Heat-resistant protein glutaminase

Abstract

The objective is to provide a protein glutaminase with improved heat resistance. [Solution] A protein glutaminase comprising a polypeptide as shown in any of the following (1) to (3) has improved heat resistance: (1) a polypeptide comprising a specific amino acid sequence; (2) a polypeptide comprising one or more amino acid residues substituted, added, inserted, or deleted in the specific amino acid sequence, and exhibiting heat resistance equivalent to that of the polypeptide comprising the specific amino acid sequence; and (3) a polypeptide comprising the specific amino acid sequence having a sequence identity of 76% or more to the specific amino acid sequence, and exhibiting heat resistance equivalent to that of the polypeptide comprising the specific amino acid sequence.

Description

Technical Field

[0001] The present invention relates to a heat-resistant protein glutaminase. More specifically, the present invention relates to a heat-resistant protein glutaminase, DNA encoding the heat-resistant protein glutaminase, a recombinant vector, a transformant, an enzyme agent, a method for producing the heat-resistant protein glutaminase, and uses of the heat-resistant protein glutaminase.

Background Art

[0002] Protein glutaminase is an enzyme that deamidates the γ-amide group and β-amide group of glutamine residues and asparagine residues in proteins. Specifically, protein glutaminase derived from Chryseobacterium gleum JCM2410 strain and protein glutaminase derived from Chryseobacterium proteolyticum 9670 strain (Patent Document 1) are known.

[0003] When a protein is treated with protein glutaminase, a carboxyl group is generated, so it is considered that an increase in the negative charge of the protein results in an increase in the solubility and water dispersibility of the protein, and an improvement in the emulsifying power and emulsifying stability of the protein by changing the higher-order structure of the protein.

[0004] For this reason, protein glutaminase is used in technologies to modify the functional properties of various proteins. For example, Patent Document 2 discloses a method for modifying proteins by acting on them with protein glutaminase and transglutaminase, and describes how treating the raw meat with protein glutaminase and transglutaminase in the production of yogurt using milk, tofu using soy milk, and noodles using wheat improved the smoothness of the resulting foods. Furthermore, Patent Document 3 discloses a method for producing processed meat products using arginine or its salt and protein glutaminase, and describes how treating the raw meat with arginine or its salt and protein glutaminase in the production of sausages, ham, hamburgers, fried chicken, pork cutlets, and char siu improved the texture of the resulting processed foods. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2010 / 029685 [Patent Document 2] International Publication No. 2009 / 113628 [Patent Document 3] International Publication No. 2012 / 060409 [Overview of the project] [Problems that the invention aims to solve]

[0006] The effect of protein glutaminase in denaturing proteins is recognized as producing various desirable functional properties in protein materials such as foods, and it is expected that protein glutaminase will continue to be widely used in food processing. On the other hand, conventional protein glutaminases have inherent thermal properties, so the temperature conditions for processing proteins are inevitably limited according to the thermal properties of the protein glutaminase. Therefore, in order to expand the applications of protein glutaminase to a wider range of uses, it is desirable to further improve its heat resistance.

[0007] Therefore, the present invention aims to provide a protein glutaminase with improved heat resistance. [Means for solving the problem]

[0008] The inventors have discovered a protein glutaminase with improved heat resistance through screening from a library of unknown bacterial strains owned by the applicant. The present invention is completed based on this finding. That is, the present invention provides the invention in the following embodiments.

[0009] Item 1. Protein glutaminase consisting of a polypeptide as shown in any of the following (1) to (3): (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, (2) A polypeptide having one or more amino acid residues substituted, added, inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1 or 2, and exhibiting heat resistance equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, and (3) A polypeptide having an amino acid sequence of 76% or more in the amino acid sequence shown in SEQ ID NO: 1 or 2, and exhibiting heat resistance equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2. Item 2. The DNA encoding the protein glutaminase described in Item 1. Item 3. An expression cassette or recombinant vector containing the DNA described in Item 2. Item 4. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in Item 3. Item 5. A method for producing protein glutaminase according to Item 1, comprising the step of culturing the transformant described in Item 4. Item 6. An enzyme preparation containing the protein glutaminase described in Item 1. Item 7. A protein modifier containing the protein glutaminase described in Item 1. Item 8. A method for producing a modified protein material, comprising the step of reacting the protein glutaminase described in Item 1 with the protein material. Item 9. The method for producing the protein material according to item 8, wherein the protein material is edible. [Effects of the Invention]

[0010] According to the present invention, a protein glutaminase with improved heat resistance is provided. [Brief explanation of the drawing]

[0011] [Figure 1] This is an SDS-PAGE image of the purified protein glutaminase fraction derived from strain 57594 of Chryseobacterium sp. [Figure 2] This is an SDS-PAGE image of the purified protein glutaminase fraction derived from strain 61798 of Chryseobacterium sp. [Figure 3] These are the temperature stability test results for protein glutaminase derived from strains 57594 and 61798 of Chryseobacterium sp. [Figure 4] These are the results of optimal temperature tests for protein glutaminase derived from strains 57594 and 61798 of Chryseobacterium sp. [Figure 5]Results of the pH stability test of protein glutaminase derived from strains 57594 and 61798 of Chryseobacterium sp. [Figure 6] Results of the optimum pH test of protein glutaminase derived from strains 57594 and 61798 of Chryseobacterium sp.

