Protein arginine deiminase

Abstract

The present invention relates to the production of the enzyme protein arginine deiminase and the use of such enzyme.

Description

PROTEIN ARGININE DEIMINASEFieldThe present invention relates to the production of the enzyme protein arginine deiminase (PAD) and the use of such enzyme. The invention also relates to new PAD enzymes. The invention further relates to proteins and peptides comprising citrulline residues.BackgroundIn a world with a growing population there is an increased demand of proteins. To respond to this growing demand of proteins, there is a need to look at wider applications of proteins. In addition, it is desired to look for plant proteins as an alternative for animal proteins, since it is considered that plants are a more sustainable source of proteins than animals. The use of plant proteins in food is still limited in part because of poor digestibility, taste and nutritional value.An important factor in making plant-based protein food products is their taste and mouthfeel. Vegetable proteins suffer from impaired taste such as bitterness and increased mouthfeel even at low protein content. Also, many plant protein beverages contain very low amounts of proteins making them less nutritious and less tasty foods.The enzyme protein arginine deiminase (PAD) can be used to improve properties of plantbased food products (W02008 / 000714, WO2017 / 009100 and WO2019 / 233910).W02008 / 000714 (DSM) describes the isolation and identification of the secreted PAD from the fungus Fusarium graminearum. It is suggested that potentially secreted PAD’S can be found in the fungi Chaetomium globosum, Phaeosphaeria nodorum and the bacteria Streptomyces scabies and Streptomyces clavuligerus. Although many new applications were identified for PAD, see for example WO2017 / 009100 and WO2019 / 233910 (both DSM), enzyme expression needs to be further improved to obtain more commercially interesting production levels.There is a need to have increased PAD production levels. There is also a need to have alternative PAD enzymes.Surprisingly, the inventors of the present invention show that improved levels of PAD expression can be obtained by using PAD sequences from specific fungal sources or by using engineered variants of the previously disclosed Fusarium graminearum PAD. The inventors also disclose new PAD protein sequences.FiguresFigure 1 : vector pGBTOP-18Figure 2: BochPAD_02 pH curveFigure 3: Thermostability BochPAD_02Figure 4: Michaelis menten curve BochPAD_02SEQ ID NO: 1 PAD protein sequence from Trichoderma harzianum including the pro-sequenceSEQ ID NO: 2 PAD protein sequence from Verticillium longisporum including the pro-sequenceSEQ ID NO: 3 PAD protein sequence from Bionectra ochroleuca including the pro-sequenceSEQ ID NO: 4 PAD protein sequence from Fusarium longipes including the pro-sequenceSEQ ID NO: 5 PAD protein sequence from Fusarium solani species complex including the prosequenceSEQ ID NO: 6 PAD protein sequence from Akanthomyces lecanii RCEF 1005 including the prosequenceSEQ ID NO: 7 PAD protein sequence from Fusarium incarnatum-equiseti species complex including the pro-sequenceSEQ ID NO: 8 PAD protein sequence from Ophiocordyceps australis including the pro-sequenceSEQ ID NO: 9 PAD protein sequence from Verticillium longisporum including the pro-sequenceSEQ ID NO: 10 PAD protein sequence from wildtype Fusarium graminearum including the prosequenceSEQ ID NO: 11 protein sequence from Fusarium graminearum variant FgraPADmut_01 ,FgraPADmut_02 and FgraPADmut_03 including the pro-sequenceSEQ ID NO: 1 -11 do not comprise a signal sequenceSEQ ID NO: 12 polynucleotide sequence encoding the protein of SEQ ID NO: 1SEQ ID NO: 13 polynucleotide sequence encoding the protein of SEQ ID NO: 2SEQ ID NO: 14 polynucleotide sequence encoding the protein of SEQ ID NO: 3SEQ ID NO: 15 polynucleotide sequence encoding the protein of SEQ ID NO: 4SEQ ID NO: 16 polynucleotide sequence encoding the protein of SEQ ID NO: 5SEQ ID NO: 17 polynucleotide sequence encoding the protein of SEQ ID NO: 6SEQ ID NO: 18 polynucleotide sequence encoding the protein of SEQ ID NO: 7SEQ ID NO: 19 polynucleotide sequence encoding the protein of SEQ ID NO: 8SEQ ID NO: 20 polynucleotide sequence encoding the protein of SEQ ID NO: 9SEQ ID NO: 21 polynucleotide sequence encoding the protein of SEQ ID NO: 10SEQ ID NO: 22 polynucleotide sequence encoding the protein of SEQ ID NO: 11SEQ ID NO: 23 Fusarium graminearum wild type signal sequence MHLLNGKTAAVALALLNSCNASEQ ID NO: 24 A. niger signal sequence MVKSILASVFFAATALASEQ ID NO: 25 A. niger signal sequence MSFRSLLALSGLVCTGLAThe invention provides a method for producing protein arginine deiminase (PAD) an isolated and / or recombinant PAD polypeptide a Fusarium graminearum PAD polypeptidea composition comprising any of the herein claimed PAD polypeptide a method for converting arginine residues to citrulline residues a method for modifying at least one characteristic of a protein comprising food use of any of the herein claimed PAD polypeptides for modifying at least one characteristic of a protein comprising foodDefinitionsThe term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the ISP coding sequence such that the control sequence directs the production of an RNA or an mRNA and optionally of a polypeptide translated from said (m)RNA.The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of mRNA and / or a polypeptide, either in vitro or in a host cell. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Examples of control sequences are transcription initiation sequences, termination sequences, promoters, leaders, signal peptides, propeptides, prepropeptides, or enhancer sequences; Shine-Delgarno sequences, repressor or activator sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. Signal sequences used for optimizing expression are described in W02010 / 121933. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be optimized to their specific purpose.The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post transcriptional modification, translation, post- translational modification, and secretion.A host cell as defined herein is an organism suitable for genetic manipulation and one which may be cultured at cell densities useful for industrial production of a target product, such as a PAD polypeptide according to the present invention. A host cell may be a host cell found in nature or a host cell derived from a parent host cell after genetic manipulation or classical mutagenesis. Advantageously, a host cell is a recombinant host cell. A host cell may be a prokaryotic, archaebacterial or eukaryotic host cell. A prokaryotic host cell may be, but is not limited to, a bacterial host cell. A eukaryotic host cell may be, but is not limited to, a yeast, a fungus, an amoeba, an algae, a plant, an animal, or an insect host cell.A nucleic acid or polynucleotide sequence is defined herein as a nucleotide polymer comprising at least 5 nucleotide or nucleic acid units. A nucleotide or nucleic acid refers to RNA and DNA. The terms “nucleic acid” and “polynucleotide sequence” are used interchangeably herein. A nucleic acid or polynucleotide sequence is defined herein as a nucleotide polymercomprising at least 5 nucleotide or nucleic acid units. A nucleotide or nucleic acid refers to RNA and DNA.The term “polypeptide” refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. The term “protein” as used herein is synonymous with the term “polypeptide” and may also refer to two or more polypeptides. Thus, the terms “protein” and “polypeptide” can be used interchangeably. Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, and the like) to add functionality. Polypeptides exhibiting activity in the presence of a specific substrate under certain conditions may be referred to as enzymes. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given polypeptide may be produced.The term “isolated polypeptide” as used herein means a polypeptide that is removed from at least one component, e.g. other polypeptide material, with which it is naturally associated. The isolated polypeptide may be free of any other impurities. The isolated polypeptide may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art. An isolated polypeptide may be produced by a recombinant host cell.A “mature polypeptide” is defined herein as a polypeptide in its final form and is obtained after translation of a mRNA into polypeptide and post-translational modifications of said polypeptide. Post-translational modifications include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation and removal of leader sequences such as signal peptides, propeptides and / or prepropeptides by cleavage.The term “promoter” is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a nucleic acid sequence to initiate transcription. Suitable bacterial promotors are for instance disclosed in in WO- A1 -2004 / 074468.The term “recombinant” when used with reference to a nucleic acid or protein indicates that the nucleic acid or protein has been modified in its sequence if compared to its native form by human intervention. The term “recombinant” when referring to a cell, such as a host cell, indicates that the genome of the cell has been modified in its sequence if compared to its native form by human intervention. The term “recombinant” is synonymous with “genetically modified”.Sequence identity, or sequence homology are used interchangeable herein. In order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length,for example over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. Longden.l. and Bleasby.A. Trends in Genetics 16, (6) pp276 — 277, http: / / emboss.bioinformatics.nl / ). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. The optional parameters used are a gapopen penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms. After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .A “synthetic molecule”, such as a synthetic nucleic acid or a synthetic polypeptide is produced by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, variant nucleic acids made with optimal codon usage for host organisms of choice.A synthetic nucleic acid may be optimized for codon use, preferably according to the methods described in W02006 / 077258 and / or W02008000632, which are herein incorporated by reference. W02008 / 000632 addresses codon-pair optimization. Codon-pair optimization is amethod wherein the nucleotide sequences encoding a polypeptide that have been modified with respect to their codon-usage, in particular the codon-pairs that are used, are optimized to obtain improved expression of the nucleotide sequence encoding the polypeptide and / or improved production of the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence. Those skilled in the art will know that the codon usage needs to be adapted depending on the host species, possibly resulting in variants with significant homology deviation from SEQ ID NO: 2, but still encoding the polypeptide according to the invention.As used herein, the terms “variant” or “mutant” can be used interchangeably. They can refer to either polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and / or inversions, at one or more locations relative to a reference sequence. Variants can be made for example by site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombination approaches known to a skilled person in the art. Variant genes of nucleic acids may be synthesized artificially by known techniques in the art.The amino acids are referred herein with their single letter code known to a person skilled in the art and can be found in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001 .The amino acids are A= Ala = Alanine; R = Arg = Arginine; N = Asn = Arginine; D=Asp= aspartic acid, C= Cys = Cystein; E = Glu = Glutamic acid; Q = GLn = Glutamine; G = Gly = Glycine; H = His = Histidine; I = He = Isoleucine; L = Leu = Leucine; K = Lys = Lysine; M = Met = Methionine; F = Phe = Phenylalanine; P = Pro = Proline; S = Ser = Serine; T = Thr = Threonine; W = Trp = Tryptophan; V = Vai = Valine.Detailed descriptionIn a first aspect, the invention provides a method for producing protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses(i) a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9.The terms protein arginine deiminase (PAD), peptidyl arginine deiminase (PAD), PAD and PAD polypeptide are used interchangeably herein. Protein or peptidyl arginine deiminases belong to a family of enzymes (EC 3.5.3.15) which convert peptide or protein bound arginine into peptide or protein bound citrulline. This process is called deamination or citrullination. In the reaction from arginine to citrulline, one of the terminal nitrogen atoms of the arginine side chain is replaced by an oxygen. The reaction uses one water molecule and yields ammonia as a side product(htp: / / en.wikipedia.org / wiki / Citrullination). Whereas arginine is positively charged at a neutral pH, citrulline is uncharged.Typically, the herein described method for producing a PAD will result in an enzyme preparation comprising PAD as well as other proteins. The resulting enzyme preparation may comprise other (added) components, like a salt, buffer, preservative and / or polyol which will be discussed in more detail later. Hence, herein provided is a method for producing an enzyme preparation comprising protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses(i) a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incamatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9.In step (a) of the described method, a recombinant host strain capable of secreting PAD is cultivated. The cultivating step is performed under conditions suitable to produce PAD. I.e. suitable conditions such as a suitable growth medium, temperature and pH are used which allow the recombinant host strain to grow, produce and secrete PAD. The skilled person is very well capable of determining such conditions. Surprisingly, the optimal fermentation temperature is quite narrow with an optimum at 30°C. Preferably, the temperature in step (a) is 30°C. As shown herein within the experimental part, the use of low levels of copper during fermentation results in higher PAD activity at the end of fermentation, improved stability of the PAD enzyme as well as an improved overall (process) yield. Preferably, the copper concentration is below 6 mg / L, more preferably below 3 mg / L and most preferably below 1 mg / L fermentation medium.Preferably, provided herein is a method wherein step (a) comprises cultivating said recombinant host strain in a fermentation medium comprising a copper concentration above 0 mg / L and below 1 mg / L (preferably above 0.01 mg / L and below 1 mg / L). The reference to the copper concentration is when added as CuSO4 (5 aq).Also provided herein is a method wherein step (a) comprises cultivating said recombinant host strain in a fermentation medium comprising a copper concentration above 0 mg / L and below 1 mg / L (preferably above 0.01 mg / L and below 1 mg / L) and wherein the fermentation temperature is in the range of 29 to 31 degrees Celsius, preferably the fermentation temperature is 30 degrees Celsius.In step (b) the produced and secreted PAD is recovered by techniques known to the skilled person. Such techniques are for example separation of growth medium from the used host cell, filtration steps and / or purification steps. Other examples of such techniques are described in Example 7. Optionally, step (b) comprises addition of a suitable amount of EDTA. A suitable amount of EDTA can easily be determined by the skilled person and is described in the experimental part herein and is for example the addition of 1 mM EDTA to the end of fermentation broth.The recombinant host strain used in a method of the invention expresses(i) a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9.Surprisingly, as shown in the experimental part herein, a subset of wildtype PAD polypeptides can be expressed at (much) higher protein levels when compared to the prior art expression of Fusarium graminearum PAD under identical conditions. Therefore, the PAD expressed by the host strain is preferably a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis.Some of the mentioned strains (from which PAD is derived) are also known by other names, for example:Trichoderma harzianum is also known as Hypocrea lixii, Trichaderma harzianum, Trichoderma harzianum Rifai, 1969, Trichoderma harizianum or Trichorderma harzianumVerticillium longisporum is also known as Verticillium dahliae var. longisporum, Verticillium dahliae var. longisporum C. Stark 1961 or Verticillium longisporum (C. Stark)Bionectra ochroleuca is also known as Clonostachys rosea, Bionectria ochroleuca Gliocladium roseum, Bionectria aureofulva, Nectria aureofulva or Nectria gliocladioidesFusarium longipes, is also known as Fusarium longipes Wollenw. & Reinking, 1925, Fusarium equiseti var. longipes, is also known as Fusarium scirpi var. longipes Fusarium solani species complex also known as Nectria haematococca complex or anamorphs of Nectria haematococca Ophiocordyceps australis is also known as Cordyceps australis or Cordyceps unilateral var. australisPreferably, said PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis comprises an amino acid sequence which has at least 80% sequence identity to respectively SEQ ID NO: 1 to 9. The different sequences are explained in more detail above under the heading “Sequence listing”.Surprisingly, the inventors of the present invention have identified that these wildtype PAD polypeptide sequences comprise an internal pro-sequence of approximately 35-50 amino acids which is not present in the secreted active PAD polypeptide. Pro-sequences in secreted fungalenzymes (e.g. phopholipases) are known to protect the inside of the cell from harmful activities. It is hypothesized by the inventors that the intracellular toxicity of PAD activity in the cell is prevented by shielding the enzyme active site by an internal pro-sequence, which is cleaved and further processed during secretion. Therefore, part of the invention is that the synthesis of secreted fungal PADs begins with the transcription and translation of their corresponding genes as a PAD prepropeptide within the fungal cell. The resulting full-length protein is known as the pre-protein or pre-propeptide. The pre-protein PAD contains a signal peptide at its N-terminus, which acts as a molecular tag into the secretory pathway and translocates the propeptide into the endoplasmatic reticulum (ER). Within current knowledge and models, in the ER, processes like protein folding, disulfide bridge formation and I or glycosylation can take place. Subsequently, the folded preprotein is transported from ER into Golgi and subsequently secretory vesicles. Within the Golgi apparatus, the secreted protein may undergo additional modifications, such as further glycosylation, phosphorylation and / or sulfation, etc.... It is in this trans-Golgi network (late-Golgi / vesicles) that the pre-protein PAD likely undergoes proteolytic processing by the mono- and dibasic selective Kex2 protease, resulting in a cleavage at the kex-site (C-terminal end of the internal pro-sequence), which results in a large (approx. 50 kDa) and a small protein domain (approx. 10 kDa), ready to be secreted into the extracellular environment. After kex processing the prosequence at the C- terminus of the large subunit could be further processed proteolytically to further remove additional amino acids of the prosequence, which could happen intracellularly in late-Golgi / vesicles and / or extracellularly. As a result, a mature and active PAD enzyme is transported into the extracellular space and I or the PAD dimeric polypeptide is further processed extracellularly on the large subunit to generate an active and mature PAD in the extracellular space. The transport vesicles carrying the secreted protein fuse with the plasma membrane, releasing the protein into the extracellular environment. Once outside the fungal cell, the secreted active form of PAD consists of a large (approx. 50 kDa) and a small (approx. 10 kDa) domain and can interact with its target molecules or exert its biological function. As an additional or alternative option for PAD activation, the full length, unprocessed PAD propeptide is secreted to the extracellular matrix and further processing of the mono- and dibasic selective Kex2 site and proteolytical processing of the propeptide to further remove additional amino acids of the C-terminal prosequence present in the large subunit is done extracellularly.More preferably, said polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis comprises an amino acid sequence which does not comprise an internal pro-sequence and which has at least 80% sequence identity to respectively SEQ ID NO: 1-9. More detailed information on the PAD protein sequence is provided below. Alternatively, the below described isolated and / or recombinant PAD polypeptides (see also claims 10-12 herein) can also be used to describe the PAD polypeptide produced by the herein described PAD production methods.More preferred (based on PAD protein expression level) in the herein described and claimed method are a PAD polypeptide selected from Trichoderma harzianum and Bionectra ochroleuca. Hence, herein provided is a method for producing (an enzyme preparation comprising) protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses a PAD polypeptide from Trichoderma harzianum or from Bionectra ochroleuca,Preferably, said PAD polypeptide selected from the group of Trichoderma harzianum and Bionectra ochroleuca comprises an amino acid sequence which has at least 80% sequence identity to respectively SEQ ID NO: 1 or 3.More preferably, said polypeptide selected from the group of Trichoderma harzianum and Bionectra ochroleuca comprises an amino acid sequence which does not comprise an internal prosequence and which has at least 80% sequence identity to respectively SEQ ID NO: 1 or 3.Most preferred (based on PAD protein expression level) in the herein described and claimed method is a PAD polypeptide from Bionectra ochroleuca. Hence, herein provided is a method for producing (an enzyme preparation