Patent Description:
A wild-type α1-<NUM>/<NUM> fucosidase has been isolated from Bifidobacterium longum subsp. infantis ATCC <NUM> (SEQ ID No. <NUM> of <CIT>, <NPL>), <NPL>); for its crystal structure see <NPL>)). This fucosidase is encoded by a DNA sequence of <NUM> nucleotides as set forth in the '<NUM> patent, encoding a sequence of <NUM> amino acids. According to the '<NUM> patent, human milk oligosaccharides ("HMOs") can be synthesized by contacting an oligosaccharide containing precursor with this wild-type fucosidase and then isolating a modified oligosaccharide containing precursor. The protein according to SEQ ID No. <NUM> of <CIT> is referred to as SEQ ID No. <NUM> in the present application.

<CIT> relates to methods of making mixtures of oligosaccharides by enzyme mediated methods, with one of the possible enzymes used being the wild-type α1-<NUM>/<NUM> fucosidase isolated from Bifidobacterium longum subsp. infantis ATCC <NUM>.

However, the wild-type α1-<NUM>/<NUM> fucosidase has not been entirely suitable for making fucosylated oligosaccharides, particularly fucosylated HMOs. Mutants of the enzyme have therefore been sought preferably having increased transfucosidase synthetic activity and/or decreased hydrolytic activity and/or increased thermostability, especially increased transfucosidase synthetic activity, decreased hydrolytic activity and increased thermostability.

<NPL>)) describes the conversion of an α-L fucosidase from Thermotoga maritima into an α-L-transfucosidase by directed evolution.

The present invention relates to a mutated α1-<NUM>/<NUM> transfucosidase having increased thermostability, and increased transfucosidase synthetic performance, and/or significantly reduced hydrolytic activity, compared to the wild type α1-<NUM>/<NUM> transfucosidase of SEQ ID No. <NUM>, having.

According to another aspect, the invention relates to a process for making a mutated α1-<NUM>/<NUM> transfucosidase mentioned above comprising the steps of:.

Also, a method for synthesizing a fucosylated carbohydrate is provided comprising the step of reacting a fucosyl donor and a carbohydrate acceptor in the presence of a mutant α1-<NUM>/<NUM> transfucosidase mentioned above to transfer the fucosyl residue of the fucosyl donor to the carbohydrate acceptor.

The first aspect of the invention relates to a mutated α1-<NUM>/<NUM> transfucosidase having increased thermostability, and increased transfucosidase synthetic performance, and/or significantly reduced hydrolytic activity, compared to the wild type α1-<NUM>/<NUM> transfucosidase of SEQ ID No. <NUM>, having:.

Thereby, a mutated α1-<NUM>/<NUM> transfucosidase can be obtained providing, in comparison with the wild-type α1-<NUM>/<NUM> fucosidase of SEQ ID No. <NUM>:.

The term "practically undetectable hydrolysis of the fucosylated product" preferably means that if hydrolysis of the fucosylated product by the mutated α1-<NUM>/<NUM> transfucosidase of the present invention occurs, the presence of the hydrolysis products in the sample is below the detection level. The skilled person is aware of the limit of detection of the different analytical methods. Typically, enzyme hydrolysis experiments are followed by HPLC. Under the conditions used (see e.g. Examples <NUM> and <NUM>) the hydrolysis products at below a concentration of about <NUM> % cannot be detected.

Accordingly, the present invention provides a mutated α1-<NUM>/<NUM> transfucosidase having a sequence identity of at least <NUM> % to SEQ ID No.<NUM>, and.

and
increased transfucosidase synthetic performance in a reaction between a fucosyl donor and an acceptor to produce a fucosylated product, and/or significantly reduced, preferably practically undetectable, hydrolytic activity towards the fucosylated product of such a reaction and enhanced thermostability, comparing to the wild-type α1-<NUM>/<NUM> fucosidase of SEQ ID No. <NUM>.

The polypeptide fragment from amino acid position <NUM> to <NUM> of SEQ ID No.<NUM> has been identified as the conserved domain (a sequence alignment representing a protein domain conserved during molecular evolution of the α-L-fucosidase superfamily) of the α1-<NUM>/<NUM> fucosidase from Bifidobacterium longum subsp. infantis ATCC <NUM> by the Conserved Domain Database of the National Center for Biotechnology Information (http://www. α-Fucosidases containing the conserved domain of the α1-<NUM>/<NUM> fucosidase from Bifidobacterium longum subsp. infantis ATCC <NUM> with a sequence identity of at least <NUM> % are listed in Table <NUM>.

