JP7649100B2 - How to produce ammonia - Google Patents

Description

The present invention broadly relates to a method for producing ammonia, etc.

Ammonia is widely used as a raw material for nitrogen-based fertilizers for agricultural crops and food, contributing to increased production of agricultural crops and food, and continuing to support today's growing population. Most ammonia is produced industrially using the Haber-Bosch process, but this process requires high temperatures and pressures, and requires a large amount of energy, which creates problems with the large environmental impact. Ammonia production methods that do not rely on the Haber-Bosch process are also being considered, and one example is an ammonia production method that uses nitrogen-fixing bacteria such as soil bacteria. However, this method has issues with the rate at which ammonia is produced by the nitrogen-fixing bacteria and the safety of the bacteria used.

On the other hand, today, as energy and global environmental issues become more serious, biomass has been attracting attention from the perspective of renewable energy and carbon neutrality.

The main components of biomass are carbohydrates, proteins, etc. Although carbohydrate-based biomass such as starch-based and wood-based biomass is used to produce bioethanol, protein-rich biomass such as food waste is not suitable as a raw material for bioethanol production, and most of it is not used.

JP 2010-046026 A Patent No. 4615126

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

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.

When glutaminase is expressed intracellularly, it is separated and purified from the bacterial culture or cell lysate. However, other intracellular enzymes, such as glutamate dehydrogenase, may also be present. Therefore, when using intracellularly expressed glutaminase to produce ammonia, the generated ammonia is quickly converted into other amino acids by other intracellular enzymes. In particular, yeast has a high nitrogen assimilation ability, making it difficult to obtain high concentrations of ammonia.

In addition, in the inventors' research into ammonia production methods from biomass, it became clear that glucose, which is present in high concentrations in biomass, inhibits the ammonia production reaction.

In view of the above, the present invention aims to provide a novel method for producing ammonia that has a low environmental impact.

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

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 includes food or waste thereof.
[4] The method according to [3], wherein the food or food waste comprises soybeans, processed soybeans 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 hydrolysate 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 includes 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 according to any one of [1] to [18].
[20] The product according to [19], which 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] The method according to [21], wherein the glutamic acid is used in the production of a food product.
[23] The method according to [22], wherein the food is soy sauce.
[24] A method for enhancing glutamic acid in a food product, comprising the steps of:
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 one 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.

The present invention provides an environmentally friendly method for producing ammonia, since it uses protein-containing materials such as protein-rich biomass as raw materials.

In addition, according to the present invention, by using a surface-displaying bacterial cell 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.

By using surface-displaying bacteria that display glutaminase, it is possible to produce not only ammonia but also glutamate at the same time.

Furthermore, according to the present invention, by using a glucose transporter or glutamine synthetase disrupted strain, ammonia can be produced efficiently even when the biomass contains glucose.

FIG. 1-1 to FIG. 1-4 show the nucleotide sequence of pULD1. FIG. 1-1 to FIG. 1-4 show the nucleotide sequence of pULD1. FIG. 1-1 to FIG. 1-4 show the nucleotide sequence of pULD1. FIG. 1-1 to FIG. 1-4 show the nucleotide sequence of pULD1. FIG. 2 shows the evaluation of ammonia productivity of the pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain in Example 1. FIG. 3 shows the evaluation of ammonia productivity of the pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain in Example 2. 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. FIG. 5 shows the evaluation of ammonia productivity of the DH10B strain, the ΔglnA strain, and the ΔglnAΔptsG strain in Example 4. FIG. 6 shows the evaluation of glutamic acid productivity of the pULD1-Glutaminase recombinant strain in Example 5. FIG. 7 shows the evaluation of ammonia and glutamic acid productivity of the pULD1-Glutaminase recombinant strain in Example 6.

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.

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

(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.

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

(Genetically modified microorganisms)
As used herein, "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.

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, if the end product is one that can be applied to the human body, such as medicine, food, or cosmetics, it is preferable that the microorganism is not harmful to the human body.

