Xanthan is a well-known polysaccharide that has a cellulose-like -1 ,4-glucan backbone structure and further comprises (3- 1 ) linked a-D-mannose-(2- 1 )- -D- glucuronic acid-(4- 1 )- -D-mannose side chains that are typically located on every second glucose unit (Jansson, Kenne and Lindberg, 1975). Xanthan can be obtained from natural sources. Exemplarily, Xanthomonas bacteriae are used in the production of xanthan, in particular those of the species Xanthomonas campestris.

When dissolved in water, xanthan solutions exhibit pseudo-plastic properties, even at low xanthan concentrations. These properties are highly stable over a wide pH and temperature range, making xanthan suitable for many food applications. Today, xanthan is already used in food industry as food stabilizer and rheological modifier.

However, its applicability in many fields is still severely hampered by some considerably drawbacks with respect of its rheological properties. It has been considered that the presence of too many acetyl residues conjugated to the xanthan in some positions may influence the desired rheological properties negatively. Naturally occurring xanthan typically bears one or more acetyl residues conjugated to one or both of the mannose moieties. In fact, recent studies have revealed that approximately 85 to 90 % of all inner mannose units are acetylated at the O-6 position and about 50 to 70 % of all terminal mannose units are substituted with a pyruvic acid acetal. Additionally, about 5 to 25 % of all terminal mannose units can be substituted with an acetyl group (Stankowski, Mueller and Zeller, 1993; Kool et al., 2013b).

The acetyl and pyruvate groups of xanthan are known to have a great influence on the viscosity of xanthan solutions and its stability towards the addition of salts and changes in acidity (Morrison, Clark, Talashek and Yuan, 2004; Shatwell, Sutherland, Dea and Ross-Murphy, 1990). Lowering the degree of acetylation results in xanthan solutions with better and more stable physical properties and in improved interactions with galactomannans (Morrison, Clark, Talashek and Yuan, 2004; Shatwell, Sutherland, Ross-Murphy and Dea, 1990).

It is known that the chemical removal of acetyl groups may improve the properties of the xanthan and its interactions with galactomannans. To date, acetyl groups are removed using an acidic or, in particular, alkali treatment (Bradshaw, Nisbet, Kerr and Sutherland, 1983; Pinto, Furlan and Vendruscolo, 201 1 ; Tako and Nakamura, 1984).

However, such method based on synthetic chemical means is a very rough procedure and bears several severe drawbacks. First, the removal of acetyl residues is entirely unspecific and random and cannot be controlled properly. The rheological properties of the xanthan that will be obtained from such a method can previously not even be roughly assessed. Secondly, in the chemical reaction toxic and unwanted agents, such as exemplarily alkaline agents, are used that have to be thoroughly removed from the obtained xanthan after the removal of acetyl residues is completed. This requires additional laborious method steps. Thirdly, removal of acetyl residues by synthetical chemical means often also results in a partly degradation of the xanthan's backbone what is entirely unwanted.

An alternative method to control the acetyl levels in xanthan is the use of specific Xanthomonas strains and/or fermentation conditions for the xanthan production (Flores Candia and Deckwer, 1999; Hassler and Doherty, 1990; Peters, Suh, Schumpe and Deckwer, 1993).

Altering the production conditions of xanthan, however, influences the pyruvate levels in the produced xanthan. As the pyruvate groups are also of great importance for xanthans functionality (Sandford, Pittsley, Knutson, Cadmus, Watson and Jeanes, 1977; Shatwell, Sutherland, Ross-Murphy and Dea, 1990; Smith, Symes, Lawson and Morris, 1984), these techniques are also not applicable to modify only the acetyl content of xanthan.

Therefore, there is still a need for a method for mild and selective removal of acetyl groups from the xanthan side chains in a controlled way. Furthermore, there is also a need for xanthans with improved technical properties and broadened applicability.

Surprisingly, as laid out in detail below, a method for mild and selective removal of acetyl groups from the xanthan side chains in a controlled way was found that entirely does not affect the xanthan backbone. Additionally, xanthan with an altered acetylation pattern and improved technical properties was obtained.

In a first aspect, the present invention therefore relates to a method for producing xanthan with an altered acetylation pattern, wherein an enzyme site-specifically cleaving acetyl residues off mannose moieties of acetylated xanthan is contacted with acetylated xanthan.

As used herein, the terms "method", "process", "procedure" and "technique" may be understood exchangeably and in their broadest sense.

"Xanthan" in the context of the present invention may be understood in the broadest sense as any xanthan known in the art. Preferably, xanthan is a polysaccharide comprising a -1 ,4-glucan backbone and further comprises on approximately every second glucose unit a (3- 1 ) linked a-D-mannose-(2- 1 )-β-ϋ- glucuronic acid-(4- 1 )- -D-mannose side chain. Naturally occurring xanthan typically bears one or more acetyl residues conjugated to one or both of the mannose moieties. An exemplary generic structure of xanthan is depicted in Figure 1A. Further examples of xanthan repeating units are depicted in Figure 1 B.

The xanthan in the context of the present invention is acetylated xanthan. Herein, the term "acetylated xanthan" may be understood in the broadest sense as any xanthan molecule comprising at least one acetyl residue, preferably at least two, more preferably at least five, even more preferably at least ten, even more preferably at least 50 acetyl residues, even more preferably at least 100 acetyl residues, even more preferably at least 1000 acetyl residues or even more residues. More preferably, acetylated xanthan comprises at least one acetyl residue, preferably at least two, more preferably at least five, even more preferably at least ten, even more preferably at least 50 acetyl residues at mannose moieties. The inner mannose groups are those attached to the backbone of xanthan, whereas the outer are those terminal at the side chains. Highly preferably, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 % or at least 90% of all inner mannose units are acetylated at the O-6 position and/or at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 % or at least 90% of all terminal mannose units may be substituted with a pyruvic acid acetal. In particular, herein, at least 10 %, at least 20 %, at least 30 %, at least 40 % or at least 50 % of all terminal mannose units may be substituted with a pyruvic acid acetal.

The term "altered acetylation pattern" as used herein refers to removing and/or adding of acetyl residues on the xanthan. This may overall result in xanthan with a reduced number of acetyl residues, with an essentially equal number of acetyl residues or in an increased number of acetyl residues.

Preferably, the number of acetyl residues in the site-specific mannose moieties of the xanthan is reduced, in particular at least 1 .1 -fold, at least 1 .5-fold, at least 2- fold, at least 5-fold, at least 10-fold or at least 20-fold.

Alternatively or additionally, incubating the mixture may or may not comprise conjugating one or more acetyl residue(s) to xanthan at specific positions in the xanthan.

Herein, the terms "site-specifically cleaving off' may be understood in the broadest sense as the selectively preferred removal of acetyl residue(s) at particular positions. Preferably, at the specific positions, the yield of removal of acetyl residue(s) is at least 1 .1 -fold, at least 1 .5-fold, at least 2-fold, at least 5-fold, at least 10-fold or at least 20-fold more effective that at other positions in the acetylated xanthan.

The term "enzyme" as used herein may be understood in the broadest sense as any macromolecule, i.e., a molecule of a molecular weight (MW) of more than 500 Da, than catalyzes one or more chemical reaction(s). In the context of the present invention, an enzyme catalyzes the cleavage of the ester bond of acetyl residues off mannose moieties of the xanthan. In this context, acetylated xanthan may serve as the substrate (also: the educt) and xanthan with an altered acetylation pattern may be the product. The person skilled in the art will immediately notice that, depending on the concentration ratios between substrates and educts, the kinetic of the reaction may vary and that the reaction may even be reversed, wherein upon reversal of the reaction the designations "substrate" and "educt" may also be reversed. Preferably, the enzyme is a polypeptide.

As used throughout the present invention, the terms "polypeptide", "peptide" and "protein" may be understood interchangeably in the broadest sense as any molecule comprising a strand of at least four, preferably of at least ten, more preferably of at least 20, even more preferably of at least 50 consecutive amino acids.

A polypeptide in the sense of the present invention may also include one or more posttranslational modification(s). A plurality of posttranslational modifications are well-known in the art and may comprise but may not be limited to lipidation, phosphorylation, sulfatation, glycosylation, truncation, cyclization of several amino acid moieties, cyclization of the polypeptide strand, oxidation, reduction, decarboxylation, acetylation, amidation, deamidation, disulfide bond formation, pyroglutamate formation, amino acid addition, cofactor addition (e.g., biotinylation, heme addition) and complexation of metal ions, non-metal ions, peptides or small molecules and addition of iron-sulphide clusters. Evidently, co-factors, such as the binding of ATP, ADP, NAD+, NADH+H+, NADP+, NADPH+H+, metal ions, anions, lipids, etc. may be bound to the polypeptide, irrespective on the biological influence of these co-factors.