In accordance with this invention, the terms "substantial identity" and "substantially identical" in the context of two or more nucleic acid or amino acid sequences preferably mean that the two or more sequences are the same or have at least about <NUM> % of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids (i.e. the sequences have at least about <NUM> percent (%) identity). Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST <NUM> sequence comparison algorithms with default parameters, or by manual alignment and visual inspection (see e.g. http://www. gov/BLAST/). In accordance with this invention, the percent identity of substantially identical polypeptide fragment from amino acid position <NUM> to <NUM> of SEQ ID No.<NUM>, or substantially identical amino acid sequence of SEQ ID No. <NUM>, or substantially identical nucleic acid sequences encoding the polypeptide fragment from amino acid position <NUM> to <NUM> of SEQ ID No.<NUM> or substantially identical nucleic acid sequences encoding the whole amino acid sequence of SEQ ID No.<NUM> is at least <NUM> %, preferably at least <NUM> %, more preferably at least <NUM> %, yet more preferably at least <NUM> %, and most preferably at least <NUM> %. Suitably, the definition preferably excludes <NUM> % sequence identity, such as imposing a maximum limit on the sequence identity of <NUM> %, <NUM> %, or <NUM> %, or requiring that at least one amino acid difference occurs between the sequences being compared. This definition also applies to the complement of a test sequence and to sequences that have deletions and/or additions, as well as those that have substitutions. An example of an algorithm that is suitable for determining percent identity and sequence similarity is the BLAST <NUM>. <NUM>+ algorithm, which is described in <NPL>). BLAST <NUM>. <NUM>+ is used to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.

Fucosidases transfer a fucosyl residue from a donor oligosaccharide to an acceptor. If the acceptor is another carbohydrate (mono- or oligosaccharide) then the fucosidase acts as transfucosidase (able to make a fucosylated carbohydrate product). On the other hand, the same fucosidase can transfer the same fucosyl residue, which was added to the carbohydrate acceptor previously, from the product to a water molecule, acting thus as a hydrolase. The two processes take place concurrently. The overall synthetic performance is the ratio of the transfucosidase and hydrolysis activities. If the overall synthetic performance is below <NUM>, then the hydrolysis activity is dominant, and if the overall synthetic performance is more than <NUM>, then the transfucosidase activity is dominant. The experimental overall synthetic performance of the wild-type α1-<NUM>/<NUM> fucosidase from Bifidobacterium longum subsp. infantis ATCC <NUM> is about <NUM> (as determined in the <NUM>-FL + LNnT <IMG> LNFP-III + Lac reaction, see Example <NUM>).

By comparison, the mutant fucosidases of this invention show a much higher overall synthetic performance, preferably higher than <NUM>, which means a much higher transfucosidase activity relative to hydrolytic activity. In this regard, a relatively low transfucosidase synthetic activity of a mutant of this invention can be compensated for by a significant reduction in the hydrolytic activity of the mutant that results in an improved synthetic performance (that is, the transfucosidase-mediated hydrolysis of the fucosylated product is significantly less than its transfucosidase-mediated synthesis, so that the equilibrium of the competing reactions is shifted to the product formation). Similarly, a relatively high hydrolytic activity of a mutant can be overcome by a significant improvement in its transfucosidase synthetic activity. The mutated α1-<NUM>/<NUM> fucosidases of this invention show a substantial improvement in their transfucosidase synthetic performance over the wild-type fucosidase of SEQ ID No.<NUM>, that is, at least a <NUM>-fold, preferably at least a <NUM>-fold, more preferably at least a <NUM>-fold, even more preferably a <NUM>-fold, particularly a <NUM>-fold improvement over the wild-type fucosidase. As a consequence of this increased transfucosidase synthetic performance, the amount of the mutated α1-<NUM>/<NUM> transfucosidase of the invention, used in the synthesis of a fucosylated product, can be significantly reduced and reaction times can be significantly shortened, which can lower the costs of synthesizing fucosylated oligosaccharide products, particularly fucosylated HMOs.

