefore be useful to counter diabetes, obesity and cardiovascular diseases ¹⁰ ' . Studies on the health-promoting properties of kojibiose are however hampered by the limited availability of this disaccharide ¹ . Kojibiose is naturally present in honey ¹² , beer ¹³ , sweet potato starch hydro lysate ¹⁴ , sake and koji ¹⁵ (hence the name), but the amounts are too low for extraction on larger scale. According to some authors, the isolation from the partial acetolysis of dextran from Leuconostoc mesenteroides NRRL B-1299 is currently the best method ^{4, 16} ' ¹⁷ . Yet, it is a multi-step process involving several chemical reagents like glacial acetic acid, acetic anhydride, concentrated sulphuric acid, chloroform and methanol. Chemical synthesis via modified Koenigs-Knorr reactions is still applied as well, despite the need for multiple (de)protection steps, silver or mercury catalysts and solvents like acetonitrile, and low yields ^{18 20} . Alternatively, enzymes like a- glucosidase ²¹ ' ²² , a-glucoamylase ²² ' ²³ , glucansucrase ²⁴ , dextransucrase ²⁵ and kojibiose phosphorylase ⁴ can be used, but unfortunately these often have low yields and/or generate product mixtures. Sucrose phosphorylase (SP) is another enzyme that is capable of producing kojibiose ^{26, 27} , starting from sucrose (renewable, cheap and readily available ²⁸ ) and D-glucose. It naturally catalyses the reversible phosphorolysis of sucrose into a-D-glucose 1-phosphate and D-fructose, but it can also glycosylate alternative acceptors with yields exceeding 90% ²⁹ ' ³⁰ (WO 2011/124538). In case of D-glucose as acceptor, the overall kojibiose yield is however lowered by the side-formation of maltose (4-O-a-D-glucopyranosyl-D-glucopyranose) as glucobiose product ²⁷ . There is thus a need to produce a, preferably thermostable, sucrose phosphorylase having a high selectivity of kojibiose production.

Summary of the invention

The present invention relates to an isolated sucrose phosphorylase comprising an amino acid sequence given by SEQ. ID N° 1 characterized in that it contains at least one of the following mutations: P134V, P134R, P134W, P134S, R135E, A193G, H234T, L341I, L343P, Y344R, Y344D, Y344V, Y344I, Q345S, Q345N and has an activity ratio of kojibiose over maltose formation greater than 0.5 during a transglycosylation reaction with glucose, or, a fragment of said sucrose phosphorylase containing at least one of said mutations and having said selectivity of kojibiose.

The present invention further relates to an isolated sucrose phosphorylase as described above containing the mutations L341I_Q345S or L341I_Y344A_Q345N and wherein said selectivity for kojibiose is characterized by a K/M ratio of 15 and 22, respectively.

The present invention further relates to an isolated nucleic acid encoding for a sucrose phosphorylase as described above, and, further relates to a vector comprising a nucleic acid as described above. Moreover, the present invention relates to a host cell comprising a vector as described above. The present invention also relates to the usage of of a sucrose phosphorylase to produce kojibiose wherein said sucrose phosphorylase is an isolated sucrose phosphorylase comprising an amino acid sequence given by SEQ. ID N° 1 characterized in that it contains at least one of the following mutations: P134V, P134R, P134W, P134S, R135E, A193G, H234T, L341I, D342A, L343P, Y344A, Y344R, Y344D, Y344V, Y344I, Q345A, Q345S, Q345N and has an activity ratio of kojibiose over maltose formation greater than 0.5 during a transglycosylation reaction with glucose, or, a fragment of said sucrose phosphorylase containing at least one of said mutations and having said selectivity of kojibiose.

The present invention relates to a process to produce kojibiose comprising the steps of:

- providing sucrose and/or alpha-glucose 1-phosphate as donor, and, glucose as acceptor,

-providing a sucrose phosphorylase as described above,

-bringing said donor and acceptor, and, said sucrose phosphorylase in a medium wherein the synthesis of kojibiose by said sucrose phosphorylase takes place, and

-purifying said kojibiose from said medium.

