Novel bacillus subtilis bm107 strain and use thereof

Abstract

The present invention relates to: Bacillus subtilis comprising a gene encoding a novel esterase; and a use thereof. The present invention provides a novel Bacillus subtilis which can efficiently enhance the production of butyrate by breaking down tributyrin in the body or intestine, and exhibits therapeutic and preventive effects on inflammatory bowel disease on the basis thereof.

Description

Novel Bacillus subtilis BM107 strain and its use

[0001] The present invention relates to Bacillus subtilis comprising a gene encoding a novel esterase and to uses thereof.

[0002] Butyrate, also known as buytyrate or bytyric acid, is a short-chain fatty acid produced by butyrate-producing bacteria in the intestines of humans and animals through the fermentation of dietary fiber. Recent studies have shown that butyrate exhibits various physiological effects, including anti-inflammatory and colon cancer prevention, and that butyrate may prevent or improve various diseases.

[0003] More specifically, butyrate serves as the primary energy source for colon cells and maintains the integrity of the intestinal mucosa, preventing intestinal leakiness. Furthermore, butyrate has been shown to reduce inflammation, and some studies have even linked it to a reduced risk of colon cancer. Furthermore, studies have shown that butyrate has positive effects on brain health and cognitive function, including by promoting nerve cell growth and reducing brain inflammation.

[0004] For this reason, various attempts are being made to utilize butyrate for disease prevention and treatment, or to promote health. While butyrate is produced through the fermentation of dietary fiber, the intake of dietary fiber alone is limited in producing sufficient amounts. Furthermore, because butyrate has a characteristic unpleasant odor, adding it to supplements, medicines, or foods can be difficult. Furthermore, because butyrate is a volatile substance, concentration management is difficult. Therefore, various treatments are being implemented to increase the amount of butyrate in the intestines of humans and animals.

[0005] Therefore, there is a continuous need for the development of a new butyrate-enhancing system that effectively increases intestinal butyrate levels.

[0006] The background technology of the invention has been prepared to facilitate a better understanding of the present invention. It should not be construed as an admission that the matters described in the background technology of the invention constitute prior art.

[0007] As a method for increasing intestinal butyrate levels, an intestinal butyrate-producing bacteria enhancer containing Clostridium butylicum, which produces butyrate during metabolism, as an active ingredient, has been proposed.

[0008] Although such probiotic compositions can further enhance the level of intestinal butyrate, it may be difficult to establish the target intestinal butyrate concentration, and the intestinal colonization rate of Clostridium butyricum may vary from person to person. In other words, the efficiency of intestinal butyrate production by enhancement based on butyrate-producing bacteria may vary from person to person. Furthermore, when producing the enhancer, Clostridium butyricum may require more stringent culture conditions than fast-dividing or anaerobic strains such as Escherichia coli. Furthermore, when producing and utilizing a recombinant strain with a bacteria containing a similar mechanism, an E. coli strain is used, and in this case, stability issues, including the lipopolysaccharide problem of E. coli, a Gram-negative bacterium, may be raised.

[0009] The inventors of the present invention sought to develop a new intestinal butyrate level enhancing system as a means of overcoming the limitations of the conventional intestinal butyrate-producing bacteria enhancing agents described above.

[0010] Meanwhile, the inventors of the present invention focused on Bacillus subtilis, which produces butyrate using tributyrin as a substrate.

[0011] More specifically, Bacillus subtilis is a Gram-positive bacterium without a lipopolysaccharide, has a history of being eaten, and is a safe bacterium with a history of being developed into health functional foods and pharmaceuticals.

[0012] Accordingly, the inventors of the present invention identified bacteria capable of producing butyrate from healthy individuals, and among these, discovered a Bacillus subtilis strain that exhibited no toxicity to the host and enhanced butyrate production efficiency. Furthermore, by identifying the discovered Bacillus subtilis strain, they further discovered a specific factor capable of enhancing butyrate production efficiency, namely the estB gene capable of enhancing esterase activity.

[0013] Finally, the inventors of the present invention were able to anticipate that when Bacillus subtilis containing the estB gene capable of enhancing the aforementioned esterase activity is used in the intestine, butyrate production in the intestinal environment can be effectively enhanced, and intestinal diseases can be prevented or treated from the produced butyrate.

[0014] Therefore, the problem to be solved by the present invention is to provide an estB gene encoding an esterase derived from Bacillus subtilis that can improve the intrinsic activity of esterase of a microorganism.

