Protein arginine deiminase

Abstract

The present invention relates to the production of zymoprotein arginine deiminases and the use of such enzymes.

Description

Technical Field

[0001] This invention relates to the production of enzyme protein arginine deiminase (PAD) and the use of such enzyme. The invention also relates to novel PAD enzymes. Furthermore, the invention relates to proteins and peptides containing citrulline residues. Background Technology

[0002] In a world with a growing population, the demand for protein is also increasing. To respond to this growing demand, research into the broader applications of protein is needed. Furthermore, there is a desire to find plant proteins as alternatives to animal proteins, as plants are considered a more sustainable protein source than animals. The use of plant proteins in food remains limited, partly due to their lower digestibility, taste, and nutritional value.

[0003] A key factor in creating plant-based protein foods is their taste and texture. Plant proteins suffer from impaired taste (such as bitterness) and increased mouthfeel, even at low protein content. Furthermore, many plant-based protein drinks contain very low amounts of protein, making them nutritionally poor and unpalatable.

[0004] The enzyme protein arginine deiminase (PAD) can be used to improve the properties of plant-based foods (WO2008 / 000714, WO2017 / 009100 and WO2019 / 233910).

[0005] WO2008 / 000714 (DSM) describes a fungus Fusarium gramineae Isolation and identification of secretory PADs from *Fusarium graminearum*. This indicates that in fungi... Chaetomium globosum , Phaeosphere nodules and bacteria Streptomyces scabies and Streptomyces clavuligerus Potential secretory PADs can be found in this study. Although many new applications have been identified for PADs, see, for example, WO2017 / 009100 and WO2019 / 233910 (both DSM), enzyme expression needs further improvement to achieve production levels of greater commercial interest.

[0006] There is a need to improve PAD production levels. There is also a need for alternative PAD enzymes.

[0007] Surprisingly, the inventors of this invention discovered that by using PAD sequences from a specific fungal source or by using previously disclosed... Fusarium gramineae Engineered variants of PAD can achieve improved PAD expression levels. The inventors have also disclosed a novel PAD protein sequence.

[0008] Attached Figure Figure 1 Carrier pGBTOP-18 Figure 2 pH curve of BochPAD_02 Figure 3 Thermal stability of BochPAD_02 Figure 4 Michaelis curve of BochPAD_02 sequence list SEQ ID NO: 1 contains a pro-sequence from Trichoderma harzianum PAD protein sequence SEQ ID NO: 2 contains the pre-sequence from Verticillium longisporum PAD protein sequence SEQ ID NO: 3 contains the pre-sequence from Bionectra ochroleuca PAD protein sequence SEQ ID NO: 4 contains the pre-sequence from Fusarium longipes PAD protein sequence SEQ ID NO: 5 contains the pre-sequence from Fusarium solani PAD protein sequence of species complex SEQ ID NO: 6 contains the pre-sequence from Akanthomyces lecanii RCEF 1005 PAD protein sequence SEQ ID NO: 7 contains the pre-sequence from Fusarium incarnatum-horsetail PAD protein sequence of species complex SEQ ID NO: 8 contains the pre-sequence from Ophiocordyceps australis PAD protein sequence SEQ ID NO: 9 contains the pre-sequence from Verticillium longisporum PAD protein sequence SEQ ID NO: 10 contains a pre-sequence from the wild type Fusarium gramineae PAD protein sequence SEQ ID NO: 11 contains the pre-sequence from Fusarium gramineae Protein sequences of variants FgraPADmut_01, FgraPADmut_02, and FgraPADmut_03 SEQ ID NO: 1-11 does not contain a signal sequence.

[0009] SEQ ID NO: 12 encodes the polynucleotide sequence of the protein of SEQ ID NO: 1. SEQ ID NO: 13 encodes the polynucleotide sequence of the protein SEQ ID NO: 2. SEQ ID NO: 14 encodes the polynucleotide sequence of the protein SEQ ID NO: 3. SEQ ID NO: 15 encodes the polynucleotide sequence of the protein SEQ ID NO: 4. SEQ ID NO: 16 encodes the polynucleotide sequence of the protein of SEQ ID NO: 5. SEQ ID NO: 17 encodes the polynucleotide sequence of the protein of SEQ ID NO: 6. SEQ ID NO: 18 encodes the polynucleotide sequence of the protein in SEQ ID NO: 7. SEQ ID NO: 19 encodes the polynucleotide sequence of the protein of SEQ ID NO: 8. SEQ ID NO: 20 encodes the polynucleotide sequence of the protein SEQ ID NO: 9. SEQ ID NO: 21 encodes the polynucleotide sequence of the protein in SEQ ID NO: 10. The polynucleotide sequence of the protein SEQ ID NO: 22 encoding SEQ ID NO: 11. SEQ ID NO: 23 Fusarium graminearum wild-type signal sequence MHLLNGKTAAVALALLNSCNA SEQ ID NO: 24A. Niger signal sequence MVKSILASVFFAATALA SEQ ID NO: 25A. Niger signal sequence MSFRSLLALSGLVCTGLA. Summary of the Invention

[0010] This invention provides: -A method for producing protein arginine deiminase (PAD), - Isolated and / or recombinant PAD peptides, -Fusarium graminearum PAD polypeptide, -A composition comprising any of the PAD peptides claimed herein. - A method for converting arginine residues into citrulline residues. - A method for modifying at least one characteristic of a protein-containing food. - This document claims the use of any of the PAD peptides for modifying at least one feature of a protein-containing food.

[0011] definition The term “operably linked” is defined herein as a construction in which a control sequence is appropriately placed relative to the ISP coding sequence, such that the control sequence directs the production of RNA or mRNA and, optionally, the production of a polypeptide translated from said (m)RNA.

[0012] The term "control sequence" is defined herein as encompassing all components, whether in vitro or in host cells, that are essential or advantageous for the expression of mRNA and / or polypeptides. Each control sequence may be native or exogenous to the nucleic acid sequence encoding the polypeptide. Examples of control sequences are transcription initiation sequences, termination sequences, promoters, leader sequences, signal peptides, propeptides, prepropeptides, or enhancer sequences; Shine-Delgarno sequences, repressor or activator sequences; effective 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 sequences that enhance protein secretion when needed. Signal sequences used to optimize expression are described in WO2010 / 121933. At a minimum, control sequences include promoters as well as transcription and translation termination signals. Control sequences can be optimized for specific purposes.

[0013] The term "expression" includes any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0014] As defined herein, a “host cell” is an organism suitable for genetic manipulation and capable of being cultured at a cell density suitable for the industrial production of a target product (e.g., the PAD peptide according to the invention). A host cell can be a host cell found in nature or a host cell derived from a parent host cell after genetic manipulation or classical mutagenesis. Advantageously, the host cell is a recombinant host cell. A host cell can be a prokaryotic host cell, an archaea host cell, or a eukaryotic host cell. Prokaryotic host cells can be, but are not limited to, bacterial host cells. Eukaryotic host cells can be, but are not limited to, yeast, fungi, amoebas, algae, plants, animals, or insect host cells.

[0015] Nucleic acid or polynucleotide sequences are defined herein as polymers of nucleotides containing at least five nucleotides or nucleic acid units. Nucleotides or nucleic acids refer to RNA and DNA. The terms "nucleic acid" and "polynucleotide sequence" are used interchangeably herein.

[0016] The term "polypeptide" refers to a molecule containing more than five amino acid residues linked by peptide bonds. As used herein, the term "protein" is synonymous with the term "polypeptide" and can also refer to two or more polypeptides. Therefore, the terms "protein" and "polypeptide" are used interchangeably. Polypeptides can optionally be modified (e.g., glycosylation, phosphorylation, acylation, farnesylation, isopreneation, sulfonation, etc.) to add functionality. Polypeptides that exhibit activity under certain conditions in the presence of a specific substrate can be called enzymes. It should be understood that due to the degeneracy of the genetic code, multiple nucleotide sequences encoding a given polypeptide can be produced.

[0017] As used herein, the term "isolated polypeptide" refers to a polypeptide 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, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 80%, at least 80%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, with purity determined by SDS-PAGE or any other analytical method known to those skilled in the art and suitable for this purpose. The isolated polypeptide may be produced by recombinant host cells.

[0018] “Mature polypeptide” is defined herein as the polypeptide in its final form obtained after mRNA is translated into a polypeptide and said polypeptide undergoes post-translational modifications. Post-translational modifications include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of leader sequences (e.g., signal peptides, propeptides, and / or prepropeptides) by cleavage.

[0019] The term "promoter" is defined herein as a DNA sequence that binds to RNA polymerase and guides the polymerase to the correct downstream transcription start site of a nucleic acid sequence to initiate transcription. Suitable bacterial promoters are disclosed, for example, in WO-A1-2004 / 074468.

[0020] When used in relation to nucleic acids or proteins, the term "recombinant" means that the sequence of a nucleic acid or protein has been modified by human intervention compared to its natural form. When referring to cells (such as host cells), the term "recombinant" means that the cell's genome has been modified in its sequence by human intervention compared to its natural form. The term "recombinant" is synonymous with "genetically modified."

[0021] Sequence identity or sequence homology are used interchangeably in this paper. To determine the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal comparison. To optimize the alignment between two sequences, a gap can be introduced into either of the two sequences being compared. This alignment can be performed over the full length of the sequences being compared. Alternatively, it can be performed over a shorter length, such as about 20, about 50, about 100 or more nucleic acid / base or amino acid sequences. Sequence identity is the percentage of identical matches between two sequences in the reported alignment region. The percentage of sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman-Wunsch algorithm used to align two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid and nucleotide sequences can be aligned using the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS software package (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ) was used. For protein sequences, EBLOSUM62 was used for the substitution matrix. For nucleotide sequences, EDNAFULL was used. Optional parameters used were a gap opening penalty of 10 and a gap extension penalty of 0.5. Those skilled in the art will understand that all these different parameters will produce slightly different results, but the overall percentage of identity between the two sequences will not change significantly when different algorithms are used. After alignment using the NEEDLE procedure as described above, the percentage of sequence identity between the query sequence and the sequence of this invention is calculated as follows: the number of corresponding positions showing the same amino acid or nucleotide in both sequences divided by (the total length of the alignment minus the total number of gaps in the alignment). Identity as defined herein can be obtained from NEEDLE using the NOBRIEF option and is marked as "Longest Identity" in the program's output.

[0022] The nucleic acid and protein sequences of the present invention can also be used as "query sequences" to perform searches against public databases, such as to 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. (1990) Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. For gapped alignments for comparative purposes, a Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402, can be used. When using BLAST and Gapped BLAST programs, the default parameters for the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / .

