How to produce ammonia

Abstract

To provide a novel ammonia production method with low environmental load.SOLUTION: A method for producing ammonia has a step of bringing a protein-containing material into contact with a recombinant microorganism.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates generally to a method for producing ammonia. [Background technology]

[0002] Ammonia is widely used as a raw material for nitrogen fertilizers for agricultural crops and food, contributing to increased production of agricultural crops and food, and supporting today's population growth. Most ammonia is produced industrially by the Haber-Bosch process, but this process requires high temperature and pressure conditions and a large amount of energy, which poses a problem of large environmental impact. Ammonia production methods that do not rely on the Haber-Bosch process are also being considered, and for example, an ammonia production method using nitrogen-fixing bacteria such as soil bacteria has been proposed. However, this method has issues with the ammonia production rate by nitrogen-fixing bacteria and the safety of the bacteria used.

[0003] On the other hand, in today's world where energy and global environmental issues are becoming more serious, biomass has been attracting attention from the perspectives of renewability and carbon neutrality.

[0004] Representative components of biomass are carbohydrates, proteins, etc. Although carbohydrate-based biomass such as starch-based and wood-based biomass is used for bioethanol production, protein-rich biomass such as food waste is not suitable as a raw material for bioethanol production, and most of it is not used. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2010-046026 A [Patent Document 2] Patent No. 4615126 Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors conducted research based on the idea that it would be useful if it were possible to efficiently utilize biomass protein-containing materials as a source.

[0007] Glutaminase is an enzyme that uses glutamine and water as substrates to produce one molecule of ammonia and one molecule of glutamic acid from one molecule of glutamine. Glutamic acid produced by glutaminase is an umami component and is one of the important substances in food manufacturing.

[0008] When glutaminase is expressed intracellularly, it is separated and purified from the culture solution or cell lysate, but usually, other enzymes such as glutamate dehydrogenase may also be contained in the cells. Therefore, when ammonia is produced using intracellularly expressed glutaminase, the generated ammonia is quickly converted to other amino acids by other enzymes in the cells. In particular, since yeast has a high nitrogen assimilation ability, it has been difficult to obtain ammonia at a high concentration.

[0009] Furthermore, in the study of ammonia production methods from biomass by the present inventors, it was revealed that glucose, which is contained in high concentrations in biomass, inhibits the ammonia production reaction.

[0010] In view of the above circumstances, an object of the present invention is to provide a novel method for producing ammonia with low environmental impact. [Means for solving the problem]

[0011] The present inventors discovered that the above object can be achieved by adding a microorganism in which a gene related to ammonia production has been modified to a raw material containing a protein, such as protein-rich biomass, and thus completed the present invention.

[0012] That is, the present application includes the following inventions. [1] A method for producing ammonia, comprising: A method comprising the step of contacting a protein-containing material with a genetically modified microorganism. [2] The method according to [1], wherein the protein-containing material is biomass. [3] The method according to [2], wherein the biomass comprises food or food waste. [4] The method according to [3], wherein the food or food waste comprises soybeans, soybean products, or residues thereof. [5] The method according to [4], wherein the processed product or residue includes a decomposition product. [6] The method according to [5], wherein the decomposition product contains soy sauce koji. [7] The method according to [5], wherein the hydrolyzate contains soy protein or a peptide thereof. [8] The method according to any one of [1] to [7], wherein the genetically modified microorganism has been genetically modified to display glutaminase on its cell surface. [9] The method according to [8], wherein the microorganism comprises yeast, Escherichia coli or lactic acid bacteria.

[10] The method according to [9], wherein the glutaminase displayed on the cell surface of the yeast is acid-resistant.

[11] The method according to any one of [8] to

[10] , wherein the glutaminase is derived from Aspergillus oryzae, Bacillus subtilis or Escherichia coli.

[12] The method according to

[11] , wherein the koji fungus includes Aspergillus oryzae or Aspergillus sojae.

[13] The method according to any one of [9] to

[12] , wherein the yeast comprises Saccharomyces cerevisiae, Zygosaccharomyces rouxii or Candida utilis.

