This invention relates to an α1-¾ transfucosidase having increased transfucosidase synthetic activity, decreased hydrolytic activity and/or increased thermostability.
A wild-type α1-¾ fucosidase has been isolated from Bifidobacterium longum subsp. infantis ATCC 15697 (SEQ ID No. 18 of U.S. Pat. No. 8,361,756, Sela et al. Proc. Natl. Acad. Sci. USA 105, 18964 (2008), Sela et al. Appl. Environ. Microbiol. 78, 795 (2012); for its crystal structure see Sakurama et al. J. Biol. Chem. 287, 16709 (2012)). This fucosidase is encoded by a DNA sequence of 1437 nucleotides as set forth in the '756 patent, encoding a sequence of 478 amino acids. According to the '756 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. 18 of U.S. Pat. No. 8,361,756 is referred to as SEQ ID No. 1 in the present application.
However, the wild-type α1-¾ 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.
The present invention relates to a mutated α1-¾ transfucosidase having
Preferably, the mutated α1-¾ transfucosidase comprises one or more of the following mutations:
It is also provided a mutated α1-¾ transfucosidase having
According to another aspect, the invention relates to a process for making a mutated α1-¾ 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 α1-¾ transfucosidase mentioned above to transfer the fucosyl residue of the fucosyl donor to the carbohydrate acceptor.
In a further aspect of the invention, use of a mutated α1-¾ transfucosidase mentioned for the preparation of a fucosylated carbohydrate, preferably a fucosylated human milk oligosaccharide having an α1-3 and/or a α1-4 fucosyl residue, is provided.
The first aspect of the invention relates to a mutated α1-¾ fucosidase comprising a polypeptide fragment having:
Thereby, a mutated α1-¾ fucosidase can be obtained providing, in comparison with the wild-type α1-¾ fucosidase of SEQ ID No. 1:
The term “practically undetectable hydrolysis of the fucosylated product” preferably means that if hydrolysis of the fucosylated product by the mutated α1-¾ fucosidase 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 1 and 2) the hydrolysis products at below a concentration of about 1% cannot be detected.
Accordingly, the present invention provides a mutated α1-¾ fucosidase comprising a polypeptide fragment having a sequence identity of at least 75% to a polypeptide fragment from amino acid position 56 to 345 of SEQ ID No.1, and
and/or
Moreover, the present invention provides a mutated α1-¾ fucosidase, comprising a polypeptide fragment having a sequence identity of at least 75% to a polypeptide fragment from amino acid position 56 to 345 of SEQ ID No.1, and
and/or,
The polypeptide fragment from amino acid position 56 to 345 of SEQ ID No.1 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 α1-¾ fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697 by the Conserved Domain Database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). α-Fucosidases containing the conserved domain of the α1-¾ fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697 with a sequence identity of at least 75% are listed in Table 1.
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 75% 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 75 percent (%) identity). Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection (see e.g. http://www.ncbi.nlm.nih.gov/BLAST/). In accordance with this invention, the percent identity of substantially identical polypeptide fragment from amino acid position 56 to 345 of SEQ ID No.1, or substantially identical amino acid sequence of SEQ ID No. 1, or substantially identical nucleic acid sequences encoding the polypeptide fragment from amino acid position 56 to 345 of SEQ ID No.1 or substantially identical nucleic acid sequences encoding the whole amino acid sequence of SEQ ID No.1 is preferably at least 80%, more preferably at least 85%, yet more preferably at least 90%, still even more preferably at least 92%, especially at least 93%, more especially at least 94%, even more especially at least 95%, yet even more especially at least 96%, particularly at least 97%, more particularly at least 98%, and most particularly at least 99%. Suitably, the definition preferably excludes 100% sequence identity, such as imposing a maximum limit on the sequence identity of 99.9%, 99.8%, or 99.7%, 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 2.2.20+ algorithm, which is described in Altschul et al. Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20+ 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.ncbi.nlm.nih.gov/).
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 1, then the hydrolysis activity is dominant, and if the overall synthetic performance is more than 1, then the transfucosidase activity is dominant. The experimental overall synthetic performance of the wild-type α1-¾ fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697 is about 0.03 (as determined in the 3-FL+LNnT LNFP-III+Lac reaction, see Example 1).