[0023] "Synthetic molecules," such as synthetic nucleic acids or synthetic peptides, are produced through in vitro chemical or enzymatic synthesis. This includes, but is not limited to, variant nucleic acids prepared using the optimal codon usage for a selected host organism.

[0024] Optimization of synthesized nucleic acids can be achieved through codon usage optimization, preferably according to the methods described in WO2006 / 077258 and / or WO2008000632, which are incorporated herein by reference. WO2008 / 000632 relates to codon pair optimization. Codon pair optimization is a method in which the codon usage (especially the codon pairs used) of a nucleotide sequence encoding a polypeptide is optimized to obtain increased expression of the nucleotide sequence encoding the polypeptide and / or increased yield of the encoded polypeptide. A codon pair is defined as a set of two consecutive triplets (codons) in a coding sequence. Those skilled in the art will recognize that codon usage needs to be adjusted according to the host species, which may result in variants having significant homology differences from SEQ ID NO: 2 but still encoding a polypeptide according to the invention.

[0025] As used herein, the terms “variant” or “mutant” are used interchangeably. They can refer to polypeptides or nucleic acids. Variants include substitutions, insertions, deletions, truncations, transversions, and / or inversions at one or more sites relative to a reference sequence. Variants can be prepared, for example, by site-saturation mutagenesis, scan mutagenesis, insertion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as a variety of other recombination methods known to those skilled in the art. Variant genes of nucleic acids can be synthesized artificially using techniques known in the art.

[0026] Amino acids are referred to herein by their single-letter codes, which are known to those skilled in the art and can be found in Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHLPress, Cold Spring Harbor, NY, 2001. The amino acids are: A=Ala=alanine; R=Arg=arginine; N=Asn=arginine; D=Asp=aspartic acid; C=Cys=cysteine; E=Glu=glutamic acid; Q=GLn=glutamine; G=Gly=glycine; H=His=histidine; I=Ile=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=Val=valine. Detailed Implementation

[0027] In a first aspect, the present invention provides a method for producing protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses: (i) selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group of PAD peptides; or (ii) variants with pI below 9 Fusarium gramineae PAD polypeptide.

[0028] The terms protein arginine deiminase (PAD), peptidyl arginine deiminase (PAD), PAD, and PAD polypeptide are used interchangeably throughout this document. Protein arginine deiminase, or peptidyl arginine deiminase, belongs to the family of enzymes that convert arginine, which is bound to a peptide or protein, to citrulline, which is bound to a peptide or protein (EC 3.5.3.15). This process is called deamination or citrullination. In the reaction from arginine to citrulline, one of the terminal nitrogen atoms in the arginine side chain is replaced by an oxygen atom. The reaction uses one water molecule and produces ammonia as a byproduct (http: / / en.wikipedia.org / wiki / Citrullination). Arginine carries a positive charge at neutral pH, while citrulline is uncharged.

[0029] Typically, the methods described herein for producing PAD result in an enzyme preparation containing PAD as well as other proteins. The resulting enzyme preparation may contain other (added) components, such as salts, buffers, preservatives, and / or polyols, which will be discussed in more detail later. Therefore, this document provides a method for producing an enzyme preparation containing a protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses: (i) selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group of PAD peptides that make up the group; or (ii) variants with pI below 9 Fusarium gramineae PAD polypeptide.

[0030] In step (a) of the described method, a recombinant host strain capable of secreting PAD is cultured. The culturing step is carried out under conditions suitable for PAD production. That is, suitable conditions are used that allow the recombinant host strain to grow, produce, and secrete PAD, such as suitable growth medium, temperature, and pH. These conditions can be well determined by those skilled in the art. Surprisingly, the optimal fermentation temperature is quite narrow, with an optimal temperature of 30°C. Preferably, the temperature in step (a) is 30°C. As shown herein in the experimental section, 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, and improved overall (process) yield. Preferably, the copper concentration is less than 6 mg / L, more preferably less than 3 mg / L, and most preferably less than 1 mg / L of fermentation medium.

[0031] Preferably, this document provides a method wherein step (a) comprises culturing the recombinant host strain in a fermentation medium containing a copper concentration of greater than 0 mg / L and less than 1 mg / L (preferably greater than 0.01 mg / L and less than 1 mg / L). The copper concentration is measured with reference to when added as CuSO4 (5 aq).

[0032] This article also provides a method in which step (a) includes culturing the recombinant host strain in a fermentation medium containing a copper concentration of more than 0 mg / L and less than 1 mg / L (preferably more than 0.01 mg / L and less than 1 mg / L), and wherein the fermentation temperature is in the range of 29°C to 31°C, preferably 30°C.

[0033] In step (b), the generated and secreted PAD is recovered using techniques known to those skilled in the art. Such techniques include, for example, separation of growth medium from the host cells used, filtration steps, and / or purification steps. Other examples of such techniques are described in Example 7. Optionally, step (b) includes the addition of a suitable amount of EDTA. The suitable amount of EDTA can be readily determined by those skilled in the art and is described in the Experimental section of this document, and, for example, is the addition of 1 mM EDTA at the end of the fermentation broth.

[0034] Expression of recombinant host strains used in the method of the present invention: (i) selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group of PAD peptides that make up the group; or (ii) variants with pI below 9 Fusarium gramineae PAD polypeptide.

[0035] Surprisingly, as shown in the experimental section of this paper, a subset of wild-type pad peptides can achieve the same results as in the prior art under the same conditions. Fusarium gramineae PAD is expressed at a higher (or much higher) level than other proteins. Therefore, PAD expressed by the host strain is preferably selected from... Trichoderma harzianum、Verticillium longisporum, Bionectra ochroleuca, Fusarium longipes, Fusarium solani Species complex 、 Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group consists of PAD polypeptides.

[0036] Some of the strains mentioned (from which PADs are obtained) also have other names, such as: - Trichoderma harzianum Also known as Hypocritical nonsense., Trichaderma harzianum, Trichoderma harzianum Rifai, 1969, Trichoderma harzianum or Trichordermaharzianum; - Verticillium longisporum is also known as Verticillium dahliae var.longisporum, Verticillium dahliae var. longisporum C. Stark 1961 or Verticilliumlongisporum (C. Stark); - Bionectra ochroleuca Also known as Clonostachys rosea , Bionectria ochroleuca, Gliocladium roseum, Bionectria aureofulva, Nectria aureofulva or Nectria gliocladioides; - Fusarium longipes is also known as Fusarium longipes Wollenw. & Reinking, 1925, Fusarium equiseti var. longipes, Fusarium equiseti var. longipes, and also Fusariumscirpi var. longipes; - Fusarium solani species complex, also known as Nectria haematococca complex or Nectria haematococca asexual forms (anamorphs). - Ophiocordyceps australis is also known as Cordyceps australis or Cordycepsunilateralis var. australis.

[0037] Preferably, selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The PAD polypeptides comprising the group contain amino acid sequences having at least 80% sequence identity with the corresponding SEQ ID NO: 1 to 9. The different sequences are explained in more detail under the heading “Sequence Listing” above.

[0038] Surprisingly, the inventors of this invention have determined that these wild-type PAD polypeptide sequences contain an internal pre-sequence of approximately 35-50 amino acids not present in secretory active PAD polypeptides. It is known that pre-sequences in secretory fungal enzymes (e.g., phospholipases) protect the cellular interior from harmful activities. The inventors hypothesize that the internal pre-sequence is cleaved and further processed during secretion to prevent the intracellular toxicity of PAD activity by shielding the enzyme's active site. Therefore, part of this invention involves the transcription and translation of the corresponding gene serving as the PAD prepeptide within the fungal cell. The resulting full-length protein is referred to 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 for the secretory pathway and transfers the prepeptide to the endoplasmic reticulum (ER). In current knowledge and models, processes such as protein folding, disulfide bridging, and / or glycosylation can occur in the ER. Subsequently, the folded preprotein is transported from the ER to the Golgi apparatus, and then to secretory vesicles. Within the Golgi apparatus, the secreted protein can undergo further modifications, such as further glycosylation, phosphorylation, and / or sulfation. It is within this trans-Golgi network (late Golgi / vesicle) that the preprotein PAD may undergo proteolytic processing by the mono- and di-base-selective Kex2 protease, resulting in cleavage at the kex site (the C-terminus of the internal pre-sequence), which yields a large protein domain (approximately 50 kDa) and a small protein domain (approximately 10 kDa), ready for secretion into the extracellular environment. Following kex processing, further proteolytic processing of the pre-sequence at the C-terminus of the large subunit can further remove additional amino acids from the pre-sequence; this can occur intracellularly within the late Golgi / vesicle and / or extracellularly. As a result, a mature and active PAD enzyme is transported to the extracellular space and / or the PAD dimer polypeptide undergoes further extracellular processing on the large subunit to produce an active and mature PAD in the extracellular space. Transport vesicles carrying the secreted proteins fuse with the plasma membrane, releasing the proteins into the extracellular environment. Once outside the fungal cell, the secretory active form of PAD consists of a large (approximately 50 kDa) and a small (approximately 10 kDa) domain, and can interact with its target molecules or perform its biological function. As an additional or alternative option for PAD activation, the full-length, unprocessed PAD propeptide is secreted into the extracellular matrix, where further processing at the single- and dibase-selective Kex2 sites, as well as proteolytic processing of the propeptide, is completed extracellularly to remove additional amino acids from the C-terminal pro-sequence present in the large subunit.

[0039] More preferably, selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The polypeptides of the group comprise an amino acid sequence that does not contain an internal pre-sequence and has at least 80% sequence identity with the corresponding SEQ ID NO: 1-9. More detailed information about the PAD protein sequence is provided below. Alternatively, isolated and / or recombinant PAD polypeptides described below (see also claims 10-12 herein) may also be used to describe PAD polypeptides produced by the PAD production method described herein.

[0040] More preferred among the methods described and claimed herein (based on PAD protein expression levels) is selected from... Trichoderma harzianum and Bionectra ochroleuca The present invention provides a method for producing a protein arginine deiminase (PAD) or an enzyme preparation containing a protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses a protein derived from PAD. Trichoderma harzianum Or from Bionectra ochroleuca PAD multipeptide.

[0041] Preferably, selected from Trichoderma harzianum and Bionectra ochroleuca The PAD polypeptide of the group comprises an amino acid sequence having at least 80% sequence identity with the corresponding SEQ ID NO: 1 or 3.

[0042] More preferably, selected from Trichoderma harzianum and Bionectra ochroleuca The polypeptide of the group comprises an amino acid sequence that does not contain an internal pre-sequence and has at least 80% sequence identity with the corresponding SEQ ID NO: 1 or 3.