[14] The method according to any one of [1] to

[13] , further comprising producing glutamic acid.

[15] The method according to any one of [1] to [7], wherein the genetically modified microorganism is a disruptant of the glnA and / or ptsG genes.

[16] The method according to

[15] , wherein the microorganism expresses the DgsA and / or kivD genes.

[17] The method according to

[15] or

[16] , wherein the microorganism comprises Escherichia coli, Bacillus subtilis or Enterococcus faecalis.

[18] The method according to any one of

[15] to

[17] , wherein the contacting step is carried out in the presence of glucose at a concentration of 5 mM or more.

[19] A method for producing a product using ammonia as a raw material, the method comprising a step of producing ammonia using the method described in any one of [1] to

[18] .

[20] The product according to

[19] , wherein the product is a yeast extract raw material or an agricultural fertilizer.

[21] A method for producing glutamic acid, comprising: A method comprising the step of contacting a protein-containing material with a microorganism that has been genetically modified to display glutaminase on its cell surface to produce glutamic acid.

[22] Glutamic acid is used in the production of food products according to the method described in

[21] .

[23] The method according to

[22] , wherein the food is soy sauce.

[24] A method for enhancing glutamic acid in a food product, comprising: A method comprising the step of contacting a protein-containing material with a microorganism that has been genetically modified to display glutaminase on its cell surface to produce glutamic acid.

[25] The method according to

[24] , wherein the food is soy sauce.

[26] The method according to

[24] or

[25] , wherein the protein-containing material is biomass.

[27] The method according to

[26] , wherein the biomass comprises soybeans or processed products thereof.

[28] The method according to

[27] , wherein the processed product comprises soy sauce koji.

[29] The method according to

[27] , wherein the processed product contains soy protein or a peptide thereof.

[30] The method according to any of

[24] to

[29] , wherein the glutaminase is derived from Aspergillus oryzae.

[31] The method according to

[30] , wherein the koji fungus is Aspergillus oryzae or Aspergillus sojae.

[32] The method according to any one of

[24] to

[31] , wherein the microorganism is a yeast selected from Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Candida utilis. Effect of the Invention

[0013] According to the present invention, since a protein-containing material such as protein-rich biomass is used as a raw material, an environmentally friendly method for producing ammonia can be provided.

[0014] Furthermore, according to the present invention, by using a surface-displaying bacterium that displays glutaminase on the cell surface as a biocatalyst, it is possible to prevent nitrogen assimilation of the produced ammonia, and thus to efficiently produce ammonia. Another advantage of glutaminase is that it is located on the cell surface, which improves acid resistance.

[0015] By using a surface-displaying bacterial cell that displays glutaminase, not only ammonia but also glutamic acid can be produced simultaneously.

[0016] Furthermore, according to the present invention, by using a glucose transporter- or glutamine synthetase-disrupted strain, ammonia can be produced efficiently even when glucose is contained in the biomass. [Brief description of the drawings]

[0017] [Figure 1-1] 1-1 to 1-4 show the nucleotide sequence of pULD1. [Figure 1-2] 1-1 to 1-4 show the nucleotide sequence of pULD1. [Figure 1-3] 1-1 to 1-4 show the nucleotide sequence of pULD1. [Figure 1-4] 1-1 to 1-4 show the nucleotide sequence of pULD1. [Diagram 2] FIG. 2 shows the evaluation of ammonia productivity of the pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain in Example 1. [Diagram 3] FIG. 3 shows the evaluation of ammonia productivity of the pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain in Example 2. [Figure 4] FIG. 4 shows the evaluation of ammonia productivity of the DH10B strain, the ΔglnA strain, the ΔptsG strain, and the ΔglnAΔptsG strain in Example 3. [Diagram 5] FIG. 5 shows the evaluation of ammonia productivity of the DH10B strain, the ΔglnA strain, and the ΔglnAΔptsG strain in Example 4. [Figure 6] FIG. 6 shows the evaluation of glutamic acid productivity of the pULD1-Glutaminase recombinant strain in Example 5. [Figure 7] FIG. 7 shows the evaluation of ammonia and glutamic acid productivity of the pULD1-Glutaminase recombinant strain in Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] (Protein-containing materials) As used herein, the term "protein-containing material" refers to a material containing protein, and refers to any biologically derived protein-containing material, including not only animal-derived protein-containing materials such as meat, fish, shellfish, and eggs, and plant-derived protein-containing materials such as grains and vegetables, but also microbial-derived protein-containing materials. Examples of such materials include, but are not limited to, biomass, such as waste generated during food production, particularly during processing. Biomass may contain carbohydrate components such as starch in addition to protein.