Suitably, the mutant fucosidases of the invention are non-natural fucosidases, that is, they are not made in nature or naturally-occurring, but are made as a result of chemical synthesis, genetic engineering or similar methods in the laboratory, resulting in synthetic mutant fucosidases.

The combination of mutations as disclosed above imparts not only a further improved transfucosidase synthetic performance to the mutated enzyme but an enhanced stability, particularly temperature stability.

The α1-<NUM>/<NUM> transfucosidase comprises an amino acid sequence that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM> as described above, and an amino acid mutation:.

In this aspect, at position <NUM> Trp (W) is substituted by Phe or Tyr; at position <NUM> Ser (S) is substituted by Glu (E); at position <NUM>, Ala (A) is substituted by Asn, His or Phe; at position <NUM>, Glu (E) is substituted by His (H); and at position <NUM>, Glu (E) is substituted by Arg (R).

Preferably, the α1-<NUM>/<NUM> transfucosidase comprises, more preferably consists of, the sequence of SEQ ID NO <NUM> having mutations:.

In a more preferred embodiment, the α1-<NUM>/<NUM> transfucosidase comprises an amino acid sequence that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM> as described above, and mutation to the amino acid sequence at position <NUM> and <NUM> plus a further position selected from <NUM> and <NUM>.

In a more preferred embodiment, the α1-<NUM>/<NUM> transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No. <NUM> having mutations to the amino acid sequence at positions <NUM> and <NUM> plus a further position selected from <NUM> and <NUM>.

Preferably, the α1-<NUM>/<NUM> transfucosidase of this first aspect comprises a further mutation at the amino acid position selected from <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and more preferably from <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

α-Fucosidases having at least about <NUM> percent sequence identity to SEQ ID No. <NUM>, are listed in Table <NUM>.

Even more preferably, the α1-<NUM>/<NUM> transfucosidase comprises an amino acid sequence that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM> as described above, and amino acid mutations:.

Even more preferably, the α1-<NUM>/<NUM> transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No.<NUM> as described above having mutations:.

The above combination of mutations imparts not only a further improved transfucosidase synthetic performance to the mutated enzyme but a further enhanced stability, particularly temperature stability while maintaining further improved transfucosidase synthetic performance. The mutations at <NUM> and <NUM> are the following:.

and at <NUM>, <NUM>, <NUM>, <NUM> and <NUM> are preferably the following:.

In the first aspect of the invention, the mutated α1-<NUM>/<NUM> fucosidase that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM>, within the invention as set out above, has a mutation at amino acid position <NUM>, and optionally also <NUM>, to provide significantly or completely suppressed hydrolytic activity. In this regard, at position <NUM>, Ala (A) is preferably replaced by Phe (F), Asn (N) or His (H) and at position <NUM>, Val (V) is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K). The suppressed hydrolytic activity is beneficial because the mutated enzyme then does not significantly degrade the donor and/or the product by hydrolysis. As a result, the transfucosidase reaction is no longer kinetically controlled, and a much better synthesis/hydrolysis ratio (meaning a better synthetic performance) can be achieved. Mutation at one or both, preferably both, of the above amino acid positions can provide at least a <NUM>-fold, preferably at least a <NUM>-fold, more preferably at least a <NUM>-fold reduced hydrolytic activity towards the fucosylated products.

In addition, according to a certain embodiment, a mutated α1-<NUM>/<NUM> fucosidase that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM>, and, within the invention as set out above, comprises mutation of.

The combination of mutations as disclosed above imparts not only a significantly reduced, preferably practically undetectable, hydrolysis of the fucosylated product but an enhanced stability, particularly temperature stability.

Preferably, in the mutation of the amino acid at position <NUM> and optionally also <NUM>, at position <NUM>, Ala (A) is replaced by Phe (F), Asn (N) or His (H) and/or at position <NUM>, Val (V) is replaced by Arg (R), Glu (E), His (H) or Lys (K).

According to the second aspect of the invention, a method is provided for making a mutated α1-<NUM>/<NUM> transfucosidase of the first aspect of the invention, comprising the steps of:.