[0043] More preferred (based on PAD protein expression levels) among the methods described and claimed herein is derived from... Bionectra ochroleuca The present invention provides a method for producing a protein arginine deiminase (PAD) or an enzyme preparation containing a protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses a protein derived from PAD. Bionectra ochroleuca PAD peptides.

[0044] Preferably, from Bionectra ochroleuca The PAD polypeptide comprises an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 3.

[0045] More preferably, from Bionectra ochroleucaThe polypeptide comprises an amino acid sequence that does not contain an internal pre-sequence and has at least 80% sequence identity with SEQ ID NO: 3.

[0046] As described above, the recombinant host strain used in the method of the present invention expresses: (i) selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group of PAD peptides that make up the group; or (ii) variants with pI below 9 Fusarium gramineae PAD polypeptide.

[0047] Surprisingly, as shown in the experimental section of this paper, it is possible to increase [the effectiveness of] PAD peptides by introducing mutations. Fusarium gramineae Expression levels of PAD peptides. Wild type Fusarium gramineae PAD peptides possess a relatively high protein pI. Introducing mutations that lower the total protein pI yields PAD protein with increased levels. PAD surface charge (pI) variants are compared with existing technologies under the same conditions. Fusarium gramineae PAD expression was at a much higher level than protein expression.

[0048] Preferably, variants with a pI below 9 Fusarium gramineae PAD peptides are variants with pI below 8, 7, or 6.5. Fusarium gramineae PAD polypeptide.

[0049] suitable Fusarium gramineae The PAD variant polypeptide has an amino acid sequence that, when compared with PAD of SEQ ID NO: 10, contains at least substitutions of amino acid residues corresponding to amino acid positions 29, 76, 92, 107, 153, 190, 219, 251, 365, 439, 467, and 560, as defined with reference to SEQ ID NO: 10. Preferably, the variant... Fusarium gramineae The PAD polypeptide has at least 80% sequence identity with SEQ ID NO: 10.

[0050] Suitable substitutions are N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251D, N365D, Q439E, Q467E, and N560D. By adding these mutations, SEQ ID NO: 11 is derived from SEQ ID NO: 10.

[0051] The experimental section describes three variants. Fusarium gramineaeThe PAD peptides (FgraPADmut_01, FgraPADmut_02, and FgraPADmut_03) all contain the mutations mentioned above and have a pI of 6.3. The variants differ in the signal sequences used.

[0052] As can be clearly seen from the embodiments herein, PAD peptides with relatively high pI can be expressed at higher levels by lowering their pI. Therefore, the present invention also provides a method for producing protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses a variant peptide with a pI lower than 9. Preferably, the variant PAD peptide with a pI lower than 9 is a variant PAD peptide with a pI lower than 8, 7, or 6.5.

[0053] In yet another embodiment, the recombinant host strain in any of the above methods contains a nucleic acid sequence encoding the following polypeptide: (i) selected from Trichoderma harzianum、Verticillium longisporum、Bionectra ochroleuca、Fusarium longipes、Fusarium solani Species complex 、Akanthomyces lecanii RCEF 1005、Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group of PAD peptides that make up the group; or (ii) variants with pI below 9 Fusarium gramineae PAD polypeptide.

[0054] The nucleic acid sequence is operatively linked to one or more control sequences that can guide the expression of the PAD polypeptide in host cells.

[0055] Preferably, such nucleic acid sequences encode any one of the amino acid sequences SEQ ID NO: 1 to 9 or 11, or amino acid sequences having at least 80% sequence identity with the corresponding SEQ ID NO: 1 to 9 or 11. The different sequences have been explained in more detail above.

[0056] Suitable nucleic acid sequences are shown in SEQ ID NO: 12 to 20 or 22, or nucleic acid sequences that have at least 80% identity with any one of SEQ ID NO: 12 to 20 or 22.

[0057] Preferably, the nucleic acid sequence further comprises a nucleic acid sequence encoding a signal sequence, wherein the signal sequence is preferably a signal sequence having an amino acid sequence SEQ ID NO: 23, 24 or 25.

[0058] The method of this invention uses a recombinant host strain. The host cell can be a prokaryotic host cell, an archaea host cell, or a eukaryotic host cell. The prokaryotic host cell can be a bacterial host cell. The eukaryotic host cell can be yeast, fungi, amoebas, algae, plant, or animal cells, such as mammalian or insect cells. The eukaryotic cell can be a fungal cell, such as a yeast cell, etc. White , Little Hansen , Kluyveromyces , Peach , Saccharomyces , Schizosaccharomyces or Yarrow Yeast cells can originate from [a specific genus of cells]. Kluyveromyces lactis , Saccharomyces cerevisiae , Hansenula polymorpha , Yarrowia lipolytica and Peach shepherd , Candida krusei Eukaryotic cells can be filamentous fungal cells.

[0059] Preferably, the host cell is a filamentous fungal host cell, preferably Aspergillus The host cells of filamentous fungi of the genus, more preferably Aspergillus niger .

[0060] Filamentous fungi can be host cells Eumycota and Oomycota The subfamily comprises any filamentous cell form (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK). Filamentous fungi are characterized by a hyphal wall composed of chitin, cellulose, dextran, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs via hyphal elongation, and carbon catabolism is obligate aerobic.

[0061] Filamentous fungi can be host cells Trichocomaceae Any filamentous form of cell belonging to the taxonomic unit (as defined by Houbraken and Samson in Studies in Mycology 70: 1–51. 2011). In another preferred embodiment, the filamentous fungal host cell can be classified as belonging to Trichocomaceae Classification units Trichocomaceae , Thermoascaceae Any filamentous form of cell belonging to any of the three families of Aspergillaceae. Suitable host cells for filamentous fungi can be, for example, those described in Houbraken and Samson, 2011 (ibid.). Figure 1 The second branch described: Aspergillus Those in it.

[0062] Suitable filamentous fungal host cells for use in this invention include, but are not limited to, those that are suitable for use in this invention. 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 .

[0063] Preferred filamentous fungal cells belong to Acremonium、Aspergillus、Chrysosporium、 Myceliophthora、Penicillium、Talaromyces、Rasamsonia、Thielavia、Fusarium or Trichoderma genus Species, and most preferably 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 The species. A more preferred host cell belongs to... Aspergillus Genus, more preferably, the host cell belongs to the species. Aspergillus niger When the host cell according to the invention is Aspergillus niger The host cell is preferably CBS 513.88, CBS 124.903 or a derivative thereof.

[0064] Several filamentous fungal strains are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Collection (DSM), the Central Bureau Voor Schimmelcultures (CBS), the Agricultural Research Service Patent Culture Collection, the Northern Regional Research Center (NRRL), and the All-Russian Collection of Microorganisms of the Russian Academy of Sciences (VKM, RCM) in Moscow, Russia. In the context of this invention, useful strains may include… Aspergillus niger CBS 513.88, CBS 124.903 Aspergillus oryzae ATCC 20423, IFO4177, 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 ATCC48272 Trichoderma reesei ATCC 26921 or ATCC 56765 or ATCC 26921 Aspergillus sojae ATCC11906 Myceliophthora thermophila C1, Garg 27K, VKM-F 3500 D, Chrysosporium from Lucknow C1, Garg 27K, VKM-F 3500 D, ATCC44006 and their derivatives.

[0065] Preferred filamentous fungal host cells (such as...) A. nigerThe host cell may contain one, more, or all of the following modifications: nonribosomal peptide synthase deficiency, preferably nonribosomal peptide synthase npsE deficiency (see WO2012 / 001169); pepA deficiency; glucosylamylase (glaA) deficiency; acid-stable α-amylase (amyA) deficiency; neutral α-amylase (amyBI and amyBII) deficiency; oxalate hydrolase (oahA) or oreE deficiency; one or more toxins (preferably ochratoxin and / or fumonisin) deficiency; prtT deficiency; hdfA deficiency; SEC 61 modification containing the S376W mutation, wherein serine 376 is replaced by tryptophan, and / or contains an adapted amplicon as defined in WO2005 / 123763 and / or WO2011 / 009700. These and other possible host modifications are also described in WO2012 / 001169, WO2011 / 009700, WO2007 / 062936, WO2006 / 040312, WO2004 / 070022, WO2013 / 135729, WO2014 / 013074, WO2014 / 013073, and WO2018 / 166943.

[0066] Preferably, the host cell is a filamentous fungal host cell, preferably Aspergillus The host cells of filamentous fungi of the genus, more preferably Aspergillus niger As described above, among the methods described and claimed herein, those selected are more preferred. Trichoderma harzianum and Bionectra ochroleuca The present invention provides a method for producing a protein arginine deiminase (PAD) or an enzyme preparation containing a protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses a protein derived from PAD. Trichoderma harzianum Or from Bionectra ochroleuca The PAD polypeptide, wherein the host cell is Aspergillus niger .

[0067] Preferably, selected from T richoderma harzianum and Bionectra ochroleuca The PAD polypeptide of the group comprises an amino acid sequence having at least 80% sequence identity with the corresponding SEQ ID NO: 1 or 3, and the host cell is Aspergillus niger .

[0068] More preferably, selected from Trichoderma harzianum and Bionectra ochroleuca The polypeptide of the group comprises an amino acid sequence that does not contain an internal pre-sequence and has at least 80% sequence identity with the corresponding SEQ ID NO: 1 or 3, and the host cell is Aspergillus niger .

[0069] More preferred (based on PAD protein expression levels) among the methods described and claimed herein is derived from... Bionectra ochroleuca The PAD polypeptide, and the host cell is Aspergillus niger Therefore, this document provides a method for producing protein arginine deiminase (PAD) or an enzyme preparation containing protein arginine deiminase (PAD), comprising: (a) culturing a recombinant host strain capable of secreting PAD, and (b) recovering PAD, wherein the recombinant host strain expresses protein arginine deiminase (PAD) from protein arginine deiminase (PAD). Bionectra ochroleuca The PAD polypeptide, and the host cell is Aspergillus black .

[0070] Preferably, from Bionectra ochroleuca The PAD polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3, and the host cell is Aspergillus niger .

[0071] More preferably, from Bionectra ochroleuca The polypeptide comprises an amino acid sequence that does not contain an internal pre-sequence and has at least 80% sequence identity with SEQ ID NO: 3, and the host cell is Aspergillus black .

[0072] Those skilled in the art know how to use one or more nucleic acid constructs or expression vectors of the present invention to transform cells.