[0019] As used herein, "protein" refers to proteins derived from organisms as described above, but may also include their degradation products, such as oligopeptides, peptides, and amino acids.

[0020] (biomass) As used herein, "biomass" broadly means a renewable, organic resource derived from a living organism, excluding fossil resources. Among such organic resources, materials containing nitrogen-containing compounds such as proteins, amino acids, urea, and uric acid are preferred because they generate ammonia through decomposition. Examples of biomass containing nitrogen-containing compounds include food and beverages and their waste products. The scope of food and beverages and waste products may vary depending on the type of biomass. For example, in the case where the living organism is soybeans, soybeans themselves and processed products such as soy milk are considered to be included in food and beverages, but the soybean meal that is the residue of soybeans may be discarded and used as feed, or may be eaten as soybean pulp, and food and waste cannot be clearly distinguished. Therefore, biomass as used herein may include not only waste products but also food and beverages.

[0021] Food and food waste include those that have been fermented by microorganisms and those that have been decomposed by hydrolysis, etc. Taking soybeans as an example, an example of a fermented product is soy sauce koji, and decomposition products include soybean protein and its peptides.

[0022] (Genetically Modified Microorganisms) As used herein, the term "genetically modified microorganism" refers to a microorganism that has been transformed to produce ammonia extracellularly, preferably a microorganism that has been transformed to display glutaminase on the cell surface (hereinafter also referred to as a "glutaminase surface-displaying" microorganism), or a microorganism in which a gene that adversely affects ammonia production has been destroyed. Examples of such microorganisms include yeast, Escherichia coli, and lactic acid bacteria. As long as the microorganisms are capable of producing ammonia, one or more strains of the same or different species can be used simultaneously.

[0023] The type of microorganism can be appropriately determined by a person skilled in the art depending on the efficiency of ammonia production and the end use of the ammonia. For example, when the end product is one that can be applied to the human body, such as medicine, food, or cosmetics, the microorganism is preferably one that is not harmful to the human body.

[0024] Although not intended to be limiting, examples of yeast that can be used in foods and beverages include Saccharomyces cerevisiae, Zygosaccharomyces rouxii, Candida utilis, etc. These yeasts can also be suitably used when transformed to display glutaminase on the cell surface.

[0025] It is preferable that the glutaminase has a property of high ammonia production efficiency even under harsh conditions. Examples of such glutaminase include glutaminase derived from koji mold such as Aspergillus oryzae and Aspergillus sojae, Bacillus subtilis, and Escherichia coli. Genes encoding glutaminase derived from Aspergillus oryzae (SEQ ID NO: 8-10) and gene encoding glutaminase derived from Escherichia coli (SEQ ID NO: 11) are shown in Table 1 below. [Table 1]

[0026] Aspergillus sojae and Bacillus subtilis have orthologs of the genes in Table 1.

[0027] Genetic recombination using the glutaminase gene can be carried out by a conventional method. For example, by incorporating the glutaminase gene into a vector such as a bacteriophage, cosmid, or a plasmid used for transformation of a prokaryotic or eukaryotic cell, a host cell corresponding to each vector is transformed to display glutaminase on its cell surface. Since ammonia is produced outside the bacterial cell by glutaminase displayed on the cell surface, nitrogen is not assimilated unless it is taken up into the bacterial cell, enabling efficient production.