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

The glutaminase is preferably one that 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.

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

Genetic recombination using the glutaminase gene can be carried out by standard methods. For example, by incorporating the glutaminase gene into a vector such as a bacteriophage, cosmid, or a plasmid used for transformation of prokaryotic or eukaryotic cells, the 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.

Although not intended to be limiting, other examples of microorganisms that can be used in foods and beverages include Bacillus subtilis, Enterococcus faecalis, and Escherichia coli. 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.

Disruption of the above-mentioned genes means disrupting part or all of the target gene, for example deleting 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.

The microorganism may be further transformed with another gene to express a gene involved in increasing the efficiency of ammonia production, or a mutation may be introduced to destroy a gene involved in decreasing the production efficiency. 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.

(Method of producing ammonia)
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.

When glucose is contained in the biomass, ammonia production is inhibited as the concentration increases, but by disrupting the ptsG gene and the glnA gene, a decrease in production efficiency can be prevented. Therefore, the above contact step can be carried out even 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.

(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.

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

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

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

The sequence information of the primers used in the examples is shown in Table 2.

(Creation of yeast that displays glutaminase on its surface)

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

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 from Escherichia coli DH5α strain (Takara Bio) as a template. The resulting DNA fragment and pULD1 were treated with restriction enzymes XhoI and BglII (TOYOBO), and then ligated to create the plasmid pULD1-Glutaminase.

[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)) to perform 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.

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

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 in a shaking incubator (TAITEC BR-23; the same applies below) at 240 rpm and 37°C, and samples were taken at 0, 4, 8, and 24 hours after the reaction.

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

As shown in Figure 2, in the control pULD1 recombinant strain, almost no 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 analysis value/theoretical maximum value of ammonia (120 mM ammonia).

As mentioned above, the reaction efficiency exceeded 100%, at approximately 109%, possibly due to the elution of glutamic acid from within the yeast cells.

(Ammonia production from biomass using glutaminase-displaying yeast)

In this example, we investigated ammonia production by yeast that displays glutaminase on its surface, using soybean pulp as biomass.

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. 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 and 200 rpm for 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). To the filtrate, pULD1-Glutaminase recombinant strain and pULD1 recombinant strain cultured overnight in SC (-Uracil) medium were added so that the OD600 of the medium became 1. These were reacted in a shaker at 240 rpm and 37°C for 10 minutes, then heated in a block incubator at 80°C for 15 minutes, after which the ammonia concentration was measured.

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 yeast that displays glutaminase on its surface.

(Ammonia production from biomass using glnA, ptsG-disrupted E. coli)

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

[Preparation of ΔglnA and ΔptsG strains]
Gene disruption of Escherichia coli DH10B strain (Takara Bio) was performed by a method known as the 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)).

First, DH10B strain transformed with 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.

To delete the glnA and ptsG genes, PCR was performed using the plasmid pKD13 as a template with a combination of primers ΔglnA_Fw (sequence number 3) and ΔglnA_Rv (sequence number 4) and a combination of primers ΔptsG_Fw (sequence number 5) and ΔptsG_Rv (sequence number 6). The resulting PCR products were transformed into the above competent cells using a Gene Pluser II (registered trademark) electroporator (Biorad) under conditions of 600 Ω, 1.35 kV, and 10 μF.

The transformed strains were then statically cultured overnight in SOC medium, and the next day in LB medium (plus kanamycin), yielding ΔglnA and ΔptsG strains, each with a kanamycin resistance gene inserted via homologous recombination. The resulting strains were then further cultured at 37°C to eliminate pKD46.

[Preparation of ΔptsGΔglnA strain]
The prepared ΔptsG strain was pre-cultured 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. The pellet after centrifugation was washed with cold water and cold 10% glycerol aqueous solution, and then stored at -80°C as competent cells.

To transform the competent cells with the plasmid pCP20, a Gene Pulser II electroporator was used 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 remove pCP20.

The ΔptsG strain was produced using the same procedure as for producing the ΔglnA strain, to produce the ΔglnAΔptsG strain.

