WO2024032886A1

WO2024032886A1 - Glutaminase

Info

Publication number: WO2024032886A1
Application number: PCT/EP2022/072483
Authority: WO
Inventors: Tobias STADELMANN
Original assignee: Picea Biosolutions Gmbh
Priority date: 2022-08-10
Filing date: 2022-08-10
Publication date: 2024-02-15

Abstract

The present invention is directed to a novel protein glutaminase.

Description

Glutaminase

The present invention relates to a new protein glutaminase, a process for its preparation and its use for the deamidation of, in particular, vegetable proteins in order to improve their suitability as substitutes for animal proteins, in particular by increasing their solubility.

In recent years, the use of plant proteins in the food industry has become increasingly important. Vegetable proteins are significantly more resource-efficient in terms of their eco-balance: greenhouse gases, water consumption, water pollution and land use, and are therefore becoming increasingly popular with young consumers. They thus enable new sustainable nutrition concepts and make an important contribution to climate protection (reduction of greenhouse gases) and to CO2 savings. Market reports forecast the total volume of the vegetable protein market to be between 150 and 200 billion euros by 2030. A major challenge for the use and processing of plant proteins in food is that many plant proteins contain amide groups in their natural protein sequence, which negatively affect solubility, emulsification, foaming and gelling. Therefore, the food industry has developed physical and chemical methods for deamidation, which, however, involve negative side reactions, such as protein hydrolysis.

Enzymatic processes for deamidation have also been developed. However, the protein glutaminases available so far are very expensive. In addition, the glutaminase activity of the commercially available protein glutaminases is comparatively low. Another disadvantage of the protein glutaminases available so far is their low stability under usual storage conditions. The combination of these three factors means that the use of protein glutaminases for the deamidation of, for example, plant proteins in the food industry is very costly and, due to the low stability of the available protein glutaminases under usual storage conditions, also requires a high logistical effort. A further disadvantage of the currently commercially available protein glutaminases is that they require a chromatographic purification after their production in a cell culture in order to purify them. This required chromatographic purification step further increases the costs of these glutaminases.

A further disadvantage of the currently commercially available protein glutaminases is that they are usually not stable in liquid form. Accordingly, it is required to transport and store them in spray-dried form. Since the production of the proteins in liquid form and the subsequent spray-drying are usually carried out at different places, further logistic effort is necessary in order to handle these commercially available protein glutaminases. Several methods for the expression of recombinant protein glutaminases have already been described in the state of the art. Basically, a distinction must be made between expression in prokaryotes (such as E. coli) and expression in eukaryotes (such as Pichia Pastoris). Expression systems in prokaryotes, such as E. coli, are very easy to handle and allow the inexpensive production of large quantities of recombinant proteins. However, in these expression systems, the proteins are usually not produced in soluble form, but as inclusion bodies, which must first be renatured. Further, expression systems like E. Coli are not organisms suitable for the production of enzymes to be used in the food industry, since side products like lipopolysaccharides have to be separated by complicated chromatographic purifications. Expression systems in eukaryotes, such as Pichia Pastoris, are often more complicated to handle, but have the advantage that the proteins are present as soluble proteins in the supernatant.

Methods for producing recombinant glutaminase eukaryotic expression systems are described in the prior art, whereby these constructs are extracted as pre-pro- proteins. In this regard, the prior art describes that these pre-pro proteins are activated only after proteolytic cleavage with exogenous peptidases. This leads to the disadvantage that the exogenously added endopeptidases must first be removed chromatographically, as these peptidases would otherwise lead to degradation of the protein substrates during the deamidation reactions. Against this background, the object underlying the present invention was on the one hand on providing protein glutaminases that are superior to the available protein glutaminases in terms of their deamidation activity and in terms of their storage stability. In addition, the protein glutaminase should be as simple and inexpensive to produce as possible and, in particular, should be free of endopeptidases.

Furthermore, the object underlying the present invention was providing a method for the expression of protein glutaminase in a eukaryotic expression system, such as Pichia Pastoris, in which the protein glutaminase can be obtained from the expression supernatant in a simple manner without the need for complex purification steps, such as chromatographic purification.

The term "protein glutaminase" used in this specification means a protein which has activity for catalyzing hydrolysis of D-glutamine to D-glutamic acid and ammonia.

The term „solid formulation" is preferably a formulation, which contains less than 15% (w/w), preferably less than 10% (w/w), especially less than 8% (w/w) of water.

The object is solved according to the invention by a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, as well as homologues, fragments and parts thereof, for use as a protein glutaminase.

In this invention, using a previously established bioinformatic method in public databases, putative protein glutaminases were first expressed in E. coli. Subsequently, these recombinantly expressed protein glutaminases were tested for deamidation activity on natural substrates using a wheat gluten suspension. Active protein glutaminases were then recombinantly expressed in a food-grade expression system (Pichia Pastoris) and secreted into the culture supernatant of the medium and also tested for activity. The advantage of the Pichia Pastoris expression system is that there is no measurable endopeptidase activity in the culture supernatant and therefore expensive chromatographic methods do not have to be used during downstream processing. A particular focus is on the expression of the constructs as so-called pre-pro-peptides of the protein glutaminase, whereby it has only recently been described in the literature that these are only activated after proteolytic cleavage with exogenous peptidases. Surprisingly, the pro-glutaminase constructs expressed in Pichia pastoris are active and do not need to be activated by the addition of endopeptidases. Accordingly, no exogenously added endopeptidases has to be removed chromatographically, which reduces process costs.

The protein glutaminase according to the present invention expressed in Pichia pastoris has a strong glycosylation, which is quite typical for proteins expressed in Pichia pastoris. Surprisingly, despite the strong glycosylation, the protein glutaminase according to the present invention expressed in Pichia pastoris has a very high enzyme activity.

Accordingly, another advantage of the protein glutaminase (PG) from Chitinophaga sp. is that it has a faster solubilisation of natural proteins (deamidation) and also a higher degree of deamidation than already known protein glutaminases. Faster deamidation as well as a higher degree of deamidation are of great interest in the food industry.

For enzyme activity determination and protein determination a commercially available ammonium kit (Ammonium assay, Sigma-Aldrich, SKU 1147520001) as well as a commercially available BCA Kit (Thermofisher) were applied to determine the specific enzyme activity of the protein glutaminases. Wheat gluten was used as a substrate. The protein content was determined as described in the manual from Thermofisher. For the enzyme activity determination, a 5% (w/v) wheat gluten suspension in 100 mM MES buffer at pH 6.0 was incubated at 50°C for a given time. The Protein Glutaminase was inactivated by transferring 400 pl of the wheat gluten suspension to 80 pl of 2 M TCA in a 1.5 mL reaction tube. Subsequently, the sample was frozen at -20°C to avoid acid deamidation or stored on ice and analyzed the same day for its ammonium content. Furthermore, the PG from Chitinophaga sp. in a liquid formulation (PBS, pH 7.4) is still measurably active after 8 weeks of storage at 6-8 °C, whereas the PG from Bacillus helcegones has already completely lost its activity and the PG from Chryseobacterium proteolyticum has less than < 40 % residual activity.