Mode for Carrying Out the Invention

[0012] Hereinafter, the present invention will be described in detail. Except in the sequence listing, the 20 types of amino acid residues in the amino acid sequence may be represented by one-letter abbreviations. That is, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine (Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P.

[0013] In this specification, the amino acid sequence shown has the N-terminus at the left end and the C-terminus at the right end.

[0014] In this specification, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "acidic amino acids" include aspartic acid and glutamic acid. "basic amino acids" include lysine, arginine, and histidine.

[0015] As used herein, "substitution" includes not only the case where an artificial substitution of an amino acid residue is introduced, but also the case where a natural substitution of an amino acid residue is introduced, that is, the case where the amino acid residues are originally different. In this specification, the substitution of an amino acid residue may be an artificial substitution or a natural substitution, but an artificial substitution is preferred.

[0016] 1. Protein glutaminase The protein glutaminase of the present invention consists of a polypeptide shown in any one of the following (1) to (3).

[0017] (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) A polypeptide in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 or 2, and which exhibits heat resistance equivalent to that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; and (3) A polypeptide in which the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 is 76% or more in the amino acid sequence shown in SEQ ID NO: 1 or 2, and which exhibits heat resistance equivalent to that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0018] The polypeptides shown in (1) to (3) have protein glutaminase activity and improved heat resistance.

[0019] The polypeptide shown in (1) is a protein glutaminase derived from Chryseobacterium sp., and the polypeptides shown in (2) and (3) are variants of the polypeptide shown in (1). The polypeptides of (1) to (3) include not only polypeptides obtained by artificial substitution, but also polypeptides originally having such an amino acid sequence.

[0020] In the polypeptide of (2) above, the modification of the amino acids introduced may include only one type of modification (e.g., only substitution) from among substitution, addition, insertion, and deletion, or it may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide of (2) above, the number of amino acid differences at any difference site may be one or several, for example, 1 to 80, preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, or 1 to 30, more preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0021] Furthermore, in the polypeptide of (3) above, the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 or 2 should be 76% or more, but preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and particularly preferably 99% or more.

[0022] Here, in the polypeptide of (3) above, the sequence identity for each amino acid sequence shown in SEQ ID NO: 1 or 2 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 1 or 2. Furthermore, "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0023] In the polypeptides described in (2) and (3) above, the amino acids at positions 176 (cysteine), 217 (histidine), and 237 (aspartic acid) in the amino acid sequences shown in SEQ ID NOs: 1 and 2 are considered to be protein glutaminase activity catalytic residues; therefore, it is desirable not to introduce substitutions or deletions at these sites.

[0024] In the polypeptides of (2) and (3) above, when an amino acid substitution is introduced for SEQ ID NO: 1 or 2, a preferred form of the introduced amino acid substitution is a conservative substitution. Specifically, substitutions in the polypeptides of (2) and (3) above include, for example, substitution of the amino acid before substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution of the amino acid before substitution with another noncharged amino acid if the amino acid before substitution is an acidic amino acid, and substitution of the amino acid before substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0025] In the polypeptides of (2) and (3) above, "exhibiting heat resistance equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2" means that when heat resistance, i.e., relative protein glutaminase activity at 65°C, is measured, the relative protein glutaminase activity is equivalent to that of the polypeptide of (1) above. Specifically, when the relative protein glutaminase activity of the polypeptide of (1) above is set to 1, the relative protein glutaminase activity of the polypeptide is approximately 0.8 to 1.2. The relative protein glutaminase activity at 65°C refers to the relative amount (%) of the protein glutaminase activity of the enzyme exposed to the 65°C temperature condition, when the protein glutaminase activity of the enzyme exposed to the 4°C temperature condition is set to 100%.

[0026] 2. DNA The DNA of the present invention is DNA that encodes the protein glutaminase described in "1. Protein Glutaminase" above (hereinafter also referred to as "the predetermined protein glutaminase"). The DNA of the present invention includes DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4. The base sequence shown in SEQ ID NO: 3 is a gene that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 above, and the base sequence shown in SEQ ID NO: 4 is a gene that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 above.

[0027] The DNA of the present invention is not limited to the above sequence, and any DNA having a base sequence with 76% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, even more preferably 95% or more, and particularly preferably 99% or more homology to the base sequence shown in Sequence ID No. 3 or 4 is also included in the DNA of the present invention, as long as it encodes a polypeptide having protein glutaminase activity.

[0028] Here, DNA "homology" is calculated using publicly available or commercially available software with an algorithm that compares a reference sequence to a query sequence. Specifically, BLAST, FASTA, or GENETYX (manufactured by Genetics Co., Ltd.) can be used, and these can be used with their default parameters set.

[0029] Furthermore, in response to the aforementioned substitutions, additions, insertions, or deletions of amino acid sequences, a nucleotide sequence in which several bases are substituted, added, inserted, or deleted in the nucleotide sequence described in Sequence ID No. 3 or 4 is also included in the DNA of the present invention, as long as it encodes a polypeptide having protein glutaminase activity.

[0030] Furthermore, DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having the base sequence described in SEQ ID NO: 3 or 4 is also included in the DNA of the present invention, insofar as it encodes a polypeptide having protein glutaminase activity.

[0031] Here, stringent conditions refer to the conditions under which hybridization is performed by incubating a nylon membrane immobilized with DNA together with a probe at 65°C for 20 hours in a solution containing 6×SSC (1×SSC is made by dissolving 8.76g of sodium chloride and 4.41g of sodium citrate in 1 liter of water), 1% SDS, 100 μg / mL salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficol (in this specification, "%" means "w / v").

