Uses and methods for generating PAPS from cyclo-PAPS using a phytase

Abstract

The present disclosure relates to uses and methods implementing a phytase for converting adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (cyclo-PAPS) into 3'- phosphoadenosine-5'-phosphosulfate (PAPS) useful for sulfating a substrate.

Description

[TITLE] USES AND METHODS FOR GENERATING PAPS FROM CYCLO-PAPS USING A PHYTASE [TECHNICAL FIELD]

[0001] The present disclosure relates to a new route, i.e. an alternative and cost- effective process to produce 3′-phosphoadenosine 5′-phosphosulfate (PAPS) starting from adenosine 2′,3′-cyclic phosphate 5′-phosphosulfate (cyclo-PAPS). It further relates to phytases able to convert cyclo-PAPS into PAPS. Also, the present disclosure relates to methods for sulfation of substrates, e.g. for synthesizing heparin compounds based thereon. [TECHNICAL BACKGROUND]

[0002] Sulfation is a conjugation process involved in numerous biological processes including synthesis of proteins, peptides or glycosaminoglycans (GAGs), detoxification, hormone regulation, molecular recognition, cell signaling, or viral entry into cells (see for a review Chapman E. et al., Angew. Chem., 2004, 43(27): 3526-48).

[0003] The sulfation reaction needs a sulfotransferase (SULT) enzyme as a catalyst and a co-substrate as a sulfuryl (or sulfo group or sulfate group) donor. PAPS dependent Sulfotransferases (SULTS) are a family of enzymes that transfer the sulfate group from universal sulfate donor PAPS (3′-phosphoadenosine 5′-phosphosulfate) onto usually a hydroxyl or amine group of a target substrate. There is a large number of substrates including proteins, hormones (steroidal and thyroid) and various glycosaminoglycans (GAG) (chondroitin sulfate, heparan sulfate, …).

[0004] As an example, heparin is a highly sulfated polymeric linear chain with repeating disaccharide units comprised of an uronic acid (glucuronic or iduronic) and a glucosamine. Heparin is widely used as an anticoagulant e.g. in the treatment of thromboembolic diseases either as unfractionated heparin, or as the starting material for the synthesis of low-molecular-weight heparins (LMWH).

[0005] In the body, heparin synthesis is complex, involving a large number of enzymes including O-sulfotransferases (2OSTs, 6OSTs and 3OSTs). The successful expression of recombinant heparin biosynthetic enzymes (Fu et al., Adv Drug Deliv Rev.2016;97:237-249) has rendered possible the bioengineering of heparins but it requires stoichiometric amounts of the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS).

[0006] However, PAPS which, as already said, is a universal sulfate donor and source of sulfate for PAPS dependent sulfotransferases is a very expensive molecule that has been an obstacle to the large-scale production of enzymatically sulfated products.

[0007] PAPS is commercially available, but only at small scale and at high prices depending on the supplier: from 2500 to 101000 k€ / kg (see the product commercialized by Sigma, Glycan Therapeutics and Yamasa). It appears that fully enzymatic or chemo- enzymatic processes are commonly used for its production.

[0008] As previously described e.g. in US 4,169,011, a chemo-enzymatic process is as shown below:

[0009] First and in the chemical part of the process, adenosine or 5’-AMP is converted to cyclo-PAPS. Then, cyclo-PAPS is converted to PAPS by opening the cyclic 2’,3’-phosphate through a nucleophilic addition of water. At this stage, both PAPS and iso- PAPS can be obtained, wherein PAPS is the most active sulfate donor:

[0010] In order to be specific and to obtain only PAPS, this reaction has almost exclusively been described using a selective phosphatase. To the best knowledge of the inventors, all examples in the literature of this reaction describe the use of Ribonuclease T2 (RNase T2). Although this enzyme allows a very selective ring-opening, it is very expensiveto use and its cost is the principal contributor to the overall cost associated with PAPS production according to the chemo-enzymatic pathway described above.

[0011] As an example, the ribonuclease T2 (RNase T2) may be the recombinant enzyme from Aspergillus oryzae sold by Creative Enzymes®or Worthington biochemical®.

[0012] Therefore, there is a need to optimize the conversion of cyclo-PAPS into PAPS.

[0013] There is a need to have enzymes usable in bioprocess to convert cyclo- PAPS into PAPS.

[0014] There is a need to have enzymes with phosphatase activity to convert cyclo- PAPS into PAPS.

[0015] There is a need to have phytases, such as Aspergillus niger phytases, with phosphatase activity to convert cyclo-PAPS into PAPS.

[0016] There is a need to have phytases, such as Aspergillus niger phytases, with good phosphatase activity and lower price.

[0017] There is a need to have methods for sulfation of substrate with lower cost and / or improved yield.

[0018] There is a need to have a method for producing bioengineered heparin with lower cost and / or improved yield.

[0019] There is a need to have a method for sulfation of substrate or preparing bioengineered heparin products which can use a new enzyme to convert cyclo-PAPS to PAPS.

[0020] There is a need to produce PAPS at low cost and in large quantity, thereby avoiding PAPS regeneration via PAP recycling, which is costly and time-consuming.

[0021] The present has for purpose to meet all or part of these needs. [SUMMARY]

[0022] According to one of its objects, the present disclosure relates to the use of a phytase to convert adenosine 2′,3′-cyclic phosphate 5′-phosphosulfate (cyclo-PAPS) into 3’-phosphoadenosine-5’-phosphosulfate (PAPS).

[0023] As shown in the Examples illustrating the present disclosure, the inventors have surprisingly found that said specific phosphatases called Phytases, which canonicalreaction is the conversion of phytic acid to inositol, are able to convert cyclo-PAPS into PAPS, in an efficient manner and without significant formation of side-products.

[0024] PAPS that is produced using phytases to convert cyclo-PAPS to PAPS can be advantageously used in biocatalytic sulfation reactions.

[0025] PAPS that is produced using phytases to convert cyclo-PAPS to PAPS can be advantageously used in the biocatalytic synthesis of heparin and more generally in any sulfation reactions involving an enzyme working with this cofactor.

[0026] The present disclosure provides advantageously a source of PAPS at low cost and high yield, allowing the large-scale synthesis of sulfated substrates such as heparin products.

[0027] Furthermore, the present disclosure provides phytases to convert cyclo- PAPS into PAPS, which can be easily obtained.

[0028] The present disclosure provides advantageously methods for obtaining sulfated substrate, such as heparin, at high-yield and low cost, allowing an efficient industrial scale-up while avoiding the need to recycle PAP (3′(2′)-phosphoadenosine-5′- phosphate) into PAPS.

[0029] According to one of its objects, the present disclosure relates to the use of a phytase for generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from adenosine 2′,3′-cyclic phosphate 5′-phosphosulfate (cyclo-PAPS).

[0030] According to one of its objects, the present disclosure relates to a method for generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from adenosine 2′,3′-cyclic phosphate 5′-phosphosulfate (cyclo-PAPS) using a phytase.

[0031] According to one of its objects, the present disclosure relates to a method of preparing PAPS comprising incubating cyclo-PAPS with at least one phytase in a reaction medium.

[0032] A phytase as disclosed herein may be obtained from a fungus. According to one embodiment, a phytase as disclosed herein originates from Ascomycete fungi or basidiomycetes. According to a specific embodiment, a phytase as disclosed herein is from Aspergillus species, e.g. from Aspergillus niger. Alternatively, a phytase as disclosed herein may originate from bacteria, e.g. Escherichia coli.

[0033] A phytase as disclosed herein may be a native enzyme, endogenously produced by an organism. Alternatively, a phytase as disclosed herein is a recombinant enzyme, possibly a non-naturally occurring enzyme, produced by a host cell or organism.

[0034] In some embodiments, the cells or organisms producing the phytase may produce not only one phytase but a mixture of phytases. As an example, Aspergillus niger produces at least two phytases encoded by the phyA and phyB genes, respectively.

[0035] A phytase as disclosed herein may be isolated or purified.

[0036] A reaction medium adapted for a phytase as disclosed herein may have a pH from 4 to 6, optionally a pH from 4.5 to 5.5. In some embodiments, it may comprise phosphate, optionally at a concentration from 25 to 400 mM. In some embodiments, it may comprise a buffer, optionally a succinate-phosphate buffer, a citrate-phosphate buffer or a sodium phosphate buffer.

[0037] The concentration of a phytase as disclosed herein in the reaction medium depends on the phosphatase activity of the phytase (usually expressed in unity of activity / g (U / g)) and can be determined by the skilled person as explained below. It is usually expressed in unity of activity / L of the reaction medium (U / L). When using a phytase having a standardized activity per weight (e.g. 2000 U / g), said quantity can be expressed in weight / weight (%w / w or wt%), weight / volume (g / L), activity / weight (U / g) or activity / L (U / L). The concentration of a phytase as disclosed herein, optionally having a phytase activity of 2000 U / g, may be from 0.1 g / L (about 0.01 wt% or 0.01%w / w or 200 U / L) to 1 g / L (about 0.1 wt% or 0.1%w / w or 2000 U / L), optionally from 0.2 g / L (about 0.02 wt% or 0.02%w / w or 400 U / L) to 0.8 g / L (about 0.08 wt% or 0.08%w / w or 1600 U / L).

[0038] The concentration of cyclo-PAPS in the reaction medium may be from 2 g / L to 200 g / L, optionally from 30 g / L to 120 g / L.

[0039] A phytase as disclosed herein may be incubated with its substrate (cyclo- PAPS) at an appropriate temperature, optionally from 15°C to 40°C, e.g. at 25°C or at 37°C.

[0040] The incubation between a phytase as disclosed herein and its substrate (cyclo-PAPS) may be stopped after an appropriate time, optionally from 1 hour to 40 hours, e.g. not after 24 hours.

[0041] The end-product of this conversion is 3'-phosphoadenosine-5'- phosphosulfate (PAPS). As known in the art, said product can be provided as a salt, e.g. a metal salt or an amine salt. Examples of metal salt include lithium salt or sodium salt. As disclosed in US 2009 / 0118491, an amine salt may be formed between PAPS and an amine compound. Such an amine compound may have one or more amino or imino groups, may be a secondary amine, optionally having a C1-C10 hydrocarbon chain, a tertiary amine, optionally having a C1-C7 hydrocarbon chain, a polyamine or an amino acid. Said amine compound can be selected in the group consisting of: diethylamine, piperidine,trimethylamine, triethylamine, triethanolamine, putrescine, polyethyleneimine, and L- histidine. It may be in the form of a triethylamine ammonium salt.