By comparison, the mutant fucosidases of this invention show a much higher overall synthetic performance, preferably higher than 1, 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 α1-¾ fucosidases of this invention show a substantial improvement in their transfucosidase synthetic performance over the wild-type fucosidase of SEQ ID No.1, that is, at least a 50-fold, preferably at least a 100-fold, more preferably at least a 500-fold, even more preferably a 1000-fold, particularly a 2000-fold improvement over the wild-type fucosidase. As a consequence of this increased transfucosidase synthetic performance, the amount of the mutated α1-¾ 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.
According to a preferred embodiment of the first aspect of the invention, the mutated α1-¾ transfucosidase comprises a polypeptide fragment that has at least 75% sequence identity to the segment from amino acid positions 56 to 345 of SEQ ID No.1, and an amino acid mutation at least at one or more of the following amino acid positions: 134, 135, 174, 216, 221 and 282.
More preferably, the α1-¾ transfucosidase of this first aspect comprises a polypeptide domain that has a sequence identity of at least 75% to the segment from amino acid positions 56 to 345 of SEQ ID No.1, and an amino acid mutation at least at amino acid position 174, and at one or more of the following amino acid positions: 134, 135, 170, 216, 221, 236, 237, 241, 244, 245 and 282, preferably at one or more of the following amino acid positions: 134, 135, 216, 221 and 282.
Also more preferably, the α1-¾ transfucosidase of this first aspect comprises a polypeptide domain that has a sequence identity of at least 75% to the segment from amino acid positions 56 to 345 of SEQ ID No.1, and an amino acid mutation at least at amino acid position 135, and at one or more of the following amino acid positions: 134, 170, 174, 216, 221, 236, 237, 241, 244, 245 and 282, preferably at one or more of the following amino acid positions: 134, 135, 216, 221 and 282.
Even more preferably, the α1-¾ transfucosidase of this first aspect comprises a polypeptide domain that has a sequence identity of at least 75% to the segment from amino acid positions 56 to 345 of SEQ ID No.1, and an amino acid mutation at least at amino acid positions 135 and 174, and at one or more of the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably at one or more of the following amino acid positions: 134, 216, 221 and 282.
Optionally, the α1-¾ transfucosidase of this first aspect comprises a polypeptide domain that has a sequence identity of at least 75% to the segment from amino acid positions 56 to 345 of SEQ ID No.1, and an amino acid mutation at least at amino acid positions 135 and/or 174, and at one or more of the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably at one or more of the following amino acid positions: 134, 216, 221 and 282, and there is a further mutation at one or more of the following amino acid positions: 165, 168, 232, 237, 258, 260 or 274.
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.
Yet more preferably, the α1-¾ transfucosidase of this first aspect comprises polypeptide domain that has a sequence identity of at least 75% to the segment from amino acid positions 56 to 345 of SEQ ID No.1 as described above, and the following amino acid mutations, in which:
Preferably, the α1-¾ transfucosidase of this first aspect comprises the sequence of the entire polypeptide domain from amino acid position 56 to 345 of SEQ ID No. 1 having the following mutations:
Within the first aspect of the invention concerning the provision of mutated α1-¾ fucosidases having increased transfucosidase synthetic performance in a reaction between a fucosyl donor and an acceptor to yield a fucosylated product, it is preferred that the mutated α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1, and an amino acid mutation at least at one or more of the following amino acid positions: 134, 135, 170, 174, 216, 221, 236, 237, 244, 245 and 282, preferably at least at one or more of the following amino acid positions: 134, 135, 174, 216, 221 and 282.
Accordingly, the present invention provides a mutated α1-¾ fucosidase comprising an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 and
and/or
Moreover, a mutated α1-¾ fucosidase is provided comprising an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 and
and/or, preferably and
α-Fucosidases containing a substantially identical amino acid sequence of SEQ ID No.1, that is α-fucosidases having at least about 75 percent sequence identity to SEQ ID No. 1, are listed in Table 2.