In this way, the following protein, which has a high protein glutaminase activity, was provided:

SEQ ID NO 1 :

CKKSEQTPISTPADNDIVLLGNYIPFSYNVNGKEDATVGFLQSAQPFSVDPSKAANGAYVDLL KAGIDKSTPVEVYVYRNTRTIAKVKPASDEAMARYRQALVAPAKTEALPTIPSEAALTTLFNQ LKAAPIPFKFASDGCYARAHKMRQMILAAGYDADKLFVYGNLAASTGTCCVSWSYHVAPLVN VKTANGTVQQRILDPSLFTAPVAVSTWLNACRNTGCVSTANYTTTRQMPGAVYFIASTGNS PLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCPL

The object according to the invention is also solved by homologues, fragments and parts of the amino acid sequence comprising a protein according to SEQ ID NO: 1.

Sequence processing of the proteins of the invention (e.g. sequence variation) offers the possibility of developing further enzymes.

The present invention may also relate to other proteins that can be identified using the method disclosed above.

"Homologous sequences" or "homologues" means, for the purposes of the invention, that an amino acid sequence has an identity with one of the above amino acid sequences of the monomers of at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%. Preferred are 80% and 90%. Instead of the term "identity", the terms "homolog" or "homology" are used synonymously in the present description. The identity between two nucleic acid sequences or polypeptide sequences is determined by comparison using the program BESTFIT based on the algorithm of Smith, T. F. and Waterman, M. S (Adv. Appl. Math. 2: 482-489 (1981) ) with the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3. Preferably, the identity between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence/polypeptide sequence over the respective entire sequence length as calculated by comparison using the program GAP based on the algorithm of Needleman, S. B. and Wunsch, C. D. Mol. Biol. 48: 443-453) with the following parameters set for amino acids: Gap creation penalty: 8 and Gap extension penalty: 2; and the following parameters for nucleic acids Gap creation penalty: 50 and Gap extension penalty: 3.

Two amino acid sequences are identical within the meaning of the present invention if they have the same amino acid sequence.

In a further variant, the proteins according to the invention have sequences that differ from the indicated sequences by up to two or three amino acids.

Furthermore, sequences containing the above-mentioned sequences can also be used as proteins.

The protein according to the invention is further preferably characterised in that it consists of an amino acid sequence according to SEQ ID NO: 1 or homologues, fragments and parts thereof.

The protein according to the invention is further preferably characterised in that the protein is linked to a further substance, in particular to an affinity tag, preferably selected from the group consisting of poly histidine tags.

For the purposes of the invention, the linkage is a chemical bond as defined in Rompp Chemie Lexikon, 9th edition, volume 1, page 650 ff, Georg Thieme Verlag Stuttgart, preferably a principal valence bond, in particular a covalent bond. In one embodiment of the invention, the proteins of the invention are defined by their protein glutaminase activity.

In a preferred embodiment, the protein according to the invention is therefore designed to have a minimum level of 0.1 U*mg ¹ Protein Glutaminase activity such as > 0.1 U*mg ¹ - 10 U*mg ¹ Protein Glutaminase activity, such as > 10 U*mg ¹ Protein Glutaminase activity.

The present invention is also directed to the use of the protein described above as a protein glutaminase.

The present invention is also directed to a DNA which encodes a protein as described above.

In particular, the DNA according to the present invention comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11.

SEQ ID NO: 11

TGCAAGAAAAGCGAACAGACTCCGATCTCTACTCCGGCAGACAACGATATCGTTCTGCTG GGTAACTATATCCCGTTCAGCTACAACGTAAACGGTAAGGAGGATGCGACCGTTGGCTTC CTCCAGAGCGCTCAGCCGTTCTCTGTTGATCCGTCCAAAGCTGCGAACGGTGCTTATGTG GACCTGCTGAAAGCAGGTATCGATAAATCCACCCCGGTGGAAGTGTACGTGTACCGTAAC ACCCGCACTATCGCAAAGGTTAAACCGGCTAGCGACGAAGCTATGGCACGTTACCGCCAG GCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCGTCTGAAGCAGCGCTG ACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTTGCTTCTGATGGTT GCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGCTATGACGCAGATA AACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGCGTAAGCTGGTCTT ACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTTCAGCAGCGCATCC TGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTGAACGCGTGTCGTA ACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATGCCGGGTGCAGTAT ATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTACGCGCACACTAACTG TGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCGACCCTGAACTGCCC GCTGCTCGAG The present invention is also directed to recombinant vector containing a DNA as described above.

The present invention is further directed to a transformed microorganism in which is introduced the DNA as described above or a recombinant vector as described above.

The present invention is further directed to a transformed microorganism which is capable of producing glutaminase.

While there is in principle no limitation to the microorganism, it is preferred that the transformed microorganism is derived from a fungus, such as a yeast, such as Pichia pastoris or Saccharomyces cerevisiae.

The present invention is also directed to a method for producing glutaminase which comprises cultivating the microorganism in a culture medium to produce glutaminase in the culture.

The present invention is also directed a method for producing glutaminase which comprises cultivating the transformed microorganism described above in a culture medium to produce glutaminase in culture.

The present invention is further directed to a method for the preparation of a protein glutaminase, in particular as described above, that is characterized by a) Recombinant expression of a functional active microbial protein glutaminase as a Pre-Pro-Protein Glutaminase in a yeast b) secretion of the glycosylated microbial protein glutaminase in the culture broth c) absence of endopeptidase activity in the culture broth d) Concentration, intermediate storing or transport for at least 24 h up to 12 weeks of the Pichia pastoris culture broth prior to spray drying. The present invention is also directed to a protein, which is produced by such a method.

The present invention is further directed to the use of a protein as described above in a process for deaminating proteins, in particular vegetable proteins, in particular to improve their suitability as substitutes for milk proteins by increasing their solubility.

The Pre-Pro-Protein glutaminase is expressed in a yeast (or other fungal) host such as P. pastoris or S. cerevisiae. By either transforming the yeast cell with an integrative, episomal or centromere plasmid or an artificial chromosome by for example a Lithiumacetete-PEG-based transformation method. The Pre-Pro-Protein glutaminase is expressed from inducible promoters such as pAOXl or constitutive promoters such as pGAP and either secreted into the medium by adding a secretion tag (e.g. alpha mating signal or others) or expressed in the cytosol.