The present invention further relates to a process as described above wherein said purification consist of a yeast treatment.

Description of the invention

The present invention relates to a systematic mutagenesis study of the active site of the sucrose phosphorylase of Bifidobacterium adolescentis resulting in the identification of only a few mutations that surprisingly can improve the activity ratio of kojibiose over maltose formation (K/M ratio) to values higher than 0.5. Combining mutations L341I_Y344A_Q345N into one sequence yields the highest selectivity, corresponding to a K/M ratio of 22. However, the double mutant L341I_Q.345S produces kojibiose two times faster than this triple mutant and has a K/M ratio of 15, which is still enough to generate highly pure kojibiose. A further and simple purification procedure consists of a yeast treatment to consume the remaining glucose, fructose and sucrose.

Hence, the present invention relates to an isolated sucrose phosphorylase comprising an amino acid sequence given by SEQ ID N° 1 characterized in that it contains at least one of the following mutations: P134V, P134R, P134W, P134S, R135E, A193G, H234T, L341I, D342A, L343P, Y344A, Y344R, Y344D, Y344V, Y344I, Q345A, Q345S and Q345N and having a K/M ratio of kojibiose over maltose formation greater than 0.5 during a transglycosylation reaction with glucose as acceptor, or, a fragment of said sucrose phosphorylase containing at least one said mutations and having said selectivity of kojibiose.

More specifically, the present invention relates to an isolated sucrose phosphorylase comprising an amino acid sequence given by SEQ. ID N° 1 characterized in that it contains at least one of the following mutations: P134V, P134R, P134W, P134S, R135E, A193G, H234T, L341I, L343P, Y344R, Y344D, Y344V, Y344I, Q345S, Q345N and has an activity ratio of kojibiose over maltose formation greater than 0.5 during a transglycosylation reaction with glucose, or, a fragment of said sucrose phosphorylase containing at least one of said mutations and having said selectivity of kojibiose.

The term 'sucrose phosphorylase comprising an amino acid sequence given by SEQ ID N° 1' refers to an amino acid sequence comprising an amino acid sequence encoded by a nucleic acid having GenBank accession Number AF543301 as described by Sprogoe et al. (2004). The latter nucleic acid corresponds to a sucrose phosphorylase gene from Bifidobacterium adolescentis. More specifically, the latter term refers to a sucrose phosphorylase encoded by the sucrose phosphorylase gene from Bifidobacterium adolescentis LMG 10502 as described by Reuter (1963) and which is synonymous to DSM20083 and ATTC15703. SEQ ID N° 1 corresponds to the following amino acid sequence (the amino acids of which at least one should be substituted according to the present invention are underlined): MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTK VDERLG SWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEE DLA GIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYI RLDAV GYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFA LPPLLLHAL STGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIH ANTHG ESQAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYVGALAGKND MELL RKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISF TWRGETS QATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVA

The terms "characterized in that it contains at least one of the following mutations: P134V, P134R, P134W, P134S, R135E, A193G, H234T, L341I, D342A, L343P, Y344A, Y344R, Y344D, Y344V, Y344I, Q345A, Q345S and Q345N" refers to the fact that specific amino acids at specific positions of wild type SEQ. ID N° 1 (i.e. at amino acid positions 134, 135, 193, 234, 341, 342, 343, 344 and/or 345) have been substituted by other specific amino acids. The latter substitutions can be obtained by any method known in the art. The latter method can be for example mutating, via for example performing site- directed mutagenesis, the sucrose phosphorylase gene from Bifidobacterium adolescentis. The term "at least one of the following mutations" indicates that the mutant sucrose phosphorylase of the present invention must contain one of the indicated list of single mutations but may also contain 2, 3, 4,...,16, 17 or all of the in total 18 single mutations, or, any combination of said list of single mutations which results in mutant sucrose phosphorylases containing multiple (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10...) mutations. Preferred -but non-limiting- examples of specific combinations of 2, 3, 4, 5, 6 and 7 of said list of mutations are given in Table 1. The terms "having an activity ratio of kojibiose over maltose formation greater than 0.5 during a transglycosylation reaction with glucose as acceptor" refers to the fact that sucrose phosphorylases are capable of producing kojibiose ^{26, 21} , starting from sucrose and D-glucose. It naturally catalyses the reversible phosphorolysis of sucrose into a-D- glucose 1-phosphate and D-fructose, but it can also glycosylate alternative acceptors with yields exceeding 90% ²⁹ ' ³⁰ (WO 2011/124538) . In case of D-glucose as acceptor, the overall kojibiose yield is however lowered by the side-formation of maltose (4-O-a-D- glucopyranosyl-D-glucopyranose) as glucobiose product ²⁷ . For example, the wild-type and thermostable SP from Bifidobacterium adolescentis (WO 2011/124538) displays a K/M ratio of only 0.5, meaning that the velocity of maltose formation is twice as high as the velocity of kojibiose formation. The mutants of the present invention as indicated above have a K/M ratio that is greater than 0.5 (i.e. 0.6, 0.7, 0.8, 0.9, 1.0,..., 2.0..., 3.0..., 4.0,...10.0,...20.0,...30.0...) meaning that the mutants show a velocity of formation of kojibiose that is increased relative to the velocity of maltose formation, when compared to the wild-type enzyme.

The terms "a fragment of said sucrose phosphorylase containing at least one said mutations and having said selectivity of kojibiose" 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 the activity of said mutant sucrose phosphorylases of the present invention (i.e. the velocity of kojibiose formation is increased relative to the velocity of maltose formation when compared to the wild-type enzyme). Such fragment must thus contain at least one of the indicated list of single mutations or a combination thereof as indicated above and 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. The present invention preferably relates to mutants of the present invention have a K/M ratio that is greater than 10.0 (i.e. 10.0,...15.0,...20.0...) meaning that the mutants show a velocity of formation of kojibiose and a velocity of maltose formation which is significantly higher and lower, respectively compared to corresponding velocities of the wild-type enzyme. Hence, and more specifically, the present invention relates to an isolated sucrose phosphorylase as described above containing the mutations L341I_Q345S or L341I_Y344A_Q345N and wherein said selectivity for kojibiose is characterized by a K/M ratio of 15 and 22, respectively. The present invention further relates to an isolated nucleic acid encoding for a sucrose phosphorylase as described above. An example of a nucleic acid encoding for mutant sucrose phosphorylase of the present invention (SEQ. ID N° 2) is as follows (the codons of which at least one should be mutated according to the present invention are underlined):