More preferably, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% with SEQ ID No.1, and an amino acid mutation at least at amino acid position 174, and at one or more of the following amino acid positions: 134, 135, 170, 216, 221, 236, 237, 241, 244, 245 and 282, preferably at one or more of the following amino acid positions: 134, 135, 216, 221 and 282.
Also more preferably, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1, and an amino acid mutation at least at amino acid position 135, and at one or more of the following amino acid positions: 134, 170, 174, 216, 221, 236, 237, 241, 244, 245 and 282, preferably at one or more of the following amino acid positions: 134, 135, 216, 221 and 282.
Even more preferably, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1, and an amino acid mutation at least: at amino acid positions 135 and 174, and at one or more of the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably at one or more of the following amino acid positions: 134, 216, 221 and 282.
Optionally, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1, and an amino acid mutation at least: at amino acid positions 135 and/or 174, and at one or more of the following amino acid positions: 134, 170, 216, 221, 236, 237, 241 and 282, preferably at one or more of the following amino acid positions: 134, 216, 221 and 282, and there is a further mutation at one or more of the following amino acid positions: 165, 168, 232, 237, 258, 260, 274 and 413.
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.
Preferably, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 as described above, and an amino acid mutation:
Preferably, the α1-¾ transfucosidase comprises, more preferably consists of, the sequence of SEQ ID NO 1 having mutations:
In this aspect, at position 135 Trp (W) is preferably substituted by Ala, Asp, Asn, Glu, Gln, His, Phe, Leu, Lys, Val or Tyr, more preferably Phe or Tyr; at position 168 Ser (S) is preferably substituted by Glu (E); at position 174, Ala (A) is preferably substituted by Arg, Asn, Cys, Glu, Ile, His, Leu, Lys, Met, Phe, Trp, Tyr or Val, more preferably Asn, His or Phe; at position 237, Glu (E) is preferably substituted by His (H); and at position 413, Glu (E) is substituted by Arg (R).
In a more preferred embodiment, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 as described above, and an amino acid mutation:
In a more preferred embodiment, the α1-¾ transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No.1 as described above having mutations:
In a more preferred embodiment, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 as described above, and an amino acid mutation:
In a more preferred embodiment, the α1-¾ transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No.1 having mutations:
In a more preferred embodiment, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 as described above, and mutations to the amino acid sequence at three positions selected from 135, 168, 174 and 413.
In a more preferred embodiment, the α1-¾ transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No. 1 having mutations to the amino acid sequence at three positions selected from 135, 168, 174 and 413.
Even more preferably, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 as described above, and an amino acid mutations:
Even more preferably, the α1-¾ transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No.1 as described above having mutations:
Even more preferably, the α1-¾ transfucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 as described above, and an amino acid mutations:
Even more preferably, the α1-¾ transfucosidase comprises, more preferably consists of, the sequence of SEQ ID No.1 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 135, 165, 174, 232, 258, 260 and 274 are preferably the following:
Another embodiment of the first aspect of the invention relates to a mutated α1-¾ fucosidase that comprises a polypeptide fragment that has a sequence identity of at least 75% to the segment from amino acid position 56 to 345 of SEQ ID No.1, and a mutation of at least at amino acid position 174 or 282, preferably at least at both amino acids, to provide significantly or completely suppressed hydrolytic activity. In this regard, at position 174, Ala (A) is preferably replaced by Phe (F), Asn (N) or His (H) and/or at position 282, 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 100-fold, preferably at least a 1000-fold, more preferably at least a 10000-fold reduced hydrolytic activity towards the fucosylated products.
Accordingly, a mutated α1-¾ fucosidase is provided having
and/or
In addition, according to a certain embodiment, a mutated α1-¾ fucosidase that has a sequence identity of at least 75% to the fragment from amino acid position 56 to 345 of SEQ ID No.1, and 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.
Therefore a mutated α1-¾ fucosidase is also provided, having
and/or
when compared to the protein according to SEQ ID No. 1.
Preferably, the mutated α1-¾ fucosidase comprises an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1, mutation of at least the amino acid at position 174 or 282, preferably at least both amino acid positions, more preferably at position 174, Ala (A) is preferably replaced by Phe (F), Asn (N) or His (H) and/or at position 282, Val (V) is preferably replaced by Arg (R), Glu (E), His (H) or Lys (K).