A basic production process for fermentation of Pichia pastoris is described in the Invitrogen Life Technologies "Pichia Fermentation Guidelines". Basic parameters for the fermentation of Pichia pastoris are:

Temperature: 30°C, minimum amount of dissolved oxygen > 20% and an optimal pH for growth of 5.0 - 6.0 or as an alternative to inhibit neutral proteases pH 3.0. The gassing rate should be in the range of 0.1 to 1.0 vvm. Compressed air, synthetic air, or pure oxygen can be used for gassing. Fermentation typically takes place in the Basal Salts medium. Care must also be taken to ensure that the methanol concentration ideally does not exceed a maximum concentration of 1-2% (v/v), as this is toxic to Pichia pastoris.

For fermentation in a bioreactor, a single colony and Pichia pastoris from the agar plate is used in baffled shake flasks for inoculation. The pre-culture is then grown in MGY or MNGY medium. The pre-culture fermentation is performed at 30°C, 250 - 300 rpm, 16 - 24 h to an OD₆oo nm = 2 - 6. The bioreactor is typically inoculated with 5 - 10% of the initial fermentation volume. The batch phase typically lasts for 18 - 24 h. Subsequently, a 50% - 100% (w/v) glycerol feeding is started to increase the cell density. Subsequently, the AOX promoter is replaced by methanol feeding with PTMi trace elements. A detailed overview of Recombinant Protein Production in Yeast may be found in the textbook from Roslyn M. Bill (Editor), Recombinant Protein Production in Yeast, Springer Protocols. Special. In particular, on the chapters dealing with molecular biology and bioreactor fermentation as well its optimization for recombinant protein production. Further fermentation protocols may be found on www.pichia.com.

The present invention is explained in more detail below with non-limiting examples:

Examples

1. Heterologous Protein Glutaminase Expression in E. coli

In order to produce the protein glutaminase according to Seq ID NO: 1, Seq ID NO: 2 and Seq ID NO: 3, respectively, Seq ID NO: 11, Seq ID NO: 12 and Seq ID NO: 13, respectively were ordered as gene fragments from Twist Bioscience, cloned into pET-28a(+) by Ncol and Xhol restriction sites. Plasmids were then transformed in Escherichia coli BL21(DE3) cells for protein expression. A single colony was picked into a 5 ml LB pre-culture supplemented with 25 pg*ml ¹ kanamycin and grown overnight at 37°C and 220 rpm until saturation. The next day, the cells were diluted 1: 100 in 500 ml LB medium supplemented with 25 pg*ml ¹ kanamycin and grown at 37°C and 220 rpm until an optical density OD₆oo of 0.4 was reached. Then, cells were induced with 0.5 mM IPTG, and grown at 30°C and 220 rpm overnight. Cells were harvested by centrifugation, lysed with lysing buffer (50 mM Tris-HCL pH 7.5, 200 mM NaCI, 1 mM DTT, 1 mM PMSF, and 0.3 mg/ml lysozyme) and cleared by ultracentrifugation. Cleared lysates were frozen at -80 °C.

2. Heterologous Protein Glutaminase expression in Pichia Pastoris

Seq ID NO: 11, Seq ID NO: 12 and Seq ID NO: 13 were amplified with Q5 Polymerase from New England Biolabs (Frankfurt, Germany) and cloned into the Pichia Pastoris expression vector pAOXlZeoR (see Figure 6 for vector map (Geneious)) containing flanking A0X1 promotor and terminator sites and a zeocin resistance marker by Bsal restriction sites. Plasmids were transformed into Pichia Pastoris ce\\s with the Pichia EasyComp Kit (Thermo Fisher Scientific, Waltham (MA), USA). Four colonies for each construct were picked and incubated in 1 ml BMGY in a 24 well plate for 24 h at 28-30°C in a shaking incubator (250- 300 rpm). The next day, cells were diluted to an OD₆oo of 1 in 1 ml BMMY medium in a 24 well plate and incubated at 28-30°C in a shaking incubator (250-300 rpm) for 72 hours with 0.5 % methanol (>99%) supplementation every 24 h supernatants were analyzed by SDS-PAGE gelelectrophoresis.

3. Shaking flask cultivation, Protein Glutaminase concentration, desalting, protein quantification and SDS-PAGE of recombinantly expressed Protein Glutaminases in Pichia Pastoris

The shaking flask production of the recombinantly produced Protein Glutaminase (PG) in P. pastoris was performed as follows. BMGY medium (10-50 mL) was transferred in a 100-250 mL baffled flask with a single transformed colony, cells from a glycerol stock, or a liquid culture from a recent BMGY cultivation (dilution 1:500). The incubation of the P. pastoris was performed at 28-30°C in a shaking incubator (250-300 rpm) until the culture reached an OD₆oo of 6.0-10.0. Subsequently, the Pichia Pastoris cells were harvested at 1,000-4,000 x g for 5-10 minutes at 4°C and after a washing step deploying BMMY medium transferred to BMMY (starting OD₆oo = 1.0) and further methanol (> 99%) was added to a final concentration of 1% (v/v) to the BMMY medium at least every 24 h. After 72 h - 96 h of shaking flask cultivation, the fermentation was terminated by centrifugation. The Protein Glutaminase containing culture supernatant was obtained after a centrifugation step (20,000 x g; 15 min; Sigma 1-16, Sigma Zentrifugen, Osterode am Harz, Germany). Subsequently, the culture supernatant was sterile filtered using a bottle top filter (Bottle-Top-Filter Nalgene™ Rapid- Flow™, PES-Membrane, sterile, Faust Laborbedarf, Schaffhausen, Schweiz). After the sterile filtration, the containing supernatant was concentrated ~25-fold using Pierce™ Protein Concentrator PES, 10K MWCO, 20-100 mL (Thermo Fisher, Waltham (MA), USA) and finally desalted to phosphate buffered saline (PBS, 137 mM NaCI, 2.7 mM KCI and 12 mM total phosphate, pH 7.4).

Protein determination using BCA

The protein content of the concentrated and desalted Protein glutaminases was determined using a commercially available protein determination kit from Thermo Fisher (Waltham (MA), USA; Pierce™ BCA Protein Assay Kit; Catalog number: 23225). The protein determination was performed according to the manufacturer instructor:

1. Pipette 25 pL of each standard or unknown sample replicate into a microplate well (working range = 20 - 2,000 pg*mL ^-1).

2. Add 200 pL of the BCA solution (mixture of 50 parts of solution A and 1 part of solution B) to each well and mix plate thoroughly on a plate shaker for 30 seconds.

3. Cover plate and incubate at 37°C for 30 minutes.

4. Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader.