[0032] For detailed procedures regarding hybridization, refer to Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)), etc.

[0033] An example of a method for obtaining the DNA of the present invention by hybridization is shown below, but the method for obtaining the DNA of the present invention is not limited to the following.

[0034] First, DNA obtained from a suitable gene source is linked to a plasmid or phage vector according to standard procedures to create a DNA library. This library is introduced into a suitable host, and the resulting transformants are cultured on a plate. The grown colonies or plaques are transferred to a nitrocellulose or nylon membrane, and after denaturation treatment, the DNA is immobilized on the membrane. This membrane is then incubated in a solution of the above composition containing a probe pre-labeled with 32P, etc., under the above stringent conditions, and hybridization is performed. As the probe, a polynucleotide encoding all or part of the amino acid sequence described in SEQ ID NO: 1 or 2 can be used.

[0035] After hybridization is complete, nonspecifically adsorbed probes are washed away, and clones that have formed hybrids with the probes are identified by autoradiography or other methods. This procedure is repeated until hybrid-forming clones can be isolated. Finally, from the obtained clones, the gene encoding the protein with the desired enzyme activity is selected. Gene isolation can be performed by known polynucleotide extraction methods such as the alkaline method.

[0036] The DNA of the present invention can also be isolated from microorganisms that produce the specified protein glutaminase described above. For example, using genomic DNA from Chryseobacterium sp. as a template, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using primers or probes designed based on known amino acid sequence information considering gene degeneracy, or primers or probes designed based on known base sequence information.

[0037] The DNA of this invention encompasses various types of DNA derived from codon degeneracy. Artificially creating various types of DNA encoding the same amino acid sequence can be easily done using known genetic engineering techniques. For example, in the production of genetically engineered proteins, if the codons used on the original gene encoding the target protein are infrequently used in the host, the protein expression level may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon utilization frequency for the host without altering the encoded amino acid sequence.

[0038] As an indicator of codon utilization frequency, the sum of the host-optimal codon utilization frequencies for each codon can be adopted. An optimal codon is defined as the codon with the highest utilization frequency among codons corresponding to the same amino acid. Codon utilization frequency is not particularly limited as long as it is optimized for the host, but the following are examples of optimal codons in E. coli. F: Phenylalanine (ttt), L: Leucine (ctg), I: Isoleucine (att), M: Methionine (atg), V: Valine (gtg), Y: Tyrosine (tat), Stop codon (taa), H: Histidine (cat), Q: Glutamine (cag), N: Asparagine (aat), K: Lysine (aaa), D: Aspartic acid (gat), E: Glutamic acid (gaa), S: Serine (agc), P: Proline (ccg), T: Threonine (acc), A: Alanine (gcg), C: Cysteine ​​(tgc), W: Tryptophan (tgg), R: Arginine (cgc), G: Glycine (ggc).

[0039] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known techniques such as the Kunkel method and the Gapped duplex method, as well as mutation introduction kits utilizing site-directed mutagenesis, such as the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneArt™ Site-Directed Mutagenesis PLUS System (Invitrogen), and the TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, PrimeSTAR Mutagenesis Basal Kit, etc.: Takara Bio).

[0040] The base sequence of DNA can be confirmed by sequencing using conventional methods. For example, it can be done by dideoxynucleotide chain termination (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using a suitable DNA sequencer.

[0041] To confirm whether the obtained DNA encodes the target protein glutaminase, the determined base sequence can be compared with the base sequence described in SEQ ID NO: 3 or 4. Alternatively, the amino acid sequence predicted from the determined base sequence can be compared with the amino acid sequence described in SEQ ID NO: 1 or 2.

[0042] 3. Expression cassette or recombinant vector An expression cassette or recombinant vector containing DNA encoding the specified protein glutaminase (hereinafter also referred to as "the expression cassette of the present invention" or "the recombinant vector of the present invention") can be obtained by linking a promoter and a terminator to the DNA of the present invention, or by inserting the expression cassette of the present invention or the DNA of the present invention into an expression vector.

[0043] The expression cassette or recombinant vector of the present invention may, as regulatory factors, include a promoter and a terminator, as well as, if necessary, transcription elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites. These regulatory factors only need to be operably ligated to the DNA of the present invention. Operable ligation means that the various regulatory factors that regulate the DNA of the present invention and the DNA of the present invention are ligated in a manner that allows them to function in a host cell.

[0044] The recombinant vector of the present invention is preferably constructed from a phage, plasmid, or virus capable of autonomously proliferating within a host for genetic recombination. Such expression vectors are well known, and commercially available examples include pQE vectors (Qiagen Co., Ltd.), pDR540, pRIT2T (GE Healthcare Biosciences Co., Ltd.), and pET vectors (Merck KGaA). The expression vector can be used in any combination with a host cell. For example, when using Escherichia coli as the host cell, examples include a combination of a pET vector and the DH5α strain, a combination of a pET vector and the BL21(DE3) strain, or a combination of a pDR540 vector and the JM109 strain.

[0045] 4. Transformed organism A transformant (hereinafter also referred to as "the transformant of the present invention") can be obtained by transforming a host using the expression cassette or recombinant vector of the present invention.

[0046] The host used to produce the transformant is not particularly limited as long as it is capable of gene introduction, the expression cassette or recombinant vector is stable, autonomously replicates, and expresses the traits of the gene containing the DNA of the present invention. Suitable examples include bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, and the Pseudomonas genus such as Pseudomonas putida; yeast, etc., but animal cells, insect cells, plants, etc. may also be used.

