NOVEL FLAVONOIDS O-A-GLUCOSYLATED ON THE B CYCLE, METHOD FOR THE PRODUCTION THEREOF AND USES
20170107242 ยท 2017-04-20
Assignee
- Institut National De La Recherche Agronomique (Paris, FR)
- INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE TOULOUSE (Toulouse, FR)
- Centre National de la Recherche Scientifique (Paris Cedex 16, FR)
Inventors
- Sandrine Morel (Auzeville-Tolosane, FR)
- Isabelle Andre (Toulouse, FR)
- Yoan Brison (Toulouse, FR)
- Emmanuelle Cambon (Montpellier, FR)
- Yannick Malbert (Saint-Alban, FR)
- Denis Pompon (Pechabou, FR)
- Magali Remaud-Simeon (Ramonville, FR)
- Philippe Urban (Ramonville-saint-Agne, FR)
Cpc classification
C12P19/60
CHEMISTRY; METALLURGY
A61P1/04
HUMAN NECESSITIES
A61K31/7048
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
C07H1/00
CHEMISTRY; METALLURGY
C07H15/203
CHEMISTRY; METALLURGY
C12P19/46
CHEMISTRY; METALLURGY
C07D311/30
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
A61P9/14
HUMAN NECESSITIES
International classification
C07H15/203
CHEMISTRY; METALLURGY
A61K31/7048
HUMAN NECESSITIES
Abstract
The invention relates to a method for producing derivatives of O--glucosylated flavonoid, comprising at least one step of incubating a glucansucrase with a flavonoid and at least one sucrose, the flavonoid being a flavonoid which is monohydroxylated or hydroxylated in a non-vicinal manner on the B cycle. The invention also relates to novel O--glucosylated flavonoid derivatives, and to the use thereof.
##STR00001##
Claims
1. A process for producing O--glucosylated flavonoid derivatives, comprising at least one step of incubating a glucansucrose with a flavonoid and at least one sucrose, in which: (A) said flavonoid is of formula (I) below: ##STR00010## in which the C ring represents a ring chosen from the group consisting of the rings of formula (II), (III), (IV) or (V) below: ##STR00011## in which: one of the R.sub.1, R.sub.2 or R.sub.3 groups represents a B ring of formula (VI) below: ##STR00012## in which: (a) just one of the groups chosen from R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12 represents a hydroxyl group, the other groups among R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); or (b) R.sub.8 and just one of the groups chosen from R.sub.10, R.sub.11 and R.sub.12 represent a hydroxyl group, R.sub.9 and the other groups among R.sub.10, R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); or (c) R.sub.9 and just one of the groups chosen from R.sub.11 and R.sub.12 represent a hydroxyl group, the R.sub.8 and R.sub.10 groups, and the other group among R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.r C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); or (d) R.sub.10 and R.sub.12 represent a hydroxyl group, the R.sub.8, R.sub.9 and R.sub.11 groups, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.19 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); or (e) R.sub.8, R.sub.10 and R.sub.12 represent a hydroxyl group, the R.sub.9 and R.sub.11 groups, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); the R.sub.1, R.sub.2 and R.sub.3 groups which do not represent a B ring of formula (VI), which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched C.sub.1-C.sub.6 alkyl; an OH group; a C.sub.1-C.sub.3 amine; a COOH group; C(O)O(C.sub.2-C.sub.3); a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); R.sub.1, R.sub.2 and R.sub.3, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.2-C.sub.3 amine; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); or the R.sub.1 and R.sub.1 groups when R.sub.1 does not represent a B ring of formula (VI), or R.sub.2 and R.sub.2 groups when R.sub.2 does not represent a B ring of formula (VI), or R.sub.3 and R.sub.3 groups when R.sub.3 does not represent a B ring of formula (VI), together form an O group; R.sub.4, R.sub.5, R.sub.6 and R.sub.7, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; an OH; COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); and (B) said glucansucrose being chosen from the group comprising: a sequence having at least 80% identity with the sequence SEQ ID NO:1, said sequence having an amino acid X.sub.1 representing an amino acid chosen from the group consisting of A, C, E, F, G, H, I, K, M, N, P, Q, S, T, V and Y; a sequence having at least 80% identity with the sequence SEQ ID NO:2, said sequence having an amino acid X.sub.2 representing an amino acid chosen from the group consisting of A, C, D, F, G, H, K, L, M, N, P, S, V and Y; a sequence having at least 80% identity with the sequence SEQ ID NO:3, said sequence having an amino acid X.sub.3 representing an amino acid chosen from the group consisting of A, C, G, I, K, M, N and W; a sequence having at least 80% identity with the sequence SEQ ID NO:4, said sequence having an amino acid X.sub.4 representing an amino acid chosen from the group consisting of C, I, N, P, V and W; a sequence having at least 80% identity with the sequence SEQ ID NO:5, said sequence having an amino acid X.sub.5 representing an amino acid chosen from the group consisting of A, C, D, G, I, K, L, M, R, V and W; a sequence having at least 80% identity with the sequence SEQ ID NO:6, said sequence having an amino acid X.sub.6 representing an amino acid chosen from the group consisting of C, G, Q, S and T; a sequence having at least 80% identity with the sequence SEQ ID NO:7, said sequence having an amino acid X.sub.7 representing an amino acid chosen from the group consisting of A and G; a sequence having at least 80% identity with SEQ ID NO:8; a sequence having at least 80% identity with SEQ ID NO:9, said sequence having an amino acid X.sub.9 representing an amino acid chosen from the group consisting of C, I and L; a sequence having at least 80% identity with SEQ ID NO:10; a sequence having at least 80% identity with SEQ ID NO:11; and a sequence having at least 80% identity with SEQ ID NO:12, said sequence having amino acids X.sub.9, X.sub.10, X.sub.11, X.sub.12 and X.sub.13, with: (i) X.sub.9 representing, independently of X.sub.10, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of G, S, V, C, F, N, I, L and W; X.sub.10 representing, independently of X.sub.9, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of L, I, H, Y and F; with the exception of the case where X.sub.9 represents W and X.sub.10 represents F; X.sub.11 representing A; X.sub.12 representing F; and X.sub.13 representing L; (ii) X.sub.9 representing W; X.sub.10 representing F; X.sub.11 representing, independently of X.sub.9, X.sub.10, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of E and A; X.sub.12 representing, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.13, an amino acid chosen from the group consisting of L and F; and X.sub.13 representing L; with the exception of the case where X.sub.11 represents A and X.sub.12 represents F; or (iii) X.sub.9 representing W; X.sub.10 representing F; X.sub.11 representing A; X.sub.12 representing, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.13, an amino acid chosen from the list consisting of A, R, D, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y and V; and X.sub.13 representing, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.12, an amino acid chosen from the list consisting of A, R, D, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y and V.
2. The process as claimed in claim 1, wherein just one of the groups chosen from R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12, preferably R.sub.10, represents a hydroxyl group, the other groups among R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; and a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s); the R.sub.8, R.sub.9, R.sub.11 and R.sub.12 groups preferably representing hydrogen atoms.
3. The process as claimed in claim 2, wherein R.sub.10 represents a hydroxyl group and R.sub.8, R.sub.9, R.sub.11 and R.sub.12 represent hydrogen atoms.
4. The process as claimed in claim 1, wherein: R.sub.8 and just one of the groups chosen from R.sub.10, R.sub.11 and R.sub.12, preferably R.sub.10, represent a hydroxyl group, R.sub.9 and the other groups among R.sub.10, R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s).
5. The process as claimed in claim 4, wherein R.sub.10 represents a hydroxyl group and R.sub.9, R.sub.11 and R.sub.12 represent hydrogen atoms.
6. The process as claimed in claim 1, wherein the C ring represents a ring of formula (II) or (IV) as defined in claim 1.
7. The process as claimed in claim 1, wherein R.sub.1 represents a B ring of formula (VI) as defined in claim 1.
8. The process as claimed in claim 1, wherein R.sub.1 and R.sub.2 represent hydrogen atoms, R.sub.2 represents a hydrogen atom or an OH group, and R.sub.3 and R.sub.3 together form an O group.
9. The process as claimed in claim 1, wherein two of the R.sub.4, R.sub.5, R.sub.6 and R.sub.7, groups represent a hydroxyl group, the other two groups being as defined in claim 1.
10. The process as claimed in claim 1, wherein R.sub.5 and R.sub.7 represent hydrogen atoms.
11. The process as claimed in claim 1, wherein said flavonoid is of formula (VII), (VIII) or (IX) below: ##STR00013##
12. The process as claimed in claim 1, wherein the glucansucrose is chosen from the group comprising: a sequence having at least 80% identity with the sequence SEQ ID NO:1, said sequence having an amino acid X.sub.1 representing an amino acid chosen from the group consisting of H, N or S; a sequence having at least 80% identity with the sequence SEQ ID NO:2, said sequence having an amino acid X.sub.2 representing an amino acid chosen from the group consisting of A, C, F, L, M, S or V; a sequence having at least 80% identity with the sequence SEQ ID NO:3, said sequence having an amino acid X.sub.3 representing an amino acid chosen from the group consisting of A and N; a sequence having at least 80% identity with the sequence SEQ ID NO:4, said sequence having an amino acid X.sub.4 representing an amino acid chosen from the group consisting of C, I, N, P, V or W; a sequence having at least 80% identity with the sequence SEQ ID NO:5, said sequence having an amino acid X.sub.5 representing an amino acid chosen from the group consisting of C, K, R or V; a sequence having at least 80% identity with the sequence SEQ ID NO:9, said sequence having an amino acid X.sub.8 representing an amino acid chosen from the group consisting of C or L; and a sequence having at least 80% identity with the sequence SEQ ID NO:12, said sequence having amino acids X.sub.9, X.sub.10, X.sub.11, X.sub.12 and X.sub.13, with: (i) X.sub.9 representing an amino acid chosen from the group consisting of G, V, C and F; X.sub.10 representing F; X.sub.11 representing A; X.sub.12 representing F; and X.sub.13 representing L; (ii) X.sub.9 representing, independently of X.sub.10, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of S, N, L and I; X.sub.10 representing, independently of X.sub.9, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of L, I, H and Y; X.sub.11 representing A; X.sub.12 representing F; and X.sub.13 representing L; (iii) X.sub.9 representing W; X.sub.10 representing F; X.sub.11 representing A or E; X.sub.12 representing L; and X.sub.13 representing L; or said sequence having at least 80% identity with SEQ ID NO:12 is the sequence SEQ ID NO:13.
13. An O--glycosylated flavonoid derivative obtained by means of the process as defined in as defined in claim 1, wherein: the C ring represents the ring of formula (IV) in which the R.sub.1 group represents a B ring of formula (VI); and at least the B ring is O--glycosylated.