[0028] Other examples of microorganisms that can be used in foods and beverages include, but are not limited to, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and the like. These microorganisms can also be used favorably when creating strains in which genes that adversely affect ammonia production, such as genes encoding glutamine synthetase and genes encoding proteins that constitute the phosphotransferase system (PTS), have been disrupted. An example of a gene that encodes glutamine synthetase is glnA. Another example of a gene that encodes proteins that constitute the PTS is the ptsG gene, which encodes domains A, B, and C of enzyme II (EII). By disrupting these genes, it becomes possible to efficiently produce ammonia even if the biomass contains glucose.

[0029] The above-mentioned gene disruption means disrupting a part or all of a target gene, for example, deleting a part or all of the glnA gene, or modifying the gene so that it does not function normally by inserting another gene, such as a drug resistance gene, into the middle of the glnA gene.

[0030] The microorganism may be further transformed with another gene so that a gene involved in increasing the efficiency of ammonia production is expressed, or a mutation may be introduced so that a gene involved in decreasing the production efficiency is destroyed. Genes involved in increasing the efficiency of ammonia production include DgsA, a transcription factor involved in glucose catabolite repression, and kivd, an amino acid decarboxylase.

[0031] (Ammonia production method) The contact step between the protein-containing material and the genetically modified microorganism is carried out under conditions necessary for the genetically modified microorganism to produce ammonia, which conditions can be appropriately determined by a person skilled in the art depending on the type of microorganism used.

[0032] When glucose is contained in the biomass, ammonia production is inhibited as the glucose concentration increases, but the decrease in production efficiency can be prevented by disrupting the ptsG gene and the glnA gene. Therefore, the contact step can be carried out in the presence of high concentrations of glucose, for example, 5 mM or more, preferably 10 mM or more, and more preferably 50 mM or more.

[0033] (Uses of ammonia) The produced ammonia can be suitably used for various applications, for example, as a raw material for food and beverage products such as yeast extract, basic chemicals such as nitric acid, and nitrogen fertilizers such as ammonium sulfate.

[0034] (glutamic acid production) When a glutaminase surface-displaying microorganism is used, glutamic acid can be further produced outside the bacterial cell.

[0035] (Uses of glutamic acid) Glutamic acid can be suitably used as a raw material for soy sauce and umami seasonings. EXAMPLES

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0037] The sequence information of the primers used in the examples is shown in Table 2. [Table 2] EXAMPLES

[0038] (Preparation of glutaminase surface-displaying yeast)

[0039] [Construction of a plasmid for expressing glutaminase] For expression of cell surface-displayed glutaminase, plasmid pULD1 prepared according to the method disclosed in International Publication No. 2010 / 101158 was used, and URA3 marker was used as a selection marker. The nucleotide sequence of pULD1 (SEQ ID NO: 7) is shown in Figures 1-1 to 1-4.

[0040] First, a DNA fragment (SEQ ID NO: 11) of the glutaminase gene (YbaS) was amplified by PCR using a pair of primers pULD1-Glutaminase_Fw (SEQ ID NO: 1) and pULD1-Glutaminase_Rv (SEQ ID NO: 2) with DNA of Escherichia coli DH5α strain (Takara Bio) as a template. The obtained DNA fragment and pULD1 were treated with restriction enzymes XhoI and BglII (TOYOBO) and ligated to create the plasmid pULD1-Glutaminase.

[0041] [Transformation] Using a Frozen-EZ Yeast Transformation II (registered trademark) kit (Zymo Research), the plasmid pULD1-Glutaminase and the plasmid pULD1 were introduced into the budding yeast (Saccharomyces cerevisiae) W303-1a strain (National BioResource Project (NBRP)) for transformation. Colonies obtained by transformation were selected on SC (uracil-free (hereinafter also referred to as "-Uracil")) medium plates to obtain pULD1-Glutaminase recombinant strains and pULD1 recombinant strains.

[0042] [Evaluation of glutaminase surface-displaying yeast] In this example, the ability of surface-displayed glutaminase to obtain ammonia from glutamine was evaluated.

[0043] The pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain, which had been cultured overnight in SC(-Uracil), were added to a reaction solution containing 200 mM MES buffer (pH 5.5) and 120 mM or 179 mM glutamine, so that the OD600 of the medium was 1. These were reacted at 240 rpm and 37°C in a shaking incubator (TAITEC BR-23; the same applies below), and samples were taken at 0, 4, 8, and 24 hours after the reaction.