Step (a) can be carried out in a conventional manner by making a mutant DNA sequence encoding the mutated α1-<NUM>/<NUM> transfucosidase of the invention, from a DNA sequence encoding a protein comprising a polypeptide that has a sequence identity of at least <NUM> % to SEQ ID No. <NUM>, or comprising, preferably consisting of, the entire SEQ ID No. <NUM>. In step (b) the so-mutated DNA sequence is then introduced at the gene level by usual molecular-biological methods. The DNA sequence of the enzyme variants can be cloned in an expression vector which can be introduced in an appropriate host expression strain such as E. coli, containing DNA plasmids with the required information for regulation of expression of the enzyme variant. The sequence encoding the enzyme variant can be placed under the control of an inducible promoter. As a result, by adding an inducer, the expression of the enzyme variant can be controlled (generally, isopropyl-β-D-thiogalactopyranoside (IPTG) is used). The so-transformed host cells are then cultured in conventional nutrient media (e.g. Lennox broth, minimal medium M9) and induced with IPTG. After expression, the biomass can be harvested by centrifugation. The mutated enzyme can be isolated from the biomass after appropriate cell lysis and purification. In this process, conventional centrifugation, precipitation, ultrafiltration and/or chromatographic methods can be used.

According to the third aspect of the invention, a method is provided for synthesizing a fucosylated carbohydrate by reacting a fucosyl donor and a carbohydrate acceptor in the presence of a mutated α1-<NUM>/<NUM> transfucosidase of the first aspect of the invention, whereby the fucosyl residue of the fucosyl donor is transferred to the carbohydrate acceptor.

In the following paragraphs, the expression "may carry" is equivalent with the expression "optionally carries", and the expression "can be substituted" is equivalent with the expression "is optionally substituted".

The carbohydrate acceptor used in the third aspect of the invention can be any mono- or oligosaccharide, preferably an oligosaccharide of <NUM>-<NUM> monosaccharide units that the mutated a1-<NUM>/<NUM> fucosidase is able to accept. The oligosaccharide acceptor preferably contains a N-acetyl-glucosamine unit which forms a N-acetyl-lactosaminyl (Galpβ1-4GlcNAcp) or a lacto-N-biosyl (Galpβ1-3GlcNAcp) fragment with an adjacent galactose and/or it contains a glucose unit which is advantageously at the reducing end and preferably has a free <NUM>-OH group. More preferably, the oligosaccharide acceptor having <NUM>-<NUM> units comprises a N-acetyl-lactosaminyl or lacto-N-biosyl moiety and is of formula <NUM>, or is a lactose derivative of formula <NUM>
<CHM>.

Preferably, compounds of formula <NUM> are of formulae 1a or 1b
<CHM>.

More preferably, compounds of formulae 1a and 1b have one or more of the following linkages and modifications:.