ATGAAAAATAAAGTCCAACTGATTACCTATGCGGATCGTCTGGGTGATGGCACCATT AAAAGC ATGACGGACATCCTGCGTACCCGCTTTGATGGCGTTTATGACGGTGTCCACATTCTGCCG TTTT TCACCCCGTTTGATGGCGCCGACGCAGGTTTCGATCCGATCGACCATACCAAAGTTGATG AAC GTCTGGGTAGCTGGGATGACGTCGCCGAACTGTCTAAAACCCACAACATTATGGTGGATG CA ATCGTTAATCACATGTCATGGGAATCGAAACAGTTCCAAGATGTTCTGGCCAAAGGCGAA GAA TCAGAATATTACCCGATGTTTCTGACCATGAGCAGCGTGTTCCCGAACGGTGCCACGGAA GAA GACCTGGCAGGCATTTATCGTCCGCGTCCGGGTCTGCCGTTTACCCACTACAAATTCGCA GGC AAAACGCGTCTGGTCTGGGTGTCTTTTACCCCGCAGCAAGTGGACATCGATACGGACAGT GAT AAAGGTTGGGAATATCTGATGTCCATTTTCGACCAAATGGCGGCCAGCCATGTGTCTTAT ATC CGCCTGGATGCGGTTGGCTACGGTGCCAAAGAAGCAGGCACCAGTTGCTTTATGACCCCG AA AACGTTCAAACTGATTTCCCGTCTGCGCGAAGAAGGCGTGAAACGCGGTCTGGAAATTCT GAT CGAAGTCCACAGCTATTACAAAAAACAGGTGGAAATCGCGTCTAAAGTGGATCGCGTTTA TGA CTTTGCCCTGCCGCCGCTGCTGCTGCATGCCCTGAGTACCGGCCACGTCGAACCGGTGGC CCA TTGGACGGATATTCGTCCGAACAATGCTGTTACCGTCCTGGATACGCATGACGGCATCGG CGT TATTGATATCGGTTCAGATCAGCTGGACCGCTCGCTGAAAGGTCTGGTGCCGGACGAAGA TG TGGATAACCTGGTGAATACGATTCATGCGAACACCCACGGCGAATCACAGGCAGCTACCG GT GCGGCGGCATCGAACATTGATCTGTACTCGGTTAATAGTACCTACTACTCCGCTCTGGGC TGT AACG ATCAG CACTAC ATCG CTG CG CGTG CG GTG CAGTTTTTCCTG CCG GGTGTTCCG C AAGTC TATTACGTGGGCGCCCTGGCAGGTAAAAATGATATGGAACTGCTGCGCAAAACCAACAAT GG TCGTGACATTAACCGCCATTATTACAGCACGGCCGAAATCGATGAAAATCTGAAACGTCC GGT GGTTAAAGCACTGAACGCTCTGGCGAAATTTCGCAATGAACTGGATGCTTTTGACGGCAC CTT CTCATATACCACGGATGACGATACGAGTATTTCCTTTACCTGGCGTGGTGAAACGTCGCA GGC CACCCTGACGTTCGAACCGAAACGCGGCCTGGGTGTTGATAATACCACGCCGGTCGCAAT GCT GGAATGGGAAGACAGTGCTGGTGACCATCGCTCCGACGACCTGATCGCTAATCCGCCGGT TG TTGCG The present invention also relates to a vector comprising a nucleic acid as described above.

The present invention further relates to a host cell comprising a vector as described above.

The term 'nucleic acid' as used herein corresponds to the sucrose phosphorylase gene from Bifidobacterium adolescentis, and preferably from Bifidobacterium adolescentis LMG 10502. Said nucleic acids can be incorporated in appropriate vectors such as plasmids and appropriate host cells such as Escherichia coli can be transfected with said vectors.

The present invention relates to the usage of a sucrose phosphorylase as described above to produce kojibiose.

More specifically, the present invention relates to the usage of a sucrose

phosphorylase to produce kojibiose wherein said sucrose phosphorylase is an isolated sucrose phosphorylase comprising an amino acid sequence given by SEQ. ID N° 1 characterized in that it contains at least one of the following mutations: P134V, P134R, P134W, P134S, R135E, A193G, H234T, L341I, D342A, L343P, Y344A, Y344R, Y344D, Y344V, Y344I, Q345A, Q345S, Q345N and has an activity ratio of kojibiose over maltose formation greater than 0.5 during a transglycosylation reaction with glucose, or, a fragment of said sucrose phosphorylase containing at least one of said mutations and having said selectivity of kojibiose.

More specifically, the present invention relates to a process to produce kojibiose comprising the steps of:

-providing sucrose and/or alpha-glucose 1-phosphate as donor, and, glucose as acceptor,

-providing a sucrose phosphorylase as described above, -bringing said donor and acceptor, and, said sucrose phosphorylase in a medium wherein the synthesis of kojibiose by said sucrose phosphorylase takes place, and

-purifying said kojibiose from said medium. The term providing sucrose and/or alpha-glucose 1-phosphate as donor means that sucrose or alpha-glucose 1-phosphate or a mixture of both may be used as donor in order to glucosylate the acceptor glucose via the mutant sucrose phosphorylases of the present invention.

The term providing a sucrose phosphorylase means providing a mutant sucrose phosphorylase of the present invention. A non-limiting example of the latter 'providing' encompasses: a) mutating the sucrose phosphorylase wt gene from Bifidobacterium adolescentis contained in a vector such as a plasmid, b) transforming a host cell such as E. coli with said mutated genes, c) checking the desired mutations by sequencing each construct, d) growing individual colonies, e) lysing said colonies and f) purifying the mutant sucrose phosphorylases of interest.