[0042] It is understood that the selected salt of cyclo-PAPS will determine which salt of PAPS will be produced.

[0043] In some embodiments, PAPS prepared according to the method as disclosed above is in the form of a triethylammonium salt (TEA), when starting from cyclo-PAPS TEA salt.

[0044] The end-product of this conversion, i.e. PAPS, may be isolated from the reaction medium and further purified. It can be in the form of powder, or a liquid (e.g. a solution) which can be further dried or lyophilized.

[0045] According to one of its objects, the present disclosure relates to a kit for preparing PAPS, the kit comprising at least:

[0046] a phytase as disclosed herein in a first container; and

[0047] cyclo-PAPS in a second container.

[0048] In some embodiments, a kit may further comprise a buffer as disclosed above, advantageously comprising phosphate.

[0049] In some embodiments, in a kit as disclosed herein, the buffer may be selected in the group comprising a succinate-phosphate buffer, a citrate-phosphate buffer or a sodium phosphate buffer.

[0050] According to one of its objects, the present disclosure relates to a method of preparing PAPS from adenosine or 5’-AMP, comprising the following steps:

[0051] Phosphorylation of adenosine or 5’-AMP into PAP and iso-PAP

[0052] Conversion of PAP and iso-PAP into cyclo-PAPS

[0053] Conversion of cyclo-PAPS into PAPS using a phytase as disclosed herein, optionally according to a method as disclosed above.

[0054] According to one of its objects, the present disclosure relates to the use of PAPS so prepared as disclosed herein, for sulfating a substrate.

[0055] According to one of its objects, the present disclosure relates to a method for sulfating a substrate, comprising at least a step of contacting said substrate to be sulfated with:

[0056] at least a sulfotransferase

[0057] a sulfo group donor comprising PAPS prepared according to the method as disclosed above

[0058] in conditions suitable for a transfer of the sulfo group from the sulfo group donor to said substrate.

[0059] In uses or methods as disclosed herein a substrate may be sulfated with one or a plurality of sulfotransferases to carry out a plurality of sulfations.

[0060] In a method as disclosed herein a plurality of sulfations may be carried out concomitantly or sequentially.

[0061] In a method as disclosed herein a step of converting cyclo-PAPS into PAPS may be carried out concomitantly with the sulfation or separately.

[0062] A method as disclosed herein may further comprise a step of recovering the sulfated substrate.

[0063] A use or a method as disclosed herein may be for preparing any sulfated substrate including heparin products.

[0064] According to one of its objects, the present disclosure relates to a method for sulfating a substrate or for preparing a heparin product comprising a step of generating PAPS from cyclo-PAPS using a phytase as disclosed herein, optionally according to a method as disclosed above. [DESCRIPTION OF THE FIGURES]

[0065] Figure 1 represents:

[0066] (Figure 1A) the chromatogram of PAPS, cyclo-PAPS, 3’-PAP and 5’-AMP.

[0067] (Figure 1B) the chromatogram of a phytase catalyzed conversion of cyclo- PAPS into PAPS, after 2.5 h incubation with 0.1 g / L Sumizyme®Phytase (from Aspergillus niger) at pH 5.5 in a succinate-phosphate buffer.

[0068] Figure 2 represents the cyclo-PAPS (■) and the PAPS (▲) area% during a phytase catalyzed conversion of cyclo-PAPS into PAPS as a function of time, obtained with a Sumizyme®Phytase (from Aspergillus niger) used under optimized conditions.

[0069] Figure 3 represents the HPLC chromatogram of PAPS as obtained by the new procedure according to the present disclosure. [DETAILED DESCRIPTION]Definitions

[0070] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. “A” and “an” mean “at least one”, unless the content clearly dictates otherwise.

[0071] The terms “about” or “approximately” as used herein refer to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term “about” refers to ±10% of a given value. However, whenever the value in question refers to an indivisible object, such as a molecule or other object that would lose its identity once subdivided, then “about” refers to ±1 of the indivisible object.

[0072] Within the disclosure, the expression “sulfotransferase” (noted SULT or ST) intends to refer to an enzyme that catalyzes the sulfate conjugation of a substrate.

[0073] Within the disclosure, the expression “phosphatase” intends to refer to an enzyme belonging to a subcategory of hydrolases, that uses water to cleave a phosphoric acid ester into a phosphate ion and an alcohol. Some phosphatases are also able to use organic cyclic phosphates as a substrate, thereby generating a phosphoric acid ester instead of a phosphate ion.

[0074] Within the disclosure, the expression “phytase” intends to refer to a phosphatase enzyme whose canonical reaction is the hydrolysis of phytic acid into inorganic phosphorus.

[0075] Within the disclosure, the expression “phosphatase activity” intends to refer to the catalytic activity of a phytase opening (e.g. through a nucleophilic addition of water) the cyclic 2’, 3’-phosphate on cyclo-PAPS to generate PAPS. Said activity may result in the production of PAPS (3′-phosphoadenosine 5′-phosphosulfate; CAS number: 482-67-7), possibly iso-PAPS (2′-phosphoadenosine 5′-phosphosulfate), and the disappearance of cyclo-PAPS (adenosine 2′,3′-cyclic phosphate 5′-phosphosulfate; CAS number: 62230-90- 4).

[0076] It is understood that aspects and embodiments of the present disclosure described herein include “having,” “comprising,” “consisting of,” and “consisting essentially of” aspects and embodiments. The words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of” implies the inclusion of thestated element(s), to the exclusion of any additional elements. The term “consisting essentially of” implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic characteristic(s) of the disclosure. It is understood that the different embodiments of the disclosure using the term “comprising” or equivalent cover the embodiments where this term is replaced with “comprising only”, “consisting of” or “consisting essentially of”.

[0077] The expression “enhanced activity” with regard to an enzyme intends to mean that the enzyme has a catalytic activity, or a thermal stability or a structure stability which is enhanced compared to a reference enzyme.

[0078] Within the disclosure, the expression "isolated" with regard to a compound or entity, such as an enzyme, refers to this compound or entity in an environment different from the one in which the compound or entity may naturally occur. "Isolated" is meant to include compound or entity in samples which are substantially enriched for this compound or entity and / or in which this compound or entity is partially or substantially purified. In some cases, an isolated compound or entity (e.g. a protein, such as a phytase; a nucleic acid; a recombinant vector) is purified, e.g. it is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99%, pure.

[0079] Within the disclosure, the expression “non-naturally occurring” as used herein with regard to a nucleic acid, a peptide, polypeptide, or protein refers to any nucleic acid, peptide, polypeptide, or protein which are not found in nature.

[0080] Within the disclosure, the expression “mutant” as used herein with regard to a peptide, polypeptide, or protein refers to any peptide, polypeptide, or protein comprising at least one amino acid mutation. “Amino acid mutation” and “mutation” are used interchangeably and intend to refer to a substitution, a deletion, or an insertion of an amino acid, as compared to a wild-type, or naturally occurring, counterpart. In particular, a mutant peptide, polypeptide, or protein may comprise at least one amino acid substitution.

[0081] A “recombinant protein” as used herein intends to refer to a protein produced with a recombinant DNA. A “recombinant DNA” refers to a genetically engineered DNA molecule formed by splicing fragments of DNA from different sources or from another part of the same source, and then introduced into the recipient (host) cell. For example, a recombinant protein may be produced by inserting the corresponding coding nucleic acid in a plasmid vector and delivering the vector in a host cell suitable for the expression of the protein.

[0082] Within the disclosure, the term “significantly” used with respect to change intends to mean that the observe change is noticeable and / or it has a statistic meaning.

[0083] Within the disclosure, the term “substantially” used in conjunction with a feature of the disclosure intends to define a set of embodiments related to this feature which are largely but not wholly similar to this feature. The difference between the set of embodiments related to the given feature and the given feature is such that in the set of embodiments, the nature and function of the given feature is not materially affected.

[0084] Within the disclosure, the expression “substantially the same or greater than” used to qualify the catalytic activity of a given enzyme with respect to the catalytic activity of a reference enzyme intends to define (i) that the catalytic activity of both enzymes, when measured with same protocol and conditions, are not significantly different or (ii) that the catalytic activity of the given enzyme is significantly above the catalytic activity of the reference enzyme, when both measured with same protocol and conditions.

[0085] The terms “sulfation”, “sulfonation” and “sulfurylation” are used interchangeably and herein refer to a transfer of a sulfonate or sulfuryl group from one molecule to another.

[0086] The terms “heparin product” and “heparin compound” means any of various glycosaminoglycans which are structurally or pharmacologically related to heparin.

[0087] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0089] The list of sources, ingredients, and components as described hereinafter are listed such that combinations and mixtures thereof are also contemplated and within the scope herein.

[0090] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0091] All lists of items, such as, for example, lists of ingredients, are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of’ the list of items “and combinations and mixtures thereof.”

[0092] Referenced herein may be trade names for components including various ingredients utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g. those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein. Phytases

[0093] A phytase (myo-inositol hexakisphosphate phosphohydrolase) as disclosed herein is any type of phosphatase that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) - an indigestible, organic form of phosphorus that is found in many plant tissues, especially in grains and oil seeds - and releases a usable form of inorganic phosphorus (Pi). While phytases have been found to occur in animals, plants, fungi and bacteria, phytases have been most commonly detected and characterized from fungi (see for a review: Livia Ribeiro Corrêa and Fernandez de Araujo, Brazilian Journal of Microbiology (2020) 51: 1009-1021).

[0094] In some embodiments and according to the order of release of the phosphate groups from phytic acid (i.e.; the first phosphate position of phytic acid that is hydrolyzed), a phytase as disclosed herein may be selected among the 3-phytases (E.C.3.1.3.8), 4 / 6- phytases (E.C.3.1.3.26), and 5-phytases (E.C.3.1.3.72).

[0095] In some embodiments, a phytase as disclosed herein may be selected among the four distinct classes of phytase as characterized in the literature according to their catalytic domains: histidine acid phosphatases (HAPS), beta-propeller phytases(BPPs), purple acid phosphatases (PAPs), and protein tyrosine phosphatase-like phytases (PTP-like phytases).

[0096] In some embodiments, a phytase as disclosed herein belongs to the class of HAPs, characterized by an active site sequence motif (Arg-His-Gly-X-Arg-X-Pro) and having a two-step mechanism that hydrolyzes phytic acid (as well as some other phosphoesters).