[Evaluation of ammonia productivity of gene disruptant strains]
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.

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

As shown in Figure 4, the DH10B strain was unable to produce ammonia efficiently. However, it was shown that by simultaneously disrupting glnA, which encodes glutamine synthetase, and ptsG, which encodes the main transporter of the glucose phosphotransferase system, it was possible to produce ammonia highly efficiently from biomass containing high concentrations of glucose.

(Evaluation of ammonia productivity in gene disruption strains)

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

6 g/L disodium monohydrogen phosphate (Nacalai Tesque), 3 g/L monosodium dihydrogen phosphate (Nacalai Tesque), 0.5 g/L sodium chloride (Nacalai Tesque), 7.25 g/L yeast extract (BD Japan), 0.1 mM calcium chloride (Nacalai Tesque), and 1 mM magnesium sulfate (Nacalai Tesque) were mixed and sterilized. 10 mM or 50 mM glucose (Nacalai Tesque) was added to the medium obtained in this way, and the DH10B strain, ΔglnA strain, or ΔglnAΔptsG strain was added 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. 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.

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.

(Glutamate production using surface-displayed glutaminase)

In this example, we investigated glutamic acid production using the glutaminase surface-displaying yeast prepared in Example 1, and investigated whether it could be applied to food manufacturing.

[Preparation of soy sauce koji]
Spores of a strain of the koji mold Aspergillus sojae owned by Kikkoman Corporation were added to a solid medium containing equal amounts of heat-denatured soybeans and wheat, and koji was produced at 25 to 40° C. for 72 hours to obtain soy sauce koji.

[Evaluation of glutamic acid productivity]
0.6 mL of lactic acid and 76 mL of water were added to 60 g of the obtained soy sauce koji, and further, the pULD1-Glutaminase recombinant strain and the pULD1 recombinant strain cultured overnight in SC (-Uracil) medium were added so that the OD600 of the medium became 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 filtered with qualitative filter paper No. 2. The glutamic acid concentration of the filtrate was measured using a biosensor (Oji Scientific Instruments BF-5D).

Shoyu koji contains a large amount of glutaminase derived from koji mold, but it is inactivated by salt or acid, and is decomposed by proteases derived from koji mold. 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.

(Ammonia and glutamic acid production from soy protein hydrolysate)

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

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 medium had an OD600 of 1. These were reacted in a shaker 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 and ammonia concentrations were measured.

As shown in Figure 7, it was shown that the glutaminase-displaying yeast reacted with glutamine in the soybean hydrolysate to simultaneously produce ammonia and glutamic acid.

Claims (17)

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. The method of claim 1, wherein the protein-containing material is biomass. The method of claim 2, wherein the biomass includes food or waste products thereof. The method of claim 3, wherein the food or waste product thereof includes soybeans, processed soybeans, or residues thereof. The method of claim 4, wherein the processed product or residue includes a decomposition product. The method according to claim 5, wherein the decomposition product includes soy sauce koji. The method according to claim 5, wherein the hydrolyzate comprises soy protein or a peptide thereof. The method of claim 1, wherein the microorganism comprises yeast, Escherichia coli, or lactic acid bacteria. The method according to claim 8, wherein the glutaminase displayed on the cell surface of the yeast is acid-resistant. The method according to any one of claims 1 to 9, wherein the koji fungus includes Aspergillus oryzae or Aspergillus sojae. The method of any one of claims 8 or 9, wherein the yeast comprises Saccharomyces cerevisiae, Zygosaccharomyces rouxii, or Candida utilis. The method according to any one of claims 1 to 11, further comprising producing glutamic acid. 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. The method of claim 13, wherein the microorganism comprises Escherichia coli, Bacillus subtilis, or Enterococcus faecalis. 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 more. A method for producing a product using ammonia as a raw material, the method comprising the step of producing ammonia using the method according to any one of claims 1 to 15. 17. The method of claim 16, wherein the product is a yeast extract raw material or an agricultural fertilizer.