Even more preferably, a compound of formula 1a, 1b or <NUM> is selected from the group consisting of <NUM>'-O-fucosyllactose (<NUM>'-FL), <NUM>'-O-sialyllactose (<NUM>'-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I, Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), Galβ1-4GlcNAcβ1-3Galβ1-<NUM>[Fucα1-<NUM>]Glc, lacto-N-fucopentaose V (LNFP-V, Galβ1-3GlcNAcβ1-3Galβ1-<NUM>[Fucα1-<NUM>]Glc), Galβ1-4GlcNAcβ1-3Galβ1-<NUM>[Fucα1-<NUM>]Glc, lacto-N-hexaose (LNH, Galβ1-3GlcNAcβ1-<NUM>[Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-<NUM>[Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), para-lacto-N-hexaose (pLNH, Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), para-lacto-N-neohexaose (pLNnH, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), fucosyl-LNH I (FLNH-I, Fucα1-2Galβ1-3GlcNAcβ1-<NUM>[Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), fucosyl-LNH II (FLNH-II, Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-<NUM>[Galβ1-3GlcNAcβ1-<NUM>]Galβ1-4Glc), fucosyl-para-LNH I (FpLNH-I, Galβ1-3GlcNAcβ1-3Galβ1-<NUM>[Fuca1-<NUM>]GlcNAcβ1-3Galβ1-4Glc), fucosyl-para-LNH II (FpLNH-II, Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), Galβ1-4GlcNAcβ1-3Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4Glc, Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc, difucosyl-LNH I (DFLNH-I, Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-<NUM>[Fucα1-2Galβ1-3GlcNAcβ1-<NUM>]Galβ1-4Glc), difucosyl-para-LNH (DFpLNH, Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4Glc), difucosyl-para-LNnH (DFpLNnH, Galβ1-<NUM>[Fuca1-<NUM>]GlcNAcβ1-3Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4Glc),lacto-N-octaose (LNO, Galβ1-3GlcNAcβ1-<NUM>[Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), lacto-N-neooctaose (LNnO, Galβ1-4GlcNAcβ1-<NUM>[Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), iso-lacto-N-octaose (iLNO, Galβ1-3GlcNAcβ1-<NUM>[Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), para-lacto-N-octaose (pLNO, Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), LST a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc), LST c (NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc), sialyl-LNH (SLNH, Galβ1-3GlcNAcβ1-<NUM>[NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), sialyl-LNnH I (SLNnH-I, Galβ1-4GlcNAcβ1-<NUM>[NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), sialyl-LNnH II (SLNnH-II, Galβ1-4GlcNAcβ1-<NUM>[NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), disialyl-LNT (DSLNT, NeuAcα2-3Galβ1-<NUM>[NeuAcα2-<NUM>]GlcNAcβ1-3Galβ1-4Glc), fucosyl-sialyl-LNH (FSLNH, NeuAcα2-3Galβ1-3GlcNAcβ1-<NUM>[Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-<NUM>]Galβ1-4Glc), fucosyl-sialyl-LNH II (FSLNH-II, Fucα1-2Galβ1-3GlcNAcβ1-<NUM>[NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), disialyl-LNH I (DSLNH-I, NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>[NeuAcα2-3Galβ1-3GlcNAcβ1-<NUM>]Galβ1-4Glc), disialyl-LNH II (DSLNH-II, Galβ1-4GlcNAcβ1-<NUM>[NeuAcα2-3Galβ1-<NUM>[NeuAcα2-<NUM>]GlcNAcβ1-<NUM>]Galβ1-4Glc) and disialyl-LNnH (DSLNnH, NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>[NeuAcα2-6Galβ1-4GlcNAcβ1-<NUM>]Galβ1-4Glc), advantageously <NUM>'-FL, <NUM>'-SL, LNT, LNnT, LNFP-I, LNFP-V, LNH, LNnH, pLNH, pLNnH and DSLNT.

A mutated α1-<NUM>/<NUM> fucosidase of the first aspect of the invention demonstrates a strong α1-<NUM>/<NUM> selectivity when carrying out the method of the third aspect of the invention. As a result, the product of the reaction is an α1-<NUM>- or a α1-<NUM>-fucosyl mono- or oligosaccharide, preferably an oligosaccharide of <NUM>-<NUM> monomer units, exclusively, and no an α1-<NUM>-fucosylated product can be detected. Preferably, the mutated α1-<NUM>/<NUM> transfucosidase brings the fucosyl residue of an appropriate donor to the <NUM>-position of the glucose in an acceptor of formula <NUM>, to the <NUM>-position of the N-acetyl-glucosamine in a, preferably terminal, N-acetyl-lactosaminyl group in an acceptor of formula <NUM>, 1a or 1b, or to the <NUM>-position of the N-acetyl-glucosamine in a, preferably terminal, lacto-N-biosyl group, in an acceptor of formula <NUM>, 1a or 1b. Accordingly, a mutated α1-<NUM>/<NUM> transfucosidases of the invention is preferably used to synthesize fucosylated HMOs such as DFL, FSL, or those in which the fucosyl residue is attached to a GlcNAc moiety with α1-<NUM> or α1-<NUM> linkage, more preferably to the fucosylated HMOs listed in Table <NUM> below (for abbreviations see <NPL>).