[0015] In addition, another problem to be solved by the present invention is to provide a composition for preventing or treating inflammatory bowel disease, which can be effectively applied to the treatment or prevention of various inflammatory bowel diseases by further improving the efficiency of butyrate production by including butyrate together with the aforementioned Bacillus subtilis.

[0016] The tasks of the present invention are not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0017] To solve the above-described problem, the present invention provides a gene represented by the base sequence of SEQ ID NO: 1 encoding an esterase derived from Bacillus subtilis.

[0018] To address another challenge as described above, an esterase represented by the amino acid sequence of SEQ ID NO: 2 is provided.

[0019] According to another feature of the present invention, the esterase may decompose the substrate tributyrin to produce butyrate, but is not limited thereto.

[0020] According to another feature of the present invention, the esterase may be expressed from a gene represented by the base sequence of sequence number 1 derived from Bacillus subtilis, but is not limited thereto.

[0021] To solve another problem as described above, a recombinant vector is provided comprising a gene represented by the base sequence of SEQ ID NO: 1 encoding an esterase.

[0022] To solve another problem as described above, a host cell transformed with the recombinant vector is provided.

[0023] According to another feature of the present invention, the host cell may produce an esterase represented by the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0024] According to another feature of the present invention, the host cell may have an increased activity of the esterase compared to the wild type, but is not limited thereto.

[0025] According to another feature of the present invention, the host cell may be, but is not limited to, Escherichia coli or Bacillus subtilis.

[0026] According to another feature of the present invention, the Bacillus subtilis may be, but is not limited to, Bacillus subtilis BM107 deposited under accession number KCTC 15208BP.

[0027] To solve another problem as mentioned above, Bacillus subtilis BM107, deposited under accession number KCTC 15208BP, which has excellent butyrate production ability, is provided.

[0028] According to another feature of the present invention, the Bacillus subtilis BM107 may be a strain in which a gene represented by the base sequence of SEQ ID NO: 1 is overexpressed, but is not limited thereto.

[0029] According to another feature of the present invention, the Bacillus subtilis BM107 may decompose tributyrin to produce butyrate, but is not limited thereto.

[0030] According to another feature of the present invention, the Bacillus subtilis BM107 may have negative β-Hemolysin activity, but is not limited thereto.

[0031] According to another feature of the present invention, the Bacillus subtilis BM107 may not have resistance to at least one antibiotic selected from the group consisting of vancomycin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, and chloramphenicol, but is not limited thereto.

[0032] According to another feature of the present invention, the Bacillus subtilis BM107 may have a tributyrin decomposition rate 1.2 times higher than that of Bacillus subtilis that does not include the gene represented by the base sequence of SEQ ID NO: 1, but is not limited thereto.

[0033] According to another feature of the present invention, the Bacillus subtilis BM107 may have a tributyrin decomposition rate 17 times higher than that of Lactobacillus sp., but is not limited thereto.

[0034] According to another feature of the present invention, the Bacillus subtilis BM107 has a tributyrin decomposition rate that is 34.5 times higher than that of Weissella sp.

[0035] According to another feature of the present invention, a host cell transformed with a recombinant vector including a gene represented by the base sequence of SEQ ID NO: 1 and a pharmaceutical composition for preventing or treating inflammatory bowel disease including tributyrin are provided.

[0036] According to another feature of the present invention, the inflammatory bowel disease may be selected from the group consisting of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, conversion colitis, and Behcet's syndrome, but is not limited thereto, and may include various intestinal diseases including an inflammatory response in the intestine.

[0037] Meanwhile, according to another feature of the present invention, the composition for preventing or treating inflammatory bowel disease according to one embodiment of the present invention can be applied not only to inflammatory bowel disease but also to all diseases that can be improved by enhancing butyrate production. For example, the composition for preventing or treating inflammatory bowel disease according to one embodiment of the present invention can be applied to diseases such as cancer, obesity, diabetes, metabolic syndrome, cardiovascular disease, dementia, depression, brain disease, and osteoporosis. In addition, the composition for preventing or treating inflammatory bowel disease according to one embodiment of the present invention is not limited to the aforementioned diseases and can be expected to have an antioxidant effect in a subject.

[0038] According to another feature of the present invention, the composition may be formulated as a rectally administered form or an orally ingestible form, but is not limited thereto, and may include any of a variety of delivery methods that can be used to deliver the composition for preventing or treating inflammatory bowel disease according to an embodiment of the present invention into the body of a host to decompose tributyrin in the intestine. However, most preferably, the composition may be in a rectally administered form or an orally ingestible form.