[0097] In some embodiments, a phytase as disclosed herein belongs to the class of BPPs, characterized by a three-dimensional structure similar to a propeller with six blades.

[0098] In some embodiments, a phytase as disclosed herein belongs to the class of PAPs, characterized by having an active site motif of a purple acid phosphatase.

[0099] In some embodiments, a phytase as disclosed herein belongs to the class of PTP-like phytases, characterized by an active site sequence motif (His-Cys-(X)5-Arg).

[0100] In some embodiments, a phytase as disclosed herein may be a HAP from fungi, optionally from Aspergillus sp., e.g. Aspergillus sp. A25 (GenBank Accession Number: ADK38546.1), A. fumigatus (NCBI Reference Sequence: XP_751964.2) or A. niger (GenBank Accession Numbers: CAA78904.1 (PhyA) and AAA02934.1 (PhyB)), Penicillium chrysogenum (or notatum; NCBI Reference Sequence: XP_002561094.1) or oxalicum (GenBank Accession Number: AAL55406.1), Peniophora lycii (GenBank Accession Number: CAC48195.1), Ceriporia sp. (GenBank Accession Number: CAC48163.1) or Trametes versicolor or pubescens (GenBank Accession Number: CAC48160.1).

[0101] In some embodiments, a phytase as disclosed herein may be a PAP from animals or plants, optionally from Zea mays (NCBI Reference Sequence: NP_001149655.1), Glycine max (GenBank Accession Number: ADM32502.1), Sus scrofa, Homo sapiens (UniProtKB / Swiss-Prot Accession Number: P13686.3), or Rattus norvegicus.

[0102] In some embodiments, a phytase as disclosed herein is a BBP from bacteria, optionally from Bacillus sp., e.g. B. licheniformis (GenBank Accession Number: AOP13449.1 and AFQ59979.1), B. amylolichefaciens (GenBank Accession Number: OBR26768.1) or B. subtilis (NCBI Reference Sequence: WP_367147708.1), Pseudomonas fluorescens (GenBank Accession Number: ANV78615.1), Pseudoxanthomonas suwonensis (NCBI Reference Sequence: WP_013534878.1), Rhodanobacter fulvus (GenBank Accession Number: EIL89790.1) or Stenotrophomonas maltophilia (NCBI Reference Sequence: WP_057496850.1).

[0103] In some embodiments, a phytase as disclosed herein is a fungal phytase, e.g. from a filamentous fungus. Alternatively, a phytase as disclosed herein is from another microorganism, e.g. bacteria (e.g. from Escherichia coli) or yeast, from an animal or from a plant.

[0104] A non-limiting list of possible sources for a phytase as disclosed herein is: Aspergillus niger (e.g. NRRL 3135, 113, SK-57, N-J, NII08121, BCC 18081 (TR70), CBS 513.88, SRRC256, N-3, M94550), A. niger var. ficuum, A. ficuum (e.g. NTG-23), A. terreus (e.g. 9A-1), A. nidulans, A. fumigatus (e.g. WY-2, ATCC 34625), A. oryzae (e.g. SH18, SBS50), A. aculeatus (e.g. RCEF 4894, APF1), A. foetidus (e.g. MTCC11682), A. tubingensis, Argyrosomus japonicus (e.g. BCC 18313 (TR86)), Myceliophthora thermophila, Thermus thermophilus, Thermomyces lanuginosus (e.g. CBS 586.94), Peniophora lycii (e.g. CBS 686.96), Agrocybe pediades (e.g. CBS 900.96), Ceriporia sp. (e.g. CBS 100231), Trametes pubescens (e.g. CBS 100232), Penicillium oxalicum (e.g. PJ3), Ureaplasma parvum (e.g. BCC 17694), Neosartorya spinosa (e.g. BCC 41923), Penicillium chrysogenum (e.g. CCT1273), S. thermophile, Morchella importuna, Citrobacter braakii, Escherichia coli, Buttiauxella sp., Neurospora crassa, Lentinus edodes, Volvariella volvaceae, Flammulina vellutipes, Acrophialophora sp. including Acrophialophora levis, Humicola sp., Lichtheimia sp., Scytalidium sp.

[0105] In some embodiments, a phytase as disclosed herein is from A. niger or A. niger var. ficuum, e.g. from the strain NRRL 3135 (ATCC 66876) able to produce large amounts of extracellular phytases.

[0106] In some embodiments, a phytase as disclosed herein may be the PhyA protein (GenBank Accession Number: CAA78904.1) and / or the PhyB protein (GenBank Accession Number: AAA02934.1) from A. niger.

[0107] In some embodiments, a phytase as disclosed herein may be the phytases produced by A. niger.

[0108] In some embodiments, recombinant DNA technology is used. In other words, the corresponding gene is isolated from an organism able to produce said phytase, cloned in an expression system which is then introduced in a host. As an example, the phytase genes phyA and phyB from A. niger have been cloned. Said host can be homologous (i.e. the same organism as the one serving for the isolation of the gene) or heterologous. As an example, the phytase gene phyA (from A. niger NRRL 31235) has been successfully overexpressed in A. niger. Besides microorganisms including bacteria, fungi and yeasts, the host can be a plant or an animal.

[0109] In some embodiments, a phytase as disclosed herein is obtained from genetically modified strains.

[0110] When expressed in a heterologous host, the gene sequence encoding a phytase as disclosed herein can be modified according to the codon usage of the host.

[0111] In some embodiments, the cells or organisms producing a phytase as disclosed herein may be grown under submerged or solid conditions.

[0112] In some embodiments, the cells or organisms producing a phytase as disclosed herein may be grown on various substrates such as glucose, fructose galactose, maltose, sucrose, starch, rye flour, corn / soybean meal, wheat bran and sugarcane bagasse. In some embodiments, phytic acid or calcium phytate may be used.

[0113] In some embodiments, the cells or organisms producing a phytase as disclosed herein are grown under depression conditions, i.e., at low phosphate (Pi) concentration.

[0114] In some embodiments, a phytase as disclosed herein is secreted by the cells or organisms producing it. In some embodiments, the coding sequence of a phytase as disclosed herein is fused to a signal sequence, from the same origin or from another origin. As an example, the α-factor signal sequence of Saccharomyces cerevisiae, possibly modified according to the codon usage of the host cells or organisms, can be used.

[0115] In some embodiments, a phytase as disclosed herein may be purified from the cells or organisms which produce it. In case the cells or organisms secrete said enzyme, the phytase can be obtained from the culture medium, further purified or not.

[0116] Non-limiting examples of commercial phytases which can be used in the context of the present disclosure are:

[0117] - the Natuphos®product commercialized by BASF, which contains a phytase from A. niger var. ficuum, produced by A. niger

[0118] - the Axtra®PHY product commercialized by Danisco Animal Nutrition (DuPont), which contains a phytase from Buttiauxella sp., produced by Trichoderma reesei

[0119] - the Ronozyme®products (Hiphos and NP) commercialized by Novozymes / DSM which contain a phytase from Citrobacter braakii and Peniophora lycii, respectively, produced by A. oryzae

[0120] - the Sumizyme™ PHY product commercialized by Takabio, which is an enzyme preparation derived from a non-genetically modified strain of A. niger

[0121] - the Finase®EC product commercialized by AB Vista, which contains an E. coli-derived 6-phytase

[0122] - the PHYZYME®XP 5000G / L from Danisco Animal Nutrition (DuPont), which contains a 6‐phytase from E. coli produced by a genetically modified strain of Schizosaccharomyces pombe.

[0123] The phosphatase activity of a phytase as disclosed herein may be detected and measured according to any known method in the art. In some embodiments, the activity is measured by chromatography, e.g. by high-performance liquid chromatography (HPLC) as disclosed in the examples, which allows differentiating cyclo-PAPS and PAPS. In some embodiments, the method allows measuring the amount of PAPS released (or produced) and / or the amount of cyclo-PAPS consumed. As a reference, the Aspergillus niger phytase(s), e.g. the Sumizyme™ Phytase product, may be used.

[0124] A phytase as disclosed herein may comprise or consist of an amino acid sequence which has at least 40%, 50% or 60% identity with the amino acid sequence of the A. niger phytase(s) contained in the Sumizyme™ PHY product. A phytase as disclosed herein may comprise or consist of an amino acid sequence which has at least 40%, 50% or 60% identity with the amino acid sequence of the A. niger PhyA and / or PhyB. In some embodiments, the sequences of the phytase(s) may have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over the entire sequence.

[0125] Homology or identity of sequence may be measured using known methods. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al. (1984) Nucleic Acids Research 12, 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10).

[0126] Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915- 10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0127] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g. Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873- 5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0128] In some other embodiments, a phytase as disclosed herein may comprise further mutation(s) or amino acid substitutions, as far as they do not affect its catalytic activity, in particular its phosphatase activity.

[0129] In some embodiments, a substitution may be conservative, that is it replaces an amino acid with another amino acid of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity or hydrophobicity to the amino acids they replace. Conservative amino acid changes are well known in the art. Conservative amino acid changes may also be determined by reference to the Point Accepted Mutation (PAM) or BLOcks SUbstitution Matrix (BLOSUM) family of scoring matrices for conservation of amino acid sequence. Thus, conservative amino acid changes may be members of an equivalence group, being a set of amino acids having mutually positive scores in the similarity representation of the scoring matrix selected for use in an alignment of the reference and mutant polypeptide chains.

[0130] Alternatively, a conservative substitution may be a substitution of an amino acid of one class by an amino acid of another class but with a similar chemical structure, a similar chemical property and / or a similar side-chain volume.

[0131] Alternatively, in some embodiments, a substitution mutation may be a non- conservative mutation, which replaces the amino acid of one class with an amino acid of non-similar chemical structure, non-similar chemical property and / or non-similar side-chain volume.

[0132] The mutations may be introduced into the enzyme by using any methods known in the art, such as site directed mutagenesis of the enzyme, PCR and gene shuffling methods or by the use of multiple mutagenic oligonucleotides in cycles of site-directed mutagenesis. The mutations may be introduced in a directed or random manner. The mutagenesis method thus produces one or more polynucleotides encoding one or more different mutants, which thereafter may be screened according to the methods disclosed hereafter. Alternatively, the nucleic acids encoding the mutated enzyme disclosed herein may be obtained using any gene synthesis methods known in the art.

[0133] In some embodiments, a phytase as disclosed therein may be a hybrid protein, i.e. a complex of two or more polypeptide sequences or fragments thereof which would not normally be associated but are coupled together either by fusing the genes which encode them (gene fusions) or by chemically cross-linking the purified component parts.