comprising) protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses a PAD polypeptide from Bionectra ochroleuca,Preferably, said PAD polypeptide from Bionectra ochroleuca comprises an amino acid sequence which has at least 80% sequence identity to SEQ ID NO: 3.More preferably, said polypeptide from Bionectra ochroleuca comprises an amino acid sequence which does not comprise an internal pro-sequence and which has at least 80% sequence identity to SEQ ID NO: 3.As described above, the recombinant host strain used in a method of the invention expresses(i) a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9.Surprisingly, as shown in the experimental part herein, expression levels of the Fusarium graminearum PAD polypeptide can be increased by introducing mutations in the PAD polypeptide. The wildtype Fusarium graminearum PAD polypeptide has a relative high protein pl. By introducing mutations which reduces the overall protein pl an increased level of PAD protein is produced. PAD surface charge (pl) variants are expressed at (much) higher protein levels when compared to the prior art expression of Fusarium graminearum PAD under identical conditions.Preferably, a variant Fusarium graminearum PAD polypeptide having a pl below 9 is a variant Fusarium graminearum PAD polypeptide having a pl below 8, 7 or 6.5.A suitable Fusarium graminearum PAD variant polypeptide has an amino acid sequence which, when aligned with the PAD of SEQ ID NO: 10, comprises at least substitutions of amino acids residues corresponding to amino acids 29, 76, 92, 107, 153, 190, 219, 251 , 365, 439, 467 and 560, said positions being defined with reference to SEQ ID NO:10. Preferably, said variant Fusarium graminearum PAD polypeptide has at least 80% sequence identity with SEQ ID NQ:10.Suitable substitutions are N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251 D, N365D, Q439E, Q467E and N560D. SEQ ID NO: 11 is derived from SEQ ID NO: 10 by adding these mutations.The experimental part describes 3 variant Fusarium graminearum PAD polypeptides (FgraPADmut_01 , FgraPADmut_02 and FgraPADmut_03) which all comprise the above- mentioned mutations and have a pl of 6.3. The variants differ in the used signal sequence.It is clear from the examples herein, that PAD polypeptides with a relative high pl can be expressed at higher levels by decreasing their pl and hence the invention therefore also provides a method for producing protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses a variant polypeptide having a pl below 9. Preferably, a variant PAD polypeptide having a pl below 9 is a variant PAD polypeptide having a pl below 8, 7 or 6.5.In yet another embodiment, the recombinant host strain in any of the above-described methods comprises a nucleic acid sequence encoding(i) a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incamatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9 operably linked to one or more control sequences capable of directing the expression of said PAD polypeptide in a host cell.Preferably such a nucleic acid sequence encodes any of the amino acid sequence SEQ ID NO: 1 to 9 or 11 or an amino acid sequence which has at least 80% sequence identity to respectively SEQ ID NO: 1 to 9 or 11 . The different sequences are explained in more detail above.Suitable nucleic acid sequences are shown in SEQ ID NO: 12 to 20 or 22 or a nucleic acid sequence having at least 80 % identity to any of SEQ ID NO: 12 to 20 or 22.Preferably, said nucleic acid sequence further comprises a nucleic acid sequence encoding a signal sequence, preferably a signal sequencing having amino acid sequence SEQ ID NO: 23, 24 or 25.A method of the invention uses a recombinant host strain. A host cell may be a prokaryotic, archaebacterial or eukaryotic host cell. A prokaryotic host cell may be a bacterial host cell. A eukaryotic host cell may be a yeast, a fungus, an amoeba, an alga, a plant, an animal cell, such asa mammalian or an insect cell. A eukaryotic cell may be a fungal cell, for example a yeast cell, such as a cell of the genus Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia. A yeast cell may be from Kluyveromyces lactis, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, Candida krusei. A eukaryotic cell may be a filamentous fungal cell.Preferably the host cell is a filamentous fungal host cell, preferably a filamentous fungal host cell of the genus Aspergillus, more preferably Aspergillus niger.The filamentous fungal host cell may be a cell of any filamentous form of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.The filamentous fungal host cell may be a cell of any filamentous form of the taxon Trichocomaceae (as defined by Houbraken and Samson in Studies in Mycology 70: 1-51 . 201 1). In another preferred embodiment, the filamentous fungal host cell may be a cell of any filamentous form of any of the three families Aspergillaceae, Thermoascaceae and Trichocomaceae, which are accommodated in the taxon Trichocomaceae. Suitable filamentous fungal host cells may be those in Clade 2: Aspergillus as described in Figure 1 of Houbraken and Samson, 201 1 (supra).Suitable filamentous fungal host cells suitable for use in the invention include, but are not limited to, cells of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.Preferred filamentous fungal cells belong to a species of an Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and most preferably a species of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Fusarium venenatum, Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris or Penicillium chrysogenum. A more preferred host cell belongs to the genus Aspergillus, more preferably the host cell belongs to the species Aspergillus niger. When the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.Several strains of filamentous fungi are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), Agricultural Research Service Patent Culture Collection, Northern Regional ResearchCenter (NRRL), and All-Russian Collection of Microorganisms of Russian Academy of Sciences, (abbreviation in Russian - VKM, abbreviation in English - RCM), Moscow, Russia. Useful strains in the context of the present invention may be Aspergillus niger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011 , CBS205.89, ATCC 9576, ATCC14488- 14491 , ATCC 11601 , ATCC12892, P. chrysogenum CBS 455.95, P. chrysogenum Wisconsin54- 1255(ATCC28089), Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Rasamsonia emersonii ATCC16479, CBS393.64, IFO31232, IMI116815, Thielavia terrestris NRRL8126, Talaromyces emersonii CBS 124.902, Acremonium chrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921 , Aspergillus sojae ATCC11906, Myceliophthora thermophila C1 , Garg 27K, VKM-F 3500 D, Chrysosporium lucknowense C1 , Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof.Preferred filamentous fungus host cells such as A. niger host cells, for example possibly contain one, more or all of the following modifications: deficient in a non-ribosomal peptide synthase preferably deficient in a non-ribosomal peptide synthase npsE (see WO2012 / 001 169), deficient in pepA, deficient in glucoamylase (glaA), deficient in acid stable alpha-amylase (amyA), deficient in neutral alpha-amylase (amyBI and amyBII), deficient in oxalic acid hydrolase (oahA) or oreE, deficient in one or more toxins, preferably ochratoxin and / or fumonisin, deficient in prtT, deficient in hdfA, comprises a SEC 61 modification being a S376W mutation in which Serine 376 is replaced by Tryptophan and / or comprises an adapted amplicon as defined in W02005 / 123763 and / or WO201 1 / 009700. These and other possible host modifications are also described in WO2012 / 001169, WO2011 / 009700, WG2007 / 062936, WG2006 / 040312, WG2004 / 070022, WO2013 / 135729, WO2014 / 013074, WO2014 / 013073, WO2018 / 166943.Preferably the host cell is a filamentous fungal host cell, preferably a filamentous fungal host cell of the genus Aspergillus, more preferably Aspergillus niger. As described above, more preferred in the herein described and claimed method are a PAD polypeptide selected from Trichoderma harzianum and Bionectra ochroleuca. Hence, herein provided is a method for producing (an enzyme preparation comprising) protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses a PAD polypeptide from Trichoderma harzianum or from Bionectra ochroleuca, wherein said host cell is Aspergillus niger.Preferably, said PAD polypeptide selected from the group of Trichoderma harzianum and Bionectra ochroleuca comprises an amino acid sequence which has at least 80% sequence identity to respectively SEQ ID NO: 1 or 3 and said host cell is Aspergillus niger.More preferably, said polypeptide selected from the group of Trichoderma harzianum and Bionectra ochroleuca comprises an amino acid sequence which does not comprise an internal prosequence and which has at least 80% sequence identity to respectively SEQ ID NO: 1 or 3 and said host cell is Aspergillus niger.Most preferred (based on PAD protein expression level) in the herein described and claimed method is a PAD polypeptide from Bionectra ochroleuca and said host cell is Aspergillusniger. Hence, herein provided is a method for producing (an enzyme preparation comprising) protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses a PAD polypeptide from Bionectra ochroleuca and said host cell is Aspergillus niger.Preferably, said PAD polypeptide from Bionectra ochroleuca comprises an amino acid sequence which has at least 80% sequence identity to SEQ ID NO: 3 and said host cell is Aspergillus niger.More preferably, said polypeptide from Bionectra ochroleuca comprises an amino acid sequence which does not comprise an internal pro-sequence and which has at least 80% sequence identity to SEQ ID NO: 3 and said host cell is Aspergillus niger.Those skilled in the art know how to transform cells with the one or more nucleic acid construct or expression vector of the invention.Transformation of the filamentous fungal host cell may be conducted by any suitable known methods, including e.g. electroporation methods, particle bombardment or microprojectile bombardment, protoplast methods and Agrobacterium mediated transformation (AMT). Procedures for transformation are described by J.R.S. Fincham, Transformation in fungi. 1989, Microbiological reviews. 53, 148-170.Transformation may involve a process consisting of protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81 :1470-1474. Suitable procedures for transformation of Aspergillus and other filamentous fungal host cells using Agrobacterium tumefaciens are described in e.g. De Groot et al., Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998, 16:839-842. Erratum in: Nat Biotechnol 1998 16:1074. A suitable method of transforming Fusarium species is described by Malardier et al., 1989, Gene 78:147156 or in WO 96 / 00787. Other methods can be applied such as a method using biolistic transformation as described in: Christiansen et al., Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei. 1995, Curr Genet. 