Glycosylation, deglycosylation and SDS-PAGE

The concentrated and desalted recombinantly expressed protein glutaminases were deglycosylated applying PNGase F from New England Biolabs (Frankfurt, Germany). The deglycosylation protocol from NEB for denatured proteins was applied: For the deglycosylation, 5 pg of each glycosylated protein glutaminase were combined with 1 pl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 pl total reaction volume. Denaturation of the glycoprotein was performed by heating reaction at 100°C for 10 minutes followed by an incubation on ice for 5-10 min and a short centrifugation. Subsequently, 2 pl of 10X GlycoBuffer 2 (10X), 2 pl of 10% NP-40 and 6 pl of H2O were added. Subsequently, 1 pl of PNGase F (NEB, Frankfurt, Germany) was added and the hydrolysis was performed at 37°C for at least 1 h to max. 18 h. Subsequently, the deglycosylated protein glutaminases were analysed by an SDS-PAGE and the bands visualized by a Coomassie brilliant blue staining (GelCode™ Blue Safe Protein Stain, Thermo Fisher, Waltham (MA), USA)

As shown in Fig. 1, all recombinatly expressed Protein glutaminases are glycosylated (Lanes: 1, 3 and 5). Deglycosylation using PNGaseF leads to distinct bands on the SDS-PAGE in the range of the calculated molecular weight 30.1 kDa (Seq ID NO: 1), 33.1 kDa (Seq ID NO: 2), 33.4 kDa (Seq ID NO: 3) of the Protein Glutaminases.

4. Proteolytic activity of the Pichia Pastoris supernatant

Endopeptidases hydrolyze internal peptide bonds in the interior chain of proteins. The hydrolyses of proteins affects several functional properties of the food stuff itself such as: solubility, foaming or antioxidant activity of the released peptides. The removal of endopeptidases from food glutaminases is costly and should generally be avoided since the overall yield of the target protein (Protein Glutaminase) is reduced and additional costs are added. Therefore, Pichia Pastoris was chosen as a host which is does not contain any undesired side-activity such as endopeptidases. The endopeptidase activity in the Pichia Pastoris supernatant was evaluated using the well-established azocasein assay.

The azocasein assay was performed as described with minor modifications [1]. Azocasein (5 mg ml ^-1) was dissolved in HzOdd and subsequently mixed (1: 1) with 50 mM sodium acetate buffer pH 4.5 in order to evaluate whether aspartic endopeptidases are present in the P. pastoris culture supernatant or 50 mM Tris- HCI buffer pH 7.5 for the analysis of neutral and alkaline endopeptidases. Alcalase 2.41 (Sigma Aldrich, Schnelldorf, Germany) was applied as a positive control. The azocasein hydrolysis was initiated by adding 25 pl either of a ~25-fold concentrated Pichia Pastoris culture supernatant of a protein glutaminase or the respective 100- fold diluted Alcalase 2.41 (Sigma Aldrich, St. Louis (MO), USA) to pre-incubated (10 min, 37°C) 250 pl buffered azocasein solution (2.5 mg*ml ¹ final concentration at pH 4.5 or 7.5). The azocasein Pichia Pastoris supernatant mix was incubated at 37°C for 60 min. The reaction was terminated by TCA (2 M, 25 pl). Subsequently, the reaction mixture was centrifuged (10,000 x g, 5 min). The resulting supernatant (187 pl) was transferred to a microtiter plate and containing 62.5 pl NaOH (1 M). The absorbance was analyzed at 450 nm in a microplate spectrophotometer (FLUOstar Omega, BMG Labtech, Ortenberg, Germany).

The measured delta absorbances at 450 nm are at pH 4.5 and 7.5 are shown in Fig. 2. No endopeptidases activity was observed even after 60 min incubation of a ~25-fold concentrated Pichia Pastoris culture supernatant. However, both positive controls showed proteolytic activity at pH 4.5 as well as 7.5.

5. Visual protein solubilization test

The applicability of the novel Protein Glutaminase glutaminase need to be verified as early as possible in the development of food glutaminases. The deaminating acivity of potential Protein Glutaminases, expressed either in E. coli or in Pichia Pastoris was verified in a small scale wheat gluten solubilisation assay. For this purpose, wheat gluten protein was applied as a natural substrate to prove the applicability for potential food applications. A 5% (w/v) wheat gluten suspension in the pH range of 6-7 is generally insoluble [2]. Therefore, a 100 mM MES buffer pH 6.0 was applied for the initial screening of the deaminating activity of protein glutaminases. In brief, 975 pl of a 5% (w/v) gluten suspension in 100 mM MES buffer at pH 6 was incubated at 50°C in an Digital Shaking Drybath (Thermo Fisher Scientific, Dreieich, Germany) deploying the previously concentrated Pichia Pastoris culture supernatants for up to 24 h. In different time intervals, the 1.5 mL Eppendorf tubes containing a 5% (w/v) gluten suspension in 100 mM MES buffer (pH 6.0) were placed in a 1.5 mL reaction tube rack and settled for exactly 10 min. Subsequently, the glutaminase-containing samples were visually assessed against the blank value to determine whether solubilization of the wheat gluten occurred. As an example, the solubilization of wheat gluten with different protein glutaminases is shown in comparison to the blank in Fig. 3.

As depicted in Fig. 3. The novel Protein Glutaminase from SEQ ID NO: 1 was able to solubilize the 5% (w/v) gluten suspension in 100 mM MES buffer at pH 6 within 5 h of incubation time at dose of 25 pl and 12.5 pl Pichia Pastoris culture supernatant. After 24 h of incubation time, 3.13 pl solubilized the gluten suspension completely. The dose of 1.56 pl protein glutaminase supernatant had a positive visible effect on gluten solubilization due to the deamidation of the wheat gluten.

6. Ammonia liberation kinetic using wheat gluten as a natural substrate

The experimental proof of wheat gluten deamidation of the recombinantly produced protein glutaminases was performed using a commercially available ammonium kit (Ammonium assay, Sigma-Aldrich, SKU 1147520001). In previous experiments it was observed that the glutaminase activities for SEQ ID NO: 2 and SEQ ID NO: 3 are decreasing fast. Thus, it must be noted that the Protein Glutaminases have been stored for 6 weeks at a temperature of 4 - 6 °C prior to their application. The wheat gluten deamidation was performed as follows. The protein content of the concentrated Pichia Pastoris supernatants was determined using the previously described commercially available BCA Kit. A volume of 950 pl of a 5% (w/v) wheat gluten suspension in 100 mM MES buffer at pH 6.0 was incubated deploying two dosages of Protein glutaminase. A high dose of 0.1 mgp_iChia protein su_Pematant*ml ¹ or a low dose 0.01 mg pichia protein su_Pernatant*m l ¹ of the recombinantly produced protein glutaminases was incubated at 37°C for 24 h. Sampling was performed after 0 h, 1 h, 2 h, and 4 h. To inactivate the Protein Glutaminases, 80 pl of 2 M TCA was transferred in a 1.5 mL reaction tube and mixed with exactly 400 pl of the wheat gluten suspension and immediately frozen at -20°C to avoid acid deamidation. For sampling, the pipette tip of the was shortened by scissors, if necessary, to pipette the wheat gluten suspension. For determination of ammonium content, samples were thawed and following centrifugation at 20,000 x g for 10 min. The samples were diluted 250-fold to give a volume of 5 mL directly in a glass tube. The 5 mL volume was used to determine the ammonium content according to the manufacturer's instructions (Sigma Aldrich, SKU 1147520001). In brief, 5 mL of the dilution were added into a glass tube and mixed with 0.6 mL of Reagent NH4-1. Additionally, 1 level blue microspon (in the cap of the bottle) was added to the mixture and mixed vigorously until the reagent was completely dissolved. Afterwards, the mixture was incubated for 5 min at room temperature. Finally, 4 drops of Reagent NH4-3 were added and mixed. The results of the ammonium (NH₄ ⁺) liberation kinetic are shown in Fig. 4. As depicted in Fig. 4, the ammonia liberation of SEQ ID NO: 1 is at a dose of 0.01 mg*mL ¹ significantly higher compared to SEQ ID NO: 2 (200% increase) as well as to SEQ ID NO: 3 (48% increase). Similar results were obtained for a dose of 0.01 mg*mL ¹. For SEQ ID NO: 2, no ammonia generated could be detected. However, SEQ ID NO: 3 liberated 5.57 mg*L ¹ ammonia and SEQ ID NO: 1 liberated 13.56 mg*L ¹ ammonia. This corresponds to an increase 143% of ammonia liberation.