[0073] Transformation of filamentous fungal host cells can be performed by any suitable known method, including, for example, electroporation, particle bombardment or microparticle bombardment, protoplast methods, and... Agrobacterium Mediated transformation ( Agrobacterium (Mediated transformation, AMT). JRS Fincham, Transformation in fungi. 1989, Microbiological reviews. 53, 148-170, describes the procedure for transformation.

[0074] Transformation can involve a process consisting of protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. AspergillusThe appropriate procedure for cell transformation is described in EP 238 023 and in Yelton et al., Proceedings of the National Academy of Sciences USA, 1984, 81:1470-1474. Use Agrobacterium swelling Used for Aspergillus The appropriate procedure for the transformation of host cells with other filamentous fungi is described, for example, in the work of De Groot et al. Agrobacterium swelling -mediated transformation of filamentous fungi. Nat Biotechnol. 1998, 16:839-842. Erratum in: Nat Biotechnol 1998 16:1074. A suitable transformation Fusarium The species approach is described by Malardier et al., Gene 78:147156, 1989, or in WO 96 / 00787. Other approaches may be applied, such as the use of biological projectile transformation, as described by Christiansen et al., Biolistic transformation of the obligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei. 1995, Curr Genet. 29:100-102.

[0075] Surprisingly, as shown in the experimental section of this paper, the resulting PAD levels are comparable to those of the wild-type in the prior art under the same conditions. Fusarium gramineae The expression of PAD is improved compared to that of PAD. Therefore, (in any production method described above) the produced PAD is as high as that produced under the same conditions. A. niger When expressing SEQ ID NO: 10 Fusarium gramineae The resulting level is higher than the level of expression. The yield can be at least 2% higher, for example, 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. Biodiversity PAD peptides or variants. Fusarium gramineae PAD peptides, regardless of whether they are used in accordance with existing technologies under the same conditions... Fusarium gramineaePAD expression levels can be determined, for example, by running an SDS-PAGE gel, staining the proteins on the gel, and comparing the stained protein strips.

[0076] The experimental section shows that when free arginine or rapeseed albumin (napin) is used as a substrate, the amino acids derived from arginine... Trichoderma harzianum The specificity of this PAD (TharPAD_03) differs from that of... Bionectra ochroleuca The specificity of the PAD (BochPAD_02). When compared with the prior art PAD of WO2008 / 000714, it comes from Bionectra ochroleuca The PAD (BochPAD_02) exhibits higher specific activity for rapeseed albumin. Regarding the preference for free arginine in rapeseed albumin, the PAD derived from… Trichoderma harzianum The PAD (TharPAD_03) is equivalent to the existing PAD in WO2008 / 000714.

[0077] Regarding the results Bionectra ochroleuca (BochPAD_02): produced in any of the above preparation methods Bionectra ochroleuca When under the same conditions A.niger When expressing SEQ ID NO: 20 Fusarium gramineae Compared to the specificity of other proteins, the specificity of rapeseed albumin increased.

[0078] Preferably, the method of the present invention is carried out on a large scale using at least 10 L of suitable culture medium, more preferably at least 100 L of suitable culture medium.

[0079] Any of the above methods may include additional steps, such as purification steps using techniques known to those skilled in the art. The purification step affects the purity of the final PAD product. The resulting PAD may be a pure or purified peptidyl arginine deiminase. The pure or purified peptidyl arginine deiminase may be an enzyme that is at least 50% pure, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 80%, at least 80%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% pure, for example, the purity determined by SDS-PAGE or any other analytical method known to those skilled in the art and suitable for this purpose.

[0080] In a second aspect, the present invention provides a PAD that can be obtained by any of the methods described above. The obtained PAD is a mature heterodimeric polypeptide containing a first subunit of approximately 50 kDa and a second subunit of approximately 10 kDa.

[0081] This invention provides a variety of mature PAD peptides, namely different isolated and / or recombinant PAD peptides. Preferably, the isolated and / or recombinant PAD peptides contain... (a) A first domain having at least 80% sequence identity with amino acids 1 to 477 of SEQ ID NO: 1, and wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 478 to 481 of SEQ ID NO: 1. A second structural domain having at least 80% sequence identity with amino acids 512 to 600 of SEQ ID NO: 1, wherein the second structural domain optionally comprises 1 to 2 amino acids from amino acids 510 to 511 of SEQ ID NO: 1, and The polypeptides described herein do not contain amino acids 482 to 509 of SEQ ID NO: 1 (referred to as TharPAD_01, TharPAD_03, and TharPAD_02 in the experimental section of this document). (b) A first domain having at least 80% sequence identity with amino acids 1 to 487 of SEQ ID NO: 2, and wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 488 to 491 of SEQ ID NO: 2. The second structural domain has at least 80% sequence identity with amino acids 524 to 612 of SEQ ID NO: 2, and The polypeptides described herein do not contain amino acids 492 to 523 of SEQ ID NO: 2 (referred to as VlonPAD_01 and VlonPAD_02 in the experimental section of this document). (c) A first domain having at least 80% sequence identity with amino acids 1 to 467 of SEQ ID NO: 3, and wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 468 to 471 of SEQ ID NO: 3. The second structural domain has at least 80% sequence identity with amino acids 498 to 587 of SEQ ID NO: 3, and The polypeptides described herein do not contain amino acids 472 to 497 of SEQ ID NO: 3 (referred to as BochPAD_01, BochPAD_02 and BochPAD_03 in the experimental section of this document). (d) A first domain having at least 80% sequence identity with amino acids 1 to 483 of SEQ ID NO: 4, and wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 484 to 487 of SEQ ID NO: 4. The second structural domain has at least 80% sequence identity with amino acids 528 to 615 of SEQ ID NO: 4, and The polypeptide does not contain amino acids 488 to 527 of SEQ ID NO: 4 (referred to as FlonPAD_01 in the experimental section of this paper). (e) A first domain having at least 80% sequence identity with amino acids 1 to 489 of SEQ ID NO: 5, wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 490 to 493 of SEQ ID NO: 5. The second structural domain has at least 80% sequence identity with amino acids 543 to 630 of SEQ ID NO: 5, and The polypeptides described herein do not contain amino acids 494 to 542 of SEQ ID NO: 5 (referred to as FsolPAD_01 and FsolPAD_02 in the experimental section of this document). (f) A first domain having at least 80% sequence identity with amino acids 1 to 489 of SEQ ID NO: 6, wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 490 to 493 of SEQ ID NO: 6. The second structural domain has at least 80% sequence identity with amino acids 531 to 618 of SEQ ID NO: 6, and The polypeptides described herein do not contain amino acids 494 to 530 of SEQ ID NO: 6 (referred to as AlecPAD_01 and AlecPAD_02 in the experimental section of this document). (g) A first domain having at least 80% sequence identity with amino acids 1 to 481 of SEQ ID NO: 7, wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 482 to 485 of SEQ ID NO: 7. The second structural domain has at least 80% sequence identity with amino acids 517 to 604 of SEQ ID NO: 7, and The polypeptide described herein does not contain amino acids 486 to 516 of SEQ ID NO: 7 (referred to as FincPAD_01 in the experimental section of this document). (h) A first domain having at least 80% sequence identity with amino acids 1 to 504 of SEQ ID NO: 8, and wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 505 to 508 of SEQ ID NO: 8. The second structural domain has at least 80% sequence identity with amino acids 535 to 626 of SEQ ID NO: 8, and The polypeptide described herein does not contain amino acids 509 to 534 of SEQ ID NO: 8 (referred to as OausPAD_01 and OausPAD_02 in the experimental section of this document), or (i) a first domain having at least 80% sequence identity with amino acids 1 to 489 of SEQ ID NO: 9, and wherein the first domain optionally comprises 1 to 4 amino acids from amino acids 490 to 493 of SEQ ID NO: 9. The second structural domain has at least 80% sequence identity with amino acids 525 to 613 of SEQ ID NO: 9, and The polypeptide described herein does not contain amino acids 494 to 524 of SEQ ID NO: 9 (referred to as VertPAD_01 in the experimental section of this document).

[0082] More preferably, isolated and / or recombinant PAD peptides, which contain A first structural domain having at least 80% sequence identity with amino acids 1 to 477 of SEQ ID NO: 1, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 478 to 481 of SEQ ID NO: 1. A second structural domain, having at least 80% sequence identity with amino acids 512 to 600 of SEQ ID NO: 1, and wherein the second structural domain optionally comprises 1 to 2 amino acids from amino acids 510 to 511 of SEQ ID NO: 1. Furthermore, the polypeptide described herein does not contain amino acids 482 to 509 of SEQ ID NO: 1 (referred to as Tharap_03 in the experimental section of this document). Alternatively, isolated and / or recombinant PAD peptides, which contain A first structural domain, having at least 80% sequence identity with amino acids 1 to 467 of SEQ ID NO: 3, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 468 to 471 of SEQ ID NO: 3. The second structural domain has at least 80% sequence identity with amino acids 498 to 587 of SEQ ID NO: 3, and The polypeptide described herein does not contain amino acids 472 to 497 of SEQ ID NO: 3 (referred to as BochPAD_02 in the experimental section of this document).

[0083] The most preferred are isolated and / or recombinant PAD peptides, which contain A first structural domain, having at least 80% sequence identity with amino acids 1 to 467 of SEQ ID NO: 3, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 468 to 471 of SEQ ID NO: 3. The second structural domain has at least 80% sequence identity with amino acids 498 to 587 of SEQ ID NO: 3, and The polypeptide described herein does not contain amino acids 472 to 497 of SEQ ID NO: 3 (referred to as BochPAD_02 in the experimental section of this document).

[0084] As described above, the first domain of the PAD polypeptide optionally contains 1 to 4 amino acids of SEQ ID NO: 1 to 9. The amount of optional amino acids depends on whether (and to what extent) terminal truncation occurs.

[0085] As shown in the experimental section, BochPAD_02 (derived from SEQ ID NO: 3 by removing the pre-sequence and truncating the ends) was truncated to a certain extent.

[0086] The experimental section of this paper shows that the major macrounit (N-terminal protein portion) of BochPAD_02 was identified as LSAT…SSVS (residue 001…471). Minor C-terminal truncated variants were detected as -VS (residue 001…469), -SVS (residue 001…468), and -SSVS (residue 001…467). No N-terminal truncation was detected.

[0087] Preferably, the present invention provides isolated and / or recombinant PAD peptides, which contain... The first structural domain has at least 80% sequence identity with amino acids 1 to 471 of SEQ ID NO: 3. The second structural domain has at least 80% sequence identity with amino acids 498 to 587 of SEQ ID NO: 3, and The polypeptide does not contain amino acids 472 to 497 of SEQ ID NO: 3.