Optionally, a polypeptide in the sense of the present invention may also be conjugated or bound to one or more other molecule(s) that may preferably be one or more of the group consisting of but not limited to fusion proteins (e.g., fluorescent proteins (e.g., cyan fluorescent protein (CFP), green fluorescent protein (GFP) or yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCherry, etc.)), one or more lipids or lipidoids, one or more small molecule dyes (e.g., fluorescent dyes (e.g., a Cy dye (e.g., Cy3, Cy5, Cy5.5, Cy 7), an Alexa dye (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750), a Visen dye (e.g. VivoTag680, VivoTag750), an S dye (e.g., S0387), a DyLight fluorophore (e.g., DyLight 750, DyLight 800), an IRDye (e.g., IRDye 680, IRDye 800), a fluorescein dye (e.g., fluorescein, carboxyfluorescein, fluorescein isothiocyanate (FITC)), a rhodamine dye (e.g., rhodamine, tetramethylrhodamine (TAMRA)) or a HOECHST dye) or UVA/is dyes (e.g., a p-nitrophenyl moiety, Coomassie Brilliant Blue G-250), one or more quantum dots, one or more binding moieties (e.g., biotin, methotrexate, glycocorticoids or moieties comprising, e.g., one or more active esters, isothiocyanates, maleimides, N-hydroxysuccinimidyl esters, glutaraldehyde derivatives, carbodiimide derivatives or combinations thereof), one or more insoluble and/or soluble polymers (e.g., polyethylene glycol (PEG), hydroxypropyl methacrylate (HPMA), polyethylene imine (PEI)), one or more antibodies or derivatives thereof (e.g., Fab fragments, single chain antibodies, diabodies, triabodies, flexibodies, tandabs), one or more peptides (e.g., cell-penetrating peptides (CPPs), protein transduction domains (PTDs), signal transduction peptides, etc.), one or more spin labels, one or more enzyme labels (e.g., penicillinase, horseradish peroxidase, alkaline phosphatase), micro- or nanobeads (e.g., functionalized silica beads, polysaccharide-based beads), polymersomes and/or liposomes. Further, the polypeptide may optionally be radioactively labeled by, e.g., one or more of the group consisting of but nut limited to ³ H, ³² P, ³⁵ S, ¹⁴ C, ⁹⁹ Tc or lanthonoids (e.g., ⁶⁴ Gd). Further, the polypeptide may optionally be spin-labeled by, e.g., ² H and/or ¹³ C. Contacting in the sense of the present invention may be contacting by any means. Preferably contacting is performed by dissolving or emulsifying the acetylated xanthan in the same batch as the polypeptide or derivative thereof of the present invention, wherein the batch further comprises a liquid solvent. Highly preferably, the liquid solvent is an aqueous solvent, in particular an aqueous buffer. Such aqueous buffer preferably maintains the enzymatic activity of the polypeptide or derivative thereof of the present invention.

Preferably, in the method of the present invention, in general, the xanthan is not obtained from genetically modified organisms (GMOs), in particularly no organisms are used in which the xanthan acetylation has been genetically influenced by any technical means such as by amending the acetylation pattern by knocking out or down one or more genes encoding for enzymes responsible for acetylation and/or deacetylation. When no xanthan obtained from GMOs is used, this may have several advantages. Typically, in GMOs, one or more genes involved in acetylating mannose are completely deleted what may on the one hand result in deacetylated xanthan, but does not allow intermediate levels of acetylation at either position on mannose, merely complete absence or presence of acetyl residues in possible. In contrast, the present invention has the significant improvement in specific and optionally partly removal of acetyl residues. Furthermore, xanthan with altered acetylation pattern produced in the absence of GMOs may have higher consumer acceptance.

According to the present invention, the enzyme may cleave acetyl residues off any mannose. In a preferred embodiment, the enzyme site-specifically cleaves acetyl residues off the inner mannose and/or off the outer mannose moieties of acetylated xanthan.

Therefore, the enzyme may site-specifically cleave acetyl residues off the inner mannose. An example for such enzyme is AXE3 which is an enzyme that selectively cleaves acetyl residues off the inner mannose. Alternatively, the enzyme may site-specifically cleave acetyl residues off the outer mannose. An example for such enzyme is YesY which is an enzyme that selectively cleaves acetyl residues off the outer mannose. Alternatively, the enzyme may site- specifically simultaneously cleave acetyl residues off the inner and outer mannose, therefore may be bispecific for acetyl residues at inner and outer mannose moieties.

Therefore, the enzyme may be any enzyme that site-specifically cleaves acetyl residues off the inner and/or outer mannose, therefore any enzyme bearing a site- specific mannose deacetylating activity.

As used herein, the term "deacetylating" may be understood in the broadest sense as removing acetyl residues from the xanthan molecule. Herein, the xanthan molecule may also be designated as "bare xanthan molecule". The person skilled in the art will immediately notice that removing acetyl residues refers to the cleavage of the C-O bond in the ester group between the acetyl residues and the xanthan backbone typically resulting in an increased number of hydroxyl groups on the xanthan. Removing acetyl residues from the xanthan does not necessary mean that the acetate anions liberated thereby are removed from the sample. As used herein, the terms "acetyl residue", "acetyl group" and "acetyl residue" may be understood interchangeably. The enzyme may enzyme site-specifically cleave acetyl residues off the inner and/or the outer mannose moieties of acetylated xanthan.

In a preferred embodiment, the enzyme site-specifically cleaves acetyl residues off the inner mannose moieties of acetylated xanthan.

In this context, an enzyme site-specifically cleaving acetyl residues off the inner mannose may be any enzyme having this feature known in the art.

In a more preferred embodiment, the enzyme cleaving acetyl residues off the inner mannose is an acetyl xylan esterase (AXE), preferably a fungal AXE, more preferably an AXE from Chrysosporium lucknowense, in particular a polypeptide of at least 80% homology to SEQ ID NO: 1 or a functional derivative thereof having at least 10 % of its xanthan-deacetylating activity.

Myceliophtora thermophila has previously also been designated as Chrysosporium lucknowense. Therefore these designations may be understood exchangeably throughout the present invention.

As used herein, Chrysosporium lucknowense is preferably, Chrysosporium lucknowense C1 (corresponding to Myceliopthora thermophila C1 ).

Throughout the present invention SEQ ID NO: 1 , refers to the following amino acid sequence:

MKLLGKLSAALALAGSRLAAAHPVFDELMRPTAPLVRPRAALQQVTNFGSNPSN TKMFIYVPDKLAPNPPIIVAIHYCTGTAQAYYSGSPYARLADQKGFIVIYPESPYSG TCWDVSSRAALTHNGGGDSNSIANMVTYTLEKYNGDASKVFVTGSSSGAMMTN VMAAAYPELFAAGIAYSGVPAGCFYSQSGGTNARNSSCANGQINSTPQVWAKM VFDMYPEYDGPRPKMQIYHGSADGTLRPSNYNETIKQWCGVFGFDYTRPDTTQ ANSPQAGYTTYTWGEQQLVGIYAQGVGHTVPIRGSDDMAFFGL

Herein, the first 21 amino acids may be considered as a signal peptide. Therefore, the mature polypeptide may optionally also start at the amino acid moiety in position 22 of the above sequence.

Preferably, the polypeptide of the present invention comprises an amino acid sequence of at least 80% homology to SEQ ID NO: 1 , more preferably of at least 85% homology to SEQ ID NO: 1 , even more preferably of at least 90% homology to SEQ ID NO: 1 , even more preferably of at least 95% homology to SEQ ID NO: 1 and in particular an amino acid sequence of SEQ ID NO: 1 . Highly preferably, the polypeptide of the present invention consists of an amino acid sequence of at least 80% homology to SEQ ID NO: 1 , more preferably of at least 85% homology to SEQ ID NO: 1 , even more preferably of at least 90% homology to SEQ ID NO: 1 , even more preferably of at least 95% homology to SEQ ID NO: 1 and in particular of an amino acid sequence of SEQ ID NO: 1 .

A polypeptide comprising SEQ ID NO: 1 or a derivative thereof preferably cleaves off acetyl residues located at the inner mannose.

In an alternative preferred embodiment, the enzyme site-specifically cleaves acetyl residues off the outer mannose moieties of acetylated xanthan.

Herein, an enzyme site-specifically cleaving acetyl residues off the outer mannose may be any enzyme having this feature known in the art. In a more preferred embodiment, the enzyme cleaving acetyl residues off the outer mannose is a pectin acetylesterase, preferably a rhamnogalacturonan acetylesterase, more preferably a rhamnogalacturonan acetylesterase of the YES cluster of Bacillus subtilis, in particular a polypeptide of at least 80% homology to SEQ ID NO: 2 or a functional derivative thereof having at least 10 % of its xanthan-deacetylating activity.