7. Storage stability test using the o-phthaldehyde assay (OPA)

The detection of ammonium in water samples has been established in the literature a long time ago [3]. Furthermore, the application of the OPA assay for the determination of the degree of hydrolysis is also well established [4]. Thus, the deamidation in proteinacous suspensions should be possible as well. However, in order to analyse the deamidation of natural protein substrates, some points must be considered:

1. the culture supernatants must be free of endopeptidase activity

2. the addition of TCA must not interfere with the determination of the ammonium content

3. An individual blank must be carried for each sample (glutaminase expression), since secreted glutaminase already deaminates the complex protein media components during cultivation.

The examination of the Pichia Pastoris culture supernatants for endopeptidase activity were negative (no endopeptidase activity, see azocasein example) for all three expressed PGs. In addition, an ammonium chloride calibration curve showed that the addition of 40 pl of 2 M TCA to the ammonium chloride standards only caused a dilution effect which was visible in the slope of the calibration curves, however, did not affect the detection of the NH₄ ⁺. Furthermore, for each glutaminase, the respective blank was carried along by adding 40 pl of 2 M TCA to the substrate prior to the addition of the respective Protein Glutaminase. The o-phthaldelhyde reagent was prepared as follows: 1.5 g L^-1 OPA, 3 g L^-1 DTT (dithiothreitol) and 11.25% (v/v) methanol were dissolved in 120 mM sodium tetraborate decahydrate buffer (adjusted to pH 9.6 with NaOH) for the storage stability of the 3 PGs. The previously concentrated (~25-fold) and to PBS desalted Protein Glutaminases were applied after a 3-month storage at 6- 8 °C for a wheat gluten deamidation test. For this purpose, 950 pl of a 5% (w/v) wheat gluten suspension in 100 mM MES buffer at pH 6.0 was incubated with 25 pl each of the recombinantly produced protein glutaminases for 24 h at 50°C. After 24 h incubation, 200 pl of the wheat gluten suspension was transferred to a 1.5 mL reaction tube containing 40 pl of 2 M Trichloroacetic acid (TCA) and centrifuged at 12,400 x g for 10 min. Following, 25 pl of the ammonia containing supernatant was transferred to a 96-well microtiter plate (Microtitration plates ROTILABO® U-profil, Carl Roth, Karlsruhe, Germany) and mixed with 175 pl of the OPA reagent. The derivatisation was performed for exactly 15 min at room temperature. The absorption was analyzed at 340 nm in a microplate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). The results of the storage stability test are shown in Fig.5. As shown in Fig. 5, the recombinantly expressed Protein Glutaminase from SEQ ID NO: 11 liberated significantly more ammonia after 3 months of storage in PBS at 6-8°C compared to the presently described Protein Glutaminases from SEQ ID NO: 12 and SEQ ID NO: 13. Surprisingly, the PG from SEQ ID NO: 11 deamidated the gluten suspension about 300% more than the PG from SEQ ID NO: 12 and about 900% more than the PG from SEQ ID NO: 13. These results indicate that the PG from SEQ ID NO: 11 is significantly more stable at 6-8°C in a liquid formulation compared to the PGs from SEQ ID NO: 12 and SEQ ID NO: 13.

8. Application of truncated Pro-Peptide-Protein Glutaminase variants

The wheat gluten deamidation of the recombinantly produced truncated Pro- Peptide-Protein glutaminases was performed using a commercially available ammonium kit (Ammonium assay, Sigma-Aldrich, SKU 1147520001). The wheat gluten deamidation was performed as follows. The protein content of the concentrated Pichia Pastoris supernatants was determined using the previously described commercially available BCA Kit. A volume of 950 pl of a 5% (w/v) wheat gluten suspension in 100 mM MES buffer at pH 6.0 was incubated using the truncated Pro-Peptide-Protein glutaminases. A dose of 0.1 mgp_iChia protein supernatant*ml ¹ was incubated at 50°C for 24 h. The inactivation of the Protein Glutaminases was performed by the addition of 80 pl of 2 M TCA to 400 pl of the wheat gluten suspension. Afterwards, the sample was immediately frozen at -20°C to avoid acid deamidation. For sampling, the pipette tip of the was shortened by scissors, if necessary, to pipette the wheat gluten suspension. For determination of ammonium content, samples were thawed and following centrifugation at 20,000 x g for 10 min. The samples were diluted 50-fold to give a volume of 5 mL directly in a glass tube. The determination of the liberated ammonium was performed as described in example 6 using a commercially available kit (Ammonium assay, Sigma-Aldrich, SKU 1147520001). Surprisingly, it was found that all 7 truncated variants were recombinantly expressed and showed enzymatic activity, which was shown by the deamidation of a 5% (w/v) wheat gluten suspension (table 1). However, as indicated in the literature, a truncation of SEQ ID NO: 1 did not lead to an increase in ammonium liberation. Surprisingly it was found, that the truncation of the Pro-peptide results in a lower protein expression yield and thus in a reduced ammonia liberation. It 's important to emphasize the role of the Pro- Peptide of the protein glutaminases in the recombinant expression and secretion of Protein Glutaminases in Pichia pastoris. Unlike already described in the literature, Protein Glutaminases, which are recombinantly expressed in Pichia pastoris are surprisingly active without the need of exogenous peptidases as described [5]. Moreover, the expression of the mature protein glutaminase is not favourable in terms of expression yield.