29:100-102.Surprisingly, as shown in the experimental part herein, the levels of the produced PAD can be increased when compared to the prior art expression of wildtype Fusarium graminearum PAD under identical conditions. Hence, the produced PAD - in any of the above-described production methods - is expressed at higher levels when compared to the produced level of Fusarium graminearum of SEQ ID NO: 10 when expressed in A. niger under identical conditions. The yield may be at least 2% higher, such 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or at least 1000% higher. Whether or not a biodiversity PAD polypeptide or a variant Fusarium graminearum PAD polypeptide is expressed at a higher level when compared to the prior art expression of Fusarium graminearum PAD underidentical conditions, can for example be determined by running an SDS PAGE gel, staining the proteins on the gel and comparing the stained protein bands.The experimental part shows that the specificity of the PAD from Trichoderma harzianum (TharPAD_03) differs from the specificity of the PAD from Bionectra ochroleuca (BochPAD_02) when free arginine or napin are used as a substrate. The PAD from Bionectra ochroleuca (BochPAD_02) has a higher specific activity on napin when compared to the prior art PAD of W02008 / 000714. The PAD from Trichoderma harzianum (TharPAD_03) is - in respect of preference for free arginine of napin- comparable to the prior art PAD of W02008 / 000714.In respect of the produced Bionectra ochroleuca (BochPAD_02): the produced Bionectra ochroleuca - in any of the above described production methods - has an increased specificity for napin when compared to specificity of Fusarium graminearum of SEQ ID NO: 20 when expressed in A.niger under identical conditions.Preferably, a method of the invention is performed on large scale using at least 10 L of suitable medium, more preferably at least 100 L of suitable medium.Any of the above-described methods may comprise additional steps such as for example a purification step using techniques known to the skilled person. Purification steps have an impact on the purity of the final obtained PAD product. The produced PAD may be a pure or purified peptidyl arginine deiminase. A pure of purified peptidyl arginine deiminase is an enzyme that may be at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure for instance as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.In a second aspect, the invention provides PAD obtainable by any of the above-described methods. The obtained PAD is a mature heterodimer polypeptide comprising a first subunit of approx. 50 kDa and a second subunit of approx. 10 kDa.The invention provides multiple mature PAD polypeptides, i.e. different isolated and / or recombinant PAD polypeptides. Preferably, an isolated and / or recombinant PAD polypeptide comprising(a) a first domain comprising at least 80% sequence identity to amino acids 1 to 477 of SEQ ID NO:1 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 478 to 481 of SEQ ID NO:1 , a second domain comprising at least 80% sequence identity to amino acids 512 to 600 of SEQ ID NO: 1 and wherein said second domain optionally comprises 1 to 2 amino acids of amino acids 510 to 511 of SEQ ID NO:1 , andwherein said polypeptide does not comprise amino acids 482 to 509 of SEQ ID NO: 1 (in the experimental part herein referred to as TharPAD_01 , TharPAD_03 and TharPAD_02),(b) a first domain comprising at least 80% sequence identity to amino acids 1 to 487 of SEQ ID NO:2 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 488 to 491 of SEQ ID NO:2, a second domain comprising at least 80% sequence identity to amino acids 524 to 612 of SEQ ID NO: 2 and wherein said polypeptide does not comprise amino acids 492 to 523 of SEQ ID NO: 2 (in the experimental part herein referred to as VlonPAD_01 and VlonPAD_02),(c) a first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO:3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO:3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3 (in the experimental part herein referred to as BochPAD_01 , BochPAD_02 and BochPAD_03),(d) a first domain comprising at least 80% sequence identity to amino acids 1 to 483 of SEQ ID NO:4 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 484 to 487 of SEQ ID NO:4, a second domain comprising at least 80% sequence identity to amino acids 528 to 615 of SEQ ID NO: 4 and wherein said polypeptide does not comprise amino acids 488 to 527 of SEQ ID NO: 4 (in the experimental part herein referred to as FlonPAD_01),(e) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:5 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:5, a second domain comprising at least 80% sequence identity to amino acids 543 to 630 of SEQ ID NO: 5 and wherein said polypeptide does not comprise amino acids 494 to 542 of SEQ ID NO: 5 (in the experimental part herein referred to as FsolPAD_01 and FsolPAD_02),(f) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:6 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:6, a second domain comprising at least 80% sequence identity to amino acids 531 to 618 of SEQ ID NO: 6 and wherein said polypeptide does not comprise amino acids 494 to 530 of SEQ ID NO: 6 (in the experimental part herein referred to as AlecPAD_01 and AlecPAD_02),(g) a first domain comprising at least 80% sequence identity to amino acids 1 to 481 of SEQ ID NO:7 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 482 to 485 of SEQ ID NO:7, a second domain comprising at least 80% sequence identity to amino acids 517 to 604 of SEQ ID NO: 7 and wherein said polypeptide does not comprise amino acids 486 to 516 of SEQ ID NO: 7 (in the experimental part herein referred to as FincPAD_01),(h) a first domain comprising at least 80% sequence identity to amino acids 1 to 504 of SEQ ID NO:8 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 505 to 508 of SEQ ID NO:8, a second domain comprising at least 80% sequence identity to amino acids 535 to 626 of SEQ ID NO: 8 and wherein said polypeptide does not comprise amino acids 509 to 534 of SEQ ID NO: 8 (in the experimental part herein referred to as OausPAD_01 and OausPAD_02), or(i) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:9 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:9, a second domain comprising at least 80% sequence identity to amino acids 525 to 613 of SEQ ID NO: 9 and wherein said polypeptide does not comprise amino acids 494 to 524 of SEQ ID NO: 9 (in the experimental part herein referred to as VertPAD_01).More preferred are an isolated and / or recombinant PAD polypeptide comprising a first domain comprising at least 80% sequence identity to amino acids 1 to 477 of SEQ ID NO:1 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 478 to 481 of SEQ ID NO:1 , a second domain comprising at least 80% sequence identity to amino acids 512 to 600 of SEQ ID NO: 1 and wherein said second domain optionally comprises 1 to 2 amino acids of amino acids 510 to 511 of SEQ ID NO:1 , and wherein said polypeptide does not comprise amino acids 482 to 509 of SEQ ID NO: 1 (in the experimental part herein referred to as TharPAD_03), or an isolated and / or recombinant PAD polypeptide comprising a first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO: 3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO: 3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3 (in the experimental part herein referred to as BochPAD_02).Most preferred is an isolated and / or recombinant PAD polypeptide comprisinga first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO: 3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO: 3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3(in the experimental part herein referred to as BochPAD_02).As described above, the first domain of the PAD polypeptide optionally comprises 1 to 4 amino acids of SEQ ID NO: 1 to 9. The amount of optional amino acids depends on whether or not (and to what extent) ragging has taken place.As shown in the experimental part, BochPAD_02 (derived from SEQ ID NO: 3 by removal of pro-sequence and ragging) is subjected to some ragging.It is shown in the experimental part herein that the major large unit (N-terminal protein part) of BochPAD_02 was determined to be LSAT ... SSVS (residue 001 ... 471). Minor C- terminal ragging variants were detected being -VS (residue 001 ... 469), -SVS (residue 001 ... 468), and -SSVS (residue 001 ... 467). No ragging was detected for the N-terminus.Preferably, the invention provides an isolated and / or recombinant PAD polypeptide comprising a first domain comprising at least 80% sequence identity to amino acids 1 to 471 of SEQ ID NO: 3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3.The invention further provides a variant PAD polypeptide which is a variant Fusarium graminearum PAD polypeptide and wherein the variant Fusarium graminearum PAD polypeptide has an amino acid sequence which, when aligned with the PAD of SEQ ID NO: 10, comprises at least substitutions of amino acids residue corresponding to amino acids 29, 76, 92, 107, 153, 190, 219, 251 , 365, 439, 467 and 560, said positions being defined with reference to SEQ ID NO: 10 and wherein the variant has a pl below 9 and wherein said variant Fusarium graminearum PAD polypeptide has at least 80% sequence identity with SEQ ID NO:10. Preferably, the variant Fusarium graminearum PAD polypeptide comprises substitutions N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251 D, N365D, Q439E, Q467E and N560D, wherein said substitutions are defined with reference to SEQ ID NO: 10.Further provided is a polynucleotide encoding a biodiversity PAD polypeptide or a variant Fusarium graminearum PAD polypeptide as disclosed in the embodiments herein. In an embodiment, the polynucleotide is codon optimized. Codon optimization is known to the personskilled in the art and any method known to the person skilled in the art may be used. Preferably, the method as set forward in the example herein is used. Said method is extensively described in W02008 / 000632. Also provided is an expression vector comprising the polynucleotide as disclosed herein operably linked to at least one control sequence that directs expression of the polypeptide in a host cell. There are several ways of inserting a nucleic acid into a nucleic acid construct or an expression vector which are known to a person skilled in the art, see for instance Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001 . It may be desirable to manipulate a nucleic acid encoding a polypeptide of the present invention with control sequences, such as promoter and terminator sequences. A variety of promoters can be used that can direct transcription in the host cells of the disclosure. A promoter sequence may be derived from a highly expressed gene. Strong constitutive promoters are well known and an appropriate one may be selected according to the specific sequence to be controlled in the host cell. Examples of suitable promotors are listed in WO 2009 / 106575, including examples of suitable promotors in filamentous fungi. All of the promoters mentioned therein are readily available in the art. Any terminator which is functional in a cell as disclosed herein may be