As used herein, a rhamnogalacturonan acetylesterase may also be designated as a pectin acetyl esterase or homogalacturonan acetyl esterase as these enzymes may deacetylate the homogalacturonan regions of pectin.

Bacillus subtilis is preferably but not necessarily Bacillus subtilis of commercially available strain 168.

Throughout the present invention, SEQ ID NO: 2 refers to the following amino acid sequence:

MANHIYLAGDSTVQTYGDSTNQGGWGQFLGSHLPEHIQVINRAIGGRSSKTFVE EGRLQAILDVIEPDDWLFVQMGHNDASKNKPERYTEPYTTYKQYLKQYIAGARE KGAHPLLITPVARFHYENGVFLNDFPDYCIAMKQTAAEENVQLIDLMEKSLAFFTE KGEEKVYTYFMISEGINDYTHFTKKGANEMAKLVAKGIKELGLPLTESIIKER

Preferably, the polypeptide of the present invention comprises an amino acid sequence of at least 80% homology to SEQ ID NO: 2, more preferably of at least 85% homology to SEQ ID NO: 2, even more preferably of at least 90% homology to SEQ ID NO: 2, even more preferably of at least 95% homology to SEQ ID NO: 2 and in particular an amino acid sequence of SEQ ID NO: 2. Highly preferably, the polypeptide of the present invention consists of an amino acid sequence of at least 80% homology to SEQ ID NO: 2, more preferably of at least 85% homology to SEQ ID NO: 2, even more preferably of at least 90% homology to SEQ ID NO: 2, even more preferably of at least 95% homology to SEQ ID NO: 2 and in particular of an amino acid sequence of SEQ ID NO: 2.

A polypeptide comprising SEQ ID NO: 2 or a derivative thereof preferably cleaves off acetyl residues located at the outer mannose.

In the context of the present invention, the term "homology" may be understood as sequence homology and/or three dimensional (3D) structural homology. Preferably, homology is sequence homology.

The enzymes in the context of the present invention maybe obtained from any means known in the art. Exemplarily, the enzymes may be obtained from the organism these sequences originate from or may be heterologically expressed in another organism such as, e.g., a bacterium (e.g., E. coli), a fungal cell or an animal cell (e.g., an insect or a mammalian cell).

As used in the context of the present invention, the term "functional derivative" may be understood in the broadest sense as any polypeptide that is structurally deduced from a polypeptide of SEQ ID NO: 1 and that has at least 10 %, preferably at least 20 %, more preferably at least 30 %, even more preferably at least 40 %, even more preferably at least 50 %, even more preferably at least 60 %, even more preferably at least 70 %, even more preferably at least 80 %, even more preferably at least 90 % or even 100 % or more of the xanthan-deacetylating activity of a polypeptide of a polypeptide comprising an amino acid sequence of at least 80% homology to SEQ ID NO: 1 and SEQ ID NO: 2, respectively, in particular a polypeptide of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. As used herein, "structurally deduced" means that its sequence is structurally related, i.e., that the functional derivative comprises a sequence of at least 20, preferably of at least 30, more preferably of at least 40, even more preferably of at least 50 consecutive amino acids, even more preferably of at least 75 consecutive and even more preferably of at least 100 consecutive amino acids with a sequence homology of at least 80% to the most homologous sequence interception of a SEQ ID NO: 1 and SEQ ID NO: 2, respectively. It will be understood that a functional derivative does not necessarily have to be directly obtained from a polypeptide comprising an amino acid sequence of at least 80% homology. It may also be obtained by any biotechnological means or even by chemical peptide synthesis. Exemplarily, a functional derivative may be a mutated form of a polypeptide comprising an amino acid sequence of at least 80% homology to SEQ ID NO: 1 and SEQ ID NO: 2, respectively. A mutated form may be a polypeptide in which one or more amino acid residues have been inserted, removed and/or exchanged. Such mutated form of the polypeptide may refer to but may not be limited to the polypeptide encoded by an altered DNA or RNA sequence, a splice variant of the polypeptide or an analogue polypeptide from another species (e.g., an animal, a viral or a bacterial polypeptide), most preferably a bacterial polypeptide. An altered DNA or RNA sequence encoding for the functional derivative may occur naturally throughout the population tested or may be of artificial origin. Alternatively, the functional derivative of the polypeptide may be of synthetical origin, i.e. chemically modified and/or synthesized.

The enzymes as described above, a composition for amending the acetylation pattern of xanthan in the sense of the present invention may comprise:

(a) an enzyme site-specifically cleaving acetyl residues off the inner mannose moieties, in particular a polypeptide sequence of at least 80% homology to

SEQ ID NO: 1 or a functional derivative thereof having at least 10 % of its xanthan-deacetylating activity; and/or

(b) an enzyme site-specifically cleaving acetyl residues off the outer mannose moieties, in particular a polypeptide sequence of at least 80% homology to

SEQ ID NO: 2 or a functional derivative thereof having at least 10 % of its xanthan-deacetylating activity, Wherein (a) and (b) may be administered in combination with another or an enzyme of (a) may be administered in the absence of an enzyme of (b) or an enzyme of (b) may be administered in the absence of an enzyme of (a). In a particularly preferred embodiment, an enzyme site-specifically cleaving acetyl residues off the inner mannose moieties of acetylated xanthan and an enzyme site-specifically cleaving acetyl residues off the outer mannose moieties of said acetylated xanthan are contacted with acetylated xanthan. The person skilled in the art will immediately notice that such composition typically will further comprise a solvent, in particular an aqueous solvent such as an aqueous buffer. Optionally, the composition may further comprise one or more co- factors of the enzymes. Optionally, the composition may further comprise one or more further enzymes, in particular those amending the acetylation pattern of xanthan and/or digesting or producing xanthan. Preferably, the composition is essentially free of cells, more preferably essentially free of cells of genetically modified organisms (GMOs), in particular those GMOs in which the xanthan acetylation has been genetically influenced by any technical means such as by amending the acetylation pattern by knocking out or down one or more genes encoding for enzymes responsible for acetylation and/or deacetylation. Preferably, the composition is essentially free of toxic agents (e.g., organic solvents (e.g. chloroform, toluol, phenol, acetone, acetonitrile, etc.), heavy metals, etc.) and other unwanted agents (e.g., ethyl alcohol, glycerol, dimethyl sulfoxide (DMSO), etc.).

As used herein, the method according to the present invention may be conducted by any means known in the art and by using any methodic steps suitable for this purpose. In a preferred embodiment, the method according to the present invention comprises the steps of:

(i) providing acetylated xanthan,

(ii) contacting said acetylated xanthan with one or more enzyme(s), in particular wherein at least one enzyme is an enzyme as described above, (iii) incubating the mixture obtained from step (ii) at conditions allowing said enzyme(s) to alter the acetylation pattern of the acetylated xanthan,

(iv) recovering the xanthan with altered acetylation pattern obtained from step (iii). Providing acetylated xanthan may be performed by any means known in the art. Therefore, the xanthan may be obtained from any source, such as, e.g, obtained from a commercial supplier or obtained from fungal cells and/or bacteria.

Recovering of the xanthan with altered acetylation pattern may be performed by any means known in the art. Exemplarily, the xanthan with altered acetylation pattern obtained from the method of the present invention may be obtained by chromatographic means (e.g., size exclusion chromatography, fast protein liquid chromatography (FPLC), high performance liquid chromatography (HPLC) and/or ultra high performance liquid chromatography (UPLC)), by filtration, by electrophoresis (e.g., gel electrophoresis), by thin layer liquid chromatography, ultrafiltration and/or by precipitation. Incubating may be incubating by any means. Preferably, incubating is incubating in an aqueous solvent in particular an aqueous buffer enabling the enzymatic activity of the polypeptide or derivative thereof of the present invention.

In a preferred embodiment, the step (iii) of incubating the mixture comprises incubating at conditions wherein at least a fraction of acetylated xanthan has a disordered conformation, preferably wherein said fraction constitutes at least 5 % (w/w), more preferably at least 10 % (w/w), even more preferably at least 20 % (w/w), even more preferably at least 50 % (w/w), even more preferably at least 80 % (w/w) of the total content of xanthan.