[0134] The phytases disclosed herein may be recombinant proteins.

[0135] The phytases disclosed herein may be isolated or purified proteins. Recombinant expression

[0136] The phytases of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods.

[0137] Where a phytase is produced using recombinant techniques, the methods can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, usually a bacterial, yeast, plant, and animal cells. Methods for introduction of genetic material into host cells include, for example, transformation, electroporation, conjugation, calcium phosphate methods and the like. The method for transfer can be selected so as to provide for stable expression of the introduced phytase encoding nucleic acid. The phytase encoding nucleic acid can be provided as an inheritable episomal element (e.g. plasmid) or can be genomically integrated.

[0138] In some embodiments the hosts are filamentous fungi or yeast strains. In some embodiments, hosts are bacterial strains. In some embodiments, said bacterial strains are genetically modified to overexpress chaperones and other folding-related proteins. As an example, E. coli strains such as the strain BL21 (DE3) may be used andmodified to coexpress the chaperones GroES / EL. In some embodiments, non-microbial hosts, including microalgae, plants (e.g. wheat, Brassica napus, Zea mays) and animals (e.g. pigs or fishes such as Oryzias latipes) may be used.

[0139] Non-limiting examples of hosts which may be used to produce a phytase as disclosed herein are: A. niger (e.g. NW205), Fusarium venenatum, Saccharomyces cerevisiae (e.g. INVSc1), A. oryzae (e.g. RIB40 niaD−, A1560), Pichia pastoris (e.g. GS115, KM71), Escherichia coli (e.g. BL21 DE(3)), Kluyveromyces lactis, Penicillium griseoroseum, (e.g. T73), Trichoderma reesei, Ogataea thermomethanolica.

[0140] The present disclosure provides nucleic acids, including isolated or recombinant nucleic acids, that comprise a nucleotide sequence encoding phytases as disclosed herein. In some embodiments, the present disclosure provides a nucleic acid (or nucleotide sequence) encoding a phytase as disclosed herein. In some embodiments, the nucleotide sequence is operably linked to a transcriptional control element, e.g. a promoter.

[0141] In some instances, a nucleic acid comprising a nucleotide sequence encoding a phytase may be present in an expression vector. In some embodiments, the present disclosure provides a recombinant expression vector comprising a nucleic acid encoding a phytase as disclosed herein. The present disclosure provides a recombinant expression vector (e.g. an isolated recombinant expression vector) that comprises a nucleotide sequence encoding a phytase of the present disclosure.

[0142] In some embodiments, the nucleotide sequence encoding the phytase is operably linked to a transcriptional control element, e.g. a promoter. The promoter is in some cases constitutive. The promoter is in some cases inducible. In some cases, the promoter is suitable for use (e.g. active) in a prokaryotic host cell. In some cases, the promoter is suitable for use (e.g. active) in a eukaryotic host cell.

[0143] In some embodiments, a promoter may be the constitutive promoter gpdA from Aspergillus nidulans.

[0144] In some embodiments, a promoter may be the methanol-induced AOX (alcohol oxidase) promoter from Pichia pastoris.

[0145] Suitable vectors for transferring phytase encoding nucleic acid can vary in composition.

[0146] Integrative vectors can be conditionally replicative or suicide plasmids, bacteriophages, and the like. The constructs can include various elements, including for example, promoters, selectable genetic markers (e.g. genes conferring resistance toantibiotics (for instance kanamycin, erythromycin, chloramphenicol, or gentamycin)), origin of replication (to promote replication in a host cell, e.g. a bacterial host cell), and the like. The choice of vector will depend upon a variety of factors such as the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially.

[0147] In one example, the vector is an expression vector based on episomal plasmids containing selectable drug resistance markers and elements that provide for autonomous replication in different host cells (e.g. in both E. coli and N. meningitidis). One example of such a "shuttle vector" is the plasmid pFPIO (Pagotto et al. (2000) Gene 244: 13-19).

[0148] According to some embodiments, the pFF1 plasmid may be used to harbor the sequence encoding a phytase as disclosed herein.

[0149] Constructs (recombinant vectors) can be prepared by, for example, inserting a polynucleotide of interest into a construct backbone, typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination or site-specific recombination. Typically, homologous recombination is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence, while site- specific recombination can be accomplished through use of sequences that facilitate site- specific recombination (e.g. cre-lox, att sites, etc.). Nucleic acid containing such sequences can be added by, for example, ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence.

[0150] Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome. Vectors are amply described in numerous publications well known to those in the art, including, e.g. Short Protocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expression of the nucleic acids encoding the protein of interest, may provide for propagating the subject nucleic acids, or both.

[0151] Examples of vectors that may be used include but are not limited to those derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example,plasmid vectors such as pBR322, pUC 19 / 18, pUC 118, 119 and the M13 mp series of vectors may be used. pET21 is also an expression vector that may be used. Bacteriophage vectors may include λgtl0, λgtl l, λgtl8-23, λΖΑΡ / R and the EMBL series of bacteriophage vectors. Further vectors that may be utilized include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.

[0152] For expression of a protein of interest, an expression cassette may be employed. Thus, the present disclosure provides a recombinant expression vector comprising a subject nucleic acid. The expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to a phytase or may be derived from exogenous sources. In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g. T7, and the like).

[0153] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest. A selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector. In addition, the expression construct may include additional elements. For example, the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition, the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

[0154] Any of a number of suitable host cells can be used in the production of phytases. In general, the protein of interest described herein may be expressed in prokaryotes or eukaryotes, e.g. bacteria such as Escherichia coli in accordance with conventional techniques. Thus, the present disclosure further provides an in vitro host cell, which comprises a nucleic acid encoding a phytase as disclosed herein. Host cells for production (including large scale production) of a protein of interest can be selected from any of a variety of available host cells.

[0155] Examples of host cells for expression include those of a prokaryotic or eukaryotic unicellular organism, such as bacteria (e.g. Escherichia coli strains), yeast (e.g. Saccharomyces cerevisiae, Pichia spp., and the like), and may include host cells originally derived from a higher organism such as insects, vertebrates, e.g. mammals. Suitable bacteria include but are not limited to BL21 Competent E. coli, BL21(DE3) Competent E. coli, NEB Express Competent E. col, NEB Express Iq Competent E. coli, T7 Express Competent E. coli, T7 Express Iq Competent E. coli, T7 Express lysY Competent E. coli, T7 Express lysY / Iq Competent E. coli, T7 Express Crystal Competent E. coli, SHuffle Express Competent E. coli, SHuffle T7 Express Competent E. coli, SHuffle T7 Express lysY Competent E. coli, SHuffle T7 Competent E. coli, NiCo21(DE3) Competent E. coli, Lemo21(DE3) Competent E. coli. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g. American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g. ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g. ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g. ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g. ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.). In some cases, bacterial host cells and yeast host cells are of particular interest for production of the protein of interest.

[0156] As previously mentioned, hosts cells may be from A. niger (e.g. NW205), Fusarium venenatum, Saccharomyces cerevisiae (e.g. INVSc1), A. oryzae (e.g. RIB40 niaD−, A1560), Pichia pastoris (e.g. GS115, KM71), Escherichia coli (e.g. BL21 DE(3)), Kluyveromyces lactis, Penicillium griseoroseum, (e.g. T73), Trichoderma reesei or Ogataea thermomethanolica. Isolation and purification

[0157] Isolation and purification of phytases can be accomplished according to methods known in the art. For example, a phytase can be isolated from a lysate of cells, possibly genetically modified to express a phytase, or from a synthetic reaction mix, by immunoaffinity purification, which generally involves contacting the sample with a phytase antibody, washing to remove non-specifically bound material, and eluting specifically bound phytase. Isolated phytase can be further purified by dialysis and other methods normally employed in protein purification methods. In one example, the phytase can be isolated using chromatographic methods.

[0158] In some embodiments, a phytase as disclosed above is secreted by the cells or the organism which produce it. In such a case, the phytase is retrieved in the extracellular fraction, e.g. in the culture medium, which renders it easier to purify.

[0159] Phytases can be prepared in substantially pure or substantially isolated form. Purified phytases can be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g. less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed polypeptides. Kits

[0160] In some embodiments, the disclosure relates to a kit for preparing PAPS.

[0161] A kit for preparing PAPS may comprise at least:

[0162] one phytase as disclosed herein in a first container; and

[0163] cyclo-PAPS in a second container.

[0164] A kit as disclosed herein may contain more than one phytase, all mixed in the same container or each being packaged in a separate container.

[0165] A kit as disclosed herein may be used for converting cyclo-PAPS into PAPS.

[0166] The kit may further comprise instructions for preparing PAPS.

[0167] A kit may further contain a buffer suitable for the catalytic activity of the enzyme. The buffer may be packaged with a phytase as disclosed herein or may be packaged in a separate container. A suitable buffer for the reaction may be, for example, a buffer comprising phosphate such as succinate-phosphate, citrate-phosphate and sodium phosphate buffers. A suitable pH is from about 4.0 to about 6.0, and about 5.5.

[0168] In some embodiments, the kit may comprise at least one further enzyme. An additional enzyme may be a glycosyltransferase, an N-deacetylase / N-sulfotransferase, a C5-epimerase, or an O-sulfotransferase (OST) enzyme, such as for example 2-OST, 3- OST, 3-OST-1, 3-OST-3, 6-OST, 6-OST-1, 6-OST3. When a kit contains two or more enzymes, each enzyme may be packaged in a separate container.

[0169] In some embodiments, the disclosure relates to a kit for sulfating a substrate.

[0170] A kit as disclosed herein may be used for sulfating a polysaccharide. A kit as disclosed herein may be used for synthesizing a sulfated substrate. A kit as disclosed herein may be used for producing a sulfated heparin.

[0171] The kit may further comprise instructions for sulfating a substrate, for example a polysaccharide. The instructions may concern the synthesis of heparin.

[0172] A kit may further contain a buffer suitable for the catalytic activity of the further enzyme(s), for example a sulfotransferase. The buffer may be packaged with asulfotransferase as disclosed herein or may be packaged in a separate container. A suitable buffer may be, for example, TRIS-buffer, sodium phosphate buffer, and potassium phosphate buffer. A suitable pH is from about 6.0 to about 7.5, and about 7.0. Phosphatase activity of phytases and screening methods

[0173] A phytase as disclosed herein may have a cyclic phosphate opening activity for converting cyclo-PAPS into PAPS.