The second aspect of the invention relates to a mutated α1-¾ fucosidase comprising an amino acid sequence that has a sequence identity of at least 75% to SEQ ID No.1 and at least one mutation at amino acid position 165, 168, 232, 237, 258, 260, 274 or 413. The so-mutated α1-¾ fucosidase shows enhanced stability in comparison to the protein of SEQ ID No.1, preferably enhanced thermostability, which allows the synthesis of a fucosylated product to be carried out effectively under more stringent conditions, particularly higher temperatures, which frequently leads to faster reaction times.
Concerning this second aspect, the mutated α1-¾ transfucosidase preferably comprises the following mutations:
Accordingly, a mutated α1-¾ fucosidase is provided having
and/or
Preferably, the α1-¾ transfucosidase of this second aspect comprises at least two amino acid mutations at positions selected from 165, 168, 232, 237, 258, 260, 274 or 413.
Preferably, the α1-¾ transfucosidase of this second aspect comprises at least two amino acid mutations, one of which is at position 413, and the other is selected from the group consisting of position 165, 168, 232, 237, 258, 260 and 274.
More preferably, the α1-¾ transfucosidase of this second aspect comprises at least two amino acid mutations at positions selected from 168, 237 and 413.
Even more preferably, the α1-¾ transfucosidase of this second aspect comprises at least two amino acid mutations, one of which is at position 413, and the other is selected from 168 and 237.
According to the third aspect of the invention, a method is provided for making a mutated α1-¾ transfucosidase of the first or second 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 α1-¾ transfucosidase of the invention, from a DNA sequence encoding a protein comprising a polypeptide fragment that has a sequence identity of at least 75% to the fragment of amino acid positions 56 to 345 of SEQ ID No. 1, or comprising the fragment of amino acid positions 56 to 345 of SEQ ID No. 1, or comprising a polypeptide that has a sequence identity of at least 75% to SEQ ID No. 1, or comprising, preferably consisting of, the entire SEQ ID No. 1. 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 fourth 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 α1-¾ transfucosidase of the first or second 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 fourth aspect of the invention can be any mono- or oligosaccharide, preferably an oligosaccharide of 3-10 monosaccharide units that the mutated α1-¾ 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 3-OH group. More preferably, the oligosaccharide acceptor having 3-10 units comprises a N-acetyl-lactosaminyl or lacto-N-biosyl moiety and is of formula 1, or is a lactose derivative of formula 2
wherein R1 is fucosyl or H,
R2 is selected from N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue,
R3 is H or N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue, and
each R4 independently is sialyl or H,
with the proviso that at least one of R1 or R4 is not H.
Preferably, compounds of formula 1 are of formulae 1a or 1 b
More preferably, compounds of formulae 1 a and 1 b have one or more of the following linkages and modifications:
Even more preferably, a compound of formula 1a, 1b or 2 is selected from the group consisting of 2’-O-fucosyllactose (2′-FL), 3′-O-sialyllactose (3′-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I, Fucαl-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc, lacto-N-fucopentaose V (LNFP-V, Galβ1-3GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc), Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc, lacto-N-hexaose (LNH, Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]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, Fucα1-2Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), fucosyl-LNH II (FLNH-II, Galβ1-4[Fucα1-3]GlcNAcβ1-6[Galβ1-3GlcNAcβ1-3]Galβ1-4Glc), fucosyl-para-LNH I (FpLNH-I, Galβ1-3GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc), fucosyl-para-LNH II (FpLNH-II, Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc, Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc, difucosyl-LNH I (DFLNH-I, Galβ1-4[Fucα1-3]GlcNAcβ1-6[Fucα1-2Galβ1-3GlcNAcβ1-3]Galβ1-4Glc), difucosyl-para-LNH (DFpLNH, Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc), difucosyl-para-LNnH (DFpLNnH, Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc), lacto-N-octaose (LNO, Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), lacto-N-neooctaose (LNnO, Galβ1-4GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), iso-lacto-N-octaose (iLNO, Galβ1-3GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]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, Galβ1-3GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), sialyl-LNnH I (SLNnH-I, Galβ1-4GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), sialyl-LNnH II (SLNnH-II, Galβ1-4GlcNAcβ1-6[NeuAcα2-6Galβ1-4GlcNAcβ1-3]Galβ1-4Glc), disialyl-LNT (DSLNT, NeuAcα2-3Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc), fucosyl-sialyl-LNH (FSLNH, NeuAcα2-3Galβ1-3GlcNAcβ1-3[Galβ1-4[Fucα1-3]GlcNAcβ1-6]Galβ1-4Glc), fucosyl-sialyl-LNH II (FSLNH-II, Fucα1-2Galβ1-3GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc), disialyl-LNH I (DSLNH-I, NeuAcα2-6Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-3GlcNAcβ1-3]Galβ1-4Glc), disialyl-LNH II (DSLNH-II, Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-3[NeuAcα2-6]GlcNAcβ1-3]Galβ1-4Glc) and disialyl-LNnH (DSLNnH, NeuAcα2-6Galβ1-4GlcNAcβ1-6[NeuAcα2-6Galβ1-4GlcNAcβ1-3]Galβ1-4Glc), advantageously 2′-FL, 3′-SL, LNT, LNnT, LNFP-I, LNFP-V, LNH, LNnH, pLNH, pLNnH and DSLNT.
A mutated α1-¾ fucosidase of the first or second aspect of the invention demonstrates a strong α1-¾ selectivity when carrying out the method of the fourth aspect of the invention. As a result, the product of the reaction is an α1-3- or a α1-4-fucosyl mono- or oligosaccharide, preferably an oligosaccharide of 3-10 monomer units, exclusively, and no an α1-2-fucosylated product can be detected. Preferably, the mutated α1-¾ transfucosidase brings the fucosyl residue of an appropriate donor to the 3-position of the glucose in an acceptor of formula 2, to the 3-position of the N-acetyl-glucosamine in a, preferably terminal, N-acetyl-lactosaminyl group in an acceptor of formula 1, 1a or 1b, or to the 4-position of the N-acetyl-glucosamine in a, preferably terminal, lacto-N-biosyl group, in an acceptor of formula 1, 1a or 1 b. Accordingly, a mutated α1-¾ 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 α1-3 or α1-4 linkage, more preferably to the fucosylated HMOs listed in Table 3 below (for abbreviations see Urashima et al. Milk Oligosaccharides, Nova Science Publishers, NY, 2011, Table 4 in pp. 14-25).
The fucosyl donor used in the fourth aspect of the invention can be any fucosyl compound from which the mutated α1-¾ fucosidase is able to transfer the fucosyl residue to a carbohydrate acceptor as described above. Accordingly, the fucosyl donor can be an α1-3 or α1-4 fucosyl saccharide, preferably of 3 or 4 monosaccharide units including the fucosyl residue, more preferably 3-FL or DFL, or a compound of formula 3
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
wherein Ra is independently H or alkyl, or two vicinal Ra groups represent a ═C(Rb)2 group, wherein Rb is independently H or alkyl, Rc is independently selected from the group consisting of alkoxy, amino, alkylamino and dialkylamino, Rd 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 3 is selected from the group consisting of phenoxy-, p-nitrophenoxy-, 2,4-dinitrophenoxy-, 2-chloro-4-nitrophenoxy-, 4,6-dimethoxy-1,3,5-triazin-2-yloxy-, 4,6-diethoxy-1,3,5-triazin-2-yloxy-, 2-ethyl-5-methyl-3-oxo-(2H)-furan-4-yloxy-, 5-ethyl-2-methyl-3-oxo-(2H)-furan-4-yloxy- and 2,5-dimethyl-3-oxo-(2H)-furan-4-yloxy-group. Advantageously, the fucosyl donor is 3-FL or DFL.