In this context, the acetylated xanthan used in the context of the present invention may be entirely ordered, partly disordered or entirely disordered as can be measured by circulardicroism (CD) spectroscopy. Preferably, at least a fraction of acetylated xanthan has a disordered conformation. The terms "disordered" and "open" conformation may be understood interchangeably. This is the conformation that automatically is formed in increasing amounts in the xanthan upon employing high temperatures and/or low concentrations of dissolved molecules, in particular low salt concentrations. The presence of a disordered conformation is of particular benefit when an enzyme cleaving off the inner mannose, in particular an acetyl xylan esterase as described above is used in the method. Disordering the xanthan conformation may be achieved by any means known in the art. Therefore, exemplarily, the temperature at which the xanthan is incubated with the enzymes may be above a certain threshold and/or the concentration of dissolved molecules may be below a certain threshold.

When performing the method of the present invention, incubating the mixture may be performed at any temperature above a certain threshold still suitable for the enzymes of the present invention to be active. Accordingly, in a preferred embodiment, the step (iii) of incubating the mixture comprises incubating at a temperature of at least 10°C, preferably of at least 20°C, more preferably of at least 30°C in particular of 40°C or higher.

The temperature may be constant over the entire incubation period or may vary over time. The incubation time required for the desired result will depend on the degree of altering the acetylation pattern of the xanthan to be achieved, the concentration of the polypeptide or derivative of the present invention employed, the specific activity of the polypeptide or derivative of the present invention employed, the concentration of the xanthan, the degree and pattern of acetylation of the substrate xanthan, the pH, the salt concentration, the co-factor concentrations, the presence of potential inhibitors and not the least the temperature.

Additionally or alternatively, in a preferred embodiment, the step (iii) of incubating the mixture comprises, incubating in an environment comprising a total concentration of dissolved molecules other than xanthan and enzymes of not more than 100 mM, more preferably of not more than 75 mM, even more preferably of not more than 50 mM, even more preferably of not more than 25 mM, in particular incubating in an environment essentially free of dissolved molecules other than xanthan and enzymes.

Essentially free of dissolved molecules other than xanthan and enzymes preferably means that the total concentration of said molecules is not higher than 10 mM, more preferably not higher than 5 mM, even more preferably not higher than 4 mM, even more preferably not higher than 3 mM, even more preferably not higher than 2 mM, even more preferably not higher than 1 mM, even more preferably not higher than 0.5 mM, even more preferably not higher than 0.2 mM, in particular not higher than 0.1 mM. This concentration-dependent effect of dissolved molecules on the deacetylation yields may be also found for some of the employable enzymes only. Exemplarily for enzyme such as, e.g., AXE3, salt concentrations may influence the reaction whereas they may have a far lower influence on other enzymes such as, e.g., YesY. So, YesY may work nicely on xanthan having a closed conformation as is typically found in a salt-containing environment.

In this context, particularly, the molecules other than xanthan and enzymes may be dissolved salt ions and/or small molecular (i.e., having a MW < 500 Da) organic molecules (e.g., glycerol, dimethyl sulfoxide (DMSO) and/or sugars). It will be apparent that cofactors of the enzymes, even when having a MW not more than 500 Da may nevertheless be optionally present during incubation. Accordingly, in particular the salt concentration present during incubation may influence the efficacy of the enzyme(s) of the present invention. It may be below 1 mM, between 1 and 10 mM, between 10 and 50 mM or between 50 and 100mM.

The step of incubating may be performed as long as required to obtain the xanthan with altered acetylation pattern of interest.

Typically, the step (iii) of incubating the mixture comprises incubating for at least one hour, preferably for at least five hours, more preferably for at least ten hours, in particular for at least a day.

When incubating, the pH may be in any range suitable for the polypeptide or derivative thereof of the present invention.

In a preferred embodiment, the step (iii) of incubating the mixture comprises incubating at an essentially neutral pH.

An essentially neutral pH is a pH between pH 5 and 9, preferably between pH 6 and 8, more preferably between pH 6 and 7.8, even more preferably between pH 6.5 and 7.5, in particular around pH 7.

The xanthan obtainable from the method of the present invention bears special technical properties, e.g., improved rheology properties of solutions compared to the educt xanthan. Therefore, in a further aspect, the present invention relates to xanthan obtainable from a method of the present invention as described in detail above. In the context of the xanthan of the present invention, the definitions given in the context of the method above also apply.

As mentioned above, this xanthan has an altered acetylation pattern. This may be any altered acetylation pattern.

In a preferred embodiment, in the xanthan according to the present invention at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the inner mannose moieties are deacetylated, in particular wherein essentially all inner mannose moieties are deacetylated, optionally wherein the outer mannose moieties are essentially not deacetylated or also deacetylated. Therefore, in a more preferred embodiment, in the xanthan according to the present invention at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the inner mannose moieties are deacetylated, in particular wherein essentially all inner mannose moieties are deacetylated, wherein the outer mannose moieties of said xanthan are essentially not deacetylated.

In an alternative embodiment, in this xanthan, also the outer mannose moieties of said xanthan are essentially deacetylated.

In a further preferred embodiment, in the xanthan according to the present invention at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the outer mannose moieties are deacetylated, in particular wherein essentially all outer mannose moieties are deacetylated, optionally wherein the inner mannose moieties are essentially not deacetylated or also deacetylated.

Therefore, in a more preferred embodiment, in the xanthan according to the present invention at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the outer mannose moieties are deacetylated, in particular wherein essentially all outer mannose moieties are deacetylated, wherein the inner mannose moieties of said xanthan are essentially not deacetylated.

In an alternative embodiment, in this xanthan, also the inner mannose moieties of said xanthan are essentially deacetylated.

In a particularly preferred embodiment, in the xanthan according to the present invention at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the inner and outer mannose moieties are deacetylated, in particular wherein essentially all inner and outer mannose moieties are deacetylated.

In an alternatively preferred embodiment, in the xanthan according to the present invention not more than 5 %, not more than 10 %, not more than 20%, not more than 30%, not more than 40% or not more than 50 of the inner and outer mannose moieties are deacetylated, in particular wherein between about 5 and about 90 % or between about 5 and about 50 % or between about 10 and about 70 % of the acetyl moieties are removed from xanthan are deacetylated.

As mentioned above, the xanthan according to the present invention bears special technical properties, e.g., improved rheology properties of solutions compared to the educt xanthan and to xanthans known in the art. Depending on the enzymes employed in the method, the incubation conditions (e.g., incubation time, incubation temperature, total concentration of dissolved molecules in the used buffer, pH of the used buffer) and the ratio of enzymes to another, the person skilled in the art being aware of the present invention is enabled to produce xanthan with a well-defined acetylation pattern. Herein, the person skilled in the art will be able to chose a special enzyme composition and adjust the incubation conditions accordingly to obtain the desired xanthan. Many of these xanthans with special characteristics have novel and improved properties not found in the art so far.

Therefore, in a further aspect, the present invention refers to xanthan, wherein at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the inner mannose moieties are free of acetyl residues, in particular wherein essentially all inner mannose moieties are free of acetyl residues, optionally wherein at least 10% or at least 50% of the outer mannose moieties bear at least one acetyl residue.

In a preferred embodiment of this aspect, at least 10% or at least 50% of the outer mannose moieties bear at least one acetyl residue.

A still further aspect of the present invention relates to xanthan, wherein at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the outer mannose moieties are free of acetyl residues, in particular wherein essentially all outer mannose moieties are free of acetyl residues, optionally wherein at least 10% or at least 50% of the inner mannose moieties bear at least one acetyl residue.

In a preferred embodiment of this aspect, at least 10% or at least 50% of the inner mannose moieties bear at least one acetyl residue.

In a yet further aspect, the present invention relates to xanthan, wherein at least 10 %, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% of the inner and outer mannose moieties are free of acetyl residues, in particular wherein essentially all mannose moieties are free of acetyl residues. Accordingly, in a preferred embodiment, the xanthan is essentially free of acetyl residues at specific mannose moieties and/or at all mannose moieties.

This may be understood in the broadest sense. Accordingly, the xanthan may be deacetylated at specific mannose residues only or may further be also deacetylated at other positions. It may even be essentially entirely deacetylated. Preferably, it is essentially deacetylated at specific mannose residues and still bears acetyl residues at other positions. The xanthan may be deacetylated at the inner mannose residues and still bear acetyl residues at other positions including the outer mannose. Alternatively, the xanthan may be deacetylated at the outer mannose residues and still bear acetyl residues at other positions including the inner mannose. Alternatively, the xanthan may be deacetylated at the inner and outer mannose residues and still bear acetyl residues at other positions. Due to its improved technical properties, the xanthan obtainable from the method of the present invention, said xanthan also has an improved applicability in a plenty of technical applications.

Accordingly, a yet further aspect of the present invention relates to the use of the xanthan according to the present invention for modifying rheology properties, viscosity properties, thickening, gelling, preventing the formation of ice crystals, stabilizing suspensions and/or emulsions, preserving, moistening and/or hydrating skin. In the context of the use of the xanthan of the present invention, the definitions given in the context of the use, method and xanthan obtainable from said method above also apply.