[0044] The reaction solution was heated in a block incubator (BI516 manufactured by Astec Co., Ltd.; the same applies below) at 80° C. for 15 minutes. The ammonia concentration was measured using an F-kit ammonia (manufactured by JK International Co., Ltd.).

[0045] As shown in Figure 2, in the control pULD1 recombinant strain, little ammonia production was observed during the reaction time. On the other hand, a significant increase in ammonia concentration was observed in the pULD1-Glutaminase recombinant strain. In particular, when the reaction solution containing 120 mM glutamine was reacted for 24 hours, the reaction efficiency was approximately 109%. This indicates that the surface-displayed glutaminase efficiently produced ammonia from glutamine. The above reaction efficiency was calculated based on the ammonia analytical value / theoretical maximum value of ammonia (120 mM ammonia).

[0046] As mentioned above, the reason why the reaction efficiency exceeded 100%, such as about 109%, may be due to the elution of glutamic acid from within the yeast cells. EXAMPLES

[0047] (Ammonia production from biomass using glutaminase-displaying yeast)

[0048] In this example, ammonia production by glutaminase surface-displaying yeast was investigated using soybean pulp as biomass.

[0049] First, 10 g of soybean pulverization was performed using a mixer (IFM-700G, manufactured by Iwatani Co., Ltd.), and 70 g of 50 mM MES buffer (pH 5.5) was added to the mixture. Furthermore, 1.5 g of Protin SD-AY10 (manufactured by Amano Enzyme Co., Ltd.) and Proteax (manufactured by Amano Enzyme Co., Ltd.) were added, and the mixture was reacted in a shaking incubator at 55°C, 200 rpm, and 48 hours. The decomposition liquid of the soybean pulverization was filtered using qualitative filter paper No. 2 (manufactured by ADVANTEC Co., Ltd.; the same applies below). The pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain, which had been cultured overnight in SC (-Uracil) medium, were added to the filtrate so that the OD600 of the medium was 1. The mixture was reacted in a shaking incubator at 240 rpm and 37°C for 10 minutes, heated in a block incubator at 80°C for 15 minutes, and then the ammonia concentration was measured.

[0050] As shown in Figure 3, in the control strain, no significant change in ammonia concentration was observed before and after the reaction. On the other hand, in the pULD1-Glutaminase recombinant strain, the ammonia concentration more than doubled after 10 minutes of reaction. This also demonstrated that efficient ammonia production from biomass is possible by using glutaminase surface-displaying yeast. EXAMPLES

[0051] (Ammonia production from biomass using glnA, ptsG disrupted Escherichia coli)

[0052] In this example, we investigated the establishment of bacterial cells that efficiently produce ammonia even when biomass contains glucose.

[0053] [Construction of ΔglnA and ΔptsG strains] The gene disruption method for Escherichia coli DH10B strain (Takara Bio) was carried out by a method called one-step inactivation method. Details of this method and the plasmids used are known to those skilled in the art (see, for example, Masami Yamada, Environ. Mutagen Res., 25:87-92 (2003); Kirill A. Datsenko and Barry L. Wanner, PNAS, vol.97(12): 6640-6645 (2000)).

[0054] First, the DH10B strain transformed with the plasmid pKD46 by the heat shock method was cultured overnight at 37°C in LB medium, and 2 mL of the culture was inoculated into 50 mL of LB medium (+10 mM arabinose). When the OD600 reached 0.5, the medium was cooled on ice. After centrifugation, the pellet was washed with cold water and cold 10% glycerol solution, and then stored at -80°C as competent cells.

[0055] To delete the glnA and ptsG genes, PCR was performed using the plasmid pKD13 as a template and a combination of primers ΔglnA_Fw (SEQ ID NO: 3) and ΔglnA_Rv (SEQ ID NO: 4) and a combination of primers ΔptsG_Fw (SEQ ID NO: 5) and ΔptsG_Rv (SEQ ID NO: 6). The PCR products obtained were transformed into the above-mentioned competent cells using a Gene Pluser II (registered trademark) electroporator (Biorad) under conditions of 600 Ω, 1.35 kV, and 10 μF.