The term 'medium' refers to any suitable medium known in the art which allows -when the mutant enzyme of the present invention, acceptor and donor are added to said medium-the synthesis of kojibiose by a sucrose phosphorylase of the present invention. An example of such a medium is the 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

The term "purifying" relates to any purification method known in the art but preferably relates to a purification consisting of a yeast treatment such as a treatment with baker's yeast.

The present invention thus specifically relates to a process as described above wherein said purification consists of a yeast treatment. After said treatment, the yeast can be removed via centrifugation and the supernatant can be evaporated. The kojibiose solution can then be cooled down in order to obtain pure kojibiose crystals which can be washed with ethanol. The present invention will further be illustrated by the following non-limiting example. Example

Engineering of sucrose phosphorylase for the selective production of kojibiose METHODS AND MATERIALS

Mutagenesis was performed on the constitutive expression plasmid pCXP34h ³¹ containing the sucrose phosphorylase gene from Bifidobgcterium gdolescentis LMG 10502. Saturation libraries were generated using either the Quikchange™ ³² protocol or the protocol described by Sanchis et al. ³³ , while site-directed mutagenesis was performed with the latter. In all cases, the mutated DNA was transformed in E. coli CGSC 8974 (Coli Genetic Stock Center, New Haven, CT, USA). For each library, the constructs were subjected to nucleotide sequencing (AGOWA Sequence Service, Berlin, Germany) in order to confirm that the desired mutations were indeed introduced and to exclude the presence of undesirable mutations. Individual colonies were picked, grown and lysed as described previously ³⁴ , with the difference that the lysis buffer composition was altered (1 mg/ml lysozyme, 0.1 mM PMSF, 50 mM Na ₂ S0 ₄ , 4 mM MgS0 ₄ and 1 mM EDTA in 50 mM MOPS buffer pH 7.0). Screening was performed by incubating crude cell extract with substrate solution in low well microtiter plates at 37°C. Interesting mutants were then produced at Erienmeyer scale and purified by Ni-affinity chromatography ³⁵ for further characterization at a temperature of 58°C. All reactions were performed with a donor concentration of 100 mM (sucrose or a-D-glucose 1-phosphate) and acceptor concentration of 200 mM (glucose) and were analysed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) (Dionex ICS-3000, Thermo Scientific). The K/M ratio was defined as the ratio of initial velocity of kojibiose formation and the initial velocity of maltose formation. The production of kojibiose was performed by incubating 2 mg/mL of mutant L341I_Q345S with 0.5M sucrose and 0.5M glucose in 0.1L of MOPS buffer at pH 7 and 55 °C for 24h, after which the enzyme was inactivated by heating the mixture to 95 °C during 10 min. Next, the pH was adjusted to 5, and 30 g/L baker's yeast (Algist Bruggeman) was added. After 8 h incubation at 30 °C and 50 rpm on a rotary shaker, the yeast was removed by centrifugation (5 000 g, 4 °C, 20 min), and the obtained supernatant was evaporated at 50 °C to a brix of 48 (Atago hand refractometer) using a rotary evaporator (Buchi, Rotavapor R-134). Next, the kojibiose solution was slowly cooled to 22 °C over a period of 4 h, and subsequently stored during 16 h at 22 °C. The obtained crystals were washed with ethanol (3 times 10 mL), and dried during 24 h in vacuo. The purity of the obtained crystals was analysed by HPAEC-PAD (Dionex ICS-3000, Thermo Scientific) and Ion-Moderated Partition chromatography (aminex HPX-87H, Bio- Rad).