14. A compound of formula (X) below: ##STR00014## in which X.sub.14 represents a chain consisting of at least two -glucoside groups, and X.sub.15 and X.sub.16, which may be identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C.sub.1-C.sub.6 alkyl; a C(O)O(C.sub.2-C.sub.3) group; and a chain consisting of from 1 to 600 000 -glucoside groups.
15. A compound of formula (XI) below: ##STR00015## in which X.sub.17 represents a chain consisting of from 1 to 600 000 -glucoside groups, and X.sub.18 and X.sub.19, which may be identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C.sub.1-C.sub.6 alkyl; a C(O)O(C.sub.2-C.sub.3) group; and a chain consisting of from 1 to 600 000 -glucoside groups.
16. A process for inhibiting oxidation, comprising the use of at least one O--glycosylated flavonoid derivative as defined in either of claims 14 and 15.
17. An O--glycosylated flavonoid derivative as defined in either of claims 14 and 15, for the pharmaceutical use thereof in the treatment and/or prevention of hepatotoxicity, allergies, inflammation, ulcers, tumors, menopausal disorders, or neurodegenerative diseases.
18. The O--glycosylated flavonoid derivative as defined in either of claims 14 and 15, for the pharmaceutical use thereof as a veinotonic.
19. A process for converting light into electricity repelling insects, bleaching, destroying pests, killing fungi or fungal spores and/or killing bacteria comprising the use of an O--glycosylated flavonoid derivative as defined in either of claims 14 and 15.
Description
DESCRIPTION OF THE FIGURES
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[0175] Structure of the m/z ion at 353.0667, signature of a glucosylation of each of the two positions 5 and 7 of the A ring of apigenin.
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[0177] Fragmentation of the diglucosylated form on position 4 of the B ring of apigenin resulting in the m/z ion at 269.0451.
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DETAILED DESCRIPTION OF THE INVENTION
[0191] In order to make available novel O--glucosylated flavonoids, the applicant has developed a novel process for the synthesis of novel structures of -glucoflavonoids specifically glycosylated on non-vicinal hydroxyls, in particular of the B ring. This process uses mutated specific glucansucrases, identified by the applicant, capable of performing such a glucosylation.
[0192] These specific enzymes require for this only the presence of sucrose, a renewable and inexpensive agricultural resource. In this respect, a process according to the invention is advantageously inexpensive.
[0193] Glucansucrases of the Invention
[0194] The present invention relates firstly to a process for producing O--glucosylated flavonoid derivatives, comprising at least one step of incubating an enzyme of the invention with a flavonoid of formula (I) and at least one sucrose.
[0195] As previously indicated, the enzymes of the invention are advantageously capable of glucosylating flavonoids at the level of non-vicinal hydroxyl function(s), in particular present on the B ring.
[0196] These enzymes consist more particularly of glucansucrases belonging to families 13 and 70 of the glycoside hydrolases (GH13 and GH70).
[0197] The glucansucrases belonging to family 13 are naturally produced by bacteria of the Deinococcus, Neisseria or Alteromonas genera.
[0198] The glucansucrases belonging to family 70 are for their part naturally produced by lactic acid bacteria of the Leuconostoc, Lactobacillus, Streptococcus or Weissela sp. genera.
[0199] As previously indicated, various wild-type glucansucrases of family 13 or 70 of the glycoside hydrolases have already been used for the production of glucosylated flavonoids, but none of them has to date been described as being capable of glucosylating the flavonoids more particularly targeted in the present invention, namely those which are monohydroxylated on the B ring or which have non-vicinal hydroxyl functions on the B ring.
[0200] As it happens, as shown in the examples, the inventors have determined variants of these enzymes, mutated at the level of their flavonoid-binding site, and capable of efficiently glucosylating such compounds.
[0201] All of the wild-type or mutated enzymes described in the present application that were known to those skilled in the art had to date never been used to glucosylate flavonoids according to the invention.
[0202] The nucleotide sequence of the wild-type form of the ASNp (amylosucrase Neisseria polysaccharea) enzyme (family GH13) has the GenBank reference AJ011781.1, while its polypeptide sequence has the Uniprot reference Q9ZEU2.
[0203] The nucleotide sequence of the wild-type form of the DSR-S enzyme (derived from the Leuconostoc mesenteroides B-512F strain) has the GenBank reference 109598.
[0204] The nucleotide sequence of the wild-type form of the DSR-E enzyme (derived from the Leuconostoc mesenteroides NRRL B-1299 strain) has the GenBank reference AJ430204.1 and the Uniprot reference Q8G9Q2.
[0205] The N123-GBD-CD2 enzyme (sequence SEQ ID NO: 12) is a truncated form of the abovementioned DSR-E enzyme, as described in Brison et al., J. Biol. Chem., 2012, 287, 7915-24.
[0206] Literature references describing these mutated enzymes are indicated in tables 1 and 4. In addition, the method for obtaining the mutated enzymes is described in European patent application EP 2 100 966 A1.
[0207] The peptide sequences of the various mutated or non-mutated enzymes according to the invention are indicated in the present application. Thus, an enzyme according to the invention may be synthesized by conventional synthesis chemistry methods, that is to say homogeneous chemical syntheses in solution or in solid phase. By way of illustration, those skilled in the art may use the techniques for polypeptide synthesis in solution described by Houben Weil (1974, In methode der Organischen Chemie, E. Wunsh ed., volume 15-I and 15-II, Thieme, Stuttgart.). An enzyme according to the invention may also be chemically synthesized in the liquid or solid phase by means of successive couplings of the various amino acid residues (from the N-terminal end to the C-terminal end in liquid phase, or from the C-terminal end to the N-terminal end in solid phase). Those skilled in the art may in particular use the solid-phase peptide synthesis technique described by Merrifield (Merrifield R B, (1965a), Nature, vol. 207 (996): 522-523; Merrifield R b, (1965b), Science, vol. 150 (693):178-185).
[0208] According to another aspect, an enzyme according to the invention may be synthesized by genetic recombination, for example according to a production process comprising the following steps:
[0209] (a) preparing an expression vector into which has been inserted a nucleic acid encoding the peptide sequence of an enzyme of the invention, said vector also comprising the regulatory sequences required for the expression of said nucleic acid in a chosen host cell;
[0210] (b) transfecting a host cell with the recombinant vector obtained in step (a);
[0211] (c) culturing the host cell transfected in step b) in an appropriate culture medium;
[0212] (d) recovering the culture supernatent of the transfected cells or the cell lysate of said cells, for example by sonication or by osmotic shock; and
[0213] (e) separating or purifying, from said culture medium, or from the cell lysate pellet, the enzyme of the invention thus obtained.
[0214] In order to purify an enzyme according to the invention that has been produced by host cells transfected or infected with a recombinant vector encoding said enzyme, those skilled in the art may advantageously use purification techniques described by Molinier-Frenkel (2002, J. Viral. 76, 127-135), by Karayan et al. (1994, Virology 782-795) or by Novelli et al. (1991, Virology 185, 365-376).
[0215] Thus, glucansucrases that are usable in a process of the invention are chosen from a group comprising: [0216] a sequence having at least 80% identity with the sequence SEQ ID NO: 1, said sequence having an amino acid X.sub.1 representing an amino acid chosen from the group consisting of A, C, E, F, G, H, I, K, M, N, P, Q, S, T, V and Y; [0217] a sequence having at least 80% identity with the sequence SEQ ID NO: 2, said sequence having an amino acid X.sub.2 representing an amino acid chosen from the group consisting of A, C, D, F, G, H, K, L, M, N, P, S, V and Y; [0218] a sequence having at least 80% identity with the sequence SEQ ID NO: 3, said sequence having an amino acid X.sub.3 representing an amino acid chosen from the group consisting of A, C, G, I, K, M, N and W; [0219] a sequence having at least 80% identity with the sequence SEQ ID NO: 4, said sequence having an amino acid X.sub.4 representing an amino acid chosen from the group consisting of C, I, N, P, V and W; [0220] a sequence having at least 80% identity with the sequence SEQ ID NO: 5, said sequence having an amino acid X.sub.5 representing an amino acid chosen from the group consisting of A, C, D, G, I, K, L, M, R, V and W; [0221] a sequence having at least 80% identity with the sequence SEQ ID NO: 6, said sequence having an amino acid X.sub.6 representing an amino acid chosen from the group consisting of C, G, Q, S and T; [0222] a sequence having at least 80% identity with the sequence SEQ ID NO: 7, said sequence having an amino acid X.sub.7 representing an amino acid chosen from the group consisting of A and G; [0223] a sequence having at least 80% identity with the sequence SEQ ID NO: 8; [0224] a sequence having at least 80% identity with the sequence SEQ ID NO: 9; said sequence having an amino acid X.sub.8 representing an amino acid chosen from the group consisting of C, I and L; [0225] a sequence having at least 80% identity with the sequence SEQ ID NO: 10; [0226] a sequence having at least 80% identity with the sequence SEQ ID NO: 11; and [0227] a sequence having at least 80% identity with the sequence SEQ ID NO: 12, said sequence having amino acids X.sub.9, X.sub.10, X.sub.11, X.sub.12 and X.sub.13, with:
[0228] (i) X.sub.9 representing, independently of X.sub.10, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of G, S, V, C, F, N, I, L and W;
[0229] X.sub.10 representing, independently of X.sub.9, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of L, I, H, Y and F;
[0230] with the exception of the case where X.sub.9 represents W and X.sub.10 represents F;
[0231] X.sub.11 representing A;
[0232] X.sub.12 representing F; and
[0233] X.sub.13 representing L;
[0234] (ii) X.sub.9 representing W;
[0235] X.sub.10 representing F;
[0236] X.sub.11 representing, independently of X.sub.9, X.sub.10, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of E and A;
[0237] X.sub.12 representing, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.13, an amino acid chosen from the group consisting of L and F;
[0238] with the exception of the case where X.sub.11 represents A and X.sub.12 represents F; X.sub.13 representing L;
[0239] or
[0240] (iii) X.sub.9 representing W;
[0241] X.sub.10 representing F;
[0242] X.sub.11 representing A;
[0243] X.sub.12 representing, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.13, an amino acid chosen from the group consisting of A, R, D, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y and V; and
[0244] X.sub.13 representing, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.12, an amino acid chosen from the group consisting of A, R, D, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y and V.