A mutated α1-¾ transfucosidase of the invention comprising a polypeptide that has a sequence identity of at least 75% to SEQ ID No.1, or comprising, preferably consisting of, the sequence of SEQ ID NO 1, and mutation at amino acid position 174 and at amino acid position 135 or 168, is especially suitable for making
More preferably, in a fucosylation reaction, wherein the fucosyl donor is 3-FL and the acceptor is
the utilization of an α1-¾ transfucosidase comprising an amino acid sequence having a sequence identity of at least 75% to SEQ ID No.1, and mutation of three amino acids position 174, at positions 135 or 168, and at position 413, and optionally having further mutation at amino acid position selected from 165, 232, 258, 260 and 274,
or an α1-¾ transfucosidase comprising, more preferably consisting of, the sequence of SEQ ID No. 1 having mutations of three amino acids at position 174, at positions 135 or 168, and at position 413, and optionally having further mutation at amino acid position selected from 165, 232, 258, 260 and 274,
is especially favoured.
According to a fifth aspect of the invention, the use of a mutated α1-¾ fucosidase of the first or second aspect of the invention is provided for synthesizing a fucosylated carbohydrate, preferably an α1-3 or a α1-4 fucosyl mono- or oligosaccharide, more preferably a fucosylated HMO having an α1-3 and/or a α1-4 fucosyl residue, even more preferably those in which the fucosyl residue is attached to a Glc moiety with α1-3 linkage or to a GlcNAc moiety with α1-3 or α1-4 linkage, especially one of the fucosylated HMOs listed in the Table 3 above, particularly DFL, SFL, LNFP-II, LNFP-III, LNDFH-I, fucosyl LNnH such as Galβ1-4[Fucα1-3]GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc or Galβ1-4GlcNAcβ1-3[Galβ1-4[Fucα1-3]GlcNAcβ1-6]Galβ1-4Glc, DFLNnH or fucosylated pLNnH such as Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.
In the examples below mutants of Bifidobacterium longum subsp. infantis ATCC 15697 were tested, the position(s) of mutation is/are according to SEQ ID No. 1.
The transfucosidase activity of mutants was investigated on the 3-FL+LNnT LNFP-III+Lac reaction in which the formation of LNFP-III was followed.
The reaction was performed at 30° C. in 150 μl scale using 200 mM LNnT and 200 mM 3-FL. Samples were taken typically after 1 h, 2 h, 4 h and 20 h and the reaction was stopped by adding 390 μl of acetonitrile/water 1:1.
The hydrolytic activity of mutants was investigated in a similar procedure using LNFP-III (50 mM) as only substrate, and depletion of LNFP-III and formation of LNnT were followed over time.
HPLC conditions: Kinetex 2,6 μ HILIC 100A-column (150×4.6 mm) was used with a flow of 1.8 ml/min using 76% acetonitrile and 24% 10 mM ammonium formate buffer (pH 4). The elution of substrates and products was detected at 195 nm. 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 1 μmol of LNFP-III per min.
The hydrolytic activity of mutants was investigated according to the procedure described in Example 1.
The melting temperature (Tm) is the temperature at which 50% of the initial activity of the enzyme remains after 15 min of incubation at elevated temperatures.
Activities were measured by HPLC analysis of 3-FL+LNnT LNFP-III+Lac reaction in which the formation of LNFP-III was followed.
HPLC conditions: see above
Increasing the thermostability (Tm) of the wild type protein of SEQ ID No. 1:
Increasing the thermostability of mutants designed for increased transfucosidase synthetic performance or reduced hydrolytic activity:
A) Saturation mutagenesis was screened at positions 134, 135, 174 and 282 in the following reactions:
3-FL+LNnTLNFP-III+Lac
3-FL+LNFP-ILNDFH-I+Lac
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 150 μl), [3-FL]=200 mM, [LNnT]=200 mM, [LNFP-I]=200 mM, with 10 μl of crude enzyme extract. Conversions were measured after 15 min, 30 min, 60 min and 20 hours. The tables below show the conversions (%) after 30 min, 20 hours and the maximum conversion during the course. WT values are italic.
6
0
14
6
0
14
6
0
14
6
0
14
0
0
0
0
0
0
0
0
0
0
0
0
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNnT]=200 mM, enzyme extract=2 or 0.5 mg/ml.