[0056] The transformed strains were then cultured overnight in SOC medium, and the next day in LB medium (+kanamycin) to obtain the ΔglnA and ΔptsG strains, each of which had the kanamycin resistance gene inserted via homologous recombination. The resulting strains were then cultured at 37°C to eliminate pKD46.

[0057] [Creation of ΔptsGΔglnA strain] The prepared ΔptsG strain was precultured overnight at 37°C in LB medium (+kanamycin), and 2 mL of the strain was inoculated into 50 mL of LB medium (+kanamycin). When the OD600 reached 0.5, the medium was cooled on ice. After centrifugation, the pellet was washed with cold water and cold 10% glycerol solution, and then stored at -80°C as competent cells.

[0058] To transform the competent cells with the plasmid pCP20, transformation was performed using a Gene Pulser II electroporator under conditions of 600 Ω, 1.35 kV, and 10 μF. The resulting colonies were cultured overnight at 30°C and then subcultured at 37°C to delete pCP20.

[0059] The ΔptsG strain was prepared in the same manner as in the preparation of the ΔglnA strain, to prepare the ΔglnAΔptsG strain.

[0060] [Evaluation of ammonia productivity in gene disruptants] The DH10B strain, the ΔglnA strain, the ΔptsG strain, and the ΔglnAΔptsG strain were added to the decomposition filtrate of soybean pulp prepared in Example 2 so that the OD600 of the medium was 0.5, and the reaction was carried out in a shaking incubator at 37°C and 200 rpm for 26 hours.

[0061] The reaction solution was centrifuged. The supernatant was heated in a block incubator at 80°C for 15 minutes, and the ammonia concentration was measured. The okara decomposition solution contained approximately 70 mM glucose.

[0062] As shown in Figure 4, ammonia production was not efficient in the DH10B strain. However, it was shown that it was possible to produce ammonia highly efficiently from biomass containing high concentrations of glucose by simultaneously disrupting glnA, which encodes glutamine synthetase, and ptsG, which encodes the main transporter of the glucose phosphotransferase system. EXAMPLES

[0063] (Evaluation of ammonia productivity in gene disruptants)

[0064] In this example, the ammonia productivity of the ΔglnAΔptsG strain prepared in Example 3 was examined in the presence of different concentrations of glucose.

[0065] 6g / L disodium monohydrogen phosphate (Nacalai Tesque), 3g / L monosodium dihydrogen phosphate (Nacalai Tesque), 0.5g / L sodium chloride (Nacalai Tesque), 7.25g / L yeast extract (BD Japan), 0.1mM calcium chloride (Nacalai Tesque), 1mM magnesium sulfate (Nacalai Tesque) were mixed and sterilized. Glucose (Nacalai Tesque) was added to the medium thus obtained at 10mM or 50mM, and the DH10B strain, ΔglnA strain, or ΔglnAΔptsG strain was added so that the OD600 of the medium was 0.5, and the mixture was reacted in a shaking incubator at 37°C and 200 rpm for 26 hours. The reaction solution was centrifuged, and the supernatant was heated at 80°C for 15 minutes in a block incubator, and the ammonia concentration was measured.

[0066] As shown in Figure 5, the control strain, DH10B, was unable to produce ammonia efficiently. However, it was shown that the ΔglnAΔptsG strain was able to produce ammonia efficiently even when a high concentration of glucose was added. EXAMPLES

[0067] (Glutamic acid production using surface-displayed glutaminase)

[0068] In this example, glutamic acid production using the glutaminase surface-displaying yeast prepared in Example 1 was examined, and its applicability to food production was examined.

[0069] [Preparation of soy sauce koji] Soy sauce koji was obtained by adding spores of a strain of Aspergillus sojae owned by Kikkoman Corporation to a solid medium containing equal amounts of heat-denatured soybeans and wheat, and then koji was produced at 25-40°C for 72 hours.