[0245] According to one embodiment of the invention, a sequence having at least 80% identity with SEQ ID NO: 12 indicated above is preferably such that: [0246] (i) X.sub.9 represents, independently of X.sub.10, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of G, S, V, C, F, N, I, L and W;
[0247] X.sub.10 represents, independently of X.sub.9, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of L, I, H, Y and F;
[0248] with the exception of the case where X.sub.9 represents W and X.sub.10 represents F;
[0249] X.sub.11 represents A;
[0250] X.sub.12 represents F; and
[0251] X.sub.13 represents L;
[0252] or [0253] (ii) X.sub.9 represents W;
[0254] X.sub.10 represents F;
[0255] X.sub.11 represents, independently of X.sub.9, X.sub.10, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of E and A;
[0256] X.sub.12 represents, independently of X.sub.9, X.sub.10, X.sub.11 and X.sub.13, an amino acid chosen from the group consisting of L and F;
[0257] with the exception of the case where X.sub.11 represents A and X.sub.12 represents F;
[0258] X.sub.13 represents L;
[0259] or
[0260] is the sequence SEQ ID NO: 13.
[0261] In this sequence SEQ ID NO: 13, X.sub.9 represents W, X.sub.10 represents F, X.sub.11 represents A, X.sub.12 represents I, X.sub.13 represents I, and the aspartic acid (D) in position 432 is substituted with a glutamic acid (E).
[0262] It should be understood from this formulation that the amino acids defined as being respectively X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12 and X.sub.13 are present and as defined above in the glucansucrases of the invention having at least 80% identity with, respectively, a sequence SEQ ID NO: 1 to 7, 9 and 12, as defined above.
[0263] As shown in the examples, all the enzymes having one of these peptide sequences exhibit a capacity, statistically greater than that of the wild-type enzyme, for glucosylating the flavonoids of the invention, having non-vicinal hydroxyl functions, in particular on the B ring.
[0264] The present invention also encompasses the sequences of which the amino acid sequence has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid identity with one of the sequences SEQ ID NO: 1 to 12 as defined previously and a biological activity of the same nature.
[0265] The expression biological activity of the same nature with regard to the peptide sequences 1 to 12 is intended to mean the same capacity for glucosylating flavonoids which are monohydroxylated or hydroxylated in a non-vicinal manner on the B ring.
[0266] For the purposes of the present invention, the percentage identity between two nucleic acid or amino acid sequences is determined by comparing the two sequences optimally aligned, through a comparison window.
[0267] The part of the nucleotide sequence in the comparison window may thus comprise additions or deletions (for example gaps) compared with the reference sequence (which does not comprises these additions or these deletions) so as to obtain optimal alignment between the two sequences.
[0268] The percentage identity is calculated by determining the number of positions at which an identical nucleic base (or an identical amino acid) is observed for the two sequences compared, then by dividing the number of positions at which there is identity between the two nucleic bases (or between the two amino acids) by the total number of positions in the comparison window, then by multiplying the result by one hundred in order to obtain the percentage nucleotide (or amino acid) identity of the two sequences with respect to one another.
[0269] The optimal alignment of the sequences for the comparison may be carried out by computer using known algorithms.
[0270] Entirely preferably, the percentage sequence identity is determined using the Clustal W software (version 1.82), the parameters being set as follows: (1) CPU MODE=ClustalW mp; (2) ALIGNMENT=full; (3) OUTPUT FORMAT=aln w/numbers; (4) OUTPUT ORDER=aligned; (5) COLOR ALIGNMENT=no; (6) KTUP (word size)=default; (7) WINDOW LENGTH=default; (8) SCORE TYPE=percent; (9) TOPDIAG=default; (10) PAIRGAP=default; (11) PHYLOGENETIC TREE/TREE TYPE=none; (12) MATRIX=default; (13) GAP OPEN=default; (14) END GAPS=default; (15) GAP EXTENSION=default; (16) GAP DISTANCES=default; (17) TREE TYPE=cladogram and (18) TREE GRAPH DISTANCES=hide.
[0271] More particularly, the present invention also relates to the sequences in which the amino acid sequence has 100% amino acid identity with amino acids 225 to 450 of the sequences SEQ ID NO: 1 to 9, or 100% amino acid identity with amino acids 2130 to 2170 of the sequence SEQ ID NO: 12, and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 929/0, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 1009/0 amino acid identity with the rest of the sequences SEQ ID NO: 1 to 12 as previously defined, and a biological activity of the same nature.
[0272] Among the sequences of interest of the invention, some of them prove to be more particularly advantageous in terms of glucosylation activity.
[0273] Thus, according to one embodiment, the glucansucrases preferentially used in a process of the invention are chosen from the group comprising: [0274] a sequence having at least 80% identity with SEQ ID NO: 1, said sequence having an amino acid X.sub.1 representing an amino acid chosen from the group consisting of H, N or S; [0275] a sequence having at least 80% identity with SEQ ID NO: 2, said sequence having an amino acid X.sub.2 representing an amino acid chosen from the group consisting of A, C, F, L, M, S or V; [0276] a sequence having at least 80% identity with SEQ ID NO: 3, said sequence having an amino acid X.sub.3 representing an amino acid chosen from the group consisting of A and N; [0277] a sequence having at least 80% identity with SEQ ID NO: 4, said sequence having an amino acid X.sub.4 representing an amino acid chosen from the group consisting of C, I, N, P, V or W; [0278] a sequence having at least 80% identity with SEQ ID NO: 5, said sequence having an amino acid X.sub.5 representing an amino acid chosen from the group consisting of C, K, R or V; [0279] a sequence having at least 80% identity with SEQ ID NO: 9, said sequence having an amino acid X.sub.8 representing an amino acid chosen from the group consisting of C or L; and [0280] a sequence having at least 80% identity with SEQ ID NO: 12, said sequence having amino acids X.sub.9, X.sub.10, X.sub.11, X.sub.12 and X.sub.13, with:
[0281] (i) X.sub.9 representing an amino acid chosen from the group consisting of G, V, C and F;
[0282] X.sub.10 representing F; X.sub.11 representing A; X.sub.12 representing F; and X.sub.13 representing L;
[0283] (ii) X.sub.9 representing, independently of X.sub.10, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of S, N, L and I;
[0284] X.sub.10 representing, independently of X.sub.9, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of L, I, H and Y;
[0285] X.sub.11 representing A; X.sub.12 representing F; and X.sub.13 representing L;
[0286] (iii) X.sub.9 representing W; X.sub.10 representing F; X.sub.11 representing A or E; X.sub.12 representing L and X.sub.13 representing L; or
[0287] said sequence having at least 80% identity with SEQ ID NO: 12 is the sequence SEQ ID NO: 13.
[0288] According to one preferred mode, a sequence having at least 80% identity with SEQ ID NO: 12, having the amino acids X.sub.9, X.sub.10, X.sub.11, X.sub.12 and X.sub.13, is such that:
[0289] (i) X.sub.9 represents an amino acid chosen from the group consisting of G, V. C and F;
[0290] X.sub.10 represents F; X.sub.11 represents A; X.sub.1, represents F; and X.sub.13 represents L;
[0291] (ii) X.sub.9 represents, independently of X.sub.10, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of S and I;
[0292] X.sub.10 represents, independently of X.sub.9, X.sub.11, X.sub.12 and X.sub.13, an amino acid chosen from the group consisting of L, I and Y;
[0293] X.sub.11 represents A; X.sub.12 represents F; and X.sub.13 represents L; or
[0294] (iii) X.sub.9 represents W; X.sub.10 represents F; X.sub.11 represents A or E; X.sub.12 represents L and X.sub.13 represents L; or
[0295] said sequence having at least 80% identity with SEQ ID NO: 12 is the sequence SEQ ID NO: 13.
[0296] The mutants that are more particularly advantageous according to the invention, of SEQ ID NO: 12, are in particular indicated in example 11 of the present application.
[0297] The enzymes of which the sequences have at least 809/0 identity with SEQ ID NO 1 to 11 all in fact exhibit a glucosylation efficiency on the flavonoids of the invention which is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% greater compared respectively to an activity of 0.5+/0.5% or 4.7+/1.7% for the wild-type enzyme (see in particular tables 2, 3, 5 and 6).
[0298] The enzymes of which the sequence has at least 80% identity with SEQ ID NO 12 exhibit a glucosylation efficiency on the flavonoids of the invention which is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 409/0, 45% or 50% greater compared respectively to an activity of 20.4+/3.2% or 13.9+/4.7% for the wild-type enzyme (see in particular tables 7 and 8).
[0299] Flavonoids, Derivatives and Uses
[0300] a) Flavonoids Used in a Process of the Invention
[0301] The flavonoids specifically used in a process of the invention are of formula (I) as previously described.
[0302] According to one embodiment, just one of the groups chosen from R.sub.8, R.sub.9, R.sub.10, R.sub.13 and R.sub.12 represents a hydroxyl group,
[0303] the other groups among R.sub.8, R.sub.9, R.sub.10, R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imie; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; and a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s).
[0304] Preferably, the R.sub.10 group represents a hydroxyl group.
[0305] Preferably, the R.sub.8, R.sub.9, R.sub.11 and R.sub.12 groups represent hydrogen atoms.
[0306] According to one preferred embodiment, the R.sub.10 group represents a hydroxyl group and the R.sub.8, R.sub.9, R.sub.11 and R.sub.12 groups represent hydrogen atoms.
[0307] According to one embodiment, the C ring represents a ring of formula (II) or (IV) as previously defined. According to one embodiment, the C ring represents a ring of formula (II). According to another embodiment, the C ring represents a ring of formula (IV).
[0308] According to one embodiment of the invention, the R.sub.1 group represents a B ring of formula (VI) as previously defined.
[0309] According to one embodiment, the C ring represents a ring of formula (II) and the R.sub.1 group represents a B ring of formula (VI) as previously defined.
[0310] According to another embodiment, the C ring represents a ring of formula (IV) and the R.sub.1 group represents a B ring of formula (VI) as previously defined.
[0311] According to one embodiment, the R.sub.1, R.sub.2 and R.sub.2 groups represent hydrogen atoms, and R.sub.3 and R.sub.3 together form an O group.
[0312] According to one preferred embodiment, the R.sub.1 group represents a B ring of formula (VI), the R.sub.1, R.sub.2 and R.sub.2 groups represent hydrogen atoms, and R.sub.3 and R.sub.3 together form an O group.
[0313] According to one preferred embodiment, the C ring represents a ring of formula (II) or (IV), the R.sub.1 group represents a B ring of formula (VI), the R.sub.1, R.sub.2 and R.sub.2 groups represent hydrogen atoms, and R.sub.3 and R.sub.3 together form an O group.
[0314] According to one embodiment, two of the R.sub.4, R.sub.5, R.sub.6 and R.sub.7 groups represent a hydroxyl group, the other two groups being as previously defined. Preferably, the two groups representing a hydroxyl group are the R.sub.4 and R.sub.6 groups.