used, which are known to a person skilled in the art. Examples of suitable terminator sequences in filamentous fungi include terminator sequences of a filamentous fungal gene, for example those listed in WO 2009 / 106575.Further provided is a composition comprising a biodiversity PAD polypeptide or a variant Fusarium graminearum PAD polypeptide as disclosed in the embodiments herein and at least one component selected from milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt, an oxidant, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol and a preservative. A composition as disclosed herein may be a solid (for example a powder or a granulate) or fluid composition. A composition as disclosed herein may comprise one or more compounds selected from the group consisting of: milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt (like sodium or potassium chloride), an oxidant, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol (like glycerol) and a preservative. The term composition includes a pre-mix. A composition as disclosed herein may comprise one or more further enzyme(s) such as an amylase such as an alpha-amylase, for example a fungal alpha-amylase, a betaamylase; a glucanotransferase; a peptidase for example an exopeptidase or endopeptidase; a transglutaminase; a protein glutaminase; a cellulase; a hemicellulase, in particular a pentosanase such as xylanase; protease; a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95 / 00636; a glycosyltransferase; a peroxidase; a laccase; an oxidase, such as anhexose oxidase, a glucose oxidase, aldose oxidase, pyranose oxidase; a lipoxygenase; L-amino acid oxidase; a glucoamylase, a phytase and / or an asparaginase.In yet another aspect, the invention provides a method for converting arginine residues to citrulline residues comprising incubating a protein comprising arginine residues with a PAD polypeptide as described herein or with a (enzyme) composition as described herein.The protein comprising arginine or substrate protein (the terms are used interchangeably herein) contain at least 3 mol% of protein-bound arginine, more preferably they contain at least 6 mol% of protein-bound arginine. Examples of such substrate proteins are commercially available food proteins from animal origin, such as (skim) milk protein, whey protein, casein or egg protein. Another example of a substrate protein are plant proteins.Preferably, the invention provides a method for converting arginine residues to citrulline residues comprising incubating a plant protein comprising arginine residues with a PAD polypeptide as described herein or with a (enzyme) composition as described hereinAs used herein, the term “plant protein” refers to any protein from plant origin. Preferably, the plant protein is a protein from grains, pseudocereals, legumes, nuts, seeds or other sources such as coconut, potato, canola or tiger nut.Examples of suitable grains are barley, fonio, maize, millet, oat, rye, sorghum, teff, triticale, spelt, rice or wheat.Examples of suitable pseudograins are amaranth, buckwheat or quinoa.Examples of suitable legumes are lupin, pea, chickpea, beans (preferably faba beans), peanut or soy.Examples of suitable nuts are almond, brazil, cashew, hazelnut, macadamia, pecan, pistachio or walnut.Examples of suitable seeds are chia seed, flax seed, hemp seed, pumpkin seed, sesame seed or sunflower seed.The plant protein may comprise a blend of proteins which is prepared by mixing two or more plant protein types, for example by mixing almond and coconut proteins or by mixing almond and cashew proteins. I.e. in one of the embodiments, the solution comprising a plant protein comprises plant proteins from at least 2 different types of plants.Another example of a suitable substrate protein is a microbial protein, for example yeast extract or single cell protein for meat replacers.The step of “incubating a protein comprising arginine residues with a PAD polypeptide” can be performed at any suitable pH for any suitable time and with any suitable enzyme concentration. The skilled person is very well capable of establishing a suitable enzyme amount or a suitable incubation temperature or a suitable incubation pH or a suitable incubation time, for instance incubating protein with a peptidyl arginine deiminase at a pH of between 4 and 9, such as a pH of between 5 and 8.5, such as a pH of between 5.5 and 8, such as a pH between 6 and 7, or a pH of between 6.2 and 6.8, for instance at a pH of about 6.5. A suitable temperature at which protein isincubated with PAD may be between 20 and 60 degrees Celsius, such as a between 30 and 50, or between 35 and 45 degrees Celsius.The obtained protein comprising citrulline residues can subsequently be used to prepare a food (product). The food(product) can be any type of food(product) and include solid foods as well as a drink. Examples of such a food(product) are a plant-based beverage (i.e. plant-based dairy alternative beverages), a plant-based fermented product (i.e. plant-based yoghurt or plant-based cheese), an infant, follow-on or toddler drink or a meat or fish alternative product.Treating any of the above-mentioned protein with PAD can result in different final effects, such as organoleptic benefit, reduced allergenicity, improved emulsification properties, modified sweetness, liquorice, astringency, powdery / chalk, fulness, thickness and / or digestibility and / or protease inhibitory activity of a protein comprising food comprising.In a further aspect, the invention provides a method for modifying at least one characteristic of a protein comprising food comprising incubating a protein solution with a PAD polypeptide as described herein and optionally processing said PAD-treated protein solution into a protein comprising food.This aspect of the invention is discussed in detail in WO2019 / 233920 which is incorporated by reference herein.A preferred use of the herein disclosed PAD polypeptide is the use thereof on canola protein (also referred to as rapeseed protein). This is for example discussed in detail in WO2019 / 234137 which is incorporated by reference herein.Surprisingly, the inventors of the present invention noticed a correlation between the conversion of arginine to citrulline (as confirmed by NMR analysis) in the protein substrate napin and the effect thereof on taste. Hence, an analysis by a (trained) taste panel can be replaced by NMR analysis. The invention therefore provides a method for determining a sensorial effect of a PAD polypeptide on a protein substrate comprising incubating a protein substrate with PAD polypeptide subjecting the PAD polypeptide-treated protein substrate to NMR analysis.Also surprising is the finding by the inventors of the present invention that some of the herein described PAD polypeptides have a different specificity when compared to the prior art PAD of W02008 / 000714. More in specific, some of the herein disclosed PAD polypeptides have a higher activity on protein when compared to the prior art Fusarium graminearum PAD of W02008 / 000714 which prefers free arginine.The invention will be explained in more detail in the following example, which are not limiting the invention.ExamplesStandard genetic techniques, such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g. EP-A-0 635 574, WO 98 / 46772, WO 99 / 60102 and WO 00 / 37671 , WO90 / 14423, EP-A- 0481008, EP-A-0635 574 and US 6,265,186.Materials and MethodsStrainsWT 1 : This Aspergillus niger strain is used as a wild-type strain. This strain is deposited at the CBS Institute under the deposit number CBS 513.88.GBA 306: The construction of GBA 306 using WT1 as starting strain has been described in detail in WO201 1 / 009700. This GBA 306 strain has the following genotype: glaA, pepA, AhdfA, an adapted BamHI amplicon, AamyBII, AamyBI, and AamyA.GBA 312: The construction of GBA 312 using GBA306 as starting strain and with additional deletions of (i) fumB has been described in detail in WO2011 / 009700, (ii) of ochA and ochB has been described in detail in WO2011 / 009700, (iii) of prfT has been described in detail in WO2011 / 009700, (iv) of npsE has been described in detail in WO2012 / 001169, (v) of agsE has been described in detail in WO2014 / 013074, (vi) of amyC has been described in detail in WO2014 / 013073 and (vii) of agdA has been described in detail in WO2014 / 013073. This GBA 312 strain has the following genotype: (Ag / aA, ApepA, AhdfA, adapted BamHI amplicon, AamyBII, AamyBI, AamyA, AfumB, Aoch, AprfT, AnpsE, AagsE, AamyC, AagdA).VectorsThe pGBTOP-16 vector described in WO15177171 A1 , WO16097270 A1 , WO16 / 193292 A1 was modified to allow Golden Gate cloning (New England Biolabs). The four Bsal sites present in pGBTOP-16 were removed and two Bsal sites were introduced to allow cloning, one at the 3’- end of PglaA (promoter) fragment and one at the 5’-end of the 3’glaA (terminator) fragment. This resulted in vector pGBTOP-18 (Figure 1).PAD activity assay on ArgininePeptidyl arginine deiminase (PAD) activity is determined with 500 mM L-Arginine as substrate. The method can be performed on a clinical analyzer, or manually. The reaction takes place at 37 °C in a 200 mM MOPS buffer pH 7.0. A 100 mM phosphate buffer pH 7.0 with 0.1 % Triton X-100 is used to dilute samples at high activity. During the 10 minutes incubation PAD transforms L-arginine into L-citrulline and releases ammonia. In the presence of a- ketoglutarate and NADH the ammonia is immediately converted by glutamate dehydrogenase (GDH) in areaction that releases L-glutamate, NAD+ and water. The oxidation of NADH to NAD+ is followed spectrophotometrically at 340 nm. An ammonium sulphate reference is used to translate the decrease in A340 to the amount of ammonia produced.One PAD activity unit is the amount of enzyme required to produce 1 micromole of ammonia per minute from L-arginine, under the conditions of the test.PAD activity assay on protein sourcesThe method to determine PAD activity on protein is similar to the method described above but replacing 500 mM L-Arginine by 1 - 2% (w / w) of the target protein.Used protein sources are:Vertis™ CanolaPRO® from dsm-firmenich which is a commercial rapeseed protein isolateNapin-enriched fraction of CanolaPRO®Cruciferin-enriched fraction of CanolaPRO ®Protein determination (Bradford)The PAD enzyme is diluted with water to obtain solutions with 0.1 to 1 mg of protein per mL. Bradford reagent is added to the enzyme dilutions and the color reaction is allowed to proceed for 15 minutes at room temperature. The absorbance of the final reaction sample is determined at 595 nm after blanking the spectrophotometer with water. The A595 is translated to protein content using a calibration curve consisting of several concentrations of BSA.Sensory test on CanolaPRO ®CanolaPRO ® is dissolved in tap water (2%). Bottles are preheated to 45 °C and PAD enzyme is added (0-0.3 g of protein per g of CanolaPRO ®). Incubation (2 hours) is performed at 45 °C, subsequently enzyme inactivation is performed (5 minutes at 65 °C) and afterwards cooled on ice until use.Prior to the sensory test the samples are pre-tasted