[0088] This invention also provides variant PAD peptides, which are variants Fusarium gramineae PAD polypeptide, and wherein the variants are described Fusarium gramineae The PAD polypeptide has an amino acid sequence that, when compared with PAD of SEQ ID NO: 10, contains at least substitutions of amino acid residues corresponding to amino acid positions 29, 76, 92, 107, 153, 190, 219, 251, 365, 439, 467, and 560, as defined with reference to SEQ ID NO: 10, and wherein the variant has a pI of less than 9, and wherein the variant... Fusarium gramineae The PAD polypeptide has at least 80% sequence identity with SEQ ID NO: 10. Preferably, the variant... Fusarium gramineae The PAD polypeptide comprises substituted N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251D, N365D, Q439E, Q467E and N560D, wherein the substitution is defined with reference to SEQ ID NO: 10.

[0089] A polypeptide is also provided that encodes a biodiversity PAD polypeptide or variant as disclosed in the embodiments herein. Fusarium gramineae PAD polypeptide. In one embodiment, the polynucleotide is codon-optimized. Codon optimization is known to those skilled in the art and can be performed using any method known to them. Preferably, the method as presented in the embodiments herein is used. This method is described in detail in WO2008 / 000632. An expression vector is also provided comprising the polynucleotide disclosed herein operatively linked to at least one control sequence that directs the expression of the polypeptide in a host cell. Several methods known to those skilled in the art for inserting nucleic acids into nucleic acid constructs or expression vectors exist; see, for example, Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed.CSHL Press, Cold Spring Harbor, NY, 2001. Control sequences (e.g., promoter and terminator sequences) may be needed to manipulate the nucleic acid encoding the polypeptide of the present invention. A variety of promoters can be used, which can direct transcription in the host cells of this disclosure. Promoter sequences can be derived from highly expressed genes. Strong constitutive promoters are well known, and a suitable promoter can be selected based on the specific sequence to be controlled in the host cell. Examples of suitable promoters are listed in WO 2009 / 106575, including examples of suitable promoters in filamentous fungi. All promoters mentioned therein are readily available in the art. Any terminator known to those skilled in the art that is functional in the cells disclosed herein can be used. Examples of suitable terminator sequences in filamentous fungi include terminator sequences of filamentous fungal genes, such as those listed in WO 2009 / 106575.

[0090] A composition is also provided comprising a PAD polypeptide or variant of the biodiversity disclosed in the embodiments herein. Fusarium gramineae PAD peptides and at least one component selected from milk powder, gluten, granulated fat, additional enzymes, amino acids, salts, oxidizing agents, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartrate of monoglycerides and diacetyl tartrate of diglycerides, gums, flavoring agents, acids, starch, modified starch, humectants, polyols, and preservatives. The compositions disclosed herein may be solid (e.g., powders or granules) or fluid compositions. The compositions disclosed herein comprise at least one compound selected from the group consisting of: milk powder, gluten, granulated fat, additional enzymes, amino acids, salts (such as sodium chloride or potassium chloride), oxidizing agents, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartrate of monoglycerides and diacetyl tartrate of diglycerides, gums, flavoring agents, acids, starch, modified starch, humectants, polyols (e.g., glycerol), and preservatives. The term composition includes premixes. The compositions disclosed herein may contain one or more additional enzymes, such as amylases, such as α-amylases, such as fungal α-amylases, β-amylases; glucan transferases; peptidases, such as exopeptidases or endopeptidases; transglutaminases; protein glutaminases; cellulases; hemicellulases, especially pentosanases, such as xylanases; proteases; protein disulfide isomerases, such as those disclosed in WO 95 / 00636; glycosyltransferases; peroxidases; laccases; oxidases, such as hexose oxidases, glucose oxidases, aldose oxidases, pyranose oxidases; lipoxygenases; L-amino acid oxidases; glucosylamylases, phytases, and / or asparaginases.

[0091] In another aspect, the present invention provides a method for converting arginine residues into citrulline residues, comprising incubating a protein containing arginine residues with a PAD polypeptide as described herein or with a (enzyme) composition as described herein.

[0092] Proteins or substrate proteins containing arginine (the terms are used interchangeably herein) contain at least 3 mol% protein-bound arginine, more preferably at least 6 mol% protein-bound arginine. Examples of such substrate proteins are commercially available food proteins of animal origin, such as (skimmed) milk proteins, whey proteins, casein, or egg proteins. Another example of substrate proteins is plant proteins.

[0093] Preferably, the present invention provides a method for converting arginine residues into citrulline residues, comprising incubating a plant protein containing arginine residues with a PAD polypeptide as described herein or with a (enzyme) composition as described herein.

[0094] As used herein, the term "plant protein" refers to any protein derived from a plant source. Preferably, plant protein is protein derived from cereals, false cereals, legumes, nuts, seeds, or other sources such as coconut, potato, rapeseed, or tiger nut.

[0095] Examples of suitable grains are barley, fonio, corn, millet, oats, rye, sorghum, teff, rye, spelt, rice, or wheat.

[0096] Suitable examples of false grains are amaranth, buckwheat, or quinoa.

[0097] Suitable examples of legumes are lupins, peas, chickpeas, beans (preferably broad beans), peanuts, or soybeans.

[0098] Examples of suitable nuts are almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pistachios, or walnuts.

[0099] Examples of suitable seeds are chia seeds, flax seeds, pumpkin seeds, sesame seeds, or sunflower seeds.

[0100] Plant proteins can comprise protein blends prepared by mixing two or more types of plant proteins (e.g., by mixing almond protein and coconut protein or by mixing almond protein and cashew protein). That is, in one embodiment, the solution containing plant proteins comprises plant proteins from at least two different types of plants.

[0101] Another example of suitable substrate proteins is microbial proteins, such as yeast extracts or single-cell proteins used in meat substitutes.

[0102] The step of "incubating the protein containing arginine residues with the PAD peptide" can be performed at any suitable pH, for any suitable time, and at any suitable enzyme concentration. Those skilled in the art can very well determine the appropriate enzyme amount, or the appropriate incubation temperature, or the appropriate incubation pH, or the appropriate incubation time, for example, incubating the protein with peptidyl arginine deiminase at pH 4 to 9, such as pH 5 to 8.5, such as pH 5.5 to 8, such as pH 6 to 7, or pH 6.2 to 6.8, such as pH about 6.5. Suitable temperatures for incubating the protein with PAD can be 20°C to 60°C, such as 30°C to 50°C, or 35°C to 45°C.

[0103] The obtained protein containing citrulline residues can then be used to prepare food products. Food products can be of any type and include solid foods as well as beverages. Examples of such food products include plant-based beverages (i.e., plant-based dairy alternatives), plant-based fermented products (i.e., plant-based yogurt or plant-based cheese), infant, follow-on, or toddler beverages, or meat or fish alternatives.

[0104] Treating any of the proteins mentioned above with PAD can lead to various end effects, such as sensory benefits of protein-containing foods, reduced allergenicity, improved emulsification properties, altered sweetness, licorice flavor, astringency, powdery / chalky flavor, satiety, thickness, and / or digestibility and / or protease inhibitory activity.

[0105] In another aspect, the present invention provides a method for modifying at least one feature of a protein-containing food, the method comprising: The protein solution was incubated with the PAD peptide as described herein, and Optionally, the PAD-treated protein solution is processed into a protein-containing food.

[0106] This aspect of the invention is discussed in detail in WO2019 / 233920, which is incorporated herein by reference.

[0107] The preferred use of the PAD peptide disclosed herein is its use in canola protein (also known as rapeseed protein). This is discussed in detail, for example, in WO2019 / 234137, which is incorporated herein by reference.

[0108] Surprisingly, the inventors of this invention noted a correlation between the conversion of arginine to citrulline in the protein substrate rapeseed albumin (as confirmed by NMR analysis) and its effect on taste. Therefore, analysis performed by a (trained) taste panel can be replaced by NMR analysis. Thus, this invention provides a method for determining the sensory effects of PAD peptides on protein substrates, comprising... - Incubate the protein substrate with PAD peptides - NMR analysis of protein substrates treated with PAD peptides.

[0109] Equally surprising, the inventors of this invention have discovered that some of the PAD peptides described herein possess different specificities when compared to prior art PADs as described in WO2008 / 000714. More specifically, some of the PAD peptides disclosed herein exhibit different specificities for proteins compared to prior art PADs as described in WO2008 / 000714. Fusarium grasses PAD has higher activity compared to other compounds, and free arginine is preferred.

[0110] The following embodiments will illustrate the present invention in more detail, but these embodiments do not limit the scope of the present invention.

[0111] Example Standard genetic techniques (such as overexpression of enzymes in host cells, genetic modification of host cells, or hybridization techniques) are known methods in the art, for example, Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3) rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press Or as described in 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, for example, EP-A-0 635 574, WO 98 / 46772, WO 99 / 60102 and WO 00 / 37671, WO 90 / 14423, EP-A-0481008, EP-A-0635 574 and US 6,265,186.

[0112] Materials and methods strain WT 1: This Aspergillus niger The strain was used as a wild-type strain. It is deposited at the CBS Institute under accession number CBS 513.88.

[0113] GBA 306: The construction of GBA 306 using WT1 as the starting strain has been described in detail in WO2011 / 009700. This GBA 306 strain has the following genotype: Δ ice A, Δ pep A, Δ hdf A. Adaptability Bam HI amplicon (adapted) Bam HI amplicon), Δ amy BII, Δ amy BI and Δ amy A.

[0114] GBA 312: Using GBA306 as the starting strain and possessing (i) fum The construction of the additional missing GBA 312 for B is described in detail in WO2011 / 009700, with (ii) och A and och The construction of the additional missing GBA 312 of B is described in detail in WO2011 / 009700, with (iii) prt The construction of the additional missing T GBA 312 is described in detail in WO2011 / 009700, with (iv) nps The construction of the additional missing GBA 312 for E is described in detail in WO2012 / 001169, with (v) ags The construction of the additional missing GBA 312 for E is described in detail in WO2014 / 013074, with (vi) amy The construction of the additional missing C GBA 312 is described in detail in WO2014 / 013073, and has (vii) agd The construction of GBA 312 with additional deletion of A has been described in detail in WO2014 / 013073. This GBA312 strain has the following genotype: (Δ gla A, Δ pep A, Δ HDF A, Adaptive BamHI amplicon, Δ amy BII, Δ amy BI, Δ amy A, Δf um B, Δ och Δ prt T, Δ nps E, Δ ags E, Δ amy C, Δ agd A).