[0315] According to one embodiment, two of the R.sub.4, R.sub.5, R.sub.6 and R7 groups represent a hydroxyl group, the other two groups representing a hydrogen atom.
[0316] According to one embodiment, the R.sub.5 and R.sub.7 groups represent hydrogen atoms.
[0317] According to one preferred embodiment, the R.sub.4 and R.sub.6 groups represent a hydroxyl group and the R.sub.5 and R.sub.7 groups represent a hydrogen atom.
[0318] According to another embodiment, R.sub.8 and just one of the groups chosen from R.sub.10, R.sub.11 and R.sub.12 represent a hydroxyl group,
[0319] R.sub.9 and the other groups among R.sub.10, R.sub.11 and R.sub.12, which may be identical or different, being chosen from the group comprising a hydrogen atom; a linear or branched, saturated or unsaturated C.sub.1-C.sub.10 hydrocarbon-based group, optionally interrupted with at least one heteroatom chosen from O, N or S; a halogen atom; a C.sub.5-C.sub.9 aryl; a C.sub.4-C.sub.9 heterocycle; a (C.sub.1-C.sub.3)alkoxy group; a C.sub.2-C.sub.3 acyl; a C.sub.1-C.sub.3 alcohol; a COOH; NH.sub.2; CONH.sub.2; CHO; SH; C(O)O(C.sub.2-C.sub.3) group; a C.sub.1-C.sub.3 amine; a C.sub.1-C.sub.3 imine; a nitrile group; a C.sub.1-C.sub.3 haloalkyl; a C.sub.1-C.sub.3 thioalkyl; a C(W) group; and an O(W) group; W representing a chain consisting of from 1 to 6 glycoside(s).
[0320] Preferably, the R.sub.10 group represents a hydroxyl group.
[0321] Preferably, the R.sub.9, R.sub.11 and R.sub.12 groups represent hydrogen atoms.
[0322] According to one preferred embodiment, the R.sub.8 and R.sub.10 groups represent a hydroxyl group and the R.sub.9, R.sub.11 and R.sub.12 groups represent hydrogen atoms.
[0323] According to one embodiment, the C ring represents a ring of formula (II) or (IV), preferably (II), as previously defined.
[0324] According to one embodiment of the invention, the R.sub.1 group represents a B ring of formula (VI) as previously defined.
[0325] According to one embodiment, the R.sub.1 and R.sub.2 groups represent hydrogen atoms, R.sub.2 represents a hydrogen atom or an OH group, preferably an OH group, and R.sub.3 and R.sub.3 together form an O group.
[0326] According to one preferred embodiment, the R.sub.1 group represents a B ring of formula (VI), the R.sub.1 and R.sub.2 groups represent hydrogen atoms, R.sub.2 represents an OH group, and R.sub.3 and R.sub.3 together form an O group.
[0327] According to one preferred embodiment, the C ring represents a ring of formula (II), the R.sub.1 group represents a B ring of formula (VI), the R.sub.2 group represents an OH group, and R.sub.3 and R.sub.3 together form an O group.
[0328] According to one embodiment, a flavonoid used in a process of the invention is of formula (VII), (VIII) or (IX) below:
##STR00007##
[0329] A flavonoid of the invention may be used in a process of the invention at a sucrose to flavonoid molar ratio of between 1 and 35 000, the reaction mixture comprising at least the enzyme(s), the sucrose and the receptor flavonoid(s).
[0330] Preferably, the sucrose to flavonoid molar ratio is between 7 and 292, the reaction mixture comprising at least the enzyme(s), the sucrose and the receptor flavonoid(s).
[0331] b) O--Glucosylated Flavonoid Derivatives
[0332] The present invention also relates to certain O--glucosylated flavonoid derivatives. They are capable of being obtained from a process of the invention.
[0333] The present invention is more particularly directed toward compounds of formula (X) below:
##STR00008##
[0334] in which X.sub.14 represents a chain consisting of at least two -glucoside groups, and X.sub.15 and X.sub.16, which may be identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C.sub.1-C.sub.6 alkyl; a C(O)O(C.sub.2-C.sub.3) group; and a chain consisting of from 1 to 600 000 -glucoside groups.
[0335] As illustrated in Moulis et al. Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases, J. Biol. Chem. 2006, 281: 31254-31267, a compound according to the invention, and glucosylated using a glucansucrose in accordance with the invention, may in fact comprise a chain consisting of from 1 to 600 000 -glucoside groups.
[0336] The present invention is also directed toward compounds of formula (XI) below:
##STR00009##
[0337] in which
[0338] X.sub.17 represents a chain consisting of from 1 to 600 000 -glucoside groups, and
[0339] X.sub.18 and X.sub.19, which may be identical or different, are chosen from the group comprising a hydrogen atom; a linear or branched C.sub.1-C.sub.6 alkyl; a C(O)O(C.sub.2-C.sub.3) group; and a chain consisting of from 1 to 600 000 -glucoside groups.
[0340] c) Use of the O--Glucosylated Flavonoid Derivatives of the Invention
[0341] According to one embodiment, the O--glucosylated flavonoid derivatives of the invention may be used as an antioxidant (Heim et al., J. Nutr. Biochem., 2002, 13: 572-584).
[0342] According to one embodiment, the O--glucosylated flavonoid derivatives of the invention may be employed for the pharmaceutical use thereof in the treatment and/or prevention of hepatotoxicity, allergies, inflammation, ulcers, tumors, menopausal disorders or neurodegenerative diseases (Harborne J. et al., Phytochemistry, 2000, 55: 481-504; Quideau S. et al., Angew. Chem. Int. End. 2011, 50: 586-621).
[0343] According to one embodiment, the O--glucosylated flavonoid derivatives of the invention may be employed for the pharmaceutical use thereof as a veinotonic (Katsenis K., Curr. Vasc. Pharmacol. 2005, 3(1), 1-9).
[0344] Furthermore, according to one embodiment of the invention, the O--glucosylated flavonoid derivatives of the invention may be used as: [0345] a photovoltaic agent (see in particular in this respect the document Meng et al., Nano Lett. 2008, 8(10), 3266-72; Narayan M. R., Renew. Sust. Energ. Rev. 2012, 16, 208-215; US 2009/0071534 A1); [0346] an insect repellent (see in particular in this respect the documents JP 2002060304; JP 2003104818; Benavente-garcia et al., J. Agric. Food Chem., 1997, 45 (12), 4505-4515; Singh et al., Natural product sciences, 1997, 3(1), 49-54; Diwan and Saxena, Int. J. Chem. Sci., 2010, 8(2), 777-782; Regnault-Roger et al., J. Stored Prod Res, 2004, 40, 395-408); [0347] a bleaching agent (see in particular in this respect the document Barkat Ali Khan et al., Asian J. Chem., 2011, 23(2), pp 903-906; patent applications WO 2008140440 A1; WO 2005094770 A1; Zhu W. & Gao J., J. Invest. Dermatol. Symposium Proceedings, 2008, 13, 20-24; Kim J. H. et al., J. Invest. Dermatol., 2008, 128, 1227-1235); or [0348] a pesticide, fungicide and/or bactericide (see in particular in this respect the documents WO 2013043031, CN 102477024 and CN 101002557).
[0349] The present invention is also illustrated, without in any way being limited thereto, by the examples which follow.
EXAMPLES
Example 1: Production and Use of Recombinant Glucansucrases for Apigenin and Naringenin Glucosylation
[0350] A library of 183 variants including 174 single or double mutants, constructed from the amylosucrase of N. polysaccharea (glycoside hydrolase family GH13) and 10 variants, constructed from the glucansucrases DSR-S, ASR and -1,2 BrS (belonging to the GH70 family) were tested for their ability to glucosylate apigenin and naringenin.
[0351] The origin of the glucansucrases selected for the study is reported in tables 1 and 4.
[0352] Tables 1 and 4 in fact illustrate a certain number of the glucansucrases tested in the examples of the present text and specify: column 1: the organism from which the enzyme originates; column 2: the various wild-type enzymes tested and also the mutated positions of the active site of these wild-type enzymes in the mutated glucansucrases also tested; column 3: the major binding specificities during the synthesis of the natural polymer; column 4: the literature references in which these enzymes, both in wild-type forms and in mutated forms, have been described in the prior art.
[0353] These enzymes were used in recombinant form and are expressed in Escherichia coli.
[0354] 1.1. Enzymes Production in Microplates
[0355] All of the Escherichia coli strains overexpressing the heterologous glucansucrases of the GH13 and GI-170 families, wild-types or their mutants, are maintained in the 96-well microplate format in order to facilitate the future flavonoid glucosylation screening steps.
[0356] Starting from the source microplates, a preculture of these E. coli strains is carried out for 22 hours at 30 C., 700 rpm in 96-well microplates, in 200 l of LB culture medium supplemented with 100 g/ml of ampicillin.
[0357] These precultures are in turn used to inoculate the deep-well microplates, each well of which contains 1 ml per well of ZYM5052 auto-induction medium containing in particular 0.2% (w/v) of -lactose, 0.05% (w/v) of D-glucose, 0.5% (w/v) of glycerol and 0.05% (w/v) of L-arabinose (Studier et al., 2005).
[0358] After 22 hours of culture at 30 C. and at 700 rpm, the cell suspension is centrifuged for 20 minutes at 3000 g at 4 C. The cell pellets are resuspended in the 96-well deep-well microplates, with 300 l of phosphate buffered saline (24 mM sodium/potassium phosphate and 274 mM NaCl) containing 0.5 g/l of lysozyme and 5 mg/l of bovin pancreatic RNAse.
[0359] An incubation is then carried out for 30 minutes at 30 C. with shaking, these microplates then being stored overnight at 80 C. After thawing, the microplates are vigorously shaken and then centrifuged for 20 minutes at 3000 g at 4 C.
[0360] The centrifuged cell lysates containing the recombinant enzymes are transferred into clean deep-well 96-well microplates.
[0361] 1.2. Implementation of the Acceptor Reactions
[0362] The enzymatic extracts obtained are used to carry out the flavonoid glucosylation enzymatic screening reactions. The enzymatic activity of each centrifuged cell lysate is evaluated in the microplate format, by final weight after 30 minutes incubation in the presence of a final concentration of 146 mM of sucrose, by assaying the reducing sugars with 3,5-dinitrosalicylic acid (DNS). Finally, after dilution in ultrapure water, the absorbance is read at 540 nm.