RESULTS First, several active site residues (Y132, P134, R135, Y196, V233, L341, D342, L343, Y344, Q.345) were one by one mutated to alanine to evaluate their influence on product specificity. To that end, the K/M ratio of kojibiose and maltose formation was measured for each of the single mutants and compared with that of the wild-type enzyme. In some cases the preference for maltose formation was enhanced (like for Y196A), but for other mutants the opposite was true (e.g. P134A, D342A, Y344A and Q345A). This shows that predicting changes in product specificity is not a trivial task. Interestingly, all enzymes were still active with both sucrose and glucose 1-phosphate as glycosyl donor, meaning that their donor selectivity was not fundamentally altered.

Next, several active site positions were submitted to site-saturation mutagenesis to identify the best single mutants for kojibiose production (Table 1). The best mutant was found to be L341I, which displays a K/M ratio of 3.9 (formation of kojibiose over maltose). To further improve the enzyme's specificity, the best single mutant L341I was combined with the next best mutations, i.e. P134V, Q.345S and Q.345N. Surprisingly, the combination with of Q.345N or Q.345S increased the K/M ratio to 15-18, whereas the combination with P134V decreased the K/M ratio. The specificity of the double mutant L341I_Q.345N was further optimised by introducing an alanine at position 344, resulting in a K/M ratio of 22. Combining more than 3 mutations was also attempted but no further improvements in K/M ratio could be observed (Table 1).

Table 1 Characterization of several mutants of sucrose phosphorylase kojibiose formation K/M enzyme

(U/mg) ratio wild-type 0.152 0.5

Y132A 0.039 0.4

R135A 0.138 0.6

V233A 0.145 0.6

L341A 0.056 0.4

D342A 0.050 0.9

L343A 0.130 0.4

Y344A 0.210 1.0

Q345A 0.112 1.0

Y132A 0.039 0.4

R135A 0.138 0.6

PI 34V 0.136 2.2

P134R 0.016 1.6

P134W 0.028 2.8

P134T 0.113 0.6

P134S 0.072 1.1

R135E 0.018 0.8

R135P 0.010 0.2

R135V 0.038 0.6

A193G 0.090 1.1 A193S 0.019 0.3

A193T 0.002 0.3

A193V 0.001 0.5

H234T 0.003 1.0

L341I 0.372 3.9

L343P 0.023 1.1

Y344F 0.123 0.6

Y344R 0.069 0.8

Y344D 0.051 0.7

Y344V 0.074 1.2

Y344I 0.038 1.6

Q345S 0.138 2.2

Q345N 0.010 2.6

L341I_Q345S 0.100 14.9

L341I_Q345N 0.048 18.3

P134V_L341I 0.241 1.8

P134V_Q345S 0.145 3.9

P134V_Q345N 0.051 6.0

P134V_L341I_Q345S 0.173 6.7

P134V_L341I_Q345N 0.093 12.4

L341I_Y344A_Q345N 0.058 22.3

L341I_Y344I_Q345N 0.052 15.2

P134V_L341_Y344I_Q345N 0.056 7.5

P134V_L341I_L343P_Y344I_ Q345N 0.011 7.2

P134V_L341I_L343P_Y344V _. _Q345N 0.018 8.6

P134V_R135E_L341I_Y344I_ Q345N 0.016 5.0

P134V_L341_D342A_Y344I_ Q345N 0.001 4.4

P134V_L341I_D342A_L343P _Y344I_Q345N 0.003 6.7

P134V_R135V_L341I_L343P _Y344I_Q345N 0.006 5.1 ^# activity measurements had a coefficient of variation (CV) of less than

10%

To demonstrate the practical usefulness of the obtained mutants, the production of kojibiose was performed at a larger scale (0.1 L) and with higher substrate concentrations (0.5M sucrose and 0.5M glucose), which is preferred by the industry. To that end, the double mutant L341I/Q.345S was employed as it displays a good balance between activity and product specificity. After incubating 2 mg/mL of enzyme with the substrates for 24 h, the reaction was terminated by heating to 95 °C and contaminating carbohydrates were removed by yeast treatment. Finally, the solution was concentrated and then slowly cooled to induce crystallization. In that way, 12.4 g of crystalline kojibiose with a purity exceeding 99% could be obtained. When the wild-type enzyme was incubated under similar conditions, only 2.7 g of kojibiose was produced and we were unable to crystallize this disaccharide from the product mixture.