[0115] carrier The pGBTOP-16 vector described in WO15177171A1, WO16097270A1, and WO16 / 193292A1 was modified to allow Golden Gate cloning (New England Biolabs). Four vectors exist in pGBTOP-16. Bsa The I site was removed, and two new sites were introduced. Bsa The I site allows cloning, one at the 3' end of the PglaA (promoter) fragment and one at the 5' end of the 3' glaA (terminator) fragment. This results in the vector pGBTOP-18 ( Figure 1 ).

[0116] Assay of PAD activity of arginine Peptidylarginine deiminase (PAD) activity was determined using 500 mM L-arginine as a substrate. This method can be performed on a clinical analyzer or manually. The reaction was carried out at 37 °C in 200 mM MOPS buffer at pH 7.0. The sample was diluted with 100 mM phosphate buffer at pH 7.0 containing 0.1% Triton X-100 to achieve high activity. During a 10-minute incubation, PAD converts L-arginine to L-citrulline, releasing ammonia. In the presence of α-ketoglutarate and NADH, ammonia is immediately converted by glutamate dehydrogenase (GDH), releasing L-glutamate, NAD+, and water in the process. The oxidation of NADH to NAD+ was monitored spectrophotometrically at 340 nm. An ammonium sulfate reference was used to convert the decrease in A340 into the amount of ammonia produced.

[0117] One PAD activity unit is the amount of enzyme required to produce 1 micromole of ammonia from L-arginine per minute under test conditions.

[0118] Assay of protein-derived PAD activity The method for determining the PAD activity of proteins is similar to that described above, but 1-2% (w / w) of the target protein is used instead of 500 mM L-arginine.

[0119] The protein source used is: -Vertis™ CanolaPRO from dsm-firmenich ® It is a commercial rapeseed protein isolate. CanolaPRO ® The fraction rich in rapeseed albumin. CanolaPRO ®Fractions rich in cruciferin.

[0120] Protein assay (Bradford) Dilute the PAD enzyme with water to obtain a solution containing 0.1 to 1 mg of protein per milliliter. Add Bradford's reagent to the enzyme dilution and allow the colorimetric reaction to proceed for 15 minutes at room temperature. Measure the absorbance of the final reaction sample at 595 nm using a water blank spectrophotometer. Convert A595 to protein content using a calibration curve consisting of several concentrations of BSA.

[0121] For Canola PRO ® Sensory test CanolaPRO ® Dissolve in tap water (2%). Preheat the bottle to 45°C and add PAD enzyme (0-0.3 g protein / g CanolaPRO). ® Incubate at 45°C for 2 hours, then inactivate the enzyme (at 65°C for 5 minutes), and then cool on ice until used.

[0122] Prior to sensory testing, samples were pre-tasted and discussed by a panel to define and refine the list of properties to be used for analysis. Relevant properties of the samples were assessed in duplicate by an external panel (n=12-14) using descriptive analysis (QDA). During testing, samples were provided according to an optimal balance design, distributed over the following two weeks, and scored on an unstructured line scale of 0-100 in EyeQuestions. Principles of Good Sensory Practice were followed.

[0123] Mass spectrometry analysis PAD proteins were analyzed using a Synapt G2S time-of-flight mass spectrometer coupled to an Acquity UHPLC system (Waters). Approximately 25 ng of protein was injected onto a Waters ACQUITY BEH C4 column (5 cm long, 2.1 mm inner diameter, 300 Å pore size, 1.7 μm particle size). The column was operated at 75 °C. The eluent was LC-MS grade water (A) containing 0.1% formic acid and 90% acetonitrile / 10% LC-MS grade water (B) containing 0.1% formic acid (v / v). Proteins were eluted with a linear gradient of 3–25% B over 0.2 min, followed by a gradient of 25–55% B over 5.8 min, all at a flow rate of 400 μL / min. Large and small units were separated using gradients as described for both BochPAD_02 and TharPAD_03. The Synapt G2S was operated in full MS electrospray ionization mode, scanning from 500 to 5000 m / z in contour resolution mode at a scan time of 1 second. Leucine-enkephalin (m / z 556.2771 Da) was used as the m / z value obtained for locked-quality correction. Waters deconvolution software (MaxEnt1) was applied to the six strongest charged states of both large and small cells to calculate the average quality.

[0124] NMR spectral analysis A 20 ml sample from the CanolaPRO® 2% w / w sensory test was lyophilized and dissolved in 50 mM phosphate buffer (pH 7) until completely dissolved. The filtrate from a 50 kDa centrifugal filter was collected to enrich rapeseed albumin, not cruciferous proteins. Subsequently, the rapeseed albumin sample was buffer-exchanged with 25 mM citrate buffer (pH 3) through a 3 kDa filter to a final volume of 600 μL. A 1H-15N HSQC assay was performed to observe the Hε-Nε peak of the arginine side chain near 87 ppm on the nitrogen axis.

[0125] Tested PAD variant Table 1 provides an overview of the PAD variants tested and the experimental results obtained.

[0126] GPA528-8 is an uncodon-optimized genomic clone PAD of Fusarium graminearum, representing the PAD generated in WO08000714.

[0127] FgraPAD_01 is a codon-optimized PAD of Fusarium graminearum, and its cloning method is similar to that of the tested variant. Table 1: Overview of the tested PAD variants and the experimental data obtained *Use https: / / web.expasy.org / compute_pi / Example 1: Cloning and expression of arginine deiminase variants Used in Aspergillus niger The codon-adapted DNA sequences expressing various arginine deiminase proteins (for all “PAD variant identifier” proteins described in Table 1) were designed to include additional... Bsa Type III restriction endonuclease sites, enabling the ability to... Aspergillus Subcloning was performed on the expression vector pGBTOP-18. The translation initiation and termination sequences of the promoter were modified to the optimal sequences as detailed in WO2006 / 077258. Codon adaptation was performed as described in WO2008 / 000632.

[0128] Through repetition Bsa The digestion and ligation steps (GoldenGate cloning method (New England Biolabs, according to standard procedures) clone the DNA fragment into pGBTOP-18. The resulting vector contains an enzyme expression cassette controlled by a glucosylamylase promoter, resulting in vectors with names containing variant identifiers, such as pGBTOP-TharPAD_01, pGBTOP-BochPAD_01, etc. (see Table 1 for reference of each variant).

[0129] Subsequently, in the co-transformation protocol with pGBAAS-3, strains and methods described in WO2011 / 009700 and its references were used to transform the cells with all pGBTOP-based PAD vectors. A. niger GBA 306 was selected on acetamide-containing medium and on colonies purified according to standard procedures. Transformation and selection were performed as described in WO 98 / 46772 and WO 99 / 32617. Single, single-copy pGBTOP-PAD vector transformants expressing specific gene variants were selected by PCR as representative transformants and designated as the corresponding PAD variants, such as GBA306-TharPAD_01, GBA306-BochPAD_01, etc., and further replicated to obtain single-strain inoculum spore suspensions.

[0130] Subsequently, in the co-transformation protocol with pGBAAS-3, strains and methods described in WO2011 / 009700 and its references were used to transform pGBTOP-TharPAD_03 and pGBTOP-BochPAD_02. A. niger GBA 312 was selected on acetamide-containing medium and on colonies purified according to standard procedures. Transformation and selection were performed as described in WO 98 / 46772 and WO 99 / 32617. A single pGBTOP vector quadruple transformant expressing a specific gene variant was selected by PCR as a representative transformant, named GBA312-TharPAD_03 and GBA312-BochPAD_02, and further replicated in plates to obtain single-strain inoculum spore suspensions.

[0131] Example 2: Expression of protein arginine deiminase A. niger Fermentation of strains Preparation of fresh strains from parental strain GBA306 and GBA306-based variant PAD-producing strains A. Niger Spores were obtained, and sample material was generated by culturing the strain in 24-well plates containing 4 ml of fermentation medium (15% w / v maltose, 6% w / v bacto-soytone, 0.1% w / v NaH2PO4·H2O, 0.96% w / v NaNO3, 0.1% w / v MgSO4·7H2O, 8‰ w / v Tween-80, 2‰ w / v Basildon, 2% w / v MES, pH 6.2). After culturing for 5 days in a Microton incubator shaker (Infors AG, Bottmingen, Switzerland) at 30°C, 550 rpm, and 80% humidity, 1.5 mL of sample was collected. Mycelia were separated from the supernatant by centrifugation at 4000 g for 30 min, and the supernatant was stored at -20°C until further analysis.

[0132] Furthermore, by culturing the strains in 24-well plates containing 4 ml of fermentation medium as described above, using parental strains GBA306, GBA312, and strains GBA306-TharPAD_03, GBA312-TharPAD_03, GBA306-BochPAD_02, and GBA312-BochPAD_02... A. niger Spores are used to produce sample material. Both fermentation broth and supernatant samples are used for subsequent analysis.

[0133] Example 3: Activity test of variants The variants were grown in an 8-fold manner and analyzed for activity against arginine and protein content in MTP form as described in the “Materials and Methods” section above. Variants were selected based on a combination of the highest activity per unit protein (specific activity) and the acceptable activity per milliliter (yield): FgraPAD_01, FgraPADmut_01, FlonPAD_01, FsolPAD_01, VlonPAD_01, BochPAD_02, AlecPAD_02, TharPAD_01, FgraPADmut_02, BochPAD_01, BochPAD_03, TharPAD_03, and TharPAD_02.

[0134] Example 4: Further activity testing of the variant The selected variant from Example 3 was grown and tested on a larger scale (100 mL shake flasks). -Level of expression -Specific activity against free arginine (PADU / mg protein), and -For CanolaPRO ® Activity of protein substrates and fractions rich in rapeseed albumin and cruciferin.

[0135] Based on potential candidates for arginine activity: - Bionectria Variants, especially BochPAD_02, - Fusarium graminearum Engineering variant FgraPADmut_02, - Trichoderma harzianum (Hypocrea lixii) TharPAD_01, - Fusarium longipes Variant FlonPAD_01, - Verticillium longisporum VlonPAD_01, - Akanthomyces lecanii RCEF 1005 AlecPAD_02.

[0136] Based on the study of proteins (especially CanolaPRO) ® Possible candidates for the activity of protein fractions: - Bionectria ochroleuca BochPAD_02, - Trichoderma harzianum (Hypocrea lixii) TharPAD_01, - Fusarium graminearum FgraPADmut_02, FlonPAD_01 and AlecPAD_02, which are active for free arginine, showed significantly poor performance on protein substrates.

[0137] Further options include BochPAD_02, FgraPADmut_02, and TharPAD_03, as they are available in Canola PRO. ® Good performance and high epigenetic expression on protein substrates (especially based on additional experiments with media associated with large-scale production, not described in this paper), while maintaining genetic diversity during screening. During further fermentation at larger scales, FgraPADmut_02 performed worse than BochPAD_02 and TharPAD_03 and was not included in further work.