[0047] The transformant of the present invention can be obtained by introducing the expression cassette or recombinant vector of the present invention into host cells. The site in which the DNA of the present invention is introduced is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on a genome. Specific methods for introducing the expression cassette or recombinant vector of the present invention include, for example, recombinant vector methods and genome editing methods. The conditions for introducing the expression cassette or recombinant vector into the host may be set appropriately depending on the type of host, etc. If the host is a bacterium, for example, methods using competent cells treated with calcium ions and electroporation methods may be used. If the host is a yeast, for example, electroporation, spheroplast methods and lithium acetate methods may be used. If the host is an animal, for example, electroporation, calcium phosphate methods and lipofection methods may be used. If the host is an insect, for example, calcium phosphate methods, lipofection methods and electroporation methods may be used. If the host is a plant, for example, electroporation, Agrobacterium methods, particle gun methods and PEG methods may be used.

[0048] Whether or not the expression cassette or recombinant vector of the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0049] To confirm whether the expression cassette or recombinant vector of the present invention has been incorporated into a host by PCR, for example, the genomic DNA, expression cassette, or recombinant vector can be isolated and purified from the transformant.

[0050] The isolation and purification of expression cassettes or recombinant vectors are performed, for example, based on the lysate obtained by lysing bacteria when the host is a bacterium. Lysis is performed by treating the bacteria with a lytic enzyme such as lysozyme, and if necessary, proteases, other enzymes, and surfactants such as sodium lauryl sulfate (SDS) are used in combination.

[0051] Furthermore, physical disruption methods such as freeze-thaw cycles and French presses may be combined. DNA isolation and purification from lysates can be performed, for example, by deproteinization using phenol and protease treatments, ribonuclease treatments, alcohol precipitation, and by using commercially available kits in appropriate combinations.

[0052] DNA cleavage can be performed according to standard methods, for example, using restriction enzyme digestion. For example, type II restriction enzymes that act on specific nucleotide sequences can be used. Binding of DNA to an expression cassette or expression vector can be performed, for example, using a DNA ligase.

[0053] Subsequently, using the separated and purified DNA as a template, primers specific to the DNA of the present invention are designed and PCR is performed. The amplification product obtained by PCR is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide and SYBR Green solution, and the transformation can be confirmed by detecting the amplification product as a band.

[0054] Furthermore, PCR can be performed using primers pre-labeled with fluorescent dyes or the like to detect the amplified product. Additionally, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and confirmed by fluorescence and enzymatic reactions.

[0055] 5. Method for producing protein glutaminase The above-mentioned predetermined protein glutaminase can be obtained by a manufacturing method that includes a step of culturing the transformant of the present invention, or by a manufacturing method that includes a step of culturing a microorganism that produces the above-mentioned predetermined protein glutaminase.

[0056] The above culture conditions can be appropriately set considering the nutritional and physiological properties of the transformant or microorganism, but liquid culture is preferred. Furthermore, in the case of industrial production, aerated and stirred culture is preferred.

[0057] As a nutrient source for the culture medium, those necessary for the growth of the above-mentioned transformants or microorganisms may be used. As a carbon source, any assimilated carbon compound may be used, such as glucose, sucrose, lactose, maltose, molasses, pyruvate, etc.

[0058] Any nitrogen compound that can be assimilated can be used as a nitrogen source, such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean meal alkali extract.

[0059] In addition to carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, as well as certain amino acids and certain vitamins, may be used as needed.

[0060] The culture temperature can be appropriately set within a range in which the transformant or microorganism of the present invention can grow and produce the predetermined protein glutaminase, for example, about 10 to 40°C, preferably about 15 to 37°C. The culture should be completed at an appropriate time, timing it to coincide with the time when the predetermined protein glutaminase reaches its maximum yield, and typically the culture time is about 6 to 96 hours or 12 to 48 hours.

[0061] After culturing the above-mentioned transformants or microorganisms, the culture supernatant or bacterial cells are collected from the culture medium by methods such as centrifugation. The bacterial cells are then treated with mechanical methods such as ultrasound and French press, or with lytic enzymes such as lysozyme. If necessary, enzymes such as proteases or surfactants such as sodium lauryl sulfate (SDS) are used to solubilize them and obtain a water-soluble fraction containing the specified protein glutaminase.

[0062] Furthermore, by selecting an appropriate expression cassette or expression vector and host, the expressed predetermined protein glutaminase can be secreted into the culture medium.

[0063] The water-soluble fraction containing the predetermined protein glutaminase obtained as described above may be subjected to purification as is, or the predetermined protein glutaminase in the water-soluble fraction may be concentrated before being subjected to purification.

[0064] Concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting-out treatment, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, and acetone).

[0065] The purification process of the specified protein glutaminase described above can be carried out by appropriately combining methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0066] The specified protein glutaminase purified in this manner can be powdered by freeze-drying, vacuum-drying, spray-drying, or other methods as needed and distributed to the market.

[0067] 6. Enzyme preparations The above-mentioned specified protein glutaminase can be provided in the form of an enzyme preparation. Therefore, the present invention also provides an enzyme preparation containing the above-mentioned specified protein glutaminase as an active ingredient.