[0174] A phosphatase activity of a phytase as disclosed herein may be detected and measured according to any known method in the art. In some embodiments, the activity of the phytase is measured by chromatography, e.g. by high-performance liquid chromatography (HPLC) as disclosed in the examples, which allows to differentiate cyclo- PAPS and PAPS. In some embodiments, the method allows measuring the amount of PAPS released (or produced) and / or the amount of cyclo-PAPS consumed. As a reference, the Aspergillus niger phytase(s), e.g. the Sumizyme™ Phytase product, can be used.

[0175] In some embodiments, a method of screening and / or selecting a phytase able to convert cyclo-PAPS into PAPS may comprise the steps of:

[0176] a) contacting a phytase, for example provided in a purified form, with a sufficient amount of cyclo-PAPS, in a suitable buffer,

[0177] b) acquiring a measure representative of PAPS produced at step a),

[0178] c) contacting A. niger phytase(s), e.g. the Sumizyme™ Phytase product, with a sufficient amount of cyclo-PAPS, in a suitable buffer,

[0179] d) acquiring a measure representative of PAPS produced at step c), and

[0180] e) comparing the measures obtained at step b) and at step d).

[0181] A measure representative of PAPS produced during the reaction may be obtained by a measure of the surface of the pic corresponding to PAPS on a chromatogram, for example using a HPLC system according to manufacturer’s recommendations. The obtained measure may be expressed in arbitrary Unit of surface.

[0182] A suitable buffer for the reaction may be a citrate buffer at pH 4.5 comprising phosphate at 200 mM.

[0183] A suitable temperature of reaction may be around 25 °C.

[0184] The acquisition of the measure may be carried out 30, 60 or 90 minutes after initiation of the reaction, for example 60 minutes after initiation of the reaction.

[0185] In some embodiments, a phytase of interest as disclosed herein may have a catalytic activity for converting cyclo-PAPS into PAPS at least substantially similar to or greater than the activity of A. niger phytase(s), e.g. the Sumizyme™ Phytase product.

[0186] The phytases may be screened either after extraction and purification from the cells or organisms producing them or within the recombinant cells or organisms used to produce them.

[0187] The detection of a rate or an amount of formation of PAPS at step b) may be carried out directly by measuring the amount of PAPS, indirectly by measuring the remaining amount of cyclo-PAPS.

[0188] According to another embodiment, the activity of a phytase is determined for its canonical activity, i.e. the hydrolysis of phytic acid. This activity may give a good indication of the strength of the enzyme so that the amount of enzyme is adjusted in the reaction medium for converting cyclo-PAPS to PAPS.

[0189] In relation to the phosphatase activity of a phytase, said activity may depend on the substrate and activity assay conditions, as explained above and illustrated in the examples. Uses and methods for preparing PAPS from cyclo-PAPS

[0190] In some embodiments, a phytase as disclosed herein may be used for preparing PAPS from cyclo-PAPS.

[0191] In some embodiments, the disclosure relates to a method for preparing PAPS comprising at least a step of contacting or incubating cyclo-PAPS with a phytase as disclosed herein. In some embodiments, contacting comprises incubating cyclo-PAPS with a phytase as disclosed herein in a reaction medium.

[0192] In some embodiments, the incubation conditions are adapted to the catalytic (phosphate) activity of the phytase, i.e. to allow opening, e.g. through a nucleophilic addition of water on the cyclic 2’, 3’-phosphate moiety of cyclo-PAPS to generate PAPS.

[0193] In some embodiments, the reaction medium may comprise phosphate (PO4). In some embodiments, phosphate is at a concentration from 25 to 400 mM, e.g.200 mM.

[0194] In some embodiments, the reaction medium may comprise a buffer. In some embodiments, a buffer may be a succinate-phosphate (sodium phosphate and succinic acid) buffer, a citrate-phosphate buffer or a sodium phosphate buffer. In some embodiments, the buffer is at a concentration of 50 mM to 200 mM, e.g.100 mM.

[0195] In some embodiments, the pH of the reaction medium is adapted according to the optimal pH for the phosphatase activity of a phytase as disclosed herein. In some embodiments, the pH of reaction medium may be from 4 to 6. In some embodiments, the reaction medium may be from 4.5 to 5.5, e.g.4.5.

[0196] In some embodiments, the concentration of the phytase as disclosed herein in the reaction medium is adapted to allow an efficient and specific conversion of cyclo- PAPS into PAPS. The adequate concentration, which may also depend on other factors such as the cyclo-PAPS concentration and the incubation time, can be easily determined by the skilled person, by monitoring the generation of PAPS using a method (e.g. HPLC) as disclosed above. In some embodiments, the concentration of the phytase in the reaction medium may be from about 0.1 g / L (about 0.01 wt% or 0.01%w / w) to 12 g / L (about 1.2 wt% or 1.2%w / w). In some embodiments, the concentration of the phytase in the reaction medium may be from about 0.2 g / L (about 0.02 wt% or 0.02%w / w) to 10 g / L (about 1.0 wt% or 1.0%w / w).

[0197] In some embodiments, the concentration of the substrate, i.e. cyclo-PAPS, in the reaction medium may be from about 2 g / L to 200 g / L. In some embodiments, the concentration of the substrate, i.e. cyclo-PAPS, in the reaction medium may be from about 30 g / L to 120 g / L.

[0198] In some embodiments, the incubation temperature is adapted according to the optimal temperature for the activity of the phytase as disclosed herein. In some embodiments, the incubation temperature may be from 15°C to 40°C, e.g. room temperature (RT) or 37°C.

[0199] In some embodiments, the incubation time may be from 1 hour to several days. The adequate time, which may also depend on other factors such as the phytase concentration, can be easily determined by the skilled person, by monitoring the generation of PAPS using a method (e.g. HPLC) as disclosed above. In some embodiments, the incubation time may be inferior to 24 hours, e.g.10 hours.

[0200] In some embodiments, the reaction may be stopped by any method known in the art, e.g. by inactivating or removing the phytase.

[0201] At the end of the incubation, PAPS may be in the form of a tetra- triethylammonium salt (PAPS TEA).

[0202] The method may further comprise a step of retrieving PAPS. In some embodiments, the reaction medium containing PAPS, a phytase and phosphate issubmitted to various treatments to isolate / purify PAPS and to remove phytase and phosphate.

[0203] In some embodiments, purification of PAPS may be performed by precipitation of phosphates and / or enzyme removal, e.g. by ultrafiltration. In some embodiments, enzyme and phosphate precipitation may be performed simultaneously, e.g. by methanol addition.

[0204] In some embodiments, phytase may be removed through ultrafiltration over a 30 KDa Amicon membrane after neutralization of the reaction mixture to pH 7 using 15% NaOH. In some embodiments, phytase may be removed through Tangential Flow Filtration over a cartridge or spiral wound membrane.

[0205] In some embodiments, once the enzyme is removed, methanol is added to precipitate inorganic phosphate and buffer such as citrate. A white solid is obtained after stirring the obtained suspension, e.g. at Room Temperature followed by filtration. The filtrate may be subsequently concentrated on a rotary evaporator at 40 °C to yield an aqueous solution of PAPS. Uses and methods for preparing PAPS from adenosine or 5’-AMP

[0206] In some embodiments, a phytase as disclosed herein may be used for preparing PAPS from adenosine or 5’-AMP.

[0207] In some embodiments, a method of preparing PAPS from adenosine or 5’AMP may comprise at least the steps of:

[0208] a) Phosphorylation of adenosine or 5’-AMP into PAP and iso-PAP, using a phosphorylation agent

[0209] b) Conversion of PAP and iso-PAP into cyclo-PAPS

[0210] c) Conversion of cyclo-PAPS into PAPS using a method as disclosed above.

[0211] In step a), adenosine or 5’-AMP is phosphorylated to yield a mixture of PAP and iso-PAP. In some embodiments, phosphorylation may be performed using different phosphorylating agents such as P2Cl4O3 (Haas, T. M. et al. Chem. Commun. 2019, 55, 5339-5342), POCl3 (Collier, A. et al. Org. Biomol. Chem.2006, 4, 4526-4532) or sodium cyclo-triphosphate (P3m) (Inoue, H., Phosphorus Research Bulletin 1995, 5, 137-142). In some embodiments, this step may result in the synthesis of PAP / iso-PAP sodium salt.

[0212] In step b), the PAP / iso-PAP mixture is transformed to cyclo-PAPS. In some embodiments, this may be done in one step using Et3N-SO3 (Cherniak, R. J. Biol. Chem. 1964, 239(9), 2986-2990). In other embodiments, this may be done in two steps in whichfirst cyclo-PAP is formed by using DCC (Dicyclohexylcarbodiimide) followed by sulfonation using Et3N-SO3 (Sekura, R. Methods in Enzymology, 1981, 77, 413-415). In some embodiments, this step may result in an aqueous cyclo-PAPS solution. Uses and methods for sulfating a substrate Sulfation

[0213] In some embodiments, the disclosure relates to the use of PAPS prepared with the method as disclosed above for sulfating a substrate.

[0214] In some embodiments, the disclosure relates to a method of sulfating a substrate comprising at least a step of contacting the substrate to be sulfated with a) a sulfotransferase and b) a sulfo group donor comprising PAPS prepared with the method as disclosed above, in conditions suitable for a transfer of the sulfo group from the sulfo group donor to said substrate.

[0215] In some embodiments, the disclosure relates to a method of sulfating a substrate comprising at least a step of contacting the substrate to be sulfated with a) a sulfotransferase, b) cyclo-PAPS and c) a phytase as disclosed herein, in conditions suitable for a conversion of cyclo-PAPS into PAPS and the transfer of the sulfo group from PAPS to said substrate.

[0216] The uses or methods of the disclosure may be for synthesizing a sulfated substrate, e.g. heparin.

[0217] The method may further comprise a step of retrieving the sulfated substrate.

[0218] The disclosure relates to a method for obtaining a sulfated substrate by sulfating a substrate with at least one sulfotransferase and PAPS, said method including at least one step of converting cyclo-PAPS into PAPS by contacting said cyclo-PAPS with a phytase as disclosed herein.

[0219] According to a specific embodiment, the step of converting cyclo-PAPS into PAPS is simultaneous to the step of sulfation.

[0220] According to another embodiment, the step of converting cyclo-PAPS into PAPS and the step of sulfation are sequential. According to one embodiment, the step of converting cyclo-PAPS into PAPS precedes the step of sulfation.