[0138] Example 5: Improving PAD yield through strain and bioprocess optimization To optimize the biological process, samples from GBA306 and strains GBA306-FlonPAD_01, GBA306-TharPAD_01, and GBA306-BochPAD_02 were used. A. niger Spores were cultured in 24-well plates containing 4 mL of fermentation medium as described above at three different temperatures (26°C, 30°C, and 34°C). The activity of the supernatant samples is shown in Table 2 below.

[0139] Table 2: Activity of PAD on arginine produced at different temperatures Surprisingly, the optimal fermentation temperature range is quite narrow, with the optimal temperature being 30°C.

[0140] Further bioprocess optimization was tested by conducting laboratory-scale production of the PAD enzyme as described in this paper. The performance and PAD enzyme yield of four-copy strains GBA306-BochPAD_02 and GBA312-BochPAD_02 were compared in two different fermentation processes.

[0141] In a 0.5 L shake flask without baffles, the contents of one vial of spores (FER1: GBA306-BochPAD_02 and FER2: GBA312-BochPAD_02) were added to a 50 mL pre-medium consisting of 20 g / L glucose, 20 g / L yeast extract, and 0.4 mL / L cleniol. The pre-cultures were incubated at 30 °C and 280 rpm for 24 hours. Subsequently, 20 g of the cell suspension was transferred to a second pre-medium (the same as above, except the medium volume was 300 mL in a 2 L baffled flask). The second pre-cultures were incubated at 30 °C and 220 rpm for 24 hours. The timing of the shake flask procedure can be adjusted according to the shake flask configuration and vial viability.

[0142] Fed-batch Cultures: The fed-batch culture medium consisted of a mixture of glucose and salts. The salt fractions were composed of 1.5 g / L citric acid (monohydrate (aq)), 0.8 g / L FeSO4 (7aq), 4 g / L MgSO4 (7aq), 0.2 g / L MnSO4 (4aq), ​​0.3 g / L ZnSO4 (7aq), 6.0 g / L KCl, 0.3 g / L CaCl2 (2aq), 4.5 g / L NaH2PO4 (1aq), and 6.0 mg / L CuSO4 (5aq) (FER1) or 0.6 mg / L (FER2). The glucose concentration in the medium was 90 g / kg. The medium was divided into two portions for steam sterilization (121°C for 20 minutes), one portion containing the sugar and calcium chloride. Before sterilization (with phosphoric acid), the pH of the salt fractions was adjusted to 3.0, and after collection in the bioreactor (with ammonia), it was adjusted to 5.2.

[0143] The feed medium also consists of a mixture of glucose and salt fractions. Salt is added at the same concentration as described in the medium description. The glucose concentration is 500 g / kg. The feed medium is divided into portions and steam-sterilized (121°C for 20 minutes). The pH of the feed medium is adjusted to 3.5 before sterilization (using NaOH) and not adjusted after sterilization. The temperature is maintained at 30°C during fermentation, and the pH is maintained at 5.2 (using ammonia). The bioreactor is inoculated at a 6% inoculum ratio. The working volume is 11 L.

[0144] Fermentation was carried out in a limited-oxygen (0% DOT during these fermentations, as described in US2009017515) fed-batch mode. The term "DOT" (Dissolved Oxygen Tension) is explained in paragraph

[0055] of Example 1 in US2009017515. Glucose feeding was initiated when the residual glucose concentration in the fermentation broth reached 40 g / L and was maintained between 20 and 40 g / L throughout the fermentation process by adjusting the feed rate. At the end of fermentation, the glucose feed rate was reduced so that the glucose concentration in the fermentation broth was below 5 g / L after 120 hours. Clerol was used as an antifoaming agent and added periodically. The activity of the fermentation broth samples is shown in Table 3 below.

[0145] Table 3: PAD activity of arginine substrates for FER1 and FER2 fermentation Clearly, optimization of the production strain background and / or bioprocess conditions during fermentation has a significant impact on the increased productivity and activity of the PAD enzyme produced according to the present invention.

[0146] Example 6: Fermentation with reduced copper levels It was noted that adding 1 mM EDTA at the end of the fermentation process helped stabilize PAD activity. Copper is considered a metal ion that may affect PAD activity.

[0147] Fermentation tests were conducted with different levels of copper under essentially the conditions described in Example 5. The copper concentration in 100% medium (added as CuSO4 (5 aq)) was 6 mg / L, and the copper concentration in 10% medium was 0.6 mg / L.

[0148] Table 4: Relative PAD activity in fermentation broth at end of fermentation (EoF). Fermentation at 100% CuSO4 concentration after 120 hours was used as a reference fermentation and set as 100% PAD activity (for arginine).

[0149] Surprisingly, it can be concluded that using a reduced Cu level of 50% to 10% for the GBA312-BochPAD_02 strain during fermentation resulted in increased PAD activity, which translates into higher productivity.

[0150] Example 7: Downstream processing and enzyme stability Typical recovery methods (particularly suitable for large-scale enzyme production) include (1) harvesting, (2) fermentation broth preparation, (3) solid-liquid separation, (4) concentration, and (5) formulation. Harvesting and fermentation broth preparation are steps used to retain the fermentation broth and prepare it for the solid-liquid separation step. Solid-liquid separation can be achieved, for example, by microfiltration, centrifugation, or membrane pressure. After the solid-liquid separation step, the cell-free liquid containing the enzyme of interest is transferred to the concentration step. Enzyme concentration is usually accomplished using ultrafiltration. This step produces a concentrate rich in the enzyme of interest. This concentrate, called cCUF, is the input material for the stabilization step. The stabilization step produces an enzyme formulation designed to protect the enzyme activity from degradation mechanisms, prevent microbial spoilage, and prevent physical changes in the product (e.g., turbidity).

[0151] As described in Example 6, adding 1 mM EDTA at the end of fermentation helps stabilize PAD activity. When fermentation is carried out using lower levels of Cu, EDTA is not required or can be added at reduced levels.

[0152] When compared to a 100% Cu formulation, using a 10% Cu formulation and the selected EoF sample from Example 6 resulted in a 1.5-fold increase in downstream process yield.

[0153] Surprisingly, using the 10% Cu formulation, no loss of PAD activity was observed with the selected EoF sample from Example 6 after refrigeration for 7 days at the fresh end of fermentation broth; this avoided the need for EDTA addition to prevent PAD activity loss. The same phenomenon was observed with cCUF using the 10% Cu formulation (obtained after downstream processing using solid-liquid separation and ultrafiltration steps), which had approximately 100% residual activity after refrigeration for 7 days, compared to <80% residual activity with cCUF using the 100% Cu formulation.

[0154] The conclusion is that using lower levels of Cu during fermentation leads to higher activity at the end of fermentation, improves the stability of PAD enzymes, and improves the overall (process) yield.

[0155] Example 8: Optimal pH of BochPAD_02 To determine the optimal pH for the BochPAD_02 enzyme, manual PAD activity assays, as described in the "Materials and Methods" section, were performed at different pH values. Substrate solutions were prepared according to the method description, but the final solution pH was set to pH 5, 6, 7, 8, and 9 using NaOH / HCl. The actual pH of the reaction mixture (substrate + enzyme) was also measured and used to plot relative activity data. The results are shown in... Figure 2 middle.

[0156] Example 9: Thermal deactivation of BochPAD_02 BochPAD_02 enzyme was diluted to approximately 1.5 PADU / g in activity assay dilution buffer and transferred to the wells of a PCR plate (100 μL / well). After sealing, the plate was subjected to a thermal gradient in a PCR instrument (40–56 °C). Residual activity of the samples was analyzed in a PAD activity assay after incubation in the PCR instrument for 5, 10, 22, 30, 40, 60, and 90 minutes. Results are shown in... Figure 3 middle.

[0157] Example 10: Dynamics of BochPAD_02 Substrate preparations at concentrations ranging from 500 to 1 mM were prepared in MOPS buffer (according to the PAD activity assay protocol). PAD activity was monitored at each substrate concentration using a manual PAD activity assay in the form of MTP. The initial slope of the reaction was plotted against the substrate concentration to obtain a Michaelis-Menten curve for PAD. The results are shown in... Figure 4 middle.

[0158] Example 11: Specific activity of TharPAD_03 and BochPAD_02 against rapeseed albumin and CanolaPRO® Specific activity against free Arg BochPAD_02 and TharPAD_03 (also described in Examples 5 and 6) were produced in laboratory-scale fermentation, and their activities against arginine, rapeseed albumin, and CanolaPRO®, as well as their protein content, were manually determined in MTP form as described herein. Significant differences were found between the two variants in terms of substrate affinity.

[0159] Table 5: Specific activities of TharPAD_03 and BochPAD_02 on different substrates BochPAD_02 exhibited a slightly higher specific activity on free arginine, but this difference was much more pronounced on protein substrates. For each mg of protein added, BochPAD_02 clearly performed better on rapeseed albumin (approximately 3 times) and CanolaPRO® (approximately 2 times).

[0160] Example 12: Canola PRO processed by BochPAD_02 ® NMR analysis of rapeseed albumin fractions and its relationship with sensing Official correlation NMR of PAD variants tested: original PAD molecule from Fusarium graminearum (WO2008 / 000714), BochPAD_02, FGraPADMut_02 and TharPAD_03.

[0161] Sensory tests of PAD variants: the original PAD molecule from Fusarium graminearum (WO2008 / 000714), BochPAD_02, and TharPAD_03.

[0162] NMR experiments on rapeseed albumin fractions of rapeseed protein, either directly or after treatment with four different concentrations of the BochPAD_02 enzyme, revealed the sequence of arginine conversion at different sites within the protein. All tested PAD variants (including BochPAD_02) converted arginine residues with the same preference, but not all arginine residues were converted. Based on amino acid composition analysis, the maximum arginine conversion rate was 30–40%.

[0163] After correlating the changes in amino acid composition with the sensory results of the same samples, it was concluded that the conversion of 15-20% of total arginine to citrulline had resulted in the major sensory effects (reduction in astringency, bitterness, licorice flavor, and sweetness). Arginine residue position 38 was the first to be converted by all PAD variants and was clearly responsible for the major sensory effects. Residue position 23 was converted at higher enzyme doses, and at the highest PAD doses, residue position 50 also began to be converted to citrulline. The sequence was numbered based on homology with the rapeseed albumin Bnlb sequence described in Rico, M., Bruix, M., González, C., Monsalve, RI, & Rodríguez, R. (1996). ¹H NMR Assignment and Global Fold of Napin BnIb, a Representative 2S Albumin Seed Protein.