[0070] [Evaluation of glutamic acid productivity] 0.6 mL of lactic acid and 76 mL of water were added to 60 g of the resulting soy sauce koji, and then the pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain, which had been cultured overnight in SC (-Uracil) medium, were added so that the OD600 of the medium was 1. For comparison, 50 mg of glutaminase SD-C100S (Amano Enzyme Co., Ltd.) was added. These were reacted in a shaking incubator at 240 rpm and 25°C for 24 hours, and then filtered through qualitative filter paper No. 2. The glutamic acid concentration of the filtrate was measured using a biosensor (Oji Measurement Instruments BF-5D).

[0071] Soy sauce koji contains a large amount of glutaminase derived from Aspergillus oryzae, but it is inactivated by salt or acid, and is decomposed by Aspergillus oryzae-derived protease. As shown in Figure 6, the glutamic acid concentration did not increase even when glutaminase enzyme preparation was added. On the other hand, when the pULD1-Glutaminase recombinant strain was added, the glutamic acid concentration increased by nearly 20%. This indicates that the surface-displayed glutaminase is stable against hydrochloric acid and various enzymes, has excellent acid resistance, and is also useful for increasing glutamic acid in food production. EXAMPLES

[0072] (Ammonia and glutamic acid production from soy protein hydrolysate)

[0073] In this example, the glutaminase surface-displaying yeast prepared in Example 1 was used to examine the production of ammonia and glutamic acid from soy protein hydrolysates.

[0074] 60 μL of lactic acid and 10 mL of water were added to 3 g of Soybean Peptide Hi-Nute S (Amano Enzyme), and the pULD1-Glutaminase recombinant strain cultured overnight in SC(-Uracil) medium was added so that the OD600 of the medium became 1. These were reacted in a shaking culture machine at 240 rpm and 37°C for 24 hours, and then heated in a block incubator at 80°C for 15 minutes, after which the glutamic acid concentration and ammonia concentration were measured.

[0075] As shown in FIG. 7, it was demonstrated that the glutaminase surface-displaying yeast reacted with glutamine in the soybean hydrolysate to simultaneously produce ammonia and glutamic acid.

Claims

1. 1. A method for producing ammonia, comprising: contacting a protein-containing material with a genetically modified microorganism, The genetically modified microorganism is genetically modified to display glutaminase on its cell surface, The method, wherein the glutaminase is derived from Aspergillus oryzae, Bacillus subtilis or Escherichia coli.

2. The method of claim 1 , wherein the protein-containing material is biomass.

3. The method of claim 2 , wherein the biomass comprises food or a waste product thereof.

4. The method of claim 3 , wherein the food or waste product thereof comprises soybeans, soybean products or residues thereof.

5. The method of claim 4 , wherein the workpiece or residue comprises a decomposition product.

6. The method of claim 5 , wherein the decomposition product comprises soy sauce koji.

7. The method of claim 5 , wherein the digest comprises soy protein or a peptide thereof.

8. The method of claim 1 , wherein the microorganism comprises yeast, Escherichia coli, or lactic acid bacteria.

9. The method according to claim 8 , wherein the glutaminase displayed on the cell surface of the yeast is acid-resistant.

10. The method according to any one of claims 1 to 9, wherein the koji fungus comprises Aspergillus oryzae or Aspergillus sojae.

11. 10. The method of claim 8 or 9, wherein the yeast comprises Saccharomyces cerevisiae, Zygosaccharomyces rouxii or Candida utilis.

12. 12. The method according to any one of claims 1 to 11, wherein glutamic acid is further produced.

13. 1. A method for producing ammonia, comprising: contacting a protein-containing material with a genetically modified microorganism, The method according to claim 1, wherein the genetically modified microorganism is a disruptant of the glnA and ptsG genes.

14. 14. The method of claim 13, wherein the microorganism comprises Escherichia coli, Bacillus subtilis, or Enterococcus faecalis.

15. 14. The method of claim 13, wherein the microorganism is Escherichia coli and the contacting step is carried out in the presence of glucose at a concentration of 5 mM or greater.

16. 16. A method for producing an ammonia-based product, comprising producing ammonia using the method according to any one of claims 1 to 15.

17. 17. The method of claim 16, wherein the product is a yeast extract feedstock or an agricultural fertilizer.

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