[0363] The flavonoid acceptor reactions are then carried out in deep-well microplates, in a volume of 300 l, at final concentrations of sucrose of 146 mM and of flavonoid of 2.5 mM (apigenin) or 5 mM (naringenin) (initially dissolved in 100% DMSO), and 140 l of centrifuged cell lysate.
[0364] The final DMSO concentration in the reaction medium is 3% (v/v).
[0365] The incubation is carried out at 30 C. and at 700 rpm. After 24 hours, the enzymes are denatured at 95 C. for 15 minutes. These microplates are stored at 80 C. with a view to rapid evaluation of the flavonoid glucosylation by liquid-phase chromatography coupled to mass spectrometry (HPLC-MS or LC-MS).
[0366] 1.3. Analytical Techniques
[0367] With a view to their analyses by HPLC-MS, the extensively homogenized reaction media are diluted to 1/30th in DMSO. The separation of the flavonoids and of their glucosylated forms is carried out in reverse phase with a ProntoSIL Eurobond 533.0 mm 120-3-C18-AQ column (porosity of 120 , particle size of 3 m, C18 grafting, Bischoff Chromatography, Germany).
[0368] This column is maintained at 40 C. on a Dionex Ultimate 3000 HPLC system equipped with a UV/Vis detector. This system is coupled to a tingle quadrupole mass spectrometer (Thermo Scientific, MSQ Plus).
[0369] The mobile phase is composed of a mixture of ultrapure water (solvent A)/acetonitrile of LC-MS quality (solvent B), each containing 0.05% (v/v) of formic acid. The separation is carried out in 10 minutes by means of a gradient of solvent B defined as follows:
[0370] 0 min, 15% (v/v);
[0371] 3 min, 25% (v/v);
[0372] 6.5 min, 49.5% (v/v);
[0373] 6.6 min, 80% (v/v);
[0374] 6.8 min, 15% (v/v); and
[0375] 10 min, 15% (v/v).
[0376] The mass spectrometry ionization on the MSQ Plus equipment is carried in positive electrospray mode (ESI+) for the apigenin and negative electrospray mode (ESI) for the naringenin.
[0377] The capillary voltage is regulated at 3000 V, the cone voltage at 75 V. The source block temperature is set at 450 C.
[0378] The LC-MS/MS system used for the high-resolution mass spectrometry or MS/MS fragmentation analysis comprises an Ultimate 3000 chromatographic separation system (Dionex) coupled to a linear trap/Orbitrap hybrid mass spectrometer (LQT Orbitrap, Thermo Fischer Scientific). The mass spectrometry ionization on the LQT Orbitrap equipment is this time carried out either in positive electrospray mode (ESI+) or in negative electrospray mode (ESI).
Example 2: Determination of the Efficiencies of Apigenin Glucosylation by the Recombinant Amylosucrase from N. polysaccharea and by Variants Thereof
[0379] The reactions in the presence of acceptor were carried out by applying the conditions described in example 1.
[0380] The flavonoid glucosylation efficiency was determined from the following formula:
Glucosylation efficiency=((area of the peak of glucosylated flavonoid(s)))/((area of the peak of glucosylated flavonoid(s))+area of the peak of residual aglycone flavonoid)100
[0381] The flavonoid glucosylation efficiencies, expressed as a percentage, were calculated from the areas of the peaks of the various products analyzed, as described in example 1, by HPLC with a UV detector (340 nm) after 24 h of reaction.
[0382] The values obtained are reported in table 2.
[0383] Table 2 illustrates the apigenin glucosylation efficiency, during the screening of microplates, for the wild-type form of ASNp (recombinant amylosucrase from N. polysaccharea) and also for its 174 mutants of its active site. Along the Y-axis: the positions of mutation of the wild-type enzyme (ASNp WT); along the X-axis: the amino acid substituting that present in the sequence of the wild-type enzyme.
[0384] Thus, by way of illustration, the percentage of 1.7% indicated in row 2, column 2 was obtained using an enzyme mutated in position 226 by substitution of the amino acid R (arginine) with the amino acid A (alanine).
[0385] Each case represents a single mutation on positions R226, I228, F229, A289, F290, I330, V331, D394 and R446 or a double mutation, namely two single mutations at two of these positions.
[0386] The grey bar in each case represents the level of glucosylation efficiency relative to the most efficient mutant.
[0387] The results obtained for the wild-type enzyme are indicated above table 2 and also at the intersections R226R, I228I, F229F, A289A, F290F, I330I, V331V, D394D and R446R.
[0388] The three double mutant variants are indicated under table 2.
[0389] For the wild-form type of the ASNp enzyme (amylosucrase Neisseria polysaccharea), the glucosylation efficiency is very low (0.50.5; n=16). The glucosylation efficiencies obtained for R226R, I228I, F229F, A289A, F290F, I330I, V331V, D394D and R446R given in table 2 are also included in the range of values 0.50.5.
[0390] With an apigenin glucosylation efficiency greater than that of the wild-type enzyme (greater than 1%), a large number of mutant enzymes emerge from its screening.
[0391] More particularly, with an apigenin glucosylation efficiency greater than 5%, eight enzymes emerge more particularly from the screening.
[0392] The glucosylation efficiencies for these eight mutated enzymes are respectively the following: ASNp I228F: 9.9%; ASNp I228L: 11.1%; ASNp I228M: 5.4%; ASNp F229A: 5.6%; ASNp F229N: 5.7%; ASNp A289W: 22.1%; ASNp F290C: 5.4%; and ASNp F290K: 8.9%.
[0393] This illustrates the advantage of employing enzymes derived from site-directed engineering for the glucosylation of poorly recognized acceptors such as flavonoids which are monohydroxylated or hydroxylated in a non-vicinal manner, in particular on the B ring.
Example 3: Determination of the Efficiencies of Apigenin Glucosylation by the Glucansucrases of the GH70 Family
[0394] The glucansucrases of the GH70 family tested for their apigenin glucosylation activity are reported in table 4.
[0395] Table 4 illustrates the glucansucrases of the GH70 family (glycoside hydrolase 70) tested in the examples of the present text.
[0396] Thus, ASR C-APY-del WT represents the truncated form of ASR (alternansucrase), DSR-S vardel4N WT represents the wild-type truncated form DSR-S (dextransucrase) and, for example, DSR-S vardel4N F353T represents the truncated form of DSR-S mutated in position 353 by substitution of the amino acid F (phenylalanine) with the amino acid T (threonine).
[0397] The results of apigenin glucosylation by the glucansucrases of the GH70 family are reported in table 5.
[0398] Table 5 illustrates the apigenin glucosylation efficiency for the wild-type form of the truncated variant of DSR-S (vardel4N WT), for the truncated wild-type form of ASR (ASR C-APY-del WT), for the wild-type form of the -1,2 BrS enzyme, and for seven mutants of DSR-S vardel4N.
[0399] The grey bar in each case represents the level of glucosylation efficiency relative to the most efficient mutant.
[0400] Although the wild-type form of the truncated variant of DSR-S (DSR-S vardel4N WT) exhibits only a very low glucosylation activity (0.5%), the S512C mutant exhibits a higher apigenin glucosylation efficiency (13.9%).
Example 4: Comparison of the Apigenin Glucosylation Efficiencies for the Most Efficient Enzymes
[0401] Among the tested enzymes of the GH13 and GH70 families, nine mutants have apigenin glucosylation efficiencies greater than 5%, namely ASNp I228F, ASNp I228L, ASNp I228M, ASNp F229A, ASNp F229N, ASNp A289W, ASNp F290C, ASNp F290K and DSR-S (vardel4N S512C). These efficiencies are compared for these nine most efficient mutants, with their relative activities in the presence of sucrose alone (see
[0402] The sucrose hydrolysis activities of the wild-type, ASNp WT (GH13) or DSR-S vardel4N WT (GH70) enzymes were taken as references for calculating the relative sucrose hydrolysis activities of their respective mutants.
[0403] Although the mutants exhibit activities on sucrose alone that are lower than those of the wild-type enzymes, the glucosylation efficiencies of these same mutants are from 10 to 44 times greater than for the wild-type enzymes. More globally, the correlation coefficient between the apigenin glucosylation efficiencies and the sucrose hydrolysis activities, calculated for all the mutant enzymes of the amylosucrase from N. polysaccharea, is 0.08. This illustrates the advantage of the process for identifying enzymes that are not very active on sucrose alone but capable of glucosylating the flavonoids of the invention.
[0404] In the case of the mutant enzyme ASNp A289W, a weight concentration of 149 mg/ml of glucosylated apigenin is achieved.
[0405] This is a minimum concentration obtained in microplates. Thus, an improvement factor of 10 may be expected after optimization of the medium.
Example 5: LC-MS Analysis of the Apigenin Glucosylation Products
[0406] The nine mutants mentioned in example 4 may be classified in six categories according to the glucosylation product profile obtained by LC-MS. The superimposition of the UV chromatograms (340 nm) for a representative of each of these six profile categories (respectively ASNp A289W, DSR-S (vardel4N S512C), ASNp F290K, ASNp F290C, ASNp F229N and ASNp I228F) is presented in
[0407] The superimposition of these chromatograms demonstrates the diversity of glucosylated apigenin forms that it is possible to obtain.
[0408] The LC-MS profiles obtained for ASNp WT and the nine most efficient mutants mentioned in example 4 are represented in
[0409] The molar masses, as determined by LC-MS in example 1, of the strongest glucosylated apigenin peak for each of the nine mutants are the following:
[0410]
[0411]
[0412]
[0413]
[0414]
[0415]
[0416]
[0417]
[0418]
[0419] The wild-type enzyme has a very low glucosylation efficiency on apigenin (0.59/0). Indeed, if the apigenin standard is compared with the final products of the glucosylation reaction, the appearance, on the UV chromatogram, of several peaks, of very low strength, of glucosylated apigenin is detected (
[0420] The I228F (
[0421] The group of F229A (
[0422] Finally, the F290K mutant has a product profile that is more complex than that of the F290C mutant.
Example 6: High-Resolution LC-MS and LC-MS/MS Analysis of the Apigenin Glucosylation Products
[0423] A study was carried out, by high-resolution LC-MS and LC-MS/MS (results obtained from Imagif), on the apigenin glucosylation products obtained with the mutant enzymes ASNp A289W and DSR-S vardel4N S512C.
[0424] The apigenin glucosylation product produced by the DSR-S vardel4N S512C enzyme mutant is a monoglucosylated form (
[0425] The ASNp A289W enzyme glucosylates apigenin to give a monoglucosylated product, the retention time of which is 4.25 min (
Example 7: Determination of the Efficiencies of Naringenin Glucosylation by the Recombinant Amylosucrase from N. polysaccharea and by its Variants
[0426] The reactions in the presence of acceptor were carried out by applying the conditions described in example 1.