References

1. Sanz, M.L., Gibson, G.R. & Rastall, R.A. (2005) Influence of disaccharide structure on prebiotic selectivity in vitro. Journal of Agricultural and Food Chemistry 53(13):5192-5199

2. Nakada, T., Nishimoto, T., Chaen, H. & Fukuda, S. in Oligosaccharides in Food and Agriculture, Vol. 849 104-117 (American Chemical Society, 2003).

3. Djouzi, Z., Andrieux, C, Pelenc, V. et al. (1995) Degradation and fermentation of alpha-gluco-oligosaccharides by bacterial strains from human colon - in vitro and in vivo studies in gnotobiotic rats. Journal of Applied Bacteriology 79(2):117-127

4. Chaen, H., Nishimoto, T., Nakada, T. et al. (2001) Enzymatic synthesis of kojioligosaccharides using kojibiose phosphorylase. Journal of Bioscience and Bioengineering 92(2):177-182

5. Ugalde, R.A., Staneloni, R.J. & Leloir, L.F. (1980) Mirosomal glucosidases of rat liver - partial purification and inhibition by disaccharides. European Journal of Biochemistry 113(1):97-103 Shailubhai, K., Pratta, M.A. & Vijay, I.K. (1987) Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland. Biochemical Journal 247(3):555-562

Szumilo, T., Kaushal, G.P. & Elbein, A.D. (1986) Purification and properties of glucosidase I from mung bean seedlings. Archives of Biochemistry and Biophysics 247(2):261-271

Bause, E., Erkens, R., Schweden, J. & Jaenicke, L. (1986) Purification and characterization of trimming glucosidase I from Saccharomyces cerevisiae. FEBS Letters 206(2):208-212

Borges de Melo, E., da Silveira Gomes, A. & Carvalho, I. (2006) a- and β- Glucosidase inhibitors: chemical structure and biological activity. Tetrahedron 62(44):10277-10302

Standi, E. & Schnell, 0. (2012) Alpha-glucosidase inhibitors 2012-cardiovascular considerations and trial evaluation. Diabetes and Vascular Disease Research 9(3):163-169

Phung, O.J., Scholle, J.M., Talwar, M. & Coleman, C.I. (2010) Effect of Noninsulin Antidiabetic Drugs Added to Metformin Therapy on Glycemic Control, Weight Gain, and Hypoglycemia in Type 2 Diabetes. Jama-Journal of the American Medical Association 303(14):1410-1418

Watanabe, T. & Aso, K. (1959) Isolation of kojibiose from honey. Nature 183(4677):1740-1740

Hough, J.S., Briggs, D.E., Stevens, R. & Young, T.W. in Malting and Brewing Science, Volume II Hopped Word and Beer 776-838 (Chapman and Hall Ltd., New York, USA; 1982).

Sato, A. & Aso, K. (1957) Kojibiose (2-O-alpha-D-glucopyranosyl-D-glucose) - isolation and structure - isolation for hydrol. Nature 180(4593):984-985

Matsuda, K. (1959) Studies on the disaccharides in koji extract and sake. V. Isolation and identification of kojibiose. Nippon Nogeikagaku Kaishi33) 719-723 Matsuda, K., Watanabe, H., Fujimoto, K. & Aso, K. (1961) Isolation of nigerose and kojibiose from dextrans. Nature 191(478):278 Duke, J., Little, N. & Goldstein, I.J. (1973) Preparation of cyrstalline a-kojibiose octaacetate from dextran B-1299-S: Its conversion into p-nitrophenyl and p- isothiocyanatophenyl β-kojibioside. Carbohydrate Research 27(1):193-198 Igarashi, K., Irisawa, J. & Honma, T. (1975) Syntheses of a-D-linked disaccharides. Carbohydrate Research 39(2):341-343

Wolfrom, M.L., Lineback, D.R. & Thompson, A. (1963) Isopropyl tetra-O-acetyl- alpha-D-glucopyranoside - a synthesis of kojibiose. Journal of Organic Chemistry 28(3):860