In a preferred embodiment, the xanthan according to the present invention is for use in

(i) an ingestible, in particular gluten-free bakery products, frozen food, dairy products, liquid food, beverages and/or water-based recipes;

(ii) a personal care product, in particular a tooth-paste and/or cosmetics;

(iii) a cleaning agent;

(iv) an agent for drilling, in particular for drilling oil;

(v) an agrochemical formulation;

(vi) an additive to solid material, in particular ceramics and/or a construction material; and/or (vii) a paint.

Herein, frozen food may be exemplarily frozen vegetable, frozen fruits, ice cream, frozen fish, frozen meat and/or frozen pre-packed food.

Ingestibles may be any edible or drinkable formulations known in the. A liquid food may comprise soups, (salad) dressings, sauces and any other solutions, emulsions or suspensions used in ingestibles. A cleaning agent may be any liquid cleaning agent known in the art such as, e.g., a toilet cleaner, a dishwashing agent, a dish liquid, a laundry agent, a fabric conditioner, a softener, a light-duty agent, a dish washer additive, a cleansing agent and/or a polishing agent. According to the general inventive concept of the present invention, in a further aspect, the present invention relates to the use of an enzyme site-specifically cleaving off the acetyl residues off mannose moieties of acetylated xanthan.

In this context, in a preferred embodiment, the method of the present invention a laid out above is used.

Accordingly, in the context of the use, the definitions given in the contexts of the method and xanthan above also apply. The following figures and example are intended to illustrate the invention but not to limit the scope of protection conferred by the claims.

Figures

Figure 1 exemplifies a general structure of xanthan. Figure 1 A depicts the xanthan repeating unit as described by Kool et al. (Kool et al., 2013b). This structure shows, from top to bottom, the two glucose moieties forming the backbone of the xanthan, the inner mannose moiety, the glucuronic acid moiety and the outer mannose moiety. Figure 1 B depicts the six xanthan repeating units, as identified by HILIC-ELSD-ESI-IT-MSn. Figure 2 depicts the Expression Production of the YesY enzyme. Herein, Figure 2A depicts the Expression plasmid pET22b::YesY-Strepllc. The sequence coding for the C-terminally Strepll-tagged YesY is highlighted. Relevant structural elements are indicated: F1 ori, origin of replication; bla, β-lactamase gene; lacl, lacl repressor gene. Figure 2B depicts gels of an SDS-PAGE (left) and a Western blot analysis (right) of FPLC-purified YesY and muteins. 30 g of crude extract (CE) and 8 g of purified enzyme (pure) were loaded onto an SDS gel and electrophoretically separated. The gel was stained using Coomassie brilliant blue. For the corresponding Western blot a Strep-Tactin HRP conjugate was used to detect the Strepll-tagged enzyme.

Figure 3 shows the correlation between the xanthan conformation and the enzymatic degradation by cellulases. Diamonds: normal xanthan; triangles: acetyl- free xanthan; dots: pyruvate-free xanthan; squares: acetyl- and pyruvate-free xanthan.

Figure 4 depicts the acetic acid release from xanthan after treatment with different carbohydrate acetyl esterases after a 48 h-incubation at 40 ^° C at different solvent conditions.

Figure 5 depicts far UV circular dichroism (CD) spectra of xanthan in 0, 1 , 2, 5, and 10 mM NaCI solutions at 40 ^° C. a = the fraction of disordered conformation.

Figure 6 shows the influence of the xanthan conformation on the acetic acid release by AXE3. Here, acetic release from xanthan obtained after incubation with AXE3 for 48 h at different fraction of disordered conformation (a = the fraction of disordered conformation) is shown.

Figure 7 demonstrates that the temperature dependency of the deacetylating activity of AXE3 towards xanthan (diamonds) differing from the one towards xylan (squares). Notably, the temperature optima differ from another.

Figure 8 depicts the AXE3 modification of xanthan followed in time. Herein, Figure 8A depicts the HILIC-ELSD elution profile of unmodified xanthan and xanthan modified with AXE3 for 1 day, 2 days or 3 days. Figure 8B shows the relative abundance of the six xanthan repeating units at different levels of AXE three modification square: acetylated and pyruvulated RU; triangles: single inner mannose acetylated RU; diamonds: double acetylated RU; dots: pyruvylated RU; crosses: unsubstituted RU; stars: single outer mannose acetylated RU.

Figure 9 depicts the enzyme : substrate ratio of YesY for deacetylation of xanthan.

Figure 10 depicts the pH and temperature optima of the YesY enzyme. Herein, Figure 10A depicts the pH optimum and Figure 10B depicts the temperature optimum of the YesY enzyme. Figure 11 shows the comparison of the xanthan structure before and after modification with YesY (line below = unmodified xanthan; upper line = YesY modified xanthan)

Figure 12 depicts the mass spectrometry (MS-MS) MS ² -fragmentation pattern of the single acetylated repeating unit (eluting at 12.5 min in Figure 8A) of unmodified xanthan and 3 days AXE3 modified xanthan. Herein, n refers to fragments specific for the acetylation on the outer mannose and ^* refers to fragments specific for the acetylation of the inner mannose.

Examples

1. General Materials and Methods used Herein 1 . Chemicals and substrates

All chemicals used were, if not mentioned otherwise, of analytical grade. The xanthan used was kindly provided by DuPont (Melle, France) and characterised as described previously (Kool et al 2013a). Ninety per cent the inner mannose units are acetylated; 66% of the outer mannose units are pyruvylated and 21 % of the outer mannose units are acetylated.

2. Circular Dichroism

The far-UV CD spectra of 2 mg-mL-l xanthan in 0, 1 , 2, 5 and 10 mM NaCI solutions were measured at 20, 40 and 70°C using a Jasco-J-715 spectro- polarimeter, model J-715 (JASCO, Tokyo, Japan). A quartz cuvette with an optical path of 1 mm was used. The temperature was regulated using a Jasco PTC-348 Wl controller (JASCO, Tokyo, Japan). In the 190-300 nm wavelength region (0.2 nm resolution) 10 scans were accumulated with a scan rate of 100 nm ^» min-1 and a time constant of 0.125 s. The final spectra are the average of these scans. Prior to the spectral analysis the wavelength scans were corrected for the buffer background signal.

Previous research showed that the decrease in ellipticity (Θ) at 219 nm is almost linear with the fraction of xanthan that appears in the disordered conformation (Kool et al., 2013a). The fraction of disordered conformation (a) was, therefore, estimated using Equation 1 with: 0s= ellipticity of the sample at 219 nm; ΘΙΙ = ellipticity of a completely disordered structure at 219 nm and OF = ellipticity of a completely ordered structure at 219 nm. a = (0s - OF) / (0U- OF) (1 )

The ellipticity of OF was determined in 10 mM NaCI solutions at 15°C and the ellipticity of 0U was determined in milliQ water 70°C.

II. Production of the Enzymes

1 . Production of YesY The open reading frame coding for YesY was amplified using the primers YesY-for (5'-GCAATTCCATATGGCAAATCATATTTATCTTGCCG-3') and YesY-rev (5'- CTCCCTTTCTTTGATGATCGATTC-3'), genomic DNA from Bacillus subtilis strain 168 as the template, and a proofreading DNA polymerase (Phusion, Finnzymes). Bacillus subtilis strain 168 is commonly available from the Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig). YesY-for introduced an Ndel restriction endonuclease recognition site overlapping with the start codon and YesY-rev introduced a Sacl recognition site, removing the stop codon at the same time. The amplicon was kit-purified, cut with Ndel and Sacl, and ligated with the likewise cut and dephosphorylated expression vector pET22b-Strepllc. Such vector originates from pET-22b(+) (Novagen) and contains an additionally introduced sequence coding for the Strepll affinity tag. A scheme of the generated expression plasmid pET22b::YesY-Strepllc is shown in Figure 2A. The expression strain E. coli Rosetta2 (DE3)[pLysSRARE2] was transformed with the expression plasmid and the thus generated strain was used to produce YesY. For this the strain was grown in 500 ml_ auto-induction medium (Studier 2005) complemented with ampicillin (f.c. 100 pg/mL) and chloramphenicol (f.c. 34 g/mL). Incubation was done at 37°C for 4 h at 180 rpm and continued at 26°C for 20 h at 120 rpm. Then the cells were harvested by centrifugation and the cell pellet was resuspended in FPLC buffer (40 mM triethanolamine, 400 mM NaCI, pH 8.0) and frozen/stored over-night at -20°C. After melting, the cells were opened by intracellular lysozyme activity and by ultrasonic treatment. Then the suspension was centrifuged (45 min, 40.000 x g, 4°C) and the supernatant (= crude extract) was subjected to FPLC using a Strep-Tactin column (Qiagen). The Strepll-tagged YesY (26.3 kDa) was purified, re-buffered, and concentrated using Vivaspin columns (10.000 MWCO; Sartorius). From a 500-mL culture up to 5 mg of highly pure YesY were obtained. To verify purity and integrity of the enzyme an SDS-PAGE analysis and a Western blot were performed (Figure 2B). YesY muteins, carrying single amino acid exchanges, were likewise analyzed.