[0068] The content of the predetermined protein glutaminase in the enzyme preparation of the present invention is not particularly limited, but examples include 0.1 U / g or more, 1 U / g or more, preferably 10 U / g or more, more preferably 100 U / g or more, even more preferably 250 U / g or more, and particularly preferably 500 U / g or more, 1000 U / g or more, 1500 U / g or more, 2000 U / g or more, and 5000 U / g or more.

[0069] The enzyme preparation of the present invention may contain other components in addition to the specified protein glutaminase described above, to an extent that does not affect the effects of the present invention. Examples of other components include other enzymes other than the specified protein glutaminase described above, additives, and culture residues generated by the above manufacturing method.

[0070] Other enzymes include, for example, amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamide enzymes (other than the above active ingredients), pullulanase, etc. These other enzymes may be included individually or in combination of multiple types.

[0071] Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, antimicrobials, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include benzoates (alkali metal salts such as potassium and sodium salts), sorbates (alkali metal salts such as potassium and sodium salts), phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antimicrobials include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. These additives may be included individually or in combination.

[0072] Culture residues include components derived from the culture medium, contaminating proteins, and bacterial cell components.

[0073] The form of the enzyme preparation of the present invention is not particularly limited and can be, for example, liquid or solid (powder, granules, etc.). The composition can be prepared by generally known methods.

[0074] 7.Applications The specified protein glutaminase described above can be used for known applications of protein glutaminase. For example, the specified protein glutaminase can be used for the purpose of modifying proteins. Accordingly, the present invention also provides a protein modifier containing the specified protein glutaminase described above.

[0075] The specific methods of protein modification are not particularly limited, and any change in the protein's properties resulting from the deamidation of the γ-amide and β-amide groups of glutamine and asparagine residues in the protein, thereby generating carboxyl groups, is acceptable. Specifically, protein modification can include increasing protein solubility, increasing water dispersibility, improving emulsifying power, and improving emulsion stability.

[0076] Because the specified protein glutaminase described above has improved heat resistance, the enzyme preparation of the present invention is particularly useful when the protein to be modified is treated at a relatively high temperature and / or when it is treated under heating conditions for a long period of time. Specifically, the specified protein glutaminase described above is preferably used when the protein to be modified is treated under heating conditions of 40 to 80°C, preferably 46 to 77°C, more preferably 53 to 74°C, and most preferably 60 to 70°C.

[0077] 8. Method for producing modified protein materials As described above, the specified protein glutaminase can be used for the purpose of modifying proteins. Accordingly, the present invention also provides a method for producing a modified protein material, which includes the step of acting the specified protein glutaminase on a protein material.

[0078] In the manufacturing method of the present invention, a mixture containing a protein material and a predetermined protein glutaminase is subjected to predetermined protein glutaminase operating conditions to carry out a protein modification reaction.

[0079] The protein material is not particularly limited as long as it contains protein, and may be either edible or non-edible. Edible protein materials can be used as food or beverages, or as ingredients for manufacturing food or beverages. Non-edible protein materials can be used as materials for protein experiments, medical materials, or cosmetic materials.

[0080] Specific examples of protein materials include those obtained by processing a protein source to increase its protein content using known methods, and are appropriately selected by those skilled in the art. For example, edible protein materials include plant protein materials obtained from foods containing plant protein, and animal proteins. Examples of plant proteins include bean proteins such as soy protein, broad bean protein, pea protein, chickpea protein, mung bean protein, and lupin bean protein; grain proteins such as wheat protein, rye protein, oat protein, and corn protein; and seed proteins such as canary seed, flaxseed, almond, cashew nut, hazelnut, pecan nut, macadamia nut, pistachio, walnut, Brazil nut, peanut, coconut, hemp seed (industrial hemp), pili nut, chestnut, sesame, and pine nut. Examples of animal proteins include meat, fish meat, and egg protein. Examples of non-edible protein materials include albumin and globulin derived from biological samples such as egg white and serum.

[0081] The protein content in these protein materials is not particularly limited, but examples include 30% by weight or more, 40% by weight or more, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and even more preferably 80% by weight or more. The upper limit of the protein content in the protein material is not particularly limited, but examples include 95% by weight or less, 90% by weight or less, 85% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less.

[0082] The amount of protein material in the mixture is such that the concentration of protein contained in the protein material in the mixture is, for example, 0.1% by weight or more, 0.3% by weight or more, preferably 0.7% by weight or more, and more preferably 1.4% by weight or more. The upper limit of the concentration of protein contained in the protein material in the mixture is not particularly limited, but examples include 80% by weight or less, 60% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, 8% by weight or less, 5% by weight or less, 3% by weight or less, or 2% by weight or less.

[0083] The amount of predetermined protein glutaminase used is not particularly limited, but examples of the amount of predetermined protein glutaminase used per gram of protein contained in the protein material include 0.1 U or more, preferably 0.5 U or more, more preferably 1 U or more, even more preferably 2 U or more, even more preferably 3.5 U or more, and even more preferably 4.5 U or more. The upper limit of the amount of predetermined protein glutaminase used per gram of protein contained in the protein material is not particularly limited, but examples of 45 U or less, 35 U or less, 20 U or less, 10 U or less, 8 U or less, or 5.5 U or less.

[0084] Furthermore, the amount of a predetermined protein glutaminase used per gram of protein material can be, for example, 0.1 U or more, preferably 0.5 U or more, more preferably 1 U or more, even more preferably 2 U or more, even more preferably 3 U or more, and even more preferably 4 U or more. There is no particular upper limit to the amount of a predetermined protein glutaminase used per gram of protein material, but examples include 40 U or less, 30 U or less, 20 U or less, 10 U or less, 7 U or less, or 5 U or less.