Helferich, B. & Zirner, J. (1962) Zur synthese von tetraacetyl-hexosen mit freiem 2-hydroxyl - synthese einiger disaccharide. Chemische Berichte-Recueil 95(11):2604

Takahash.M, Shimomur.T & Chiba, S. (1969) Biochemical studies on buckwheat alpha-glucosidase. 3. Transglucosylation action of enzyme and isolation of reaction products. Agricultural and Biological Chemistry 33(10):1399

Fujimoto, H., Nishida, H. & Ajisaka, K. (1988) Enzymatic syntheses of glucobioses by a condensation reaction with alpha-glucosidase, beta-glucosidase and glucoamylase. Agricultural and Biological Chemistry 52(6):1345-1351

Cantarella, L, Nikolov, Z.L. & Reilly, P.J. (1994) Dissaccharide production by glucoamylase in aqueous ether mixtures. Enzyme and Microbial Technology 16(5):383-387

Brison, Y., Pijning, T., Malbert, Y. et al. (2012) Functional and Structural Characterization of a-(l->2) Branching Sucrase Derived from DSR-E Glucansucrase. Journal of Biological Chemistry 287(ll):7915-7924

Diez-Municio, M., Montilla, A., Javier Moreno, F. & Herrero, M. (2014) A sustainable biotechnological process for the efficient synthesis of kojibiose. Green Chemistry 16(4):2219-2226

Kitao, S., Yoshida, S., Horiuchi, T., Sekine, H. & Kusakabe, I. (1994) Formation of Kojibiose and Nigerose by Sucrose Phosphorylase. Bioscience Biotechnology and Biochemistry 58(4):790-791 Goedl, C, Sawangwan, T., Brecker, L, Wildberger, P. & Nidetzky, B. (2010) Regioselective O-glucosylation by sucrose phosphorylase: a promising route for functional diversification of a range of 1,2-propanediols. Carbohydrate Research 345(12):1736-1740

Skov, L.K., Mirza, 0., Sprogoe, D. et al. (2006) Crystal structure of the Glu328Gln mutant of Neisseria polysaccharea amylosucrase in complex with sucrose and maltoheptaose. Biocatalysis and Biotransformation 24(l-2):99-105

Desmet, T. & Soetaert, W. (2011) Broadening the synthetic potential of disaccharide phosphorylases through enzyme engineering. Process Biochemistry 47(1):11-17

Desmet, T., Soetaert, W., Bojarova, P. et al. (2012) Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require Tailored Catalysts. Chemistry-a European Journal 18(35):10786-10801

Aerts, D., Verhaeghe, T., De Mey, M., Desmet, T. & Soetaert, W. (2011) A constitutive expression system for high-throughput screening. Engineering in Life Sciences 11(1):10-19

Hogrefe, H.H., Cline, J., Youngblood, G.L. & Allen, R.M. (2002) Creating randomized amino acid libraries with the QuikChange (R) Multi Site-Directed Mutagenesis Kit. BioTechniques 33(5):1158

Sanchis, J., Fernandez, L, Carballeira, J. et al. (2008) Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Applied Microbiology and Biotechnology 81(2):387-397

Verhaeghe, T., Diricks, M., Aerts, D. et al. (2013) Mapping the acceptor site of sucrose phosphorylase from Bifidobacterium adolescentis by alanine scanning. Journal of Molecular Catalysis B: Enzymatic, 96:81-88

Aerts, D., Verhaeghe, T.F., Roman, B.I. et al. (2011) Transglucosylation potential of six sucrose phosphorylases toward different classes of acceptors. Carbohydrate Research 346(13):1860-1867 Lee, J.H., Yoon, S.H., Nam, S.H. et al. (2006) Molecular cloning of a gene encoding the sucrose phosphorylase from Leuconostoc mesenteroides B-1149 and the expression in Escherichia coli. Enzyme and Microbial Technology 39(4):612-620 Verhaeghe, T., Aerts, D., Diricks, M. et al. (2014) The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity. Applied Microbiology and Biotechnology, 98(16): 7027-7037