2. Production of AXE3 and AXE2

The AXE3 and AXE2 enzymes were produced as shown in Pouvreau et al., 201 1 . Accordingly, the ascomycetous fungus C. lucknowense C1 (accession number: VKM F-3500D of the All-Russian Collection of Microorganisms of Russian Academy of Sciences) was used for the production of hemicellulases. The full genome of C1 has been sequenced and revealed many genes encoding for cellulases, hemicellulases and accessory enzymes. Annotated genes, encoding for the putative acetyl xylan esterases Axe2 and Axe3, have been cloned into a specially designed C1 -expression host that allows production of relatively pure enzyme. The amino acid sequences have been deposited at GenBank with the accession numbers HQ324256 (Axe2) and HQ324257 (Axe3). The strains, containing extra copies of the axe2 or axe3 gene were grown aerobically in 2 L fermentors in mineral medium, containing glucose as carbon source, ammonium sulfate as nitrogen source and trace elements for the essential salts. Growth is performed under glucose limitation at pH 6.0 and 32 °C. III. Overview of all carbohydrate acetyl esterases tested

Table 1. Overview of all carbohydrate acetyl esterases tested

enzym Accession Activity known/supposed by Cazy

Origin Corresponding literature e code number literature family

AXE2 HQ324256 Myceliophthora thermophila Xylan CE-5 Pouvreau et al., 201 1 ; Hinz et

C1 2009

AXE3 HQ324257 Myceliophthora thermophila Xylan CE-1 Pouvreau et al., 201 1 ; Hinz et

C1 2009

AXE6 AAU39337.1 Bacillus licheniformis DSM13 Cephalosporin C deacetylase CE-7 -

AXE7 CAB121 12.1 Bacillus subtilis str. 168 Acetyl xylooligo-saccharide CE-7 - esterase

AXE8 YP 0066633 Arthrobacter nitroguajacolicus Cephalosporin C deacetylase unknown - 58.1 Ru61 a

CDA-II AAU39149.1 Bacillus licheniformis DSM13 Polysaccharide deacetylase CE-4 -

CDA-III AAU39762.1 Bacillus licheniformis DSM13 Polysaccharide deacetylase CE-4 -

PAE2 CAG7531 1.1 Pectobacterium carotovorum Pectin unknown -

PAE4 AAU42913.1 Bacillus licheniformis DSM13 Pectin: Rhamnogalacturonan CE-12 -

PAE5 AAU40277.1 Bacillus licheniformis DSM13 Pectin: Rhamnogalacturonan CE-12 -

PPDA NP 626539. Streptomyces coelicolor A3(2) Acetyl xylan esterase CE-4

1

YesY NP 388588. Bacillus subtilis str. 168 Pectin: Rhamnogalacturonan CE-12 Ochiai et al., 2007

1

YesT NP 388583. Bacillus subtilis str. 168 Xylan, Pectin: CE-12 Ochiai et al., 2007

1 Rhamnogalacturonan

RG-04 unknown Aspergillus aculeatus Pectin: Rhamnogalacturonan CE-12 Searle-van Leeuwen at al., 1992

PE-04 unknown Aspergillus niger Pectin: Homogalacturonan unknown Searle-van Leeuwen et al., 1996

R-06 unknown Aspergillus niger Xylan: Birch wood CE-1 Kormelink et al., 1993

Conclusions

In this research nine known carbohydrate acetyl esterases were screened for their activity towards xanthan. Acetyl Xylan Esterase 3 (AXE3) produced by Myceliophthora thermophila C1 from CE family 1 , is active towards xanthan, but only towards the disordered conformation. Characterisation of the modified xanthan showed that AXE3 is specific for the removal of the acetyl groups positioned on the inner mannose unit. After a 3-day incubation -77% of all acetyl groups on the inner mannose are removed. Although the specific activity of AXE3 towards xanthan is much lower compared to the activity towards xylan, we thereby report the first acetyl esterase that is active towards xanthan.

As it is shown that the disordered conformation is necessary for the removal of acetyl groups from the inner mannose, future research towards xanthan acetyl esterases should focus on thermophilic AEs or on AEs which are stable and active in a salt free environment. Furthermore it is recommended to start screening of known AEs which have an a-specific binding site or which are member of CE family 2, since these AEs are specific for the deacetylation of carbohydrates at the O-6 position.

IV. Use of Exemplified Enzymes

1 . Analysis of a set of Enzymes

A set of the enzymes enlisted above were used in a number of enzymatic assays. 1 .1 . Enzyme assays An overview of all carbohydrate acetyl esterases used, their origin and their known specificity is given in Table 2.

Table 2. Characteristics of the carbohydrate acetyl esterases used in this study, including abbreviation, CAZY family and references

CAZy

Activity Abbr. Origin Known activity Reference

Family

Acetyl Pouvreau

Myceliophthora Xylan

xylan AXE2 CE-5 (201 1 ); Hinz thermophila C1 02 and 03 position

esterase (2009)

AXE3 Myceliophthora Xylan CE-1 Pouvreau

AXE2 and AXE3 (Hinz et al., 2009; Pouvreau, Jonathan, Kabel, Hinz, Gruppen and Schols, 201 1 ) were kindly provided by Dyadic (Wageningen, The Netherlands). CDA-II, CDA-III, PAE2, PAE4, RG-04, PE-04 and AnAXE were expressed and purified as described elsewhere (Kormelink, Lefebvre, Strozyk and Voragen, 1993; Searle-van Leeuwen, Broek, Schols, Beldman and Voragen, 1992; Searle-van Leeuwen et al., 1996). All enzymes were desalted shortly before testing their activity towards xanthan using Micro Bio-Spin chromatography columns following the companies description (Bio-Rad Laboratories, Hercules, CA, USA).

The carbohydrate acetyl esterases were screened for their activity towards xanthan by incubating one mL of a 2 mg ^» mL-1 xanthan solution with 8-35 g enzyme at 40 ^° C for 48 h. The enzyme activity was tested in: (1 ) Millipore water, (2) 10mM NaCI solutions and (3) 50 mM citric acid buffer (pH 6). The acetic acid release was determined using a Megazyme acetic acid kit (Megazyme, Wicklow, Ireland) and expressed as percentage of the total acetic acid concentration. The protocol was downscaled to microtiter scale to enable high throughput analysis. All incubations were performed in duplicates.

Acetyl esterase AXE3, that showed activity towards xanthan, was further characterised. The precise influence of the xanthan conformation on the enzyme activity was determined by incubating 2 mg ^» mL-1 xanthan in Millipore water, 1 mM, 2 mM, 5mM and 10mM NaCI solution with 25 g of protein at 40 ^° C for 48 h. The temperature optimum was determined in a salt free environment within the temperature range 35-60°C after a 24 h-incubation. All incubations were performed in fourfold. The mode of action of the AXE3 and YesY towards xanthan was determined by structural analysis of unmodified and enzymatically modified xanthan. The modified xanthan solutions were dialysed against water and lyophilized. The obtained xanthan was redissolved in Millipore water (2 mg ^» mL-1 ) and incubated with the cellulases from Myceliophthora thermophila C1 (Dyadic Netherlands, Wageningen, The Netherlands) at 60 ^° C for 48h (Kool at al., 2013a). The obtained xanthan digests were analysed for their repeating units using HILIC-ELSD-ESI-IT- MSn as described previously (Kool et al., 2013b).

1 .2. Results and Discussion

Screening for xanthan acetyl esterase activity

The three-dimensional conformation of xanthan depends on the ionic strength and temperature of a xanthan solution (Bezemer, Ubbink, Kooker de, Kuil and Leyte, 1993; Matsuda, Biyajima and Sato, 2009). As it is known that the enzymatic hydrolysis of the xanthan backbone by cellulases strongly depends on this conformation (Kool at al., 2013a; Rinaudo and Milas, 1980), the enzyme activity of all AEs was tested at different solvent conditions. An overview of the acetic acid release from xanthan after 48 h of incubation with the different AEs is shown in Figure 4.