[0427] The flavonoid glucosylation efficiency was determined from the formula set out in example 2. The flavonoid glucosylation efficiencies, expressed as a percentage, were calculated from the areas of the peaks of the various products analyzed, as described in example 1, by HPLC with a UV detector (340 nm), after 24 h of reaction.
[0428] The values obtained are reported in table 3.
[0429] Table 3 illustrates the naringenin glucosylation efficiency, during the screening of microplates, for the wild-type form of ASNp (recombinant amylosucrase from N. polysaccharea) and also for the 174 mutants of its active site. Along the Y-axis: the positions of mutation of the wild-type enzyme (ASNp WT); Along the X-axis: the amino acid substituting that present in the sequence of the wild-type enzyme.
[0430] Thus, by way of illustration, the percentage of 2.4% indicated in row 2, column 2 was obtained using an enzyme mutated in position 226 by substitution of the amino acid R (arginine) with the amino acid A (alanine).
[0431] Each case represents a single mutation on positions R226, I228, F229, A289, F290, I330, V331, D394 and R446 or a double mutation, namely two single mutations at two of these positions.
[0432] The grey bar in each case represents the level of glucosylation efficiency relative to the most efficient mutant.
[0433] The results obtained for the wild-type enzyme are indicated at the top of table 3 and also at the intersections R226R, I228I, F229F, A289A, F290F, I330I, V331V, D394D and R446R. The results obtained for the enzymes doubly mutated on positions 289 and 290 are indicated at the bottom of table 3.
[0434] For the wild-type form of the ASNp enzyme, the glucosylation efficiency is reduced (4.71.7; n=16).
[0435] With a naringenin glucosylation efficiency greater than that of the wild-type enzyme (greater than 6.4%), a large number of mutant enzymes emerge from this screening.
[0436] More particularly, with a naringenin glucosylation efficiency greater than 10%, sixteen mutant enzymes emerge more particularly from the screening. Seven of these mutant enzymes have in particular a naringenin glucosylation efficiency greater than 20% and two of them have an efficiency greater than 50%.
[0437] The glucosylation efficiencies for these sixteen mutated enzymes are respectively the following: ASNp R226H: 13.5%; ASNp R226N: 16.0%; ASNp R226S: 14.1%; ASNp I228A: 70.2%; ASNp I228C: 30.9%; ASNp I228S: 16.4%; ASNp I228V: 12.3%; and ASNp A289C: 27.8%; ASNp A289I: 11.2%; ASNp A289N: 14.5%; ASNp A289P: 10.3%; ASNp A289V: 21.8%; ASNp F290R: 11.2%; ASNp F290V: 21.1%; ASNp A289P/F290C: 50.9%; ASNp A289P/F290L: 22.9%.
[0438] The naringenin glucosylation illustrates the advantage of employing enzymes resulting from site-directed engineering for the glucosylation of weakly recognized acceptors such as flavonoids.
Example 8: Determination of the Efficiencies of Naringenin Glucosylation by the Glucansucrases of the GH70 Family
[0439] The glucansucrases of the GH70 family tested for their apigenin glucosylation activity are listed in table 4.
[0440] The results of naringenin glucosylation by the glucansucrases of the GH70 family are reported in table 6.
[0441] Table 6 illustrates the naringenin glucosylation efficiency for the wild-type form of the truncated variant of DSR-S (vardel4N WT), for the truncated wild-type form of ASR (ASR C-APY-del WT), for the wild-type form of the -1,2 BrS enzyme and for seven mutants of DSR-S vardel4N.
[0442] The grey bar in each case represents the level of glucosylation efficiency relative to the most efficient mutant.
[0443] The wild-type form of the truncated variant of ASR (ASR C-APY-del WT) exhibits a glucosylation efficiency of 27.1%. The wild-type enzyme -1,2 BrS exhibits a naringenin glucosylation efficiency of 26.8%.
Example 9: LC-MS Analysis of the Naringenin Glucosylation Products
[0444] The eighteen mutants having a naringenin glucosylation efficiency greater than 10%, discussed in examples 7 and 8, may be classified in seven categories according to the glucosylation product profiles obtained in LC-MS. The superimposition of the UV chromatograms (340 nm) for a representative of each of these seven profile categories is represented in
[0445] The superimposition of these chromatograms demonstrates the diversity of glucosylated naringenin forms that it is possible to obtain.
[0446] The LC-MS profiles obtained for ASNp WT, the five mutant enzymes of ASNp and the two glucansucrases of the GH70 family which are the most efficient are represented in
[0447] The molar masses, as determined by LC-MS in example 1, of the strongest glucosylated naringenin peak for each of these profiles are the following:
[0448]
[0449]
[0450]
[0451]
[0452]
[0453]
[0454]
[0455]
[0456] The wild-type enzyme (
[0457] The naringenin glucosylation profiles obtained with the enzymes ASNp R226N, ASNp I228A, ASNp A289C, ASNp F290V, ASNp A289P/F290C, ASR-C-APY-del or -1,2 BrS are all distinct (
Example 10: Production, Purification and Structural Determination by NMR of 4-O--D-Glucopyranosylnaringenin by the I228A Mutant of ASNp
Production of 4-O--D-glucopyranosylnaringenin
[0458] The production of the glucosylation products is carried out with the ASNp I228A enzyme on 204 mg of naringenin. The reaction conditions are the following: final concentration of sucrose 146 mM, of naringenin 5 mM (initially dissolved in DMSO at 150 mM), PBS buffer, pH 7.2, ASNp I228A 0.5 U/ml and ultrapure water qs 145 ml. The reaction is carried out with stirring at 30 C. for 24 h. At the end of the reaction, the enzyme is heat inactivated. The reaction mixture is stored at 20 C.
Purification of 4-O--D-glucopyranosylnaringenin
[0459] A prepurification step is carried out by solid phase extraction (SPE) on a cartridge containing 5 g of C18 stationary phase. After conditioning of the column, the centrifuged reaction mixture is deposited on the column and percolates by gravity. After the steps of washing with ultrapure water, the elution is carried out with methanol. The eluate is dried under a nitrogen gas stream before being taken up in 100% DMSO at a concentration of 100 g/l.
[0460] The various glucosylated forms of naringenin are separated at ambient temperature by semi-preparative HPLC-UV on a Waters apparatus. A C18 25010 mm column fitted with a precolumn makes it possible to separate the various glucosylated forms of naringenin with an aqueous mobile phase containing 0.05% (v/v) of formic acid with a gradient of acetonitrile (B). The various steps of the gradient are the following: 0 min, 22% B; 1 min, 22% B; 17 min, 25% B; 21 min, 299/0 B; 21.5 min, 95% B; 24.5 min, 95% B; 25 min, 22% B; 27.5 min, 22% B. On the basis of the UV signal, the elution fractions are collected in an automated manner. The purity of the elution fractions is evaluated by LC-UV-MS with a C18 2504.6 mm analytical column (gradient described above).
[0461] The elution fractions containing a monoglucosylated form of naringenin which is 96% pure, eluting at a retention time of 18.4 min in semi-preparative HPLC-UV, are combined and dried using a GeneVac apparatus. The product is then dissolved in 300 l de of deuterated methanol, dried under a nitrogen gas stream and then lyophilized for 48 h.
Structural Characterization of 4-O--D-glucopyranosylnaringenin
[0462] The structural determination of this monoglucosylation product was carried out by NMR.
[0463] The 1H, COSY 1H-1H, JMod and HMBC 1H-13C spectra were recorded on a Bruker Avance 500 MHz apparatus at 298 K (500 MHz for .sup.1H and 125 MHz for .sup.13C) with a TBI z-gradient 5 mm probe. The data were acquired and processed using the TopSpin 3 software. The sample was analyzed in deuterated methanol.
[0464] The assignment of the various NMR signals is indicated on
Example 11: Glucosylation of Naringenin and of Morin by the N.SUB.123.-GBD-CD2 Enzyme and its Mutants
[0465] Single or double variants constructed from the glucansucrose N.sub.123-GBD-CD2 (belonging to the glycoside hydrolase family GH70) were tested for their ability to glucosylate naringenin and morin. The results of glucosylation of these two flavonoids, by variants of N.sub.123-GBD-CD2, are reported in tables 7 and 8.
[0466] Regarding morin, the wild-type enzyme glucosylates it with a glucosylation efficiency of 20.43.2%.
[0467] Fourteen mutants which glucosylate this flavonol more efficiently than the wild-type enzyme, namely W403G, W403S-F404L, W403V, W403C, W403F, F431I-D432E-L434I, F431L, A430E-F431L, W403F-F404I, W403C-F404I, W403N-F404Y, W403N-F404H, W403I-F404Y and W403L-F404L. The glucosylation efficiencies obtained for these mutants are represented in table 7.
[0468] Among them, nine mutants glucosylate morin with a glucosylation efficiency greater than or equal to 30% (mutants W403G, W403S-F404L, W403V, W403C, W403F, F431I-D432E-L434I, F431L, A430E-F431L and W403F-F4041). Two mutants even have a morin glucosylation efficiency greater than or equal to 40%, or even greater than or equal to 45% (mutants W403S-F404L and W403G). The best glucosylation efficiencies were obtained with the mutants W403S-F404L (49.5%) and W403G (66.7%). Morin glucosylation products were detected by LC-UV-MS (
[0469] Naringenin is glucosylated by N.sub.123-GBD-CD2 WT with a glucosylation yield of 13.94.7% (table 8).
[0470] Nine variants exhibit a glucosylation efficiency greater than 20%, namely W403I-F404Y, W403V, W403G, W403F, W403S-F404L, W403C, F431I-D432E-L434I, F431L and A430E-F431L. The glucosylation efficiencies obtained are represented in table 8.
[0471] Seven of them have a glucosylation efficiency greater than or equal to 25% (W403I-F404Y, W403V, W403G, W403F, W403S-F404L, W403C, and F431I-D432E-L434I). More particularly, three variants have a glucosylation efficiency greater than or equal to 30%, or even greater than or equal to 35% (W403G, W403F, W403I-F404Y). The best degree of conversion of 59.3% was obtained with the variant W403I-F404Y. Regarding the naringenin glucosylation products, reaction products were detected by LC-UV-MS (
[0472] Naringenin is barely glucosylated by the wild-type enzyme (14%) and most of it is monoglucosylated (13%). In particular, a variant of the W403-F404 library exhibits an increase in production of the monoglucosylated product, up to 49% with the W403I-F404Y mutant. Finally, one variant (W403S-F404L) converts 10% of the naringenin to triglucosylated compound (compared with only 1% for the wild-type enzyme).