and discussed by the panel to define and align the attribute list used for the analysis. The samples were evaluated by means of descriptive analysis (QDA) in duplicate by an external panel (n=12-14) on the relevant attributes. During the test, the samples were offered according to an optimally balanced design, spread over two subsequent weeks, and were scored on 0-100 unstructured line scales in EyeQuestion. The principles of Good Sensory Practice were followed.Mass spectrometry analysisPAD protein was analyzed with a Synapt G2S Time Of Flight mass spectrometer coupled to a Acquity UHPLC system (Waters). About 25ng of the protein was injected on a Waters ACQUITY BEH C4 column (5 cm length, 2.1 mm inner diameter, 300 A pore size, 1 .7 pm particle size). The column was operated at 75°C. Eluents were LC-MS-grade water with 0.1 % formic acid(A) and 90% acetonitrile / 10% LC-MS-grade water with 0.1 % formic acid (v / v) (B). Proteins were eluted with a linear gradient from 3 to 25% B in 0.2 minutes, followed by a gradient from 25 to 55% B in 5.8 min all at a flow rate of 400 pL / min. Using the gradient as described for both BochPAD_02 and TharPAD_03 the large and small units could be separated. The Synapt G2S was operated in Full MS Electrospray Ionization mode scanning in profile Resolution mode from 500 to 5000 m / z with 1 sec scan time. Obtained m / z values were corrected using Leu-enkephalin (m / z 556.2771 Da) as lock mass. Waters deconvolution software (MaxEntl) was applied on the 6 most intense charge states of the large and small units to calculate the average mass.NMR spectroscopy analysis20 ml Samples from the Sensory test on CanolaPRO ® 2% w / w were freeze-dried, dissolved in a 50 mM phosphate buffer pH7 was added till sample was fully dissolved and the filtrate from a 50 kDa centrifugal filter was taken to enrich the napin over cruciferin. Subsequently, napin samples were buffer exchange with 25 mM citrate buffer pH3 by a 3 kDa filter to an end volume of 600 ul. 1 H-15N HSQC experiment was performed to observe the Arginine side chain Hs- Ns peaks around 87 ppm on the nitrogen axis.Tested PAD variantsTable 1 provides an overview of the tested PAD variants as well as obtained experimental results.GPA528-8 is the non-codon optimized genomic clone PAD of Fusarium graminearum and represents the PAD produced in W008000714.FgraPAD_01 is the codon optimized PAD of Fusarium graminearum and is cloned similarly to the tested variants.Table 1 : Overview of tested PAD variants as well as obtained experimental data* using https: / / web.expasy.org / compute_pi / Example 1 : Cloning and expression of the protein arginine deiminase enzyme variantsA codon-adapted DNA sequence for expression of the various protein arginine deiminase proteins (for all “PAD variant identifier” proteins as described in Table 1) in Aspergillus niger was designed containing additional Bsal type II restriction enzyme sites to enable subcloning in the Aspergillus expression vector pGBTOP-18. The translational initiation sequence of the promoter and translational termination sequence was modified into optimal ones as detailed in W02006 / 077258. Codon adaptation was performed as described in W02008 / 000632.The DNA fragments were cloned into pGBTOP-18 by repetitive steps of Bsal digestion and ligation (GoldenGate cloning method (New England Biolabs), according to standard procedure. The resulting vectors containing the enzyme expression cassettes under control of the glucoamylase promoter resulted in vectors with names comprising the variant identifier, such as pGBTOP- TharPAD_01 , pGBTOP-BochPAD_01 , etc (for ref of respective variants see Table 1).Subsequently, A. niger GBA 306 was transformed with all pGBTOP-based PAD vectors, in a co-transformation protocol with pGBAAS-3, with strain and methods as described in WO2011 / 009700 and references therein and selected on acetamide containing media and colonies purified according to standard procedures. Transformation and selection were performed as described in WO98 / 46772 and WO99 / 32617. A single, one-copy pGBTOP-PAD vector transformant expressing the specific gene variant was selected by PCR as representative transformant and called according PAD variant, such as GBA306-TharPAD_01 , GBA306- BochPAD_01 , etc and further replica-plated to obtain single strain inoculum spore suspensions.Subsequently, A. niger GBA 312 was transformed with pGBTOP-TharPAD_03 and pGBTOP-BochPAD_02, in a co-transformation protocol with pGBAAS-3, with strain and methods as described in WO2011 / 009700 and references therein and selected on acetamide containing media and colonies purified according to standard procedures. Transformation and selection were performed as described in WO98 / 46772 and WO99 / 32617. A single, pGBTOP-vector four-copy transformant expressing the specific gene variant was selected by PCR as representative transformant and called GBA312-TharPAD_03 and GBA312-BochPAD_02 and further replica- plated to obtain single strain inoculum spore suspensions.Example 2: Fermentation of protein arginine deiminase expressing A. n / qer strainsFresh A. niger spores from the parental strain GBA306 and the GBA306-based variant PAD producing strains were prepared and used to generate sample material by cultivation of the strains in 24 deep well-plates containing 4 ml fermentation medium (15 % w / v maltose, 6 % w / v bacto-soytone, 0.1 % w / v NaH2PO4.H2O, 0.96 % w / v NaNOs, 0.1 % w / v MgSO4.7H2O, 8 %o w / v Tween-80, 2 %o w / v Basildon, 2 % w / v MES, pH 6.2). After 5 days of cultivation at 30°C, 550 rpm and 80% humidity in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) 1.5 mL samples were taken, the mycelium was separated from the supernatant by centrifugation for 30 min at 4000g and the supernatants were stored at -20°C until further analyses.In addition, A. niger spores from the GBA 306, GBA312 parental strains and strains GBA306-TharPAD_03, GBA312-TharPAD_03, GBA306-BochPAD_02 and GBA312-BochPAD_02 were used to generate sample material by cultivation of the strains in 24 deep well-plates containing 4 ml fermentation medium as described above. Both broth and supernatant sample were used for follow-up analyses.Example 3: Activity testing of variantsThe variants were grown in 8-fold and analyzed for activity on Arginine and protein content as described in the “Materials and Methods section” above in MTP format. A selection of variants was made based on highest activity per protein (specific activity) combined with acceptable activity per mL (production yield): FgraPAD_01 , FgraPADmut_01 , FlonPAD_01 , FsolPAD_01 , VlonPAD_01 , BochPAD_02, AlecPAD_02, TharPAD_01 , FgraPADmut_02, BochPAD_01 , BochPAD_03, TharPAD_03and TharPAD_02.Example 4: Further activity testing of variantsThe selected variants of Example 3 were grown on a larger scale (100 ml shake flask) and tested for expression levels, specific activity on free Arginine (PADU / mg protein), and activity on CanolaPRO® protein substrate and napin and cruciferin enriched fractions Possible candidates based on activity on Arginine:Bionectria variants, in particular BochPAD_02Fusarium graminearum engineered variant FgraPADmut_02Trichoderma harzianum (Hypocrea lixii) TharPAD_01Fusarium longipes variant FlonPAD_01Verticillium longisporum VlonPAD_01Akanthomyces lecanii RCEF 1005 AlecPAD_02Possible candidates based on activity on protein (in particular CanolaPRO® protein fractions):Bionectria ochroleuca BochPAD_02Trichoderma harzianum (Hypocrea lixii) TharPAD_01Fusarium graminearum FgraPADmut_02FlonPAD_01 and AlecPAD_02, which were active on free Arginine, performed notably poorer on protein substrate.A further selection was comprised of BochPAD_02, FgraPADmut_02 and TharPAD_03 because of their good performance on CanolaPRO® protein substrate and high apparent expression (a.o. based on an additional - not described herein) experiment based on media relevant for larger scale production), while keeping genetic diversity in the screening. During further fermentations at larger scale FgraPADmut_02 did not perform as well as BochPAD_02 and TharPAD_03, and it was not taken along in further work.Example 5: Improved production of PAD by strain and bioprocess optimizationFor optimization of the bioprocess, A. niger spores from GBA 306 and strains GBA306- FlonPAD_01 , GBA306-TharPAD_01 and GBA306-BochPAD_02 were cultured in 24 deep wellplates containing 4 ml fermentation medium as described above at three different temperatures (26°C, 30°C, 34°C). Activities of the supernatant samples are as described below in Table 2.Table 2: PAD activities on arginine produced at different temperaturesSurprisingly, the optimal fermentation temperature is quite narrow with an optimum at 30°CFurther bioprocess optimization was tested by performing a lab-scale production of PAD enzyme as described herein. The performance and PAD enzyme production of a four-copy strain of GBA306-BochPAD_02 and GBA312-BochPAD_02 were compared in two different fermentation processes.The content of one vial of spores (FER1 : GBA306-BochPAD_02 and FER2: GBA312- BochPAD_02) was added to a 50 mL pre-culture medium consisting of 20 g / L glucose, 20 g / L yeast extract and 0.4 ml / L Clerol in a non-baffled 0.5 L-shake flask. The pre-culture was grown for 24h at 30°C and 280 rpm. After this, 20 g of cell suspension was transferred to a second preculture medium (same as above, except the medium volume was 300 ml in a 2 L baffled flask). The second preculture was grown for 24h at 30°C and 220 rpm. Timing of the shake flask steps can be adapted to the shake flask configuration and the vial viability.Fed-batch Cultures:The medium for fed-batch cultures is composed of a mix of glucose and salts. The salt fraction consisted of citric acid (1 aq) 1 .5 g / L, FeSO4 (7 aq) 0.8 g / L, MgSO4 (7 aq) 4 g / L, MnSO4 (4 aq) 0.2 g / L, ZnSO4(7 aq) 0.3 g / L , KCI 6.0 g / L, CaCI2(2 aq) 0.3 g / L, NaH2PO4(1 aq) 4.5 g / L and CuSO4(5 aq) 6.0 mg / L (FER1) or 0.6 mg / L (FER2). The glucose concentration in the medium was 90 g / kg. The medium was steam-sterilized (20 min at 121 °C) in two fractions, one of which combining sugars and calcium chloride. The pH of the medium salts fraction was adjusted to 3.0 (with phosphoric acid) before sterilization and adjusted to 5.2 (with ammonia) after having pooled the fractions in the bioreactor.The feed medium is also composed of a mix of glucose and a salt fraction. Salts were added in the same concentration as described for the medium. The glucose concentration was 500 g / kg. The feed medium was steam-sterilized (20 min at 121 °C) in one fraction. The pH of the feed medium was adjusted to 3.5 (with NaOH) before sterilization and not adjusted after sterilization. The temperature during fermentation was controlled at 30°C and the pH controlled at 5.2 (with ammonia). Bioreactors were inoculated at 6% inoculum ratio. The working volume was 11 L.The fermentations were run in an oxygen-limited (reaching a DOT of 0% during these fermentations as also described in US2009017515), fed-batch mode. The term "DOT" (Dissolved Oxygen Tension) is explained in