The fucosyl donor used in the third aspect of the invention can be any fucosyl compound from which the mutated α1-<NUM>/<NUM> fucosidase is able to transfer the fucosyl residue to a carbohydrate acceptor as described above. Accordingly, the fucosyl donor can be an α1-<NUM> or α1-<NUM> fucosyl saccharide, preferably of <NUM> or <NUM> monosaccharide units including the fucosyl residue, more preferably <NUM>-FL or DFL, or a compound of formula <NUM>
<CHM>
wherein X is selected from the group consisting of azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, group A, group B, group C and group D
<CHM>
wherein Ra is independently H or alkyl, or two vicinal Ra groups represent a =C(Rb)<NUM> group, wherein Rb is independently H or alkyl, Rc is independently selected from the group consisting of alkoxy, amino, alkylamino and dialkylamino, Rb is selected from the group consisting of H, alkyl and -C(=O)Re, wherein Re is OH, alkoxy, amino, alkylamino, dialkylamino, hydrazino, alkylhydrazino, dialkylhydrazino or trialkylhydrazino,
preferably X in formula <NUM> is selected from the group consisting of phenoxy-, p-nitrophenoxy-, <NUM>,<NUM>-dinitrophenoxy-, <NUM>-chloro-<NUM>-nitrophenoxy-, <NUM>,<NUM>-dimethoxy-<NUM>,<NUM>,<NUM>-triazin-<NUM>-yloxy-, <NUM>,<NUM>-diethoxy-<NUM>,<NUM>,<NUM>-triazin-<NUM>-yloxy-, <NUM>-ethyl-<NUM>-methyl-<NUM>-oxo-(<NUM>)-furan-<NUM>-yloxy-, <NUM>-ethyl-<NUM>-methyl-<NUM>-oxo-(<NUM>)-furan-<NUM>-yloxy- and <NUM>,<NUM>-dimethyl-<NUM>-oxo-(<NUM>)-furan-<NUM>-yloxy-group. Advantageously, the fucosyl donor is <NUM>-FL or DFL.

A mutated α1-<NUM>/<NUM> transfucosidase of the invention comprising a polypeptide that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM>, or comprising, preferably consisting of, the sequence of SEQ ID NO <NUM>, and mutation at amino acid position <NUM> and at amino acid position <NUM> or <NUM>, is especially suitable for making.

More preferably, in a fucosylation reaction, wherein the fucosyl donor is <NUM>-FL and the acceptor is.

In the examples below mutants of Bifidobacterium longum subsp. infantis ATCC <NUM> were tested, the position(s) of mutation is/are according to SEQ ID No. <NUM>. Not all tested mutants are according to the invention, and those not according to the invention are indicated within the examples as "(comparative)".

The transfucosidase activity of mutants was investigated on the <NUM>-FL + LNnT <IMG> LNFP-III + Lac reaction in which the formation of LNFP-III was followed.

The reaction was performed at <NUM> in <NUM>µl scale using <NUM> LNnT and <NUM> <NUM>-FL. Samples were taken typically after <NUM>, <NUM>, <NUM> and <NUM> and the reaction was stopped by adding <NUM>µl of acetonitrile/water <NUM>:<NUM>.

The hydrolytic activity of mutants was investigated in a similar procedure using LNFP-III (<NUM>) as only substrate, and depletion of LNFP-III and formation of LNnT were followed over time.

HPLC conditions: Kinetex <NUM>,6µ HILIC 100A-column (150x4. <NUM>) was used with a flow of <NUM>/min using <NUM> % acetonitrile and <NUM> % <NUM> ammonium formate buffer (pH <NUM>). The elution of substrates and products was detected at <NUM>. For the quantification of LNnT and LNFP-III the peak areas were compared to an external standard.

The measured activity data are summarized in the table below. The synthetic performance was calculated as the ratio: synthesis [U/mg]/hydrolysis [U/mg], wherein 1U= production or hydrolysis of <NUM>µmol of LNFP-III per min.

The hydrolytic activity of mutants was investigated according to the procedure described in Example <NUM>.

The melting temperature (Tm) is the temperature at which <NUM> % of the initial activity of the enzyme remains after <NUM> of incubation at elevated temperatures.

Activities were measured by HPLC analysis of <NUM>-FL + LNnT <IMG> LNFP-III + Lac reaction in which the formation of LNFP-III was followed.

Increasing the thermostability (Tm) of the wild type protein of SEQ ID No. <NUM>:.

Increasing the thermostability of mutants designed for increased transfucosidase synthetic performance or reduced hydrolytic activity:.

A) Saturation mutagenesis was screened at positions <NUM>, <NUM>, <NUM> and <NUM> in the following reactions:.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNnT]= <NUM>, [LNFP-I]= <NUM>, with <NUM>µl of crude enzyme extract. Conversions were measured after <NUM>, <NUM>, <NUM> and <NUM> hours. The tables below show the conversions (%) after <NUM>, <NUM> hours and the maximum conversion during the course. WT values are italic.

B) In <NUM>-FL + LNnT <IMG> LNFP-III + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNnT]= <NUM>, enzyme extract= <NUM> or <NUM>/ml.