The results show that most of the AEs are not active towards xanthan. AXE3 is the only AE that released significant amounts of acetic acid from xanthan, however this enzyme showed no activity in 10mM NaCI solutions and/or in 50mM Citric acid buffer. Because the ordered helical conformation is favoured with increasing ionic strength (Morris, Rees and Young, 1977), it is likely to assume that AXE3 is only active towards the disordered xanthan conformation, possibly because the acetyl groups are only accessible when xanthan appears in the disordered conformation. If acetyl groups are indeed only accessible in the disordered conformation, only those acetyl esterases which are heat stable and/or active in a salt free environment are likely to be active towards xanthan. The activity of all AEs towards their model substrate (acetylated sugar beet pectin, acetylated xylan oligosaccharides or chitin oligosaccharides) was therefore tested in a salt free solution at 40 ^° C. None of the enzymes, except AXE3, was active at such conditions (results not shown). Assuming that the disordered conformation is indeed required for the enzymatic deacetylation of xanthan, this explains why, except for AXE3, none of the other tested AEs is active towards xanthan.

Previous research towards AXE3 and AXE2 showed that both enzymes, although from different CE families, showed similar activity towards acetylated xylan oligosaccharides (Hinz et al., 2009; Pouvreau, Jonathan, Kabel, Hinz, Gruppen and Schols, 201 1 ). AXE3 had a lower initial activity, however, the total amount of acetic acid released from acetylated xylan oligosaccharides at the end point was similar for both enzymes. It is therefore a bit unexpected that these enzymes show a different activity towards xanthan. It seems that AXE2 is hindered by the xanthan structure, whereas AXE3 is not. As described by Pouvreau (201 1 ) AXE2 was, in contrast to AXE3, not active towards the synthetic substrate p-nitrophenyl acetate. It was hypothesized that the two enzymes have a different binding site, and that AXE3 has a less specific binding site since it only requires an acetyl group for binding (Pouvreau, Jonathan, Kabel, Hinz, Gruppen and Schols, 201 1 ). Further activity tests towards different acetylated xylan substrates indeed showed that AXE2 and AXE3 are differently influences by the size and structure of a substrate (Pouvreau, Jonathan, Kabel, Hinz, Gruppen and Schols, 201 1 ). More general studies towards acetyl xylan esterases from different CE families showed that enzymes from CE family 4 and 5 indeed have a higher specificity than acetyl esterases from CE family 1 (Biely, 2012). The acetyl groups in xanthan on both the inner as well as the outer mannose unit are linked at the O-6 position. Substrate specificity tests performed on CE family 2 indicated that this specific CE family preferably deacetylates polysaccharides and/or oligosaccharides at the O-6 position (Biely, 2012; Topakas, Kyriakopoulos, Biely, Hirsch, Vafiadi and Christakopoulos, 2010). In this study no AEs from CE family 2 were tested, but further screening for xanthan acetyl esterases might focus on known AEs from this specific CE family.

Influence of the xanthan conformation on the acetic acid release by AXE3

To be able to analyse the influence of the xanthan conformation on the AXE3 activity, the conformation of xanthan at 40 ^° C in solutions with different NaCI concentrations was estimated using circular dichroism (CD) and Equation 1 . The far UV CD spectra are shown in Figure 5. Figure 6 shows the acetic acid release from xanthan after a 48 h incubation with AXE3 at different a. It is shown that with an increasing fraction of disordered conformation, more acetic acid is released. It is therefore concluded that AXE3 indeed is only active towards disordered xanthan molecules. Several studies on the xanthan conformation argued that the acetyl groups positioned on the inner mannose unit show interaction with the xanthan backbone and are, therefore, positioned at the inside of the xanthan helix (Morris, Rees and Young, 1977; Pelletier, Viebke, Meadows and Williams, 2001 ; Tako and Nakamura, 1984). These acetyl groups would therefore not by accessible for acetyl esterases when xanthan is in an ordered conformation, which explains that only the disordered xanthan structures are deacetylated by AXE3. In a recent study we showed that -20% of all acetyl groups are positioned on the outer mannose (Kool et al., 2013b). Whether these acetyl groups also fold to the inside of the helical conformation is unknown. However it is known that xanthan lyases are able to remove the outer mannose in the presence of salts (Hashimoto, Miki, Tsuchiya, Nankai and Murata, 1998; Ruijssenaars, de Bont and Hartmans, 1999). It is, therefore, likely to assume that the outer mannose is accessible for enzymes when xanthan appears in an ordered conformation. The acetyl groups on the outer mannose unit should, therefore, be accessible for enzymes independent of the xanthan conformation. Since AXE3 is only active towards disordered xanthan fragments, AXE3 could be specific for the removal the acetyl groups from the inner mannose unit. This indicates that, when screening for xanthan acetyl esterases, it is off great importance to test the activity of the enzyme in conditions supporting the disordered as well as the ordered xanthan conformation in order to not overlook any deacetylating activity.

Characterisation of the AXE3 activity towards xanthan

Temperature optimum:

The temperature optimum of AXE3 towards xanthan was determined in a temperature range from 35-60 ^° C in a salt free environment and compared to the temperature optimum observed towards acetylated xylan oligosaccharides at pH 7 (Pouvreau, Jonathan, Kabel, Hinz, Gruppen and Schols, 201 1 ). The results are shown in Figure 7. AXE3 has a clear temperature optimum towards xanthan between 50-55 ^° C, whereas a broader and lower temperature optimum (35-45 ^° C) was observed towards xylan oligosaccharides at pH 7. Because the xanthan conformation changes to a more disordered structure with increasing temperature, the temperature optimum observed for xanthan is a compromise between the optimal temperature for the enzyme and the total amount of accessible acetyl groups. The observed difference in temperature optima towards the two substrates can, therefore, be explained by the influence of the conformational behaviour of xanthan on the enzyme activity. Specific activity:

To compare the activity of AXE3 towards xanthan with the activity towards xylan, the specific activity of AXE3 towards xanthan was determined after a one day incubation at 55 ^° C in a salt free environment. The specific activity towards acetylated xylan oligosaccharides (pH 7; 40 ^° C), as determined by Pouvreau (201 1 ) is 8.3 U mg protein-1 . Towards xanthan the specific activity is 13 mU-mg protein-1 . So although the enzyme can remove acetyl groups from xanthan, the activity is really low compared to its main activity. We, therefore, conclude that the observed activity towards xanthan is only a side activity of AXE3 and that the original annotation as acetyl xylan esterase is still correct.

Characterisation of the modified xanthan:

To analyse the xanthan structure the enzymatically modified xanthan, xanthan modified with AXE3 for 1 day, 2 days or 3 days was degraded to its repeating units using cellulases. The relative abundance of the repeating units present in the modified xanthans was determined using HILIC-ELSD-MS (Kool et al., 2013b). The obtained HILIC elution profiles were compared to the profile of unmodified xanthan (Figure 8). After a one day modification with AXE3 the double acetylated RUs and the acetylated and pyruvylated RUs are decreased slightly in their abundance. The amount of single acetylated RUs, unsubstituted RUs and pyruvylated RUs slightly increased. Extending the AXE3 modification resulted in a further increase of the unsubstituted RUs and pyruvylated RUs, while all the other RUs decrease. After a 3 day modification with AXE3 almost all double acetylated RUs and acetylated and pyruvylated RUs are modified. However single acetylated RUs remain present even, after a 3 day modification with AXE3. Because both the inner as well as the outer mannose unit can be acetylated and because AXE 3 if only active towards the disordered xanthan conformation, AXE3 is hypothesized to be specific for the deacetylation of the inner mannose. The double acetylated repeating unit would then be converted to single acetylated RUs that are acetylated on the outer mannose. Simultaneously, single acetylated RUs that are acetylated on the inner mannose are converted to unsubstituted RUs. Because the two single acetylated RUs elute simultaneously (Kool et al., 2013b), this could explain that no clear decrease in the total amount of single acetylated RUs is observed in the HILIC-ELSD profile. To validate this hypothesis, the MS- fragmentation pattern of the single acetylated RUs before AXE3 modification was compared to the fragmentation pattern of these RUs after 3 days of modification. The results are shown in Figure 12. As described elsewhere (Kool et al., 2013b), some of the m/z-fragments are specific for inner mannose acetylation, while other m/z-fragments are specific for outer mannose acetylation. Figure 12 shows that the ratio in which these specific fragments are present in the fragmentation pattern is different for the unmodified and modified xanthan. Before modification 12% of all single acetylated RUs are acetylated on the outer mannose and after modification this increased to 47%. This indicates that the RUs which are acetylated on the outer mannose accumulate during AXE3 deacetylation. It is, therefore, concluded that AXE3 is indeed specific for the removal of acetyl groups on the inner mannose unit and that the acetyl groups on the outer mannose are not hydrolysed. Structural characterisation of the unmodified xanthan showed that 78% of all acetyl groups are substituted to the inner mannose. After a 3-day incubation 60% of all acetyl groups is removed by AXE3. Since only the acetyl groups from the inner mannose can be hydrolysed by AXE3, it is concluded that after a 3 day modification 77% of all possible acetyl groups are removed.