[0055] of example 1 in US2009017515. The glucose feed was started when the residual glucose concentration in the broth reached 40 g / L and was kept between 20 and 40 g / L throughout the course of the fermentation by adjustment of the feed rate. At the end of the fermentation, the glucose feed rate was reduced, in such a way that the glucose concentration in the broth was below 5 g / L after 120h. Clerol was used as antifoam and added in a periodic manner. Activities of the broth samples are as described below in Table 3.Table 3: PAD activities for FER1 and FER2 fermentations in broth on arginine substrateClearly, optimization of production strain background and / or bioprocess conditions during the fermentations has a great impact on increased productivity and increased activity produced of a PAD enzyme according to the invention.Example 6: fermentation with reduced copper levelsIt was noted that the addition of 1 mM EDTA to the end of fermentation broth helps to stabilize PAD activity. Copper is considered as a metal ion which could affect PAD activity.Fermentation tests with different levels of copper were performed, with conditions as essentially described in Example 5. Copper concentration (added as CuSO4 (5 aq)) in the media at 100% was 6 mg / L and 10% was 0.6 mg / L, respectively.Table 4: Relative PAD activity in end of fermentation (EoF) broth. The fermentation with 100% CuSC concentration after 120 h was the reference fermentation and set at 100% PAD activity (on arginine).Surprisingly, it can be concluded that the use of reduced Cu levels from 50% to 10% for the GBA312-BochPAD_02 strain during fermentation results in increased PAD activity, which would translate to a higher productivity.Example 7: downs stream processing and enzyme stabilityA typical recovery process (especially applicable for larger scale enzyme production) comprises (1) harvest, (2) broth preparation, (3) solid-liquid separation, (4) concentration, and (5) formulation. Harvest and broth preparation are steps performed to hold the fermentation broth and prepare it for the solid liquid separation step. Solid-liquid separation can for example be obtained by microfiltration, centrifugation or membrane filter press. After the solid-liquid separation step a liquid free of cells, containing the enzyme of interest, moves towards the concentration step. The concentration of the enzyme is typically done using ultrafiltration. This step produces a concentrate, enriched in the enzyme of interest. This concentrate, referred to as ccUF, is the input material for the stabilization step. The latter will produce an enzyme preparation designed to protect the activity of the enzyme against degradation mechanisms, prevent microbial spoilage and physical changes to the product (e.g. development of turbidity).As described in Example 6, addition of 1 mM EDTAto the end of fermentation broth helps to stabilize PAD activity. When fermentation is performed using lower levels of Cu, EDTA does not need to be added or can be added at reduced levels.The use of the 10% Cu recipe and selected EoF samples from example 6, led to a 1.5 fold improvement of the downstream process yield when compared to the 100% Cu recipe.Surprisingly, using the 10% Cu recipe, no PAD activity losses in fresh end of fermentation broth after 7 days of cold storage were observed using selected EoF samples from example 6; for avoidance of PAD activity losses this avoids the need for EDTA addition. The same observation was noted for ccUF (obtained after downstream processing using solid-liquid separation and ultrafiltration steps) using the 10% Cu recipe, with ~100% remaining activity levels after 7 days of cold storage, with ccUF using the 100% Cu recipe at <80% remaining activity levels.It is concluded that the use of lower levels of Cu during fermentation results in higher activity end of fermentation as well as improved stability of the PAD enzyme as well as an improved overall (process) yield.Example 8: pH optimum of BochPAD 02For determination of the pH optimum of BochPAD_02enzyme the manual PAD activity assay described in the Materials and Method section was performed at different pHs. Substrate solution was prepared according to the method description, but the pH of the final solution was set to pH 5, 6, 7, 8 and 9 with NaOH / HCl. The actual pH of the reaction mixture (substrate + enzyme) was also measured and used to plot the relative activity data. Results are shown in Fig. 2Example 9: Thermal inactivation of BochPAD 02BochPAD_02enzyme was diluted to a concentration of ~ 1 .5 PADU / g in the activity assay dilution buffer and transferred to the wells of a PCR plate (100 microL per well). After sealing, the plate was subjected to a heat gradient in a PCR machine (40-56C). Samples were analyzed for residual activity in the PAD activity assay after 5, 10, 22, 30, 40, 60 and 90 min of incubation in the PCR machine. Results are shown in Fig. 3.Example 10: Kinetics of BochPAD 02Substrate preparations of different concentrations between 500 and 1 mM were prepared in MOPS buffer (according to PAD activity assay protocol). PAD activity was monitored using the manual PAD activity assay in MTP format at each of the substrate concentrations. The initial slope of the reaction was plotted vs. the substrate concentration to obtain the Michaelis-Menten curve for PAD. Results are shown in Fig, 4.Example 11 : Specific activity of TharPAD 03 and BochPAD 02 on napin and CanolaPRO® vs. free ArgBochPAD_02 and TharPAD_03 were produced in lab-scale fermentation (as also described in Example 5 and 6) and their activity on Arginine, napin and CanolaPRO®, as well as their protein content was determined manually in MTP format as per the methods described herein. There were remarkable differences between the two variants with respect to substrate affinity.Table 5: specific activity of TharPAD_03 and BochPAD_02 on different substratesOn free Arginine it was observed that BochPAD_02 had a somewhat higher specific activity, but on protein substrate this difference was much more pronounced. BochPAD_02 is clearly the better performer on napin (by approximately a factor 3) and CanolaPRO® (by approximately a factor 2) per mg protein dosed.Example 12: NMR analysis of BochPAD 02-treated CanolaPRO napin fractions and the correlation to SensoryTested PAD variants NMR: original PAD molecule from Fusarium graminearum (W02008 / 000714), BochPAD_02, FgraPADmut_02and TharPAD_03.Tested PAD variants Sensory: original PAD molecule from Fusarium graminearum (W02008 / 000714), BochPAD_02and TharPAD_03.NMR experiments on a napin-fraction of rapeseed protein as such or treated with four different concentrations of BochPAD_02enzyme revealed in which order the Arginines were converted in the different locations of the protein. All tested PAD variants, including BochPAD_02, converted the Arginine residues with the same preference, but not all Arginine residues were converted. The maximum Arginine conversion was 30-40% based on amino acid composition analysis.After correlation of changes in amino acid composition with the Sensory results on the same samples, it was concluded that 15-20% of total Arginine to Citrulline conversion already resulted in the main sensory effect (reduced astringency, reduced bitterness, reduced liqorice, reduced sweetness). Arginine residue 38 was converted first by all PAD variants and was apparently responsible for the main Sensory effect. Residue 23 was converted in the higher enzyme dosages and at the highest PAD dosages residue 50 also started to be converted to Citrulline. Numbering based on homology to the napin Bnlb sequence described in Rico, M., Bruix, M., Gonzalez, C., Monsalve, R. I., & Rodriguez, R. (1996). H NMR Assignment and Global Fold of Napin Bnlb, a Representative 2S Albumin Seed Protein.Example 13: Mass Spectrometry analysis of exact C-terminus BochPAD 02and TharPAD 03Intact protein LC-HRMS analysis for BochPAD_02and TharPAD_03Activation of PAD is initiated by the cleavage after the amino acids ER (BochPAD_02, residue 497) or KR (TharPAD_03, residue 509). In the active state, both BochPAD_02 and TharPAD_03 are present as heterologous, non-covalently bonded dimers. Using non-native LC conditions, for both BochPAD_02 and TharPAD_03, the dimeric structure is no longer retained and instead they are present as 2 different monomers; a large unit (N-terminal protein) and a small unit (C-terminal protein). Based on the intact average mass information obtained after analysis of both units it was determined how PAD was further processed after activation.For both BochPAD_02 and TharPAD_03, the small unit contains only 1 occupied N- glycosylation site. This means that the intact average mass of this unit could be determined withoutfurther sample treatment, using intact protein LC-HRMS. However, the large unit contains 3 (BochPAD_02) and 8 (TharPAD_03) N-glycosylation sites of which multiple are occupied. The obtained MS spectra of the large unit, using intact protein LC-HRMS analysis of the sample as such, were too complex to use for deconvolution. Forthat reason, forthe determination of the intact mass of the large unit of both BochPAD_02and TharPAD_03the sample was first deglycosylated using PNGase F.PAD protein was analyzed with a Synapt G2S Time Of Flight mass spectrometer coupled to a Acquity UHPLC system (Waters). About 25ng of the protein was injected on a Waters ACQUITY BEH C4 column (5 cm length, 2.1 mm inner diameter, 300 A pore size, 1 .7 pm particle size). The column was operated at 75°C. Eluents were LC-MS-grade water with 0.1 % formic acid (A) and 90% acetonitrile / 10% LC-MS-grade water with 0.1 % formic acid (v / v) (B). Proteins were eluted with a linear gradient from 3 to 25% B in 0.2 minutes, followed by a gradient from 25 to 55% B in 5.8 min all at a flow rate of 400 pL / min. Using the gradient as described for both BochPAD_02 and TharPAD_03 the large and small units could be separated. The Synapt G2S was operated in Full MS Electrospray Ionisation mode scanning in profile Resolution mode from 500 to 5000 m / z with 1 sec scan time. Obtained m / z values were corrected using Leu-enkephalin (m / z 556.2771 Da) as lock mass. Waters deconvolution software (MaxEntl) was applied on the 6 most intense charge states of the large and small units to calculate the average mass.Based on the average masses the C-, and N-terminus of the large and small units could be determined using the given amino acid sequences of both BochPAD_02 (SEQ ID NO: 3) and TharPAD_03 (SEQ ID NO:1).For the large unit (N-terminal protein) of BochPAD_02 the mature amino acid sequence was determined to be LSAT ... SSVS (residue 001 ... 471). Minor C-terminal ragging variants were detected being -VS (residue 001 ... 469), -SVS (residue 001 ... 468), and -SSVS (residue 001 ... 467), and no ragging was detected for the N-terminus.The mature amino acid sequence for the small unit (C-terminal protein) is QAGS ... WWKS (residue 498 ... 587). N-, and C-terminal ragging is present being QAGS ... VWWK (residue 498 ... 586), AGST ... WWKS (residue 499 ... 587), and GSTI ... WWKS (residue 500 ... 587.For the large unit (N-terminal protein) of TharPAD_03 the mature amino acid sequence was determined to be IQAT ... QLHS (residue 001 ... 481). C-terminal ragging variants were detected being -S (residue 001 ... 480), -HS (residue 001 ... 479), -LHS (residue 001 ... 478) and -QLHS (residue 001 ... 477). No ragging was detected for the N-terminus.The mature amino acid sequence for the small unit (C-terminal protein) is QTTT ... GPWW (residue 510 ... 600). N-terminal ragging is present being TTTR ... GPWW (residue 51 1 ... 600), TTRY ... GPWW (residue 512 ... 600), TRYT ... GPWW (residue 513 ... 600), and RTYG ... GPWW (residue 514 ... 600).