SEQ ID NO: (-) Liberated Ammonium (mg*L ¹)

1 >150

4 77.4

5 57.8

6 40.5

7 32.9

8 30.7

9 46.8

The DNA sequences corresponding SEQ IDs NO: 4 to 10 are SEQ IDs NO: 14 to 20.

Literature:

[1] 1. Eisele T, Stressler T, Kranz B, Fischer L (2013) Bioactive peptides generated in an enzyme membrane reactor using Bacillus lentus alkaline peptidase. Eur Food Res Technol 236:483-490. https://doi.org/10.1007/s00217-012-1894-5

2. Deng L, Wang Z, Yang S, et al (2016) Improvement of Functional Properties of Wheat Gluten Using Acid Protease from Aspergillus usamii. PLoS One l l :e0160101. https://doi.org/10.1371/journal.pone.0160101

3. Meseguer-Lloret S, Molins-Legua C, Campins-Falco P (2002) Ammonium determination in water samples by using OPA-NAC reagent: A comparative study with nessler and ammonium selective electrode methods. Int J Environ Anal Chem 82:475-489. https://doi.org/10.1080/0306731021000018107

4. Merz M, Ewert J, Baur C, et al (2015) Wheat gluten hydrolysis using isolated Flavourzyme peptidases: Product inhibition and determination of synergistic effects using response surface methodology. J Mol Catal B Enzym 122:218- 226. https://doi.Org/10.1016/j.molcatb.2015.09.010

5. Ouyang X, Liu Y, Qu R, et al (2021) Optimizing Protein-Glutaminase Expression in Bacillus subtilis. Curr Microbiol 78: 1752-1762. https://doi.org/10.1007/s00284-021-02404-0

Sequences:

Amino acid sequences:

SEQ ID NO 1 :

CKKSEQTPISTPADNDIVLLGNYIPFSYNVNGKEDATVGFLQSAQPFSVDPSKAANGAYVDLL

KAGIDKSTPVEVYVYRNTRTIAKVKPASDEAMARYRQALVAPAKTEALPTIPSEAALTTLFNQ

LKAAPIPFKFASDGCYARAHKMRQMILAAGYDADKLFVYGNLAASTGTCCVSWSYHVAPLVN

VKTANGTVQQRILDPSLFTAPVAVSTWLNACRNTGCVSTANYTTTRQMPGAVYFIASTGNS

PLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCPL SEQ ID NO 2:

DSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYI

GMLRQAVKNESPVHIFLKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATL NSLFNQIKNQSCGTSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKAST GTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYA NTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGFLE

SEQ ID NO 3:

CTHDDNNEALSFTPPVVELKVPTGIYSNGDQLRISVMLSHQFYTIESKAENKGFIELIKKAINY

ESLLAFYIKEGTKEIQLVREASAEDTSRFKAVFSLVKGETAFSKLEPIIPNPQTLNGLFAKIQNA

SCSFPVKKPCISFDYPVDGCYARAHKMRQLINENGYECKKEFVYGDLRARYGVKMSNQDGC CVSWSYHVAVLLTYKDEKGVLQECIIDPSLFDTPISDSDWRKACANSSCGPVSISSFTTTPGN VYYRSPKGTLLYDDGYVNTDCVLDIFADYSGCYLPAPSTVSCGFLE

SEQ ID NO 4:

MLGNYIPFSYNVNGKEDATVGFLQSAQPFSVDPSKAANGAYVDLLKAGIDKSTPVEVYVYRN

TRTIAKVKPASDEAMARYRQALVAPAKTEALPTIPSEAALTTLFNQLKAAPIPFKFASDGCYAR

AHKMRQMILAAGYDADKLFVYGNLAASTGTCCVSWSYHVAPLVNVKTANGTVQQRILDPSL FTAPVAVSTWLNACRNTGCVSTANYTTTRQMPGAVYFIASTGNSPLYDNSYAHTNCVIAGY TGLVGCGIPPTLNCPLLE

SEQ ID NO 5:

GFLQSAQPFSVDPSKAANGAYVDLLKAGIDKSTPVEVYVYRNTRTIAKVKPASDEAMARYRQ

ALVAPAKTEALPTIPSEAALTTLFNQLKAAPIPFKFASDGCYARAHKMRQMILAAGYDADKLF VYGNLAASTGTCCVSWSYHVAPLVNVKTANGTVQQRILDPSLFTAPVAVSTWLNACRNTGC VSTANYTTTRQMPGAVYFIASTGNSPLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCPLLE

SEQ ID NO 6:

AYVDLLKAGIDKSTPVEVYVYRNTRTIAKVKPASDEAMARYRQALVAPAKTEALPTIPSEAAL

TTLFNQLKAAPIPFKFASDGCYARAHKMRQMILAAGYDADKLFVYGNLAASTGTCCVSWSY HVAPLVNVKTANGTVQQRILDPSLFTAPVAVSTWLNACRNTGCVSTANYTTTRQMPGAVYF IASTGNSPLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCPLLE

SEQ ID NO 7:

VYRNTRTIAKVKPASDEAMARYRQALVAPAKTEALPTIPSEAALTTLFNQLKAAPIPFKFASD

GCYARAHKMRQMILAAGYDADKLFVYGNLAASTGTCCVSWSYHVAPLVNVKTANGTVQQRI LDPSLFTAPVAVSTWLNACRNTGCVSTANYTTTRQMPGAVYFIASTGNSPLYDNSYAHTNC VIAGYTGLVGCGIPPTLNCPLLE

SEQ ID NO 8:

ARYRQALVAPAKTEALPTIPSEAALTTLFNQLKAAPIPFKFASDGCYARAHKMRQMILAAGYD

ADKLFVYGNLAASTGTCCVSWSYHVAPLVNVKTANGTVQQRILDPSLFTAPVAVSTWLNAC RNTGCVSTANYTTTRQMPGAVYFIASTGNSPLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCP LLE

SEQ ID NO 9:

MPTIPSEAALTTLFNQLKAAPIPFKFASDGCYARAHKMRQMILAAGYDADKLFVYGNLAAST GTCCVSWSYHVAPLVNVKTANGTVQQRILDPSLFTAPVAVSTWLNACRNTGCVSTANYTTT RQMPGAVYFIASTGNSPLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCPLLE

SEQ ID NO 10:

MFNQLKAAPIPFKFASDGCYARAHKMRQMILAAGYDADKLFVYGNLAASTGTCCVSWSYHV

APLVNVKTANGTVQQRILDPSLFTAPVAVSTWLNACRNTGCVSTANYTTTRQMPGAVYFIAS TGNSPLYDNSYAHTNCVIAGYTGLVGCGIPPTLNCPLLE

DNA sequences:

SEQ ID NO 11 :