[0085] For the activity of protein deamide enzymes (protein glutaminases), one unit (1U) of enzyme is defined as the amount of enzyme that converts 1 μmol of ammonia per minute using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as a substrate.

[0086] The operating conditions for the specified protein glutaminase are appropriately determined based on the optimal temperature and pH of the protein glutaminase used.

[0087] Among the operating conditions for the specified protein glutaminase, suitable temperature conditions include, for example, 50-80°C, preferably 60-80°C, and more preferably 63-80°C. The specified protein glutaminase has excellent heat resistance and is particularly useful when used under conditions of 65°C or higher. From this viewpoint, suitable temperature conditions include, even more preferably, 65-80°C, more preferably 65-75°C, even more preferably 65-70°C, and even more preferably 65-67°C. On the other hand, under conditions below 65°C, the specified protein glutaminase tends to show a lower relative value to its activity at the optimal temperature as the temperature decreases, but because it possesses high protein deamidation ability, it exhibits excellent protein deamidation ability even under conditions below 65°C. From this viewpoint, suitable examples of suitable temperature conditions include 50°C to less than 65°C, 55°C to less than 65°C, 60°C to less than 65°C, and 63°C to less than 65°C.

[0088] Examples of pH conditions for the action of a given protein glutaminase include 2 to 12, preferably 3 to 10, and more preferably 4 to 9.

[0089] The duration for which the specified protein glutaminase is applied is not particularly limited and can be determined appropriately according to the preparation scale, etc., but examples include 1 hour or more, preferably 8 hours or more, more preferably 16 hours or more, and even more preferably 20 hours or more. There is no particular upper limit to the range of this time, but examples include 40 hours or less, 30 hours or less, or 25 hours or less.

[0090] After the reaction is complete, the enzyme is deactivated, followed by cooling, and post-processing is performed as needed to obtain the modified protein material. [Examples]

[0091] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0092] [Test Example 1] (1) Selection of heat-resistant protein glutaminase-producing strains From the applicant's library of undisclosed bacterial strains, 57,594 and 61,798 strains of Chryseobacterium sp. were selected based on protein glutaminase activity and temperature stability, and stored in a glycerol-containing culture medium.

[0093] (2) Culture of selected strains The components listed in Table 1 were dissolved in water to the indicated concentrations, and the culture medium was prepared by autoclaving at 121°C for 30 minutes. The selected strains were inoculated into the culture medium and incubated at 120 rpm, 30°C, for 40-48 hours. After incubation, the culture medium was centrifuged to collect the bacterial cells, and only the supernatant was collected. Diatomaceous earth was added to the supernatant and filtered, and the culture filtrate was further concentrated using an ultrafiltration membrane.

[0094] (3) Purification of enzymes The concentrated solution was purified by salting out, phenyl-sepharose, and Sephacryl S-100. SDS-PAGE images of the purified concentrated fractions from the cultures of strains 57594 and 61798 are shown in Figures 1 and 2, respectively. In Figure 1, lane 7 represents the pre-purification fraction, lanes 8-10 represent the purified fraction from the strain 57594 culture, and lane 11 represents the molecular marker. In Figure 2, lane 1 represents the molecular marker, and lanes 2-4 represent the purified fraction from the strain 61798 culture. As shown in Figures 1 and 2, 18 kDa (band indicated by arrow) was confirmed as a purified product in the purified fraction.

[0095] [Table 1]

[0096] (4) Enzymatic property evaluation test The following enzymatic properties were evaluated for the 18kDa purified fractions (protein glutaminase from strain 57594 and protein glutaminase from strain 61798) obtained from the culture media of strains 57594 and 61798, respectively. The same enzymatic properties were also evaluated for the protein glutaminase (PG for comparison) derived from the existing Chryseobacterium proteoricum strain 9670.

[0097] [Table 2]

[0098] (4-1) Measurement of protein glutaminase activity N-benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly; Peptide Institute) was dissolved in 0.2 mol / L phosphate buffer (pH 6.5) and a solution prepared to 30 mmol / L was used as the substrate solution. • 0.1 mL of the enzyme solution whose activity was to be measured was placed in a test tube, left in a constant temperature water bath at 37±0.5°C for 1 minute, then 1 mL of the substrate solution, which had been left at 37±0.5°C for 10 minutes beforehand, was added and immediately mixed. • After allowing the solution to stand for 10 minutes to allow the enzymatic reaction to occur, 1 mL of 0.4 mol / L trichloroacetic acid solution was added to stop the enzymatic reaction. The measurement blank was prepared by adding 0.1 mL of enzyme solution to a test tube, followed by 1 mL of 0.4 mol / L trichloroacetic acid solution and 1 mL of substrate solution. A color reaction was performed using ammonia-Test Wako (Fujifilm Wako Pure Chemical Industries), and the amount of ammonia released by the enzymatic reaction for 10 minutes was quantified based on the absorbance value at a wavelength of 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute was defined as 1 unit (1 U), and the activity value was calculated from the amount of ammonia released by the enzymatic reaction.