TABLE-US-00001 TABLE 1 Major bonds in the Organism Glucansucrase natural polymer References Neisseria ASNp WT and 152 single -(1.fwdarw.4) Albenne C. et al., J. Biol. Chem., 2004 279(1) polysaccharea mutants and three double 726-734 (EC 2.4.1.4) mutants of the active site Champion E., 2008. Doctoral thesis, INSA, (Positions 228, 229, 289, Toulouse 290, 330, 331, 394, 446) Champion C. et al., J. Am. Chem. Soc., 2009, 131, 7379-7389 Champion C. et al., J. Am. Chem. Soc., 2012, 134, 18677-18688 EP08290238.8
TABLE-US-00002 TABLE 4 Major bonds in the Organism Glucansucrase natural polymer References Leuconostoc DSR-S vardel 4N WT -(1.fwdarw.6) Moulis. C., 2006. Doctoral mesenteroides B-512F thesis, INSA, Toulouse and (EC 2.4.1.5) Moulis C. et al., FEMS Microbiol. Lett., 2006 261 203-210 Leuconostoc Seven mutants of DSR-S -(1.fwdarw.6) Irague R. et al., Anal. Chem. mesenteroides B-512F vardel
4N: F353T or S512C or 2011 83(4) 1202-1206 (EC 2.4.1.5) F353W or H463R/T464D/S512T or H463R/T464V/S512T or D460A/H463S/T464L or D460M/H463Y/T464M/S512C Leuconostoc ASR C-APY-del -(1.fwdarw.3)/ Joucla. G., 2003. Doctoral mesenteroides NRRL B- -(1.fwdarw.6) thesis, INSA, Toulouse and 1355 Joucla G et al., FEBS Lett. (EC 2.4.1.140) 2006 580(3) 763-768 Leuconostoc Mutant of DSR-E -(1.fwdarw.2) Brison et al., J. Biol. Chem., mesenteroides NRRL B-
N.sub.123-GBD-CD2 2012, 287, 7915-24 1299
The number indicated in each case is the percentage glucosylation efficiency.
The number indicated in each case is the percentage glucosylation efficiency
[0473] Efficiencies of morin glucosylation (table 7) and of naringenin glucosylation (table 8) by the wild-type glucansucrose N.sub.123-GBD-CD2 and the best mutants resulting from the secondary screening.
[0474] Sequences:
TABLE-US-00003 SeriesSEQIDNO:1:(Proteins= mutatedsequence oftheglucansucroseASNp(AmylosucraseNeisseria polysaccharea)R226X.sub.1) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLENILLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQV GGVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAV SSYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCA AGDPLFDNFYYIFPDRRMPDQYDRTLREX.sub.1FPDQHPGGFSQLEDGRWVWT TFNSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVAFIWKQMGTS CENLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIG YNPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWT FADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVS GTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDW SQDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSN PRFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYFIQTVTAHTLQAMP FKAHDLIGKKTVSLNQDLTLQPYQVMWLEIA SeriesSEQIDNO:2:(Proteins= mutatedsequence oftheglucansucraseASNp(AmylosucraseNeisseria polysaccharea)I228X.sub.2) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREX.sub.2FPDQHPGGFSQLEDGRWVWTT FNSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVAFIWKQMGTSC ENLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIGY NPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWTF ADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVSG TAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDWS QDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPFK AHDLIGGKTVSLNQDLTLQPYQVMWLEIA SeriesSEQIDNO:3:(Proteins= mutatedsequence oftheglucansucraseASNp(AmylosucraseNeisseria polysaccharea)F229X.sub.3) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREIX.sub.3PDQHPGGFSQLEDGRWVWTT FNSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRNIDAVAFIWKQMGTS CENLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIG YNPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWT FADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVS GTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDW SQDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSN PRFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPF KAHDLIGGKTVSLNQDLTLQPYQVMWLEIA SEQIDNO:4:(Protein= mutatedsequenceofthe glucansucraseASNp(AmylosucraseNeisseria polysaccharea)A289X.sub.4) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREIFPDQHPGGFSQLEDGRWVWTTF NSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVX.sub.4FIWKQMGTSC ENLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIGY NPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWTF ADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVSG TAAALVGLAQDDPHAVDREKLLYSIALSTGGLPLIYLGDEVGTLNDDDWS QDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPFK AHDLIGGKTVSLNQDLTLQPYQVMWLEIA SeriesSEQIDNO:5:(Proteins= mutatedsequence oftheglucansucraseASNp(AmylosucraseNeisseria polysaccharea)F290X.sub.5) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREIFPDQHPGGFSQLEDGRWVWTTF NSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVAX.sub.5IWKQMGTSC ENLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIGY NPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWTF ADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVSG TAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDWS QDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPFK AHDLIGGKTVSLNQDLTLQPYQVMWLEIA SeriesSEQIDNO:6:(Proteins= mutatedsequence oftheglucansucraseASNp(AmylosucraseNeisseria polysaccharea)V331X.sub.6) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREIFPDQHPGGFSQLEDGRWVWTTF NSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVAFIWKQMGTSCE NLPQAHALIRAFNAVMRIAAPAVFFKSEAIX.sub.6HPDQVVQYIGQDECQIGY NPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWTF ADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVSG TAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDWS QDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSNP RFDGGRLVTFNTNNICHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPF KAHDLIGGKTVSLNQDLTLQPYQVMWLEIA SeriesSEQIDNO:7:(Proteins= mutatedsequence oftheglucansucraseASNp(AmylosucraseNeisseria polysaccharea)D394X.sub.7) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREIFPDQHPGGFSQLEDGRWVWTTF NSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVAFIWKQMGTSCE NLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIGYN PLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDX.sub.7IGWTF ADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRVSG TAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDWS QDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSNP RFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPFK AHDLIGGKTVSLNQDLTLQPYQVMWLEIA SEQIDNO:8:(Protein= mutatedsequenceofthe glucansucraseASNp(AmylosucraseNeisseria polysaccharea)R446Q) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYAVS SYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRCAA GDPLFDNFYYIFPDRRMPDQYDRTLREIFPDQHPGGFSQLEDGRWVWTTF NSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVAFIWKQMGTSCE NLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQIGYN PLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGWTFA DEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCQVSGT AAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDDWSQ DSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQSNPR FDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMPFKA HDLIGGKTVSLNQDLTLQPYQVMWLEIA SeriesSEQIDNO:9:(Proteins= doublymutated sequencesoftheglucansucraseASNp(Amylosucrase Neisseriapolysaccharea)A289P/F290X.sub.8) SPNSQYLKTRILDIYTPEQRAGIEKSEDWRQFSRRMDTHFPKLMNELDSV YGNNEALLPMLEMLLAQAWQSYSQRNSSLKDIDIARENNPDWILSNKQVG GVCYVDLFAGDLKGLKDKIPYFQELGLTYLHLMPLFKCPEGKSDGGYA VSSYRDVNPALGTIGDLREVIAALHEAGISAVVDFIFNHTSNEHEWAQRC AAGDPLFDNFYYIFPDRRMPDQYDRTLREIFPDQHPGGFSQLEDGRWVWT TFNSFQWDLNYSNPWVFRAMAGEMLFLANLGVDILRMDAVPX.sub.8IWKQMGT SCENLPQAHALIRAFNAVMRIAAPAVFFKSEAIVHPDQVVQYIGQDECQI GYNPLQMALLWNTLATREVNLLHQALTYRHNLPEHTAWVNYVRSHDDIGW TFADEDAAYLGISGYDHRQFLNRFFVNRFDGSFARGVPFQYNPSTGDCRV SGTAAALVGLAQDDPHAVDRIKLLYSIALSTGGLPLIYLGDEVGTLNDDD WSQDSNKSDDSRWAHRPRYNEALYAQRNDPSTAAGQIYQDLRHMIAVRQS NPRFDGGRLVTFNTNNKHIIGYIRNNALLAFGNFSEYPQTVTAHTLQAMP FKAHDLIGGKTVSLNQDLTLQPYQVMWLEIA SEQIDNO:10:(Protein= mutatedsequenceofthe truncatedglucansucraseDSR-Svardel4N-S512C TQQVSGKYVEKDGSWYYYFDDGKNAKGLSTIDNNIQYFYESGKQAKG QYVTIDNQTYYFDKGSGDELTGLQSIDGNIVAFNDEGQQIFNQYYQSENG TTYYFDDKGHAATGIKNIEGKNYYFDNLGQLKKGFSGVIDGQIMTFDQET GQEVSNTTSEIKEGLTTQNTDYSEHNAAHGTDAEDFENIDGYLTASSWYR PTGELRNGTDWEPSTDTDFRPILSVWWPDKNTQVNYLNYMADLGFISNAD SFETGDSQSLLNEASNYVQKSIEMKISAQQSTEWLKDAMAAFIVAQPQWN ETSEDMSNDHLQNGALTYVNSPLTPDANSNFRLLNRTPTNQTGEQAYNLD NSKGGFELLLANQEDNSNVVVEAEQLNWLYYLMNFGTITANDADANFDGI RVDAVDNVDADLLQIAADYFKLAYGVDQNDATANQHLSILEDWSHNDPLY VTDQGSNQLTMDDYVHTQLIWSLTKSSDIRGTMQRFVDYYMVDRSNDSTE NEAIPNYSFVRAHDCEVQTVIAQIVSDLYPDVENSLAPTTEQLAAAFKVY NEDEKLADKKYTQYNMASAYAMLLTNKDTVPRVYYGDLYTDDGQYMATKS PYYDAINTLLKARVQYVAGGQSMSVDSNDVLTSVRYGKDAMTASDTGTSE TRTEGIGVIVSNNAELQLEDGHTVTLHMGAARKNQAYRALLSTTADGLAY YDTDENAPVAYTDANGDLIFTNESIYGVQNPQVSGYLAVWVPVGAQQDQD ARTASDTTTNTSDKVFHSNAALDSQVIYEGFSNFQAFATDSSEYTNVVIA QNADQFKQWGVTSFQLAPQYRSSTDTSFLDSIIQNGYAFTDRYDLGYGTP TKYGTADQLRDAIKALHASGIQAIADWVPDQIYNLPEQELATVTRTNSFG DDDTDSDIDNALYVVQSRGGGQYQEMYGGAFLEELQALYPSLFKVNQIST GVPIDGSVKITEWAAKYFNGSNIQGKGAGYVLKDMGSNKYFKVVSNTEDG DYLPKQLTNDLSETGFTHDDKGIIYYTLSGYRAQNAFIQDDDNNYYYFDK TGHLVTGLQKINNHTYFFLPNGIELVKSFLQNEDGTIVYFDKKGHQVFDQ YITDQNGNAYYFDDAGVNILKSGLATIDGHQQYFDQNGVQVKDKFVIGTD GYKYYFEPGSGNLAILRYVQNSKNQWFYFDGNGHAVTGFQTINGKKQYFY NDGHQSKGEFIDADGDTFYTSATDGRLVTGVQKINGITYAFDNTGNLITN QYYQLADGKYMLLDDSGRAKTGFVLQDGVLRYFDQNGEQVKDAIIVDPDT NLS. SEQIDNO:11:(Protein= sequenceofthe glucansucrase-1,2BrS) MRQKETITRKKLYKSGKSWVAAATAFAVMGVSAVTTVSADTQTPVGT TQSQQDLTGQRGQDKPTTKEVIDKKEPVPQVSAQNAGDLSADAKTTKADD KQDTQPTNAQLPDQGNKQTNSNSDKGVKESTTAPVKTTDVPSKSVTPETN TSINGGQYVEKDGQFVYIDQSGKQVSGLQNIEGHTQYFDPKTGYQTKGEL KNIDDNAYYFDKNSGNGRTFTKISNGSYSEKDGMWQYVDSHDKQPVKGLY DVEGNLQYFDLSTGNQAKHQIRSVDGVTYYFDADSGNATAFKAVTNGRYA EQTTKDKDGNETSYWAYLDNQGNAIKGLNDVNGEIQYFDEHTGEQLKGHT ATLDGTTYYFEGNKGNLVSVVNTAPTGQYKINGDNVYYLDNNNEAIKGLY GINGNLNYFDLATGIQLKGQAKNIDGIGYYFDKDTGNGSYQYTLMAPSNK NDYTQHNVVNNLSESNFKNLVDGFLTAETWYRPAQILSHGTDWVASTDKD FRPLITVWWPNKDIQVNYLRLMQNEGVLNQSAVYDLNTDQLLLNEAAQQA QIGIEKKISQTGNTDWLNNVLFTTHDGQPSFIKQQYLWNSDSEYHTGPFQ GGYLKYQNSDLTPNVNSKYRNADNSLDFLLANDVDNSNPIVQAEDLNWLY YLLNFGSITTQGKENNSNFDSIRIDAVDFVSNDLIQRTYDYLRAAYGVDK NDKEANAHLSLVEAGLDAGTTTIHQDALIESDIREAMKKSLTNGPGSNIS LSNLIQDKEGDKLIADRANNSTENVAIPNYSIIHAHDKDIQDKVGAAITD ATGADWTNFTPEQLQKGLSLYYEDQRKIEKKYNQYNIPSAYALLLTNKDT VPRVYYGDMYQDDGQYMQKQSLYFDTITALMEARKQFVAGGQTINVDDNG VLTSVRFGKGANITANDIGTNETRTQGIGVVIANDPSLKLSKDSKVTLHM GAAHRNQNYRALLLTTDNGIDSYSSSKNAPVIKTDDNGDLVFSNQDINDQ LNTKVHGFLNSEVSGYLSAWVPLDATEQQDARTLPSEKSVNDGKVLHSNA ALDSNLIYEAFSNFQPMPTNRNEYTNVVIADKADTFKSWGITSFEMAPQY RSSQDKTFLDSTIDNGYAFTDRYDLGFEKPTKYGNDEDLRQAIKQLHSSG MQVMADVVANQIYNLPGKEVASTNRVDWNGNNLSTPFGTQMYVVNTVGGG KYQNKYGGEFLDKLKAAYPDIFRSKNYEYDVKNYGGNGTGSVYYTVDSKT RAELDTDTKIKEWSAKYMNGTNVLGLGMGYVLKDWQTGQYFNVSNQNMKF LLPSDLISNDITVQLGVPVTDKKIIFDPASAYNMYSNLPEDMQVMDYQDD KKSTPSIKPLSSYNNKQVQVTRQYTDSKGVSWNLITFAGGDLQGQKLWVD SRALTMTPFKTMNQISFISYANRNDGLFLNAPYQVKGYQLAGMSNQYKGQ QVTIAGVANVSGKDWSLISFNGTQYWIDSQALNTNFTHDMNQKVFVNTTS NLDGLFLNAPYRQPGYKLAGLAKNYNNQTVTVSQQYFDDQGTVWSQVVLG GQTVWVDNHALAQMQVRDTNQQLYVNSNGRNDGLFLNAPYRGQGSQLIGM TADYNGQHVQVTKQGQDAYGAQWRLITLNNQQVWVDSRALSTTIMQAMND DMYVNSSQRTDGLLNAPYTMSGAKWAGDTRSANGRYVHISKAYSNEVGNT YYLTNLNGQSTWIDKRAFTATFDQVVALNATIVARQRPDGMFKTAPYGEA GAQFVDYVTNYNQQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQRSFSPV VTKVVDYQAKIVPRTTRDGVFSGAPYGEVNAKLVNMATAYQNQVVHATGE YTNASGITWSQFALSGQEDKLWIDKRALQA SeriesSEQIDNO:12:(Protein= mutatedsequence oftheglucansucraseN.sub.123-GBD-CD2)(W403X.sub.9; F404X.sub.10;A430X.sub.11;F431X.sub.12;andL434X.sub.13) MAHHHHHHVTSLYKKAGSAAAPFTMAQAGHYITKNGNDWQYDTNGE LAKGLRQDSNGKLRYFDLTTGIQAKGQFVTIGQETYYFSKDHGDAQLLPM VTEGHYGTITLKQGQDTKTAWVYRDQNNTILKGLQNINGTLQFFDPYTGE QLKGGVAKYDDKLFYFESGKGNLVSTVAGDYQDGHYISQDGQTRYADKQN QLVKGLVTVNGALQYFDNATGNQIKNQQVIVDGKTYYFDDKGNGEYLFTN TLDMSTNAFSTKNVAFNHDSSSFDHTVDGFLTADTWYRPKSILANGTTWR DSTDKDMRPLITVWWPNKNVQVNYLNFMKANGLLTTAAQYTLHSDQYDLN QAAQDVQVATFRRIASEHGTDWLQKLLFESQNNNPSFVKQQFIWNKDSEY HGGGDAX.sub.9X.sub.10QGGYLKYGNNPLTPTTNSDYRQPGNX.sub.11X.sub.12DFX.sub.13LAN DVDNSNPVVQAENLNWLHYLMNFGTITAGQDDANFDSIRIDAVDFIHNDT IQRTYDYLRDAYQVQQSEAKANQHISLVEAGLDAGTSTIHNDALIESNLR EAATLSLTNEPGKNKPLTNMLQDVDGGTLITDHTQNSTENQATPNYSIIH AHDKGVQEKVGAAITDATGADWTNFTDEQLKAGLELFYKDQRATNKKYNS YNIPSIYALMLTNKDTVPRMYYGDMYQDDGQYMANKSIYYDALVSLMTAR KSYVSGGQTMSVDNHGLLKSVRFGKDAMTANDLGTSATRTEGLGVIIGND PKLQLNDSDKVTLDMGAAHKNQKYRAVILTTRDGLATFNSDQAPTAWTND QGTLTFSNQEINGQDNTQIRGVANPQVSGYLAVWVPVGASDNQDARTAAT TTENHDGKVLHSNAALDSNLIYEGFSNFQPKATTHDELTNVVIAKNADVF NNWGITSFEMAPQYRSSGDHTFLDSTIDNGYAFTDRYDLGFNTPTKYGTD GDLRATIQALHHANMQVMADVVDNQVYNLPGKEVVSATRAGVYGNDDATG FGTQLYVTNSVGGGQYQEKYAGQYLEALKAKYPDLFEGKAYDYWYKNYAN DGSNPYYTLSHGDRESIPADVAIKQWSAKYMNGTNVLGNGMGYVLKDWHN GQYFKLDGDKSTLPKGGRADPAFLYKVVSAWSHPQFEK SeriesSEQIDNO:13:(Protein= mutatedsequence oftheglucansucraseN.sub.123-GBD-CD2)(F431I;D432E andL434I) MAHHHHHHVTSLYKKAGSAAAPFTMAQAGHYITKNGNDWQYDTNGE LAKGLRQDSNGKLRYFDLTTGIQAKGQFVTIGQETYYFSKDHGDAQLLPM VTEGHYGTITLKQGQDTKTAWVYRDQNNTILKGLQNINGTLQFFDPYTGE QLKGGVAKYDDKLFYFESGKGNLVSTVAGDYQDGHYISQDGQTRYADKQN QLVKGLVTVNGALQYFDNATGNQIKNQQVIVDGKTYYFDDKGNGEYLFTN TLDMSTNAFSTKNVAFNHDSSSFDHTVDGFLTADTWYRPKSILANGTTWR DSTDKDMRPLITVWWPNKNVQVNYLNFMKANGLLTTAAQYTLHSDQYDLN QAAQDVQVATFRRIASEHGTDWLQKLLFESQNNNPSFVKQQFIWNKDSEY HGGGDAWFQGGYLKYGNNPLTPTTNSDYRQPGNAIEFILANDVDNSNPVV QAENLNWLHYLMNFGTITAGQDDANFDSIRIDAVDFIHNDTIQRTYDYLR DAYQVQQSEAKANQHISLVEAGLDAGTSTIHNDALIESNLREAATLSLTN EPGKNKPLTNMLQDVDGGTLITDHTQNSTENQATPNYSIIHAHDKGVQEK VGAAITDATGADWTNFTDEQLKAGLELFYKDQRATNKKYNSYNIPSIYAL MLTNKDTVPRMYYGDMYQDDGQYMANKSIYYDALVSLMTARKSYVSGGQT MSVDNHGLLKSVRFGKDAMTANDLGTSATRTEGLGVIIGNDPKLQLNDSD KVTLDMGAAFIKNQKYRAVILTTRDGLATFNSDQAPTAWTNDQGTLTFSN QEINGQDNTQIRGVANPQVSGYLAVWVPVGASDNQDARTAATTTENHDGK VLHSNAALDSNLIYEGFSNFQPKATTHDELTNVVIAKNADVFNNWGITSF EMAPQYRSSGDHTFLDSTIDNGYAFTDRYDLGFNTPTKYGTDGDLRATIQ ALHHANMQVMADVVDNQVYNLPGKEVVSATRAGVYGNDDATGFGTQLYVT NSVGGGQYQEKYAGQYLEALKAKYPDLFEGKAYDYWYKNYANDGSNPYYT LSHGDRESIPADVAIKQWSAKYMNGTNVLGNGMGYVLKDWHNGQYFKLDG DKSTLPKGGRADPAFLYKVVSAWSHPQFEK