[0221] The disclosure relates to a method for sulfating a substrate with a sulfotransferase and PAPS in conditions suitable to transfer a sulfo group from PAPS to the substrate to be sulfated and to obtain a sulfated substrate and PAP.

[0222] The methods may further comprise a step of recovering the so-formed sulfated substrate.

[0223] A method as disclosed herein may be for synthesizing a heparin.

[0224] Sulfation of a substrate may be carried out using various sulfotransferases including an O-sulfotransferase (OST) enzyme, such as for example 2-OST, 3-OST, 3-OST- 1, 3-OST-3, 6-OST, 6-OST-1, 6-OST3, or a N-sulfotransferase such as NDST1, NDST2.

[0225] Step of sulfation of a substrate and step of converting cyclo-PAPS in PAPS may be carried out sequentially or simultaneously in a one-pot reaction.

[0226] Step of converting cyclo-PAPS in PAPS may comprise providing a reaction mixture comprising cyclo-PAPS and a phytase as disclosed herein.

[0227] In some embodiments, step of sulfation of a substrate of the methods disclosed herein may comprise a plurality of sub-steps a1), a2), a3), …, during which the substrate may undergo successive enzymatically catalyzed reactions. Those reactions may be sulfation at different locations within the substrate, carried out by different sulfotransferases using PAPS as sulfo donor group. The different sulfotransferases may be, for example, different OSTs.

[0228] In some embodiments, step of sulfation of a substrate may comprise a plurality of simultaneous or sequential sub-steps a1), a2), a3), …, an), and wherein at least two sub-steps comprise each a sulfation catalyzed by a sulfotransferase using PAPS as a sulfo group donor to obtain PAP and a sulfated substrate.

[0229] An aspect of the disclosure is directed to a method for sulfation of a polysaccharide substrate. The method can be of a type wherein the sulfation of a polysaccharide substrate is catalyzed by a sulfotransferase, such as one or more OSTs, with a conversion of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to adenosine 3′,5′- diphosphate (PAP). The sulfation process can be coupled with the PAPS preparation using cyclo-PAPS and a phytase as disclosed herein.

[0230] A phytase as disclosed herein may be grafted, covalently or not, to a suitable support according to any known method in the art. Sulfo group donor

[0231] The sulfo group donor may be PAPS generated by the conversion of cyclo- PAPS using a phytase as disclosed herein. Sulfotransferases and substrates

[0232] As known in the art, there are two classes of sulfotransferases (SULTs): cytosolic and membrane-associated SULTs. Cytosolic SULTs sulfonate small endogenous and exogenous compounds, such as hormones, bioamines, drugs, and various xenobiotic agents. Membrane-associated SULTs, many of which are implicated in crucial biological processes, sulfonate larger biomolecules, such as carbohydrates and proteins.

[0233] Examples of human cytosolic sulfotransferases and their cognate substrates are listed below:

[0234] The SULT1A1 enzyme has phenols as substrates.

[0235] The SULT1A2 enzyme has phenols (low affinity) as substrates.

[0236] The SULT1A3 enzyme has catecholamines as substrates.

[0237] The SULT1B1 enzyme has thyroid hormones as substrates.

[0238] The SULT1C1 and SULT1C2 enzymes have aryl hydroxylamines as substrates.

[0239] The SULT1E1 enzyme has estrogens as substrates.

[0240] The SULT2A1 and SULT1A1 enzymes have hydroxysteroids as substrates.

[0241] Examples of representative substrates are: pNP, 2-napthol, dopamine, tyramine, minoxidol, 3,3’-T2, T3, PNP, T4, rT3, N-OH-2AFF, estradiol, DHEA, androstenediol, pregnenolone, 17α-pregnenolone.

[0242] Examples of membrane-associated sulfotransferases and their cognate substrates are listed below:

[0243] The heparan sulfate D-glucosaminyl 3-O-ST-3a and b, the heparan sulfate D-glucosaminyl 3-O-ST-1, the heparan sulfate iduronic acid 2-O-ST, the heparan sulfate D- glucosaminyl 6-O-ST have heparan sulfate as substrate.

[0244] The tyrosylprotein ST has CCR-5 and PSGL-1 as substrates.

[0245] The N-acetylglucosamine 6-O-ST has sialyl Lewis as substrates.

[0246] The chondroitin sulfate N-galactosamine 4-O-ST-1, 2, and 3 have chondroitin sulfate as substrate.

[0247] The chondroitin sulfate N-galactosamine 6-O-ST has chondroitin sulfate and keratan sulfate as substrates.

[0248] The HNK-1 glucuronic acid 3-O-ST has HNK-1 sulfate as substrate.

[0249] The N-acetylgalactosamine 4-O-ST has saccharide receptor, units on lutropin, thyrotropin, pro-opiomelanocortin as substrates.

[0250] The dermatan sulfate N-galactosamine 4-O-ST has dermatan sulfate as substrate.

[0251] The keratan sulfate galactose 6-O-ST has keratan sulfate as substrate.

[0252] The substrate may be selected in a group comprising a polysaccharide, an heparan, an heparosan sulfate, a chemically desulfated N-sulfated (CDSNS) heparin, a glycosaminoglycan (GAG), an heparan sulfate or a sulfated heparin.

[0253] A polysaccharide substrate may be partially sulfated prior to reaction mixture incubation. In some embodiments, the sulfated polysaccharide is a glycosaminoglycan (GAG), such as for example a heparan sulfate (HS). In some embodiments, the sulfated polysaccharide is an HS that is an anticoagulant-active HS, an antithrombin-binding HS, a fibroblast growth factor (FGF)-binding HS, a herpes simplex virus envelope glycoprotein D- binding HS or has a combination of these properties.

[0254] In some embodiments, a substrate to be sulfated may undergo further to sulfation at least one additional enzymatically catalyzed reaction. This or these additional reaction(s) may be carried out before or after the sulfation.

[0255] A substrate to be sulfated may be a polysaccharide substrate previously N,O- desulfated and re-N-sulfated polysaccharide, such as for example a chemically desulfated N-sulfated (CDSNS) heparin. For example, a polysaccharide, such as CDSNS, can be reacted with a particular OST in presence of PAPS to produce a sulfated polysaccharide intermediate product that can then be reacted subsequently with a different OST in presence PAPS to further sulfate the polysaccharide at different locations. This sequential process of reacting the polysaccharide substrate with different OSTs can be continued until a final polysaccharide is produced exhibiting desired biological activities.

[0256] The sulfation methods disclosed herein allows producing a multitude of sulfated substrates including polysaccharides, such as heparan sulfate molecules having varied biological activities by selecting appropriate sulfotransferases and by sequentially controlling the addition of those sulfotransferases to the reaction system to facilitate appropriate timing of sulfation of the polysaccharide. For example, heparan sulfate having specific biological activities which can be synthesized includes anticoagulant heparan sulfate, heparin, fibroblast growth factor-2-binding activity, herpes simplex virus glycoprotein D (gD)-binding HS, and fibroblast growth factor 2 (FGF2) receptor-binding HS. Only two or three enzymatic steps are required for the synthesis of each of thesebiologically-active heparan sulfate molecules. Thus, the methods disclosed herein, because of the effective and low-cost PAPS generating system, provide efficient and effective methods for the large-scale synthesis of a wide range of heparan sulfate with specific activities.

[0257] In some embodiments, the sulfated polysaccharide substrate can be a glycosaminoglycan (GAG). The specific GAGs of physiological significance are hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate (including heparin), and keratan sulfate. Thus, in some embodiments, the sulfated polysaccharide product is a HS. In some embodiments, the sulfated polysaccharide product is an anticoagulant-active HS, an antithrombin-binding HS, an FGF-binding HS, and an HSV gD-binding HS. Heparin synthesis

[0258] In some embodiments, the presently disclosed subject matter provides a method of preparing or synthesizing a heparin compound.

[0259] A method for synthesizing heparin may comprise obtaining a sulfated heparin precursor by sulfating the heparin precursor with at least one sulfotransferase and PAPS, said method including at least one step of converting cyclo-PAPS into PAPS by contacting said cyclo-PAPS with at least one phytase as disclosed herein.

[0260] A method for synthesizing heparin may comprise sulfating a heparin precursor with a sulfotransferase and PAPS in conditions suitable to transfer a sulfo group from PAPS to the heparin precursor to be sulfated and to obtain a heparin precursor and PAP.

[0261] In some embodiments, the method disclosed herein for synthetizing heparin may use heparosan as heparin precursor. Heparin may be produced by subjecting heparosan as a starting material to different steps comprising N-deacetylation, N-sulfation, C5-epimerization, 2-O-sulfation, 6-O-sulfation, 3-O-sulfation. Heparin may be produced by subjecting heparosan to one, several or all of these steps or a combination of some of these steps or all of these steps. The method for producing heparin may further comprise a depolymerization step. The implementation order of the steps in the heparin production process is not particularly limited, so long as heparin having desired properties can be obtained.

[0262] In the method disclosed herein for synthetizing heparin these steps can be performed chemically or enzymatically or can be a combination between chemically and enzymatically performed steps. An enzymatic step may be, for example, a N-sulfation, a 2- O-sulfation enzymatic step, a 3-O-sulfation enzymatic step, a 6-O-sulfation enzymatic stepor a succession or a combination of these steps. Such an enzymatic step can be performed by using, for example, a N-sulfotransferase enzyme such as NDST1 or NDST2, an O- sulfotransferase (OST) enzyme such as, for example, 2-OST, 3-OST, 3-OST-1, 3-OST-3, 6-OST, 6-OST-1, 6-OST-3. An enzymatic step for synthetizing heparin according to the method disclosed herein may be performed by more than one sulfotransferase or by a combination between one or more sulfotransferases and another enzyme, for example, a 2-OST and a C5 epimerase. The method as disclosed herein comprises at least one step with one sulfotransferase and PAPS produced according to the present disclosure.

[0263] The present disclosure will be further understood from the following non- limiting examples. The following examples are provided to describe in detail some of the representative, presently preferred methods and materials of the present disclosure. These examples are provided for purposes of illustration of the inventive concepts and are not intended to limit the scope of the invention as defined by the appended claims. [EXAMPLES] Example 1: Identification and characterization of a new enzyme able to convert cyclo PAPS into PAPS Example 1-1 Screening of enzymes working at pH 7.4 Materials & Methods Enzymes

[0264] Several commercially available enzymes (see Table 1 below) were screened for the conversion of cyclo-PAPS at a concentration of 0.2 g / L in the reaction medium. Reaction conditions

[0265] The enzymes were tested under neutral conditions (pH 7.4) in a 50 mM Tris maleate buffer at 37 °C in an amount shown in Table 1 below:

[0266] TABLE 1: EXPERIMENTAL SETUP FOR ENZYME SCREENING (pH 7.4) TRIAL 1 2 3 4 5 ENZYME Phospho- Sumizyme®Quara®Finizym®W: Lecitase®lipase C from Phytase boost: 2 lysophospho- ultra: Bacillus from phospho lipase from fusion of the cereus lipase C genes of theAspergillus enzymes Trichoderma lipase from niger from a harzianum Thermomyces genetically lanuginosus modified and the Bacillus phospho- licheni- lipase A1 from formis Fusarium oxysporum PROVIDER Thermofisher Takabio Novozymes Novozymes Novozymes AMOUNT 1% v / v 20g / L 2% v / v 2% v / v 2% v / v

[0267] The reaction was allowed to run 16 h and a 25 ^L sample was also taken after 1 h and diluted with 100 ^L of water. The enzymes were deactivated by heating the samples for 2 min at 95°C and subsequently centrifuged and analyzed by HPLC. HPLC analysis

[0268] The chromatographic conditions are as follows:

[0269] Waters Acquity Arc – PDA detector

[0270] Column Waters XSelect HSS T3, 2,1*50 mm, 2.5 µm

[0271] Eluent A = 50 mM KH2PO4and 25 mM Bu4N+HSO4-in water (pH 4,44 by means of K2HPO4)

[0272] Eluent B = CAN

[0273] Flow = 0.6 mL / min

[0274] Injection volume = 10 µL

[0275] Detection UV = 254 nm

[0276] Column temperature = 30 °C

[0277] Sample diluent = H2O

[0278] Gradient: Time (min) Flow (mL / min) %A %B 0 0.6 98 22 0.6 98 2 5 0.6 70 30 5.10 0.6 98 2 8.50 0.6 98 2 Data analysis

[0279] The activity of the tested enzyme is reflected by the % of conversion of cyclo- PAPS into PAPS.

[0280] The % area 2’-PAPS (iso-PAPS) and 3’-PAPS (PAPS) should be as close as possible to the conversion.

[0281] The ratio 3’ / 2’-PAPS, reflecting the selectivity of cyclic phosphate opening at 2’-position to yield PAPS, should be as high as possible.

[0282] Possible over-hydrolyzed products, also detectable by HPLC (see Figure 1A), are depicted below:Results

[0283] The results are depicted in Table 2 below:

[0284] TABLE 2: RESULTS FOR ENZYME SCREENING (pH 7.4) 1h 16h ENZYME Conv %Area 2’ Ratio Conv %Area 2’ Ratio cPAPS and 3’- 3’ / 2’- cPAPS and 3’- 3’ / 2’- PAPS PAPS PAPS PAPS 1 3% 3% 100% 30% 27% 100%2 100% 25% 100% 100% 0% 3 96% 90% 46% 98% 74% 28% 4 36% 36% 13% 50% 48% 34% 5 99% 87% 8% 100% 100% 0%

[0285] From this table, it can be seen that apart from Sumizyme Phytase (2) and Phospholipase C (1), low 3’ to 2’ ratios are observed. However, Phospholipase C has a low activity (30% conversion after 16h using 1 vol% of enzyme), whereas the Sumizyme Phytase gives a very fast conversion (only over-hydrolyzed products after 16h). Example 1-2 Screening of enzymes working at different optimal conditions Materials & Methods Enzymes

[0286] Several further commercially available enzymes (see Table 3 below) were screened for the conversion of cyclo-PAPS. Reaction conditions

[0287] The used experimental conditions are depicted in Table 3. The reactions are performed at 37°C.

[0288] TABLE 3: EXPERIMENTAL SETUP FOR ENZYME SCREENING (OTHER OPTIMAL CONDITIONS) TRIAL 1’ 2’ 3’ 4’ 5’ 6’ ENZYME Alkaline Phospho- Bovine PLA2 Bovine RNase T2 phospha- lipase C phosphor- G12A Rnase A from tase from diesterase II from Aspergillus Clostri- human oryzae dium perfringes PROVIDER Sigma Creative Enzymes AMOUNT 2% v / v 2% v / v^1% v / v^ ^50 mM Glycine Tris- Succinate- Tris-HCl Na Na acetate Buffer NaOH maleate PO4 acetate pH 10 7.4 5.5 8 5 5 Additive 1 mM 1 mM MgCl2 CaCl2 0.2% w / v BSA ^: very low amount Results

[0289] Results of HPLC analysis of reaction samples after 0.8, 2.5 and 19h are summarized in Table 4.

[0290] TABLE 4: RESULTS FOR ENZYME SCREENING (OTHER OPTIMAL CONDITIONS) 0.8h 2.5h 19h ENZY Conv %Are Ratio Conv %Are Ratio Conv %Are Ratio -ME cPAP a 2’ 3’ / 2’- cPAP a 2’ 3’ / 2’- cPAP a 2’ 3’ / 2’- S and PAP S and PAP S and PAP 3’- S 3’- S 3’- S PAPS PAPS PAPS 1’ 3% 0% 4% 0% 22% 0% 2’ 4% 0% 8% 0% 20% 0% 3’ 27% 22% 100 59% 46% 100 97% 69% 100 % % % 4’ 5% 3% 100 8% 3% 100 42% 9% 100 % % % 5’ 33% 1% 100 32% 2% 100 88% 2% 100 % % %6’ 100% 69% 100 100% 42% 100 100% 1% 100 % % %

[0291] From this table, it can be seen than Alkaline phosphatase and Phospholipase C from C. perfringens barely show any activity. Native Bovine phosphodiesterase II, PLA2G12A and Native Ribonuclease A do show a good selectivity for the formation of PAPS over iso-PAPS but many other reactions occur and only low amounts of PAPS are observed.

[0292] Only Ribonuclease T2 from Aspergillus oryzae (6’) which is the enzyme already used in said kind of reactions, combines good conversion with good selectivity for PAPS formation although the reactions proceeded a bit too fast. Example 1-3 Optimized conditions for the selected enzyme (phytase)

[0293] As shown in Table 5 below, a good and selective conversion of cyclo-PAPS into PAPS has been observed in the following conditions: pH of 5.5, enzyme concentration of 0.1 g / L in a succinate / phosphate buffer.

[0294] TABLE 5: RESULTS FOR PHYTASE IN OPTIMIZED CONDITIONS REACTION CONDITIONS 0.8h 2.5h 18h [PHYTASE] BUFFER Conv %Area Conv %Area Conv %Area g / L (pH) cPAPS 3’- cPAPS 3’- cPAPS 3’- PAPS PAPS PAPS 0.1 50 mM 79% 70% 98% 86% 100% 78% Succinate / Phospha-te (5.5)

[0295] The chromotogram of a phytase catalysed reaction after 2.5h at 37°C in these conditions is displayed on Figure 1B, which reveals a predominant peak corresponding to PAPS. Example 1-4 Comparison with the enzyme of the prior art (Ribonuclease T2)

[0296] The same exploration of optimized conditions was performed in relation to Ribonuclease T2, especially focused on the amount of enzyme since it was observed a veryquick conversion but also the formation of many side-products. Therefore, the %Area of 3’- PAP and 5’-APS was also monitored.

[0297] The results obtained in a sodium acetate buffer (pH 5) at 37°C are shown in Table 6.

[0298] TABLE 6: RESULTS FOR RIBONUCLEASE T2 IN OPTIMIZED CONDITIONS REACTION CONDITIONS [RNASE TIME Conv %Area %Area %Area T2] (h) cPAPS 3’-PAPS 3’-PAP 5’-APS 0.46 g / L 1.5 100% 82% 8% 10% 0.23 g / L 1.5 100% 87% 8% 5% 0.092 g / L 1.5 100% 90% 8% 2% 0.046 g / L 1 100% 90% 9% 2% 0.023 g / L 1 100% 90% 9% 1% 0.0092 g / L 1 100% 91% 9% 0% 4.6 mg / L 0.7 100% 91% 9% 0% 0.92 mg / L 0.7 100% 91% 9% 0% 0.46 mg / L 0.7 100% 92% 8% 0% 0.092 mg / L 0.7 98% 88% 12% 0% 0.046 mg / L 4.5 100% 91% 9% 0% 0.0092 6 89% 80% 9% 0% mg / L

[0299] With all enzyme quantities, a very good conversion of cyclo-PAPS is observed. However, in all conditions, around 9% of 3’-PAP is also formed, indicating that the phosphosulfate group at the 5’-position is also prone to hydrolysis with this enzyme.

[0300] Besides, as shown in Table 7, a comparison between both enzymes, taking into account the amounts of enzyme required and the costs associated with said enzymes, clearly reveals an advantage for the phytase as disclosed herein over the ribonuclease T2 according to the prior art.

[0301] TABLE 7: BALLPARK NUMBERS FOR COST CONTRIBUTION OF ENZYMES FOR THE PRODUCTION OF 1 KG OF PAPS ENZYME PHYTASE RIBONUCLEASE T2 (present disclosure) (prior art) LC YIELD 86% 91% ENZYME AMOUNT TO 500 mg 230 µg CONVERT 1g OF CYCLO-PAPS ENZYME AMOUNT TO 559 mg 243 µg OBTAIN 1g OF (3’) PAPS ENZYME COST 1 kg: 200-400 euros 25 mg: 1400 euros ENZYME COST 112-224 13608 CONTRIBUTION FOR 1 kg OF (3’) PAPS (EUROS) Example 1-5 Optimal conditions for the selected enzyme (phytase)

[0302] Optimal experimental conditions have been defined as follows:

[0303] Temperature: 25 °C

[0304] pH: 4.5

[0305] cyclo-PAPS concentration: 120 g / L

[0306] Citrate concentration: 50 mM

[0307] Phosphate concentration: 200 mM

[0308] Enzyme charge: 8 wt%.

[0309] These parameters were tested in a synthetic reaction on 21 mL scale to verify. Cyclo-PAPS and PAPS area% for this reaction are depicted in Figure 2.

[0310] The results presented in Figure 2 show that the reaction is complete after approximately 6 h and that a HPLC purity of around 80% is obtained for PAPS. Example 1-6 Purification of PAPS so obtained

[0311] After the reaction has come to completion, PAPS should be isolated in a suitable form for use. To do so, most of the citrate / phosphate buffer and phytase should be removed. The latter is removed through ultrafiltration over a 30 KDa Amicon membrane after neutralization of the reaction mixture to pH 7 using 15% NaOH.