HPLC conditions: TSK Gel amide 80 (Tosoh, 3 μm, 150×4.6mm) was used with a flow of 1 ml/min using 56% acetonitrile and 44% water. The elution of substrates and products was detected by CAD and/or UV detection at 195 nm.
The tables show the LNFP-III formation (%) as a function of time.
C) In 3-FL+LNTLNFP-II+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNT]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the LNFP-II formation (%) as a function of time.
D) In 3-FL+LNnHGalβ1-4[Fucα1-3]GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc+Galβ1-4GlcNAcβ1-3[Galβ1-4[Fucα1-3]GlcNAcβ1-6]Galβ1-4Glc+DFLNnH+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNnH]=100 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The tables show the monofucosylated and difucosylated LNnH formation (%), respectively, as a function of time.
E) In 3-FL+pLNnHGalβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=100 or 200 mM, [pLNnH]=100 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The tables show the fucosylated pLNnH formation (%) as a function of time.
F) In 3-FL+LNFP-ILNDFH-I+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNFP-I]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the LNDFH-I formation (%) as a function of time.
Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 30° C., 140 μl), [3-FL]=50 mM, [LNFP-I]=50 mM, enzyme extract=10 μl. HPLC: see Example 1. The table shows the LNDFH-I formation (%) as a function of time.
G) In DFL+LNnTLNFP-III+2′-FL reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [DFL]=200 mM, [LNnT]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the LNFP-III formation (%) as a function of time.
H) In 3-FL+2′-FLDFL+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [2′-FL]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the DFL formation (%) as a function of time.
A) In 3-FL+LNnTLNFP-III+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNnT]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the LNFP-III formation (%) as a function of time.
Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 150 μl), [3-FL]=200 mM, [LNT]=200 mM, enzyme extract=0.67 mg/ml. Samples were taken after 3, 10, 15, 20, 30, 45, 61 and 115 min. HPLC: see Example 1. The table shows the activity in [U/mg] wherein 1 U=production of 1 μmol LNFP-II per min.
B) In 3-FL+LNTLNFP-II+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNT]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the LNFP-II formation (%) as a function of time.
Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 150 μl), [3-FL]=200 mM, [LNT]=200 mM, enzyme extract=0.67 mg/ml. Samples were taken after 3, 10, 15, 20, 30, 45, 61 and 115 min. HPLC: see Example 1. The table shows the activity in [U/mg] wherein 1 U=production of 1μmol LNFP-II per min.
C) In 3-FL+pLNnHGalβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=100, [pLNnH]=100 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the fucosylated pLNnH formation (%) as a function of time.
D) In 3-FL+LNFP-ILNDFH-I+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200 μl), [3-FL]=200 mM, [LNFP-1]=200 mM, enzyme extract=0.5 mg/ml. HPLC: see Example 4 B). The table shows the LNDFH-I formation (%) as a function of time.
Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C. 150 μl), [3-FL]=100 mM, [LNFP-1]=50 mM, enzyme extract=10 μl crude extract. HPLC: see example 1. The table shows the LNDFH-I formation (%) as a function of time.
Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 30° C., 140 μl), [3-FL]=50 mM, [LNFP-1]=50 mM, enzyme extract=10 μl. HPLC: see Example 1. The table shows the LNDFH-I formation (%) as a function of time.
Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 150 μl), [3-FL]=200 mM, [LNFP-I]=200 mM, enzyme extract=0.67 mg/ml. Samples were taken after 3, 10, 15, 20, 30, 45, 61 and 115 min. HPLC: see Example 1. The table shows the activity in [U/mg] wherein 1U=production of 1 μmol LNDFH-I per min.
E) In 3-FL+2′-FLDFL+Lac reaction
The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 150 μl), [3-FL]=200 mM, [2′-FL]=200 mM, enzyme extract=0.67 mg/ml. Samples were taken after 3, 10, 15, 20, 30, 45, 61 and 115 min. HPLC: see Example 1. The table shows the activity in [U/mg] wherein 1 U=production of 1 μmol 2′-FL per min.
Number | Date | Country | Kind |
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14190374.0 | Oct 2014 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/IB2015/058197 | 10/23/2015 | WO | 00 |