[0099] (4-2) Temperature stability test The purified fraction was replaced with 0.2 mol / L phosphate buffer (pH 6.5), allowed to stand at 4°C for 10 minutes, or heated at 40°C, 50°C, 60°C, 65°C, 70°C, or 75°C for 10 minutes. Then, the enzymatic reaction was carried out using method (4-1) to measure the protein glutaminase activity. The relative amount of protein glutaminase activity of the purified fraction exposed to each heating condition was calculated as the protein glutaminase relative activity (%), with the protein glutaminase activity of the purified fraction exposed to 4°C being set to 100%. The results are shown in Figure 3. As shown in Figure 3, the relative activity of the comparative PG at 65°C was 11%, while the relative activity of the protein glutaminase from strain 57594 was 65%, and the relative activity of the protein glutaminase from strain 61798 was 66%, indicating a significant improvement in heat resistance.

[0100] (4-3) Optimal temperature test Protein glutaminase activity was measured in the same manner as in (4-1), except that the enzyme reaction temperature was changed to 37°C, 50°C, 55°C, 60°C, 65°C, or 70°C. The relative amount of protein glutaminase activity in the purified fractions exposed to each temperature condition was calculated as the relative protein glutaminase activity (%), with the protein glutaminase activity at the temperature condition showing the maximum activity of each purified fraction (60°C for the comparative PG, and 65°C for the PG derived from strain 57594 and strain 61798) set as 100%. The results are shown in Figure 4. As shown in Figure 4, while the optimal temperature for the comparative PG was 60°C, the optimal temperature for the protein glutaminases derived from strains 57594 and 61798 had improved to 65°C.

[0101] (4-4) pH stability test The purified fraction was adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 using Britton-Robinson buffer. After standing at 37°C overnight (18 hours) at each pH, ​​the enzymatic reaction was carried out using method (4-1) to measure protein glutaminase activity. The relative amount of protein glutaminase activity in the purified fraction exposed to each pH condition was calculated as the relative protein glutaminase activity (%), with the protein glutaminase activity at pH 6 set to 100%. The results are shown in Figure 5.

[0102] (4-5) Optimal pH test The pH of the substrate solution was adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 using Britton-Robinson buffer, and the enzyme reaction was carried out at each pH level. The protein glutaminase activity was measured in the same manner as in (4-1), except that the enzyme reaction was performed at each pH level. When the protein glutaminase activity at pH 6 was set to 100%, the relative amount of protein glutaminase activity in the purified fraction exposed to each pH condition was calculated as the relative protein glutaminase activity (%). The results are shown in Figure 6.

[0103] (5) Sequence Degenerate primers were designed from sequence information of Chryseobacterium bacteria presumed to be closely related to strains 57594 and 61798. Using these degenerate primers, PrimeSTAR was performed using the genomic DNA of strains 57594 and 61798 as templates. (R) DNA fragments were amplified by PCR using GXL DNA Polymerase (Takara Bio Inc.). (R) The amplified fragments were recovered using Gel and PCR Clean-up (Takara Bio Inc.), and sequence analysis was performed using degenerate primers. As a result, the amino acid sequence of protein glutaminase from strain 57594 (SEQ ID NO: 1), the nucleotide sequence around the protein glutaminase gene from strain 57594 including the structural gene encoding SEQ ID NO: 1 (SEQ ID NO: 3) (SEQ ID NO: 5), the amino acid sequence of protein glutaminase from strain 61798 (SEQ ID NO: 2), and the nucleotide sequence around the protein glutaminase gene from strain 61798 including the structural gene encoding SEQ ID NO: 2 (SEQ ID NO: 4) (SEQ ID NO: 6) were determined.

[0104] [Test Example 2] In this test example, protein glutaminase derived from strain 57594 of Example 1 and protein glutaminase derived from strain 61798 of Example 2 were used to perform modification (deamidation) reactions of various protein materials. Similarly, the protein glutaminase derived from the existing comparative example 1, Chryseobacterium proteoricum strain 9670 (comparative PG), was also used to perform the following protein modification (deamidation) reactions.

[0105] Specifically, 20 mg of protein material was added to 1 mL of 50 mM phosphate buffer (pH 7.0) and suspended. Protein glutaminase was added to a concentration of 0.23 mg / g-protein (the detailed amounts of each component are shown in Table 3), and the reaction was carried out for 24 hours at 55°C, 60°C, 65°C, or 70°C. After that, 1 mL of 0.4 M trichloroacetic acid solution was added to stop the reaction. The amount of free ammonia was quantified using Ammonia-Test Wako (Fujifilm Wako Pure Chemical Industries). The protein deamidation rate was derived by dividing the obtained amount of free ammonia by the amount of free ammonia in the protein material suspension before the addition of protein glutaminase. The amount of free ammonia in the protein material suspension before the addition of protein glutaminase was measured as follows: 3N concentrated sulfuric acid was added to the protein material suspension before enzyme addition and mixed. After treatment at 110°C for 3 hours, it was cooled and neutralized with 6N sodium hydroxide, and the supernatant was obtained by centrifugation. The amount of free ammonia in the obtained supernatant was quantified using ammonia-Test Wako (Fujifilm Wako Pure Chemical Industries). Furthermore, the multiple of the protein deamidation rate in each example, relative to the protein deamidation rate using PG in Comparative Example 1 (which was set to 1), was derived as the deamidation acceleration rate. The results are shown in Table 3.

[0106] [Table 3]

[0107] As is clear from Table 3, for all food materials, regardless of the reaction temperature, superior protein deamidation rates were observed when using the protein glutaminase derived from strain 57594 in Example 1 and the protein glutaminase derived from strain 61798 in Example 2 compared with the protein glutaminase in Comparative Example 1 (Comparative Examples 2-4) (Examples 3-8). In particular, for food materials such as peas and soybeans, a particularly high rate of protein deamidation promotion was observed when the reaction temperature was 60°C (Examples 3-6), and for albumin, a high rate of protein deamidation promotion was observed at reaction temperatures of 60°C, 65°C, 70°C, and especially at 60°C (Examples 7-8).