TGCAAGAAAAGCGAACAGACTCCGATCTCTACTCCGGCAGACAACGATATCGTTCTGCTG

GGTAACTATATCCCGTTCAGCTACAACGTAAACGGTAAGGAGGATGCGACCGTTGGCTTC

CTCCAGAGCGCTCAGCCGTTCTCTGTTGATCCGTCCAAAGCTGCGAACGGTGCTTATGTG

GACCTGCTGAAAGCAGGTATCGATAAATCCACCCCGGTGGAAGTGTACGTGTACCGTAAC

ACCCGCACTATCGCAAAGGTTAAACCGGCTAGCGACGAAGCTATGGCACGTTACCGCCAG GCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCGTCTGAAGCAGCGCTG

ACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTTGCTTCTGATGGTT

GCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGCTATGACGCAGATA

AACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGCGTAAGCTGGTCTT

ACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTTCAGCAGCGCATCC

TGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTGAACGCGTGTCGTA

ACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATGCCGGGTGCAGTAT

ATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTACGCGCACACTAACTG

TGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCGACCCTGAACTGCCC GCTGCTCGAG

SEQ ID NO 12:

GATTCTAACGGCAACCAGGAAATTAACGGTAAAGAGAAGCTGAGCGTTAACGATAGCAAA

CTGAAGGACTTCGGTAAGACTGTACCGGTAGGCATTGACGAAGAGAACGGTATGATCAAA

GTGAGCTTTATGCTGACCGCACAGTTCTATGAAATCAAACCGACCAAGGAGAACGAGCAG

TATATCGGCATGCTGCGTCAGGCGGTTAAGAACGAATCTCCGGTGCACATCTTTCTGAAG

CCGAACTCCAACGAAATTGGTAAAGTGGAATCCGCGTCTCCGGAAGATGTGCGCTACTTC

AAGACCATCCTGACCAAAGAAGTAAAGGGTCAGACTAACAAACTGGCTAGCGTGATCCCG

GACGTTGCGACCCTGAACTCTCTGTTCAACCAGATCAAGAACCAGTCTTGCGGTACTTCC

ACCGCTTCCTCTCCGTGTATTACTTTCCGTTACCCGGTGGATGGTTGCTATGCGCGCGCA

CACAAGATGCGCCAGATTCTGATGAACAACGGCTACGATTGTGAGAAACAGTTCGTATAC

GGCAACCTGAAAGCATCTACCGGTACCTGCTGTGTGGCTTGGTCTTACCACGTAGCAATC

CTGGTTTCCTACAAGAACGCAAGCGGTGTAACCGAGAAACGTATCATTGACCCGTCTCTG

TTCTCTTCCGGTCCGGTGACCGACACTGCATGGCGTAACGCATGCGTAAACACCAGCTGC

GGCTCCGCGTCCGTTAGCTCTTACGCTAACACCGCGGGTAACGTATATTACCGCTCCCCG

TCTAACTCTTACCTGTACGATAACAACCTGATCAACACCAACTGTGTTCTGACCAAATTCT

CCCTGCTGTCCGGTTGCAGCCCGTCTCCGGCTCCGGACGTGTCTTCCTGCGGCTTTCTCG AG

SEQ ID NO 13:

TGTACCCACGACGATAACAACGAAGCTCTGTCTTTCACCCCGCCGGTTGTGGAACTGAAA

GTTCCGACTGGCATTTACTCTAACGGTGACCAGCTGCGTATCTCCGTGATGCTGTCCCAC

CAGTTCTATACCATCGAAAGCAAAGCAGAGAACAAAGGCTTCATCGAGCTGATTAAGAAA GCGATTAACTATGAATCCCTGCTGGCTTTCTACATTAAAGAAGGTACCAAGGAAATTCAGC

TGGTGCGTGAGGCTTCCGCTGAGGACACTAGCCGCTTCAAAGCGGTATTCAGCCTGGTGA

AAGGCGAAACCGCGTTCTCCAAGCTGGAACCGATTATCCCGAACCCGCAGACCCTGAACG

GCCTGTTCGCGAAGATTCAGAACGCGTCTTGCTCCTTCCCGGTTAAGAAACCGTGTATCT

CTTTCGATTATCCGGTTGATGGTTGCTACGCTCGTGCTCACAAGATGCGCCAGCTGATTA

ACGAGAACGGTTACGAATGTAAGAAGGAATTCGTATATGGTGATCTGCGCGCTCGTTACG

GTGTTAAGATGTCCAACCAGGACGGCTGTTGCGTTTCTTGGTCCTATCATGTTGCAGTGC

TGCTGACTTACAAAGATGAGAAAGGCGTTCTGCAGGAATGCATTATCGACCCGAGCCTGT

TTGACACCCCGATTAGCGACTCTGACTGGCGTAAGGCTTGCGCGAACAGCTCTTGCGGTC

CGGTTTCTATCTCTTCCTTTACCACTACCCCGGGCAACGTGTACTATCGTAGCCCGAAAGG

CACCCTGCTGTACGACGATGGTTACGTTAACACCGATTGCGTGCTGGACATCTTTGCTGA

TTACTCTGGCTGCTACCTGCCGGCACCGTCCACTGTAAGCTGCGGCTTTCTCGAG

SEQ ID NO 14:

CTGCTGGGTAACTATATCCCGTTCAGCTACAACGTAAACGGTAAGGAGGATGCGACCGTT

GGCTTCCTCCAGAGCGCTCAGCCGTTCTCTGTTGATCCGTCCAAAGCTGCGAACGGTGCT

TATGTGGACCTGCTGAAAGCAGGTATCGATAAATCCACCCCGGTGGAAGTGTACGTGTAC

CGTAACACCCGCACTATCGCAAAGGTTAAACCGGCTAGCGACGAAGCTATGGCACGTTAC

CGCCAGGCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCGTCTGAAGCA

GCGCTGACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTTGCTTCT

GATGGTTGCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGCTATGAC

GCAGATAAACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGCGTAAGC

TGGTCTTACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTTCAGCAG

CGCATCCTGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTGAACGCG

TGTCGTAACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATGCCGGGT

GCAGTATATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTACGCGCAC

ACTAACTGTGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCGACCCTG

AACTGCCCGCTGCTCGAG

SEQ ID NO 15:

GGCTTCCTCCAGAGCGCTCAGCCGTTCTCTGTTGATCCGTCCAAAGCTGCGAACGGTGCT

TATGTGGACCTGCTGAAAGCAGGTATCGATAAATCCACCCCGGTGGAAGTGTACGTGTAC

CGTAACACCCGCACTATCGCAAAGGTTAAACCGGCTAGCGACGAAGCTATGGCACGTTAC CGCCAGGCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCGTCTGAAGCA

GCGCTGACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTTGCTTCT

GATGGTTGCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGCTATGAC

GCAGATAAACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGCGTAAGC

TGGTCTTACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTTCAGCAG

CGCATCCTGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTGAACGCG

TGTCGTAACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATGCCGGGT

GCAGTATATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTACGCGCAC

ACTAACTGTGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCGACCCTG

AACTGCCCGCTGCTCGAG

SEQ ID NO 16:

GCTTATGTGGACCTGCTGAAAGCAGGTATCGATAAATCCACCCCGGTGGAAGTGTACGTG

TACCGTAACACCCGCACTATCGCAAAGGTTAAACCGGCTAGCGACGAAGCTATGGCACGT

TACCGCCAGGCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCGTCTGAA

GCAGCGCTGACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTTGCT

TCTGATGGTTGCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGCTAT

GACGCAGATAAACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGCGTA

AGCTGGTCTTACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTTCAG

CAGCGCATCCTGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTGAAC

GCGTGTCGTAACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATGCCG

GGTGCAGTATATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTACGCG

CACACTAACTGTGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCGACC

CTGAACTGCCCGCTGCTCGAG

SEQ ID NO 17:

GTGTACCGTAACACCCGCACTATCGCAAAGGTTAAACCGGCTAGCGACGAAGCTATGGCA

CGTTACCGCCAGGCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCGTCT

GAAGCAGCGCTGACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTT

GCTTCTGATGGTTGCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGC

TATGACGCAGATAAACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGC

GTAAGCTGGTCTTACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTT

CAGCAGCGCATCCTGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTG AACGCGTGTCGTAACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATG

CCGGGTGCAGTATATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTAC

GCGCACACTAACTGTGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCG

ACCCTGAACTGCCCGCTGCTCGAG

SEQ ID NO 18:

GCACGTTACCGCCAGGCACTGGTAGCGCCGGCTAAGACTGAAGCGCTGCCGACTATCCCG

TCTGAAGCAGCGCTGACTACCCTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAA

TTTGCTTCTGATGGTTGCTACGCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCT

GGCTATGACGCAGATAAACTGTTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGT

TGCGTAAGCTGGTCTTACCACGTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACC

GTTCAGCAGCGCATCCTGGACCCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGG

CTGAACGCGTGTCGTAACACCGGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAG

ATGCCGGGTGCAGTATATTTTATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGC

TACGCGCACACTAACTGTGTTATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCG

CCGACCCTGAACTGCCCGCTGCTCGAG

SEQ ID NO 19:

CTGCCGACTATCCCGTCTGAAGCAGCGCTGACTACCCTGTTTAACCAGCTGAAAGCTGCG

CCGATCCCGTTCAAATTTGCTTCTGATGGTTGCTACGCACGTGCTCACAAAATGCGTCAG

ATGATTCTGGCGGCTGGCTATGACGCAGATAAACTGTTCGTGTATGGCAACCTGGCGGCT

TCTACCGGTACCTGTTGCGTAAGCTGGTCTTACCACGTTGCGCCGCTGGTGAACGTGAAA

ACCGCTAACGGTACCGTTCAGCAGCGCATCCTGGACCCGAGCCTGTTTACCGCGCCGGTT

GCAGTTTCCACTTGGCTGAACGCGTGTCGTAACACCGGTTGCGTTAGCACCGCTAACTAC

ACTACCACTCGTCAGATGCCGGGTGCAGTATATTTTATCGCGTCCACTGGCAACTCTCCG

CTGTATGACAACAGCTACGCGCACACTAACTGTGTTATCGCGGGTTACACCGGTCTGGTT

GGTTGTGGCATTCCGCCGACCCTGAACTGCCCGCTGCTCGAG

SEQ ID NO 20:

CTGTTTAACCAGCTGAAAGCTGCGCCGATCCCGTTCAAATTTGCTTCTGATGGTTGCTAC

GCACGTGCTCACAAAATGCGTCAGATGATTCTGGCGGCTGGCTATGACGCAGATAAACTG

TTCGTGTATGGCAACCTGGCGGCTTCTACCGGTACCTGTTGCGTAAGCTGGTCTTACCAC

GTTGCGCCGCTGGTGAACGTGAAAACCGCTAACGGTACCGTTCAGCAGCGCATCCTGGAC CCGAGCCTGTTTACCGCGCCGGTTGCAGTTTCCACTTGGCTGAACGCGTGTCGTAACACC

GGTTGCGTTAGCACCGCTAACTACACTACCACTCGTCAGATGCCGGGTGCAGTATATTTT

ATCGCGTCCACTGGCAACTCTCCGCTGTATGACAACAGCTACGCGCACACTAACTGTGTT

ATCGCGGGTTACACCGGTCTGGTTGGTTGTGGCATTCCGCCGACCCTGAACTGCCCGCTG

CTCGAG

Claims

1. A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, and homologues, fragments and portions thereof, for use as a protein glutaminase.

2. A protein according to claim 1, characterized in that said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or homologues having an identity of at least 80% thereof.

3. A protein according to any of the preceding claims, characterized in that the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1.

4. A protein according to any of the preceding claims, characterized in that the protein is linked to a further substance, in particular to an affinity tag, preferably selected from the group consisting of poly histidine tags.

5. A protein according to any of the preceding claims, characterized in that it has a minimum level of 0.1 U*mg ¹ Protein Glutaminase activity, such as > 0.1 U*mg^_

¹ - 10 U*mg ¹ Protein Glutaminase activity, such as > 10 U*mg ¹ Protein Glutaminase activity.

6. A DNA which encodes a protein according to any of the preceding claims.

7. A DNA according to claim 6, which comprises the nucleotide sequence shown in SEQ ID NO: 11

8. A recombinant vector containing a DNA according to any of the claims 6 or 7.

9. A transformed microorganism in which is introduced the DNA according to any of the claims 6 or 7 or recombinant vector according to claim 8.

10. The transformed microorganism according to claim 9 which is capable of producing Glutaminase.

11. The transformed microorganism according to claim 9 or 10 which is derived from a fungus, such as a yeast, such as Pichia pastoris or Saccharomyces cerevisiae.

12. A method for producing glutaminase which comprises cultivating the microorganism according to any of the claims 9 to 11 in a culture medium to produce glutaminase in the culture.

13. A method for the preparation of a protein glutaminase, in particular according to any of the claims 1 to 5, characterized by: a) Recombinant expression of a functional active microbial protein glutaminase as a Pre-Pro-Protein Glutaminase in a yeast b) secretion of the glycosylated microbial protein glutaminase in the culture broth c) absence of endopeptidase activity in the culture broth d) Concentration, intermediate storage or transport for at least 24 h up to 12 weeks of the Pichia pastoris culture broth prior to the preparation of a solid formulation

14. A protein produced by the method according to claim 11 or 12.

15. Use of a protein according to any of claims 1 to 5 and 11 to 13 in a process for deaminating vegetable proteins, in particular to improve their suitability as substitutes for milk proteins by increasing their solubility.

16. Use of a protein according to any of the claims 1 to 5 and 11 to 13 as glutaminase.