1 .3. Conclusions

In this research nine known carbohydrate acetyl esterases were screened for their activity towards xanthan. Acetyl Xylan Esterase 3 (AXE3) produced by Myceliophthora thermophila C1 from CE family 1 , is active towards xanthan, but only towards the disordered conformation. Characterisation of the modified xanthan showed that AXE3 is specific for the removal of the acetyl groups positioned on the inner mannose unit. After a 3 day incubation -77% of all acetyl groups on the inner mannose are removed. Although the specific activity of AXE3 towards xanthan is much lower compared to the activity towards xylan, we thereby report the first acetyl esterase that is active towards xanthan.

As it is shown that the disordered conformation is necessary for the removal of acetyl groups from the inner mannose, future research towards xanthan acetyl esterases should focus on thermophilic AEs or on AEs which are stable and active in a salt free environment. Furthermore it is recommended to start screening of known AEs which have an a-specific binding site or which are member of CE family 2, since these AEs are specific for the deacetylation of carbohydrates at the O-6 position. 2. First use of Enzyme AXE3

2.1 . Materials

Xanthan 80 (the standard xanthan as commercially available from Danisco A S, Copenhagen, Denmark) was used for all enzyme screening assays. Molecular composition below Xanthan:

xanthan type: Xanthan 80

Glc:Man:GlcA Molar ratio 1 : 0.88 : 0.41

acetyl content [w/w%]: 5.6

Pyruvate content [w/w%]: 4.4

Enzyme AXE3:

AXE3 was produced as described elsewhere (Hinz (2009) Journal of cereal science 50 (3) 318 -323; Pouvreau (2010) enzyme and microbial technology 49 (3) 312-320)

2.2. Enzyme assays

AXE3 was screened for its activity towards xanthan by incubating one mL of a 2 mg-nriL ^"1 xanthan solution with 25 g enzyme at 40 ^° C for 48 h. The enzyme activity was tested in: Millipore water; in 10mM NaCI solutions and in 50 mM citric acid buffer (pH 6). The acetic acid release was determined using a Megazyme acetic acid kit (Megazyme, Wicklow, Ireland) and expressed as percentage of the total acetic acid concentration. All incubations were performed in duplicates.

The influence of the xanthan conformation on the activity of AXE3 was determined by incubating 2 mg-nriL ^"1 xanthan in Millipore water, 1 mM, 2 mM, 5mM and 10mM NaCI solution with 25 g of protein at 40 ^° C for 48 h. At these conditions, the conformation of xanthan was determined to be: 58, 26, 16, 5 and 0% in the disordered conformation respectively.

The temperature optimum was determined in a salt free environment within the temperature range 35-60°C after a 24 h-incubation. The mode of action of AXE3 was determined by structural analysis of unmodified and enzymatically modified xanthan. The modified xanthan was dialysed against water and lyophilized. The obtained xanthan was resolved in Millipore water (2 mg-mL ^"1 ) and incubated with the cellulases from Myceliophthora thermophila C1 (Dyadic Netherlands, Wageningen, The Netherlands) at 60 ^° C for 48h (Kool et al., 2013a). The obtained xanthan digests were analysed for their repeating units using HILIC-ELSD-ESI-IT-MS ⁿ as described previously.

2.3. Results

As shown in Figure 8, an increasing AXE3 activity is observed with an increase in the fraction of disordered xanthan molecules. It is therefore concluded that acetyl groups are only accessible to AXE3 when xanthan appears in a disordered conformation. pH optimum

As the disordered conformation is required for AXE3 to be active, it was not possible to set the pH of xanthan solution, thus the pH optimum of AXE3 towards xanthan has not been determined.

Temperature optimum

As shown in Figure 12, the pH optimum of AXE3 towards xanthan differs from the pH optimum towards xylan. As elevated temperatures stimulate the disordered xanthan conformation, the difference in temperature optimum towards the different substrates can be explained by the requirement of a disordered xanthan conformation, whereas the activity towards xylan is not hindered by such conformation dependency.

Specific activity of the enzyme

Comparison of the xanthan structure before and after modification with AXE3 shows that the enzyme specifically removed the acetyl groups on the inner mannose unit. 2.4. Summary of findings for AXE3

AXE3 produced by Myceliophthora thermophila C1 and characterised in detail to be an example for CE-1 xylan acetyl esterases, could remove 31 % of all acetyl groups present in xanthan after an extended incubation at 40 ^° C. Activity towards xanthan was only observed when xanthan molecules were in the disordered conformation, since AXE3 was only active at salt concentrations < 5 mM. Optimal performance of the enzyme towards xanthan was observed 55°C in the complete absence of salt.

Enzymatically modified xanthan was hydrolysed using cellulases and analysed for its repeating units using UPLC-HILIC-ELSD/ESI-MS. It was shown that AXE3 is specific for the removal of the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a three day-incubation 80% of all the acetyl esters on the inner mannose were hydrolysed.

3. Use of Enzyme YesY

3.1 . Materials

Xanthan 80 (the standard xanthan as commercially available from Danisco) was used for all enzyme screening assays. Molecular composition below

Xanthan:

xanthan type: Xanthan 80

Glc:Man:GlcA Molar ratio 1 : 0.88 : 0.41

acetyl content [w/w%]: 5.6

Pyruvate content [w/w%]: 4.4

Enzyme YesY:

YesY was produced as laid out above.

3.2. Enzyme assays: YesY was screened for its activity towards xanthan by incubating one ml_ of a 2 img-imL ^"1 xanthan solution with different amounts (5.4 - 81 g) of enzyme at 40°C for 24 h. The enzyme activity was tested in: Millipore water and in 50 mM citric acid buffer (pH 6). The acetic acid release was determined using a Megazyme acetic acid kit (Megazyme, Wicklow, Ireland) and expressed as percentage of the total acetic acid concentration. All incubations were performed in duplicates.

The influence of the xanthan conformation on the activity of YesY was not determined, since the enzyme is active towards the ordered xanthan conformation.

The temperature and pH optimum are not determined using xanthan as substrate. However both are determined with pectin as substrate

- 5 mg SBP3124 was incubated with 4 g of YesY for 10 min. within the temperature range 40-80°C

- 5 mg SBP3124 was incubated with 4 g of YesY for 10 min. within the pH range 3-8

The mode of action of YesY was determined by structural analysis of unmodified and enzymatically modified xanthan (1 day modification at 40°C in 50mm citric acid buffer pH 6). The modified xanthan was dialysed against water and lyophilized. The obtained xanthan was redissolved in Millipore water (2 nng-nnL ^"1 ) and incubated with the cellulases from Myceliophthora thermophila C1 (Dyadic Netherlands, Wageningen, The Netherlands) at 60 ^° C for 48h. The obtained xanthan digests were analysed for their repeating units using HILIC-ELSD-ESI-IT- MS ⁿ as described previously.

SBP 3124 = sugar beet pectin, 31 % acetylated, 24% methylated

3.3. Results

Influence of the xanthan conformation on the activity of YesY

The AE activity towards xanthan was tested in milliQ environment and in 50 mM Citric acid buffer pH 6 by incubating 1 mg xanthan with 54 g YesY for 1 day. Enzyme activity was observed in both conditions, however the highest activity was observed in buffer solution. As the enzyme shows activity in 50 mM citric acid buffer, it is concluded that the enzyme is NOT affected by the xanthan conformation. After a 24-h incubation in citric acid buffer, 23% of all acetic acid groups is released. Incubation in milliQ resulted in the release of 8% of all acetic acid groups after a 1 day incubation.

The influence of the enzyme: substrate ratio was determined and the results were used to determine the specific activity, which showed to be 0.12 U/mg enzyme.

The pH and temperature optima of the YesY enzyme is depicted in Figure 10.

Specific activity of the YesY towards xanthan

Figure 1 1 shows the comparison of the xanthan structure before and after modification with YesY (black line = unmodified xanthan; red line = YesY modified xanthan)

All RUs which are acetylated on the inner mannose and pyruvylated on the outer mannose are still present in the xanthan digest after YesY modification. Furthermore the double acetylated repeating units are all degraded by YesY, but the amount of single acetylated RUs increases. This indicates that only the acetyl groups on the outer mannose are removed by YesY. This is as expected, since xanthan appears in an ordered conformation at the chosen enzyme incubations. The acetyl groups on the inner mannose units will therefore not be available for the enzyme.

It was shown that 23% of all acetyl groups in xanthan are removed after a 2-day incubation, indicating that YesY can completely deacetylated the terminal mannose units.

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