[0312] Once the enzyme is removed by Ultrafiltration, 2 volumes of methanol are added to precipitate inorganic phosphate and citrate. A white solid is obtained after stirring the obtained suspension for 2 h at Room Temperature followed by filtration. The filtrate is subsequently concentrated on the rotary evaporator at 40 °C to yield an aqueous solution of PAPS that can be used in Sulfotransferase catalyzed reactions. Example 1-7 Analysis of PAPS so obtained

[0313] The optimal conditions as defined in Example 1-5 were applied for generating PAPS starting from cyclo-PAPS having 89% HPLC purity. Using these conditions, > 74% of PAPS was obtained with less than 2% of cyclo-PAPS remaining in the reaction mixture.

[0314] After purification as defined in Example 1-6, the final product was characterized by HPLC. The corresponding chromatogram is depicted in Figure 3.

[0315] It can be seen that PAPS is obtained with 77% HPLC purity, main impurities are APS, PAP / iso-PAP, 5’-AMP and Adenine. None of these impurities is expected to be an inhibitor of Sulfotranferases.

[0316] A further analysis by NMR of said product was performed (data not shown) and revealed that the product is a triethylammonium salt (TEA). Example 2: Use-test of produced PAPS in 6-O-sulfation reaction

[0317] Having developed a new and efficient process for the production of PAPS, this test is to evaluate its use in a Sulfotransferase catalyzed reaction with comparison with commercially available PAPS. Since PAPS can be used as a sulfate donor in the presence of a cell free extract (CFE) containing 6-O-sulfotransferase, it was chosen to compare PAPS-Na from Yamasa with PAPS produced in the newly developed process according to the present disclosure. Materials & MethodsReagents

[0318] TABLE 8: REAGENTS FOR THE 6-O SULFATION REACTION 1 2 (PAPS-Na) (PAPS-TEA) MES BUFFER 50mM 50mM (4 Morpholine-ethanesulfonic acid) pH 7.0 NaCl 90mM 100mM PAPS-Na 6.25 mM Yamasa (96.6% purity) PAPS(Et3NH) 6.25 mM Produced according to Example 1 (77.1% purity) NS-Heparosan 6.25 Mm 6.25 Mm (2.5 g / L) (2.5 g / L) 6-OST 2.8%vol 2.8%vol (461.2 mU / mL) REACTION TIME 24h 24h Reaction conditions

[0319] The reactions were performed on a 1 mL scale by adding the reagents to a 1,5 mL Eppendorf tube and allowing the reaction to proceed at 37 °C, 700 rpm in an Eppendorf Thermomixer C. After 24 h, the reactions were stopped by heating at 95 °C for 5 min. After centrifugation for 30 min at 4 °C at 9100 g, the supernatants were treated with heparinase I,II,III and the building blocks were analyzed by HPLC as disclosed by P. Mourier et al. (Analytical Chemistry Research 3 (2015) 46-53).

[0320] Disaccharides building blocks:

[0321] ^IVa: [^UA-GlcNAc]

[0322] ^IVs: [^UA-GlcNS]

[0323] ^IIa: [^UA-GlcNAc6S]

[0324] ^IIIa: [^UA2S-GlcNAc]

[0325] ^IIIs: [^UA2S-GlcNS]

[0326] ^IIs: [^UA-GlcNS6S]

[0327] ^Ia: [^UA2S-GlcNAc6S]

[0328] ^Is: [^UA2S-GlcNS6S]

[0329] % 6-OS=

[0330] 100 − (6 − ^^^^)% = 100 − 100 × ∑^^ ^^^^∑^^ ^^^^ ×^^^ ^^^^^^^^^

[0331] where x corresponds to building blocks and ^^^^^^^^^^^^is the number of carboxyl; i corresponds to building blocks containing one 6-OH (^IVa, ^IVs, ^IIIa, ^IIIs). Results

[0332] The results are depicted in Table 9.

[0333] TABLE 9: RESULTS FROM USE-TEST OF PAPS FROM THE NEW PROCESS IN 6-O-SULFATION REACTION OF NS-HEPAROSAN 1 2 Building Block PAPS Na PAPS TEA % ^IVA 2.7 3.5 % ^IVS 4.2 6.5 % ^IIA 7.8 7.0 % ^IIIA <0.1 <0.1 % ^IIS 85.3 83.1 % ^IIIS <0.1 <0.1% ^IA <0.1 <0.1 % ^IS <0.1 <0.1 % 6OS 92 88

[0334] From the results in Table 9, it can be clearly seen that PAPS-TEA from the newly developed process according to the present disclosure gives comparable results to PAPS-Na purchased from Yamasa, confirming the quality of the PAPS so obtained and the possibility of using it in all the sulfonation reactions.[REFERENCES] Altschul SF. A protein alignment scoring system sensitive at all evolutionary distances. J Mol Evol.1993;36(3):290-300. doi:10.1007 / BF00160485 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol.1990;215(3):403-410. doi:10.1016 / S0022-2836(05)80360-2 Chapman E, Best MD, Hanson SR, Wong CH. Sulfotransferases: structure, mechanism, biological activity, inhibition, and synthetic utility. Angew Chem.2004;43(27): 3526-48. doi: 10.1002 / anie.200300631 Cherniak R, Davidson EA. Synthesis of Adenylyl Sulfate and Adenylyl Sulfate 3'- Phosphate. J. Biol. Chem.1964 ;239(9) :2986-2990 Collier A, Wagner G. A facile two-step synthesis of 8-arylated guanosine mono- and triphosphates (8-aryl GXPs). Org. Biomol. Chem.2006;4:4526-4532 Corrêa LR, de Araujo F. Fungal phytases: from genes to applications. Brazilian Journal of Microbiology.2020;51: 1009-1021. doi.org / 10.1007 / s42770-020-00289-y Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res.1984;12:387-39. doi: 10.1093 / nar / 12.1part1.387 Fu L, Suflita M, Linhardt RJ. Bioengineered heparins and heparan sulfates. Adv Drug Deliv Rev.2016;97:237-249. doi:10.1016 / j.addr.2015.11.002 Haas TM, Ebensperger P, Eisenbeis VB, Nopper C, Dürr T, Jork N, Steck N, Jessen-Trefzer C, Jessen HJ. Magic spot nucleotides: tunable target-specific chemoenzymatic synthesis. Chem. Commun.2019;55:5339-5342 Henikoff S, Henikoff JG. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A.1992;89(22):10915-10919. doi:10.1073 / pnas.89.22.10915 Inoue H, Baba Y, Tsuhako M. Phosphorylation of Adenosine 5’-monophosphate (5’-AMP) with cyclo-triphosphate and purification of the phosphorylated products. Phosphorus Research Bulletin.1995;5:137-142 Karlin S, Altschul SF. Applications and statistics for multiple high-scoring segments in molecular sequences. Proc. Natl. Acad. Sci. USA 1993;90(12):5873-5787. doi: 10.1073 / pnas.90.12.5873.Mourier P, Anger P, Martinez C, Herman F, Viskov C. Quantitative Compositional Analysis of Heparin Using Exhaustive Heparinase Digestion and Strong Anion Exchange Chromatography. Analytical Chemistry Research 2015;3:46-53 Pagotto F, Salimnia H, Dillon JAR, Totten PA. Stable shuttle vectors for Neisseria gonorrhoeae, Haemophilus spp. and other bacteria based on a single origin of replication. Gene 2000;244:13-19 Sekura, R.

[0053] Adenosine 3′-phosphate 5′-phosphosulfate. Methods in Enzymology 1981;77:413-415 US 4,169,011 Facile synthesis of 3’-phosphoadenosine 5’-phosphosulfate (PAPS) US 2009 / 0118491 STABLE SALT OF 3'-PHOSPHOADENOSINE 5'- PHOSPHOSULFATE

Claims

[CLAIMS] 1. Use of a phytase for generating 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from adenosine 2′,3′-cyclic phosphate 5′-phosphosulfate (cyclo-PAPS).

2. A method of preparing PAPS comprising incubating cyclo-PAPS with at least one phytase in a reaction medium.

3. Use according to claim 1 or a method according to claim 2, wherein the phytase is from a fungus.

4. Use according to claim 3 or a method according to claim 3, wherein the phytase is from an Ascomycete fungus.

5. Use according to claim 4 or a method according to claim 4, wherein the phytase is from Aspergillus species.

6. Use according to claim 5 or a method according to claim 5, wherein the phytase is from Aspergillus niger.

7. Use according to any of claims 1 and 3 to 6 or a method according to any of claims 2 to 6, wherein the phytase is a native or a recombinant enzyme, optionally purified.

8. A method according to any of claims 2 to 7, wherein the reaction medium has a pH from 4 to 6, optionally from 4.5 to 5.

5.

9. A method according to any of claims 2 to 8, wherein the reaction medium comprises phosphate (PO4), optionally at a concentration from 25 to 400 mM.

10. A method according to any of claims 2 to 9, wherein the reaction medium comprises a buffer, optionally a succinate-phosphate buffer, a citrate-phosphate buffer or a sodium phosphate buffer.

11. A method according to any of claims 2 to 10, wherein the concentration of cyclo- PAPS in the reaction medium is from 2 g / L to 200 g / L, optionally from 30 g / L to 120 g / L.

12. A method according to any of claims 2 to 11, wherein it comprises a further step of isolating the PAPS so prepared from the reaction medium, which is optionally further purified and / or dried.

13. A method of preparing PAPS from adenosine or 5’AMP comprising the following steps: a. Phosphorylation of adenosine or 5’AMP into PAP and iso-PAP b. Conversion of PAP and iso-PAP into cyclo-PAPS c. Conversion of cyclo-PAPS into PAPS using a phytase, optionally according to a method according to any of claims 2 to 12.

14. A method for sulfating a substrate comprising a step of generating PAPS using a method according to any of claims 2 to 13.

15. A method according to claim 14, wherein the substrate is selected in the group consisting of phenols, catecholamines, thyroid hormones, aryl hydroxylamines, hydroxysteroids, heparan sulfate, CCR-5, PSGL-1, sialyl Lewis, chondroitin sulfate, keratan sulfate, HNK-1 sulfate, saccharide receptor, units on lutropin, thyrotropin, pro- opiomelanocortin and dermatan sulfate.

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