Claims

Claims1. A method for producing protein arginine deiminase (PAD) comprising (a) cultivating a recombinant host strain which is capable of secreting PAD and (b) recovering the PAD, wherein said recombinant host strain expresses(i) a PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9.

2. A method according to claim 1 , wherein said PAD polypeptide selected from the group of Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis, comprises an amino acid sequence which has at least 80% sequence identity to respectively SEQ ID NO: 1-9.

3. A method according to claim 1 , wherein the variant Fusarium graminearum PAD polypeptide has an amino acid sequence which, when aligned with the PAD of SEQ ID NO: 10, comprises at least substitutions of amino acids residues corresponding to amino acids 29, 76, 92, 107, 153, 190, 219, 251 , 365, 439, 467 and 560, said positions being defined with reference to SEQ ID NO:10.

4. A method according to claim 3 wherein said variant Fusarium graminearum PAD polypeptide has at least 80% sequence identity with SEQ ID NO:10.

5. A method according to any of the preceding claims, wherein the recombinant host strain comprises a nucleic acid sequence encoding(i) a PAD polypeptide selected from the group of Trichoderma harzianum (Hypocrea lixii), Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani species complex, Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti species complex and Ophiocordyceps australis, or(ii) a variant Fusarium graminearum PAD polypeptide having a pl below 9 operably linked to one or more control sequences capable of directing the expression of said PAD polypeptide in a host cell.

6. A method according to claim 5, wherein said nucleic acid sequence further comprises a nucleic acid sequence encoding a signal sequence, preferably a signal sequence having the amino acid sequence of SEQ ID NO: 23, 24 or 25.

7. A method according to any of the preceding claims, wherein the host cell is a filamentous fungal host cell, preferably a filamentous fungal host cell of the genus Aspergillus, more preferably Aspergillus niger.

8. A method according to any of the preceding claims wherein the produced PAD is expressed at higher levels when compared to the produced level of Fusarium graminearum of SEQ ID NO: 10 when expressed in A.niger under identical conditions.

9. PAD obtainable by a method of any one of claims 1 to 8.

10. An isolated and / or recombinant PAD polypeptide comprising(a) a first domain comprising at least 80% sequence identity to amino acids 1 to 477 of SEQ ID NO:1 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 478 to 481 of SEQ ID NO:1 , a second domain comprising at least 80% sequence identity to amino acids 512 to 600 of SEQ ID NO: 1 and wherein said second domain optionally comprises 1 to 2 amino acids of amino acids 510 to 511 of SEQ ID NO:1 and wherein said polypeptide does not comprise amino acids 482 to 509 of SEQ ID NO: 1 ,(b) a first domain comprising at least 80% sequence identity to amino acids 1 to 487 of SEQ ID NO:2 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 488 to 491 of SEQ ID NO:2, a second domain comprising at least 80% sequence identity to amino acids 524 to 612 of SEQ ID NO: 2 and wherein said polypeptide does not comprise amino acids 492 to 523 of SEQ ID NO: 2,(c) a first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO:3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO:3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3,(d) a first domain comprising at least 80% sequence identity to amino acids 1 to 483 of SEQ ID NO:4 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 484 to 487 of SEQ ID NO:4, a second domain comprising at least 80% sequence identity to amino acids 528 to 615 of SEQ ID NO: 4 and wherein said polypeptide does not comprise amino acids 488 to 527 of SEQ ID NO: 4,(e) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:5 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:5,a second domain comprising at least 80% sequence identity to amino acids 543 to 630 of SEQ ID NO: 5 and wherein said polypeptide does not comprise amino acids 494 to 542 of SEQ ID NO: 5,(f) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:6 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:6, a second domain comprising at least 80% sequence identity to amino acids 531 to 618 of SEQ ID NO: 6 and wherein said polypeptide does not comprise amino acids 494 to 530 of SEQ ID NO: 6,(g) a first domain comprising at least 80% sequence identity to amino acids 1 to 481 of SEQ ID NO:7 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 482 to 485 of SEQ ID NO:7, a second domain comprising at least 80% sequence identity to amino acids 517 to 604 of SEQ ID NO: 7 and wherein said polypeptide does not comprise amino acids 486 to 516 of SEQ ID NO: 7,(h) a first domain comprising at least 80% sequence identity to amino acids 1 to 504 of SEQ ID NO:8 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 505 to 508 of SEQ ID NO:8, a second domain comprising at least 80% sequence identity to amino acids 535 to 626 of SEQ ID NO: 8 and wherein said polypeptide does not comprise amino acids 509 to 534 of SEQ ID NO: 8, or(i) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:9 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:9, a second domain comprising at least 80% sequence identity to amino acids 525 to 613 of SEQ ID NO: 9 and wherein said polypeptide does not comprise amino acids 494 to 524 of SEQ ID NO: 9.11 . A variant PAD polypeptide which is a variant Fusarium graminearum PAD polypeptide and wherein the variant Fusarium graminearum PAD polypeptide has an amino acid sequence which, when aligned with the PAD of SEQ ID NO: 10, comprises at least substitutions of amino acids residue corresponding to amino acids 29, 76, 92, 107, 153, 190, 219, 251 , 365, 439, 467 and 560, said positions being defined with reference to SEQ ID NO: 10 and wherein the variant has a pl below 9 and wherein said variant Fusarium graminearum PAD polypeptide has at least 80% sequence identity with SEQ ID NO:10.

12. A variant PAD polypeptide according to claim 11 , wherein said substitutions are N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251 D, N365D, Q439E, Q467E and N560D.

13. A composition comprising the PAD polypeptide of any one of claims 9 to 12 and at least one component selected from milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt, an oxidant, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol and a preservative.

14. A method for converting arginine residues to citrulline residues comprising incubating a protein comprising arginine residues with a PAD polypeptide according to any one of claims 9 to 12 or with a composition according to claim 13.

15. A method for modifying at least one characteristic of a protein comprising food comprising incubating a protein solution with the PAD polypeptide of any one of claims 9-12 and optionally processing said PAD-treated protein solution into a protein comprising food.

16. A method according to claim 14 or 15, wherein said protein is a canola protein.

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