HPLC conditions: TSK Gel amide <NUM> (Tosoh, <NUM>, <NUM> x <NUM>) was used with a flow of <NUM>/min using <NUM> % acetonitrile and <NUM> % water. The elution of substrates and products was detected by CAD and/or UV detection at <NUM>.

The tables show the LNFP-III formation (%) as a function of time.

C) In <NUM>-FL + LNT <IMG> LNFP-II + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNT]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The table shows the LNFP-II formation (%) as a function of time.

Galβ1-4GlcNAcβ1-<NUM>[Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-<NUM>]Galβ1-4Glc + DFLNnH + Lac reaction The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNnH]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The tables show the monofucosylated and difucosylated LNnH formation (%), respectively, as a function of time.

E) In <NUM>-FL + pLNnH <IMG> Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM> or <NUM>, [pLNnH]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The tables show the fucosylated pLNnH formation (%) as a function of time.

F) In <NUM>-FL + LNFP-I <IMG> LNDFH-I + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNFP-I]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The table shows the LNDFH-I formation (%) as a function of time.

Another test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNFP-I]= <NUM>, enzyme extract= <NUM>µl. HPLC: see Example <NUM>. The table shows the LNDFH-I formation (%) as a function of time.

G) In DFL + LNnT <IMG> LNFP-III + <NUM>'-FL reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [DFL]= <NUM>, [LNnT]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The table shows the LNFP-III formation (%) as a function of time.

H) In <NUM>-FL + <NUM>'-FL <IMG> DFL + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [<NUM>'-FL]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The table shows the DFL formation (%) as a function of time.

A) In <NUM>-FL + LNnT <IMG> LNFP-III + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNnT]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The table shows the LNFP-III formation (%) as a function of time.

Another test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNT]= <NUM>, enzyme extract= <NUM>/ml. Samples were taken after <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. HPLC: see Example <NUM>. The table shows the activity in [U/mg] wherein 1U= production of 1µmοl LNFP-II per min.

C) In <NUM>-FL + pLNnH <IMG> Galβ1-<NUM>[Fucα1-<NUM>]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [pLNnH]= <NUM>, enzyme extract= <NUM>/ml. HPLC: see Example <NUM> B). The table shows the fucosylated pLNnH formation (%) as a function of time.

D) In <NUM>-FL + LNFP-I <IMG> LNDFH-I + Lac reaction.

Another test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM> <NUM>µl), [<NUM>-FL]= <NUM>, [LNFP-I]= <NUM>, enzyme extract= <NUM>µl crude extract. HPLC: see example <NUM>. The table shows the LNDFH-I formation (%) as a function of time.

Another test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [LNFP-I]= <NUM>, enzyme extract= <NUM>/ml. Samples were taken after <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. HPLC: see Example <NUM>. The table shows the activity in [U/mg] wherein 1U= production of 1µmol LNDFH-I per min.

E) In <NUM>-FL + <NUM>'-FL <IMG> DFL + Lac reaction.

The test was run in sodium phosphate buffer (<NUM>, pH= <NUM>, <NUM>, <NUM>µl), [<NUM>-FL]= <NUM>, [<NUM>'-FL]= <NUM>, enzyme extract= <NUM>/ml. Samples were taken after <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. HPLC: see Example <NUM>. The table shows the activity in [U/mg] wherein 1U= production of 1µmol <NUM>'-FL per min.

Claim 1:
A mutated α1-<NUM>/<NUM> transfucosidase having increased thermostability, and increased transfucosidase synthetic performance, and/or significantly reduced hydrolytic activity, compared to the wild type α1-<NUM>/<NUM> transfucosidase of SEQ ID No. <NUM>, having:
- an amino acid sequence that has a sequence identity of at least <NUM> % to SEQ ID No.<NUM>, and
- a mutation at amino acid positions <NUM> and <NUM>, and at least one further mutation at an amino acid position selected from <NUM>, <NUM> and <NUM>, said amino acid numbering being according to SEQ ID No. <NUM>,
wherein :
- at position <NUM> Trp (W) is substituted by Phe (F) or Tyr (Y);
- at position <NUM>, Ser (S) is substituted by Glu (E);
- at position <NUM> Ala (A) is substituted by Asn (N), His (H) or Phe (F);
- at position <NUM> Glu (E) is substituted by His (H);
- at position <NUM>, Glu (E) is substituted by Arg (R).