[0164] Example 13: Precise C-terminal mass spectrometry analysis of BochPAD_02 and TharPAD_03 Complete protein LC-HRMS analysis of BochPAD_02 and TharPAD_03 PAD activation was initiated by cleavage following the amino acid ER (residue 497 in BochPAD_02) or KR (residue 509 in TharPAD_03). In the active state, both BochPAD_02 and TharPAD_03 exist as heterologous, non-covalently bonded dimers. Using non-native LC conditions, the dimer structure was no longer preserved for both BochPAD_02 and TharPAD_03; instead, they existed as two distinct monomers: a large unit (N-terminal protein) and a small unit (C-terminal protein). Based on the complete average mass information obtained after analyzing both units, the further processing of PAD after activation was determined.

[0165] For both BochPAD_02 and TharPAD_03, the small units contain only one occupied N-glycosylation site. This means that the intact average mass of the unit can be determined using intact protein LC-HRMS without further sample processing. However, the large units contain 3 (BochPAD_02) and 8 (TharPAD_03) N-glycosylation sites, many of which are occupied. Using intact protein LC-HRMS analysis of the samples themselves, the resulting MS spectra of the large units are too complex for deconvolution. Therefore, to determine the intact mass of the large units in BochPAD_02 and TharPAD_03, the samples were first deglycosylated using PNGase F.

[0166] PAD protein was analyzed using a Synapt G2S time-of-flight mass spectrometer coupled to an Acquity UHPLC system (Waters). Approximately 25 ng of protein was injected into a Waters ACQUITY BEH C4 column (5 cm long, 2.1 mm inner diameter, 300 Å pore size, 1.7 µm particle size). The column was operated at 75 °C. The eluent was LC-MS grade water (A) containing 0.1% formic acid and 90% acetonitrile / 10% LC-MS grade water (B) containing 0.1% formic acid (v / v). The protein was eluted with a linear gradient of 3–25% B over 0.2 min, followed by a gradient of 25–55% B over 5.8 min, all at a flow rate of 400 μL / min. Large and small units were separated using gradients as described for both BochPAD_02 and TharPAD_03. The Synapt G2S was operated in full MS electrospray ionization mode, scanned from 500 to 5000 m / z in profile resolution mode for 1 second. Leucine-enkephalin (m / z) was used. The m / z value (556.2771 Da) was used as the locked quality correction. The Waters deconvolution software (MaxEnt1) was applied to the six strongest charge states of the large and small cells to calculate the average quality.

[0167] Based on average quality, the C-terminus and N-terminus of the macro- and micro-units can be determined using the given amino acid sequences of BochPAD_02 (SEQ ID NO: 3) and TharPAD_03 (SEQ ID NO: 1).

[0168] 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 truncated variants were detected as -VS (residue 001…469), -SVS (residue 001…468), and -SSVS (residue 001…467), and no N-terminal truncation was detected.

[0169] The mature amino acid sequence of the small unit (C-terminal protein) is QAGS…WWKS (residues 498…587). There are N-terminal and C-terminal truncations, namely QAGS…VWWK (residues 498…586), AGST…WWKS (residues 499…587), and GSTI…WWKS (residues 500…587).

[0170] For the large unit (N-terminal protein) of TharPAD_03, the mature amino acid sequence was determined to be IQAT … QLHS (residues 001…481). C-terminal truncated variants were detected as -S (residues 001…480), -HS (residues 001…479), -LHS (residues 001…478), and -QLHS (residues 001…477). No N-terminal truncation was detected.

[0171] The mature amino acid sequence of the small unit (C-terminal protein) is QTTT…GPWW (residues 510…600). The N-terminal truncated sequences are TTTR…GPWW (residues 511…600), TTRY…GPWW (residues 512…600), TRYT…GPWW (residues 513…600), and RTYG…GPWW (residues 514…600).

Claims

1. A method for producing protein arginine deiminase (PAD), comprising: (a) A recombinant host strain capable of secreting PAD is cultured, and (b) PAD is recovered, wherein the recombinant host strain expresses: (i) selected from Trichoderma harzianum, Verticillium longisporum, Bionectra ochroleuca,Fusarium longipes,Fusarium solani Species complex Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The group of PAD peptides; or (ii) variants with pI below 9 Fusarium graminearum PAD polypeptide.

2. The method of claim 1, wherein the ingredients are selected from... Trichoderma harzianum, Verticillium longisporum,Bionectra ochroleuca,Fusarium longipes,Fusarium solani Species complex 、 Akanthomyces lecanii RCEF 1005, Fusarium incarnatum-equiseti Species complex and Ophiocordyceps australis The PAD polypeptide of the group comprises an amino acid sequence having at least 80% sequence identity with the corresponding SEQ ID NO: 1-9.

3. The method of claim 1, wherein the variant Fusarium graminearum The PAD polypeptide has an amino acid sequence that, when compared with PAD of SEQ ID NO: 10, contains at least substitutions of amino acid residues corresponding to amino acid positions 29, 76, 92, 107, 153, 190, 219, 251, 365, 439, 467, and 560, as defined with reference to SEQ ID NO:

10.

4. The method of claim 3, wherein the variant Fusarium graminearum The PAD polypeptide has at least 80% sequence identity with SEQ ID NO:

10.

5. The method according to any one of the preceding claims, wherein the recombinant host strain comprises a nucleic acid sequence encoding the following polypeptide: (i) selected from 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 The group of PAD peptides; or (ii) variants with pI below 9 Fusarium graminearum PAD polypeptide. The nucleic acid sequence is operatively linked to one or more control sequences that can guide the expression of the PAD polypeptide in host cells.

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

7. The method according to any one 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. The method according to any one of the preceding claims, wherein the generated PAD is produced under the same conditions as in... A.niger When expressing SEQ ID NO: 10 Fusarium graminearum The resulting level is expressed compared to a higher level.

9. A PAD that can be obtained by the method of any one of claims 1 to 8.

10. Isolated and / or recombinant PAD peptides, which contain (a) A first structural domain having at least 80% sequence identity with amino acids 1 to 477 of SEQ ID NO: 1, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 478 to 481 of SEQ ID NO:

1. A second structural domain, having at least 80% sequence identity with amino acids 512 to 600 of SEQ ID NO: 1, and wherein the second structural domain optionally comprises 1 to 2 amino acids from amino acids 510 to 511 of SEQ ID NO:

1. The polypeptide described therein does not contain amino acids 482 to 509 of SEQ ID NO:

1. (b) A first structural domain, having at least 80% sequence identity with amino acids 1 to 487 of SEQ ID NO: 2, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 488 to 491 of SEQ ID NO:

2. The second structural domain has at least 80% sequence identity with amino acids 524 to 612 of SEQ ID NO: 2, and The polypeptide described therein does not contain amino acids 492 to 523 of SEQ ID NO:

2. (c) A first structural domain, having at least 80% sequence identity with amino acids 1 to 467 of SEQ ID NO: 3, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 468 to 471 of SEQ ID NO:

3. The second structural domain has at least 80% sequence identity with amino acids 498 to 587 of SEQ ID NO: 3, and The polypeptide described therein does not contain amino acids 472 to 497 of SEQ ID NO:

3. (d) A first structural domain, having at least 80% sequence identity with amino acids 1 to 483 of SEQ ID NO: 4, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 484 to 487 of SEQ ID NO:

4. The second structural domain has at least 80% sequence identity with amino acids 528 to 615 of SEQ ID NO: 4, and The polypeptide described therein does not contain amino acids 488 to 527 of SEQ ID NO:

4. (e) A first structural domain having at least 80% sequence identity with amino acids 1 to 489 of SEQ ID NO: 5, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 490 to 493 of SEQ ID NO:

5. The second structural domain has at least 80% sequence identity with amino acids 543 to 630 of SEQ ID NO: 5, and The polypeptide described therein does not contain amino acids 494 to 542 of SEQ ID NO:

5. (f) A first structural domain having at least 80% sequence identity with amino acids 1 to 489 of SEQ ID NO: 6, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 490 to 493 of SEQ ID NO:

6. The second structural domain has at least 80% sequence identity with amino acids 531 to 618 of SEQ ID NO: 6, and The polypeptide described therein does not contain amino acids 494 to 530 of SEQ ID NO:

6. (g) A first structural domain having at least 80% sequence identity with amino acids 1 to 481 of SEQ ID NO: 7, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 482 to 485 of SEQ ID NO:

7. The second structural domain has at least 80% sequence identity with amino acids 517 to 604 of SEQ ID NO: 7, and The polypeptide described therein does not contain amino acids 486 to 516 of SEQ ID NO:

7. (h) A first structural domain, having at least 80% sequence identity with amino acids 1 to 504 of SEQ ID NO: 8, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 505 to 508 of SEQ ID NO:

8. The second structural domain has at least 80% sequence identity with amino acids 535 to 626 of SEQ ID NO: 8, and The polypeptide described herein does not contain amino acids 509 to 534 of SEQ ID NO: 8, or (i) A first structural domain having at least 80% sequence identity with amino acids 1 to 489 of SEQ ID NO: 9, and wherein the first structural domain optionally comprises 1 to 4 amino acids from amino acids 490 to 493 of SEQ ID NO:

9. The second structural domain has at least 80% sequence identity with amino acids 525 to 613 of SEQ ID NO: 9, and The polypeptide does not contain amino acids 494 to 524 of SEQ ID NO:

9.

11. Variant PAD peptide, which is a variant Fusarium graminearum PAD polypeptide, and wherein the variants are described Fusarium graminearum The PAD polypeptide has an amino acid sequence that, when compared with PAD of SEQ ID NO: 10, contains at least substitutions of amino acid residues corresponding to amino acid positions 29, 76, 92, 107, 153, 190, 219, 251, 365, 439, 467, and 560, as defined with reference to SEQ ID NO: 10, and wherein the variant has a pI of less than 9, and wherein the variant... Fusarium graminearum The PAD polypeptide has at least 80% sequence identity with SEQ ID NO:

10.

12. The variant PAD polypeptide of claim 11, wherein the substitution is N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251D, 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, particulate fat, additional enzymes, amino acids, salts, oxidizing agents, reducing agents, emulsifiers, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartrate of monoglycerides and diacetyl tartrate of diglycerides, gums, flavoring agents, acids, starch, modified starch, humectants, polyols, and preservatives.

14. A method for converting arginine residues into citrulline residues, comprising incubating a protein containing 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 feature of a protein-containing food, the method comprising: - Incubate the protein solution with the PAD polypeptide according to any one of claims 9-12, and - Optionally, the PAD-treated protein solution can be processed into a protein-containing food.

16. The method of claim 14 or 15, wherein the protein is canola protein.

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