[0039] In order to solve another problem as described above, the present invention provides a method for enhancing esterase activity, comprising the steps of transforming a host cell with a recombinant vector containing a gene represented by the base sequence of SEQ ID NO: 1 derived from Bacillus subtilis and the step of overexpressing the gene represented by the base sequence of SEQ ID NO: 1.

[0040] The present invention promotes the production of butyrate in host cells, and based on this, has a therapeutic and preventive effect on inflammatory bowel disease.

[0041] More specifically, the present invention provides a novel Bacillus subtilis strain capable of efficiently enhancing butyrate production by decomposing tributyrin within the body or intestines. Furthermore, the present invention can further enhance butyrate production by providing not only Bacillus subtilis but also tributyrin in a prodrug form.

[0042] In particular, since butyrate can be induced to be produced in the intestines and then directly provided to the intestines, the limitations of conventional butyrate, which was difficult to ingest and administer due to problems such as off-flavor and volatility, can be overcome.

[0043] Furthermore, the present invention can easily set a target intestinal butyrate concentration by controlling the level of the substrate tributyrin, so that the intestinal butyrate production efficiency can be improved compared to conventional butyrate-producing bacteria.

[0044] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0045] FIG. 1 is a flowchart illustrating a process for identifying a Bacillus subtilis strain comprising an esterase coding gene according to one embodiment of the present invention.

[0046] FIG. 2 is a schematic diagram of a tributyrin degradation process of Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention.

[0047] FIG. 3 shows the results of hemolysis confirmation for Bacillus subtilis, a bacterial strain containing an esterase coding gene according to one embodiment of the present invention.

[0048] FIG. 4 is a result of evaluating the tributyrin decomposition ability of a bacterial strain containing an esterase coding gene according to one embodiment of the present invention.

[0049] FIG. 5a is a result of strain selection based on the tributyrin decomposition ability of Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention.

[0050] Figure 5b is a PCR result of Bacillus subtilis containing an esterase coding gene according to one embodiment of the present invention.

[0051] Figures 6a and 6b show the results of transformed E. coli containing an esterase coding gene according to one embodiment of the present invention and the tributyrin decomposition ability thereof.

[0052] Figure 6c shows the results of a Sanger base sequence analysis for a transformed E. coli colony containing an esterase coding gene according to one embodiment of the present invention.

[0053] Figure 6d is a result of transparent ring confirmation for a transformed E. coli colony containing an esterase coding gene according to one embodiment of the present invention.

[0054] Figures 7a and 7b show experimental results for antibiotic resistance of a bacterial strain comprising an esterase coding gene according to one embodiment of the present invention.

[0055] FIG. 8a shows the results of intestinal length according to treatment with a bacterial strain, Bacillus subtilis, containing an esterase coding gene according to one embodiment of the present invention, and a prodrug composition for preventing or treating inflammatory bowel disease containing the same.

[0056] Figure 8b is an image result of H&E staining of colon tissue.

[0057] Figure 8c shows the results of intestinal butyrate concentration according to treatment with Bacillus subtilis, a bacterial strain containing an esterase coding gene according to one embodiment of the present invention, and a prodrug composition containing the same for preventing or treating inflammatory bowel disease.

[0058] When it is said that a component (e.g., a second component) is "directly connected" or "directly connected" to another component, it is understood that no other component (e.g., a third component) exists between said component and said other component.

[0059] The expression "configured to" as used in this document can be used interchangeably with, for example, "suitable for," "having the capacity to," "designed to," "adapted to," "made to," or "capable of." The term "configured to" does not necessarily mean something is "specifically designed to" in hardware. Instead, in some contexts, the expression "a device configured to" can mean that the device, together with other devices or components, is "capable of." For example, the phrase "a processor configured (or set) to perform A, B, and C" may mean a dedicated processor (e.g., an embedded processor) for performing those operations, or a general-purpose processor (e.g., a CPU or application processor) that can perform those operations by executing one or more software programs stored in a memory device.

[0060] The terms used in this document are used only to describe specific embodiments and may not be intended to limit the scope of other embodiments. The singular expression may include the plural expression unless the context clearly indicates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as commonly understood by those of ordinary skill in the art described in this document. Terms defined in general dictionaries among the terms used in this document may be interpreted as having the same or similar meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined in this document. In some cases, even if a term is defined in this document, it cannot be interpreted to exclude the embodiments of this document.