[0108] [Test Example 3] In this test, the solubilizing ability of pea protein was tested using protein glutaminase derived from strain 57594 of Example 1 and protein glutaminase derived from strain 61798 of Example 2. The same solubilizing ability test was also performed on protein glutaminase derived from the existing comparative example 1, Chryseobacterium proteoricum strain 9670 (comparative PG).

[0109] Specifically, 100 mg of pea protein material (protein content: 82% by weight) was added to 10 mL of 50 mM phosphate buffer (pH 7.0) and suspended. Protein glutaminase was then added to a total concentration of 0.23 mg / g-protein (the detailed amounts of each component are shown in Table 4), and the mixture was reacted at 65°C for 24 hours.

[0110] A 1 mL sample was withdrawn, and the reaction was stopped by adding 1 mL of 0.4 M trichloroacetic acid solution. The amount of free ammonia was quantified using Ammonia-Test Wako (Fujifilm Wako Pure Chemical Industries). The protein deamidation rate was derived by dividing the obtained amount of free ammonia by the amount of free ammonia in the protein material suspension before the addition of protein glutaminase. The results are shown in Table 4.

[0111] For the remaining samples, the reaction was stopped by immersing the containers containing the reaction composition in boiling water and subjecting them to heat treatment. Desalting was performed with purified water, and deamidated protein (modified protein) powder was obtained by freeze-drying. 2 mg of the obtained freeze-dried deamidated protein (modified protein) powder was dissolved in 1 mL of Britton-Robinson buffer with a pH of 2 to 12, and shaken at room temperature for 30 minutes. Centrifugation was performed at 15,000 × g for 5 minutes, and the dissolved protein concentration of the supernatant was measured by the Lowry method. Furthermore, the multiple of the dissolved protein concentration in each example compared to the dissolved protein concentration using PG in Comparative Example 1 (set to 1) was derived as the protein dissolution acceleration rate. The results are shown in Table 4 (in Table 4, the shaded areas indicate no data).

[0112] [Table 4]

[0113] As is clear from Table 4, superior protein deamidation rates were observed when using the protein glutaminase derived from strain 57594 in Example 1 and the protein glutaminase derived from strain 61798 in Example 2 compared to the case using the protein glutaminase of Comparative Example 1 (Comparative Example 5) (Examples 9-10). Furthermore, the solubility of the obtained modified protein was improved at all pH levels compared to the case using the protein glutaminase of Comparative Example 1 (Comparative Example 5) (Examples 9-10), and a correlation was observed with the improvement in protein deamidation rates.

[0114] [Test Example 4] In this test, the deamidation ability of ovalbumin under alkaline conditions was tested using protein glutaminase derived from strain 57594 of Example 1 and protein glutaminase derived from strain 61798 of Example 2. The same deamidation ability test was also performed on protein glutaminase derived from existing Comparative Example 1, Chryseobacterium proteoricum strain 9670 (comparative PG).

[0115] Specifically, 20 mg of ovalbumin (protein content: 81% by weight) was added to 1 mL of Britton-Robinson buffer (pH 7.0 or pH 10.0) and suspended. Protein glutaminase was then added to a concentration of 0.23 mg / g-protein (more detailed amounts of each component are shown in Table 5), and the mixture was reacted at 37°C for 24 hours. Subsequently, 1 mL of 0.4 M trichloroacetic acid solution was added to stop the reaction. The amount of free ammonia was quantified using Ammonia-Test Wako (Fujifilm Wako Pure Chemical Industries). The protein deamidation rate was derived by dividing the obtained amount of free ammonia by the amount of free ammonia in the protein material suspension before the addition of protein glutaminase. Furthermore, the multiple of the protein deamidation rate at pH 10, with the protein deamidation rate at pH 7 set to 1, was derived as the deamidation acceleration rate under alkaline conditions. The results are shown in Table 5.

[0116] [Table 5]

[0117] As is clear from Table 5, compared to the case using the protein glutaminase of Comparative Example 1 (Comparative Example 6), an improvement in the rate of protein deamidation promotion under alkaline conditions was observed when using the protein glutaminase derived from strain 57594 of Example 1 and the protein glutaminase derived from strain 61798 of Example 2 (Examples 11-12).

Claims

1. Protein glutaminase consisting of one of the polypeptides shown in (1) to (3) below: (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, (2) A polypeptide having one or more amino acid residues substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 or 2, and exhibiting heat resistance equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, and (3) A polypeptide having an amino acid sequence in the amino acid sequence shown in SEQ ID NO: 1 or 2 that exhibits 76% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 2, and that exhibits heat resistance equivalent to that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.

2. DNA encoding the protein glutaminase described in claim 1.

3. An expression cassette or recombinant vector comprising the DNA described in claim 2.

4. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in claim 3.

5. A method for producing protein glutaminase according to claim 1, comprising the step of culturing the transformant according to claim 4.

6. An enzyme preparation comprising the protein glutaminase described in claim 1.

7. A protein modifier comprising the protein glutaminase described in claim 1.

8. A method for producing a modified protein material, comprising the step of reacting a protein glutaminase described in claim 1 with a protein material.

9. The manufacturing method according to claim 8, wherein the protein material is edible.

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