[0061] The individual features of the various embodiments of the present invention can be partially or wholly combined or combined with each other, and as can be fully understood by those skilled in the art, various technical connections and operations are possible, and each embodiment can be implemented independently of each other or can be implemented together in a related relationship.

[0062] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined solely by the scope of the claims.

[0063] Below, terms used in this specification are explained for clarity of explanation.

[0064] The term "or" as used herein means "and / or" unless otherwise stated.

[0065] The term "about" as used herein refers to a typical error range for each value readily known to those skilled in the art. References herein to "about" a value or parameter include embodiments relating to that value or parameter itself. Furthermore, the term "about" refers to a range of values ​​that fall within 10% in either direction (greater than or less than) the referenced reference value, unless otherwise indicated or clear from the context.

[0066] The term "patient or subject" as used herein is used interchangeably and refers to any single animal, more preferably a human or non-human mammal (including non-human animals such as cats, dogs, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) requiring treatment. The patient referred to in various embodiments herein may be a human.

[0067] The term "esterase" as used herein refers to an enzyme that hydrolyzes ester bonds, and may be used interchangeably with esterase, ester hydrolase, and the like. Preferably, the esterase in the present specification may be tributyrin esterase. More specifically, tributyrin esterase is an enzyme that hydrolyzes the ester bond of tributyrin using tributyrin as a substrate, and butyric acid and glycerol can be obtained as products of hydrolysis.

[0068] The term "butyric acid," as used herein, refers to a short-chain fatty acid and can be interpreted as having the same meaning as butyric acid and butyric acid. However, butyric acid may be a natural product produced primarily in the large intestine or rumen through microbial fermentation of carbohydrates (cellulose, hemicellulose, and starch). Meanwhile, butyric acid can serve as an important fuel for intestinal lining cells in the intestine, contributing to intestinal health. It can also reduce intestinal inflammation, making it effective against various intestinal diseases, including inflammatory bowel disease. Furthermore, butyric acid may be effective in anticancer, neuroprotective, immune-boosting, anti-inflammatory, and even obesity reduction and diabetes prevention.

[0069] As used herein, the term "intrinsic activity" may refer to the natural activity of a microorganism. For example, "E. coli enhanced compared to intrinsic activity" may refer to E. coli with enhanced hydrolytic activity compared to the esterase activity originally possessed by the E. coli.

[0070] As used herein, the term "homology" may refer to the degree of identity with a base sequence or amino acid sequence disclosed herein. Specifically, a comparison of homology may express the homology between two or more sequences as a percentage (%). For example, through the comparison of homology, a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of an esterase according to various embodiments of the present invention may be considered to substantially retain the biological activity of the esterase.

[0071] For example, a gene consisting of a base sequence that is at least 80% identical to the base sequence of a polynucleotide encoding an esterase represented by the base sequence of sequence number 1 through homology comparison can be considered to be capable of encoding an esterase having the same biological activity as the esterase according to one embodiment of the present invention.

[0072] In addition, the esterase may include, but is not limited to, an amino acid sequence of SEQ ID NO: 2 and a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence. In addition, it is obvious that an accessory protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added is also included within the scope of the present invention, as long as it has such homology and exhibits an effect corresponding to the esterase.

[0073] Additionally, the polypeptides provided in the present invention may be esterases with enhanced intrinsic activity by being further modified, for example, through chemical modification or post-translational modification. Such modifications include, but are not limited to, pegylation, albuminylation, glycosylation, panisylation, carboxylation, hydroxylation, hasylation, carbamylation, sulfation, phosphorylation, and other polypeptide modifications known in the art.

[0074] As used herein, the term "expression" may mean that a polypeptide is expressed or produced from a gene encoding an esterase of the transformed host cell.

[0075] Separation and purification refers to separating and purifying a polypeptide into its active ingredient. For example, the process of separating and purifying a polypeptide according to one embodiment of the present invention can utilize any method known in the art without limitation, preferably ion exchange chromatography. In this case, the polypeptide may be an esterase according to various embodiments of the present invention.

[0076] The term “pharmaceutical composition for preventing or treating intestinal disease” as used herein may mean a pharmaceutical composition having a therapeutic effect on intestinal disease.

[0077] At this time, when the pharmaceutical composition is formulated as a liquid solution, it may be diluted with a pharmaceutically acceptable carrier. More specifically, a pharmaceutically acceptable carrier may refer to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. For example, in a pharmaceutical composition formulated as a liquid solution, acceptable pharmaceutical carriers may be sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. In addition, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, a diluent, a dispersant, a surfactant, a binder and a lubricant may be additionally added to form the composition for preventing or treating intestinal diseases into an injectable formulation such as an aqueous solution, suspension or emulsion, a pill, a capsule, a granule or a tablet.

[0078] Compositions for preventing or treating intestinal diseases can be administered orally or parenterally, and can also be administered by applying or spraying the composition onto the affected area. Compositions for preventing or treating intestinal diseases can also be administered parenterally, including intravenously, intraperitoneally, intramuscularly, subcutaneously, or topically.

[0079] Suitable application, spraying, and dosage of compositions for the prevention or treatment of intestinal diseases may vary depending on factors such as the formulation method, administration method, age, weight, sex, severity of disease symptoms, diet, administration time, route, excretion rate, and response sensitivity of the target animal or patient. Furthermore, a skilled physician or veterinarian can easily determine and prescribe the dosage of a composition for the prevention or treatment of intestinal diseases that is effective for the intended treatment.

[0080] Oral dosage forms containing a composition for preventing or treating intestinal diseases as an active ingredient may be tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. At this time, in order to formulate into dosage forms such as tablets and capsules, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be included, and in the case of capsule dosage forms, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included.

[0081] As used herein, the term "intestinal disease" may be at least one of inflammatory bowel disease, Clostridium difficile infection, Crohn's disease, irritable bowel disease, ulcerative colitis, Behcet's disease, tuberculous enteritis, and ischemic bowel disease. Preferably, the intestinal disease in the present specification may be, but is not limited to, inflammatory bowel disease. Furthermore, such inflammatory bowel disease may include at least one of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, transitional colitis, and Behcet's syndrome.

[0082] The term "prodrug" as used herein refers to a drug whose physical and chemical properties are adjusted by chemically changing a drug, and although it does not exhibit physiological activity itself, it can be chemically changed into the original drug or by the action of an enzyme in the body after administration and exhibit a medicinal effect. For example, a composition for preventing or treating an intestinal disease according to one embodiment of the present invention, which comprises a host cell transformed with a recombinant vector including a gene encoding an esterase represented by the base sequence of SEQ ID NO: 1 and tributyrin, can reach the intestine when orally administered as a prodrug. At this time, the esterase produced by the metabolism of host cells established in the intestine can increase the level of intestinal butyric acid by hydrolyzing tributyrin. At this time, the intestinal butyric acid level to be achieved can be easily set by controlling the level of tributyrin in the prodrug.

[0083] Meanwhile, the prodrug comprising the host cell and tributyrin can be formulated into liquid and solid pharmaceutical preparations, such as injectable solutions, suspensions, emulsions, tablets, coated tablets, coated tablet cores, capsules, solutions, troches, dispersions, pellets, granules, suppositories, hard or soft gelatin capsules, etc.

[0084] According to a feature of the present invention, a prodrug composition for preventing or treating an intestinal disease may have various compositions as long as it reaches the intestines, hydrolyzes tributyrin, and increases butyric acid levels. For example, the prodrug composition for preventing or treating an intestinal disease may be a composition comprising a host cell transformed with a recombinant vector including a gene represented by the base sequence of SEQ ID NO: 1, wherein tributyrin may be included together to improve the efficiency of butyrate production. In addition, the prodrug composition for preventing or treating an intestinal disease may be composed of an esterase having at least 80% homology to the amino acid sequence of SEQ ID NO: 2 and tributyrin. In this case, the prodrug may be encapsulated so as not to be degraded by gastric acid, antibiotics, bile acids, digestive enzymes, etc. until it reaches the intestines. Furthermore, the transformed strain may be a homologous strain capable of producing spores by itself and germinating in the intestines.

[0085]

[0086] Hereinafter, the present invention will be described in more detail through examples. However, these examples are provided merely to illustrate the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[0087]

[0088] Esterase coding gene and use thereof according to one embodiment of the present invention

[0089] Hereinafter, with reference to FIGS. 1 to 6, an esterase coding gene and its use according to one embodiment of the present invention will be described.

[0090] Figure 1 is a flowchart illustrating a process for identifying a Bacillus subtilis strain containing an esterase-encoding gene according to one embodiment of the present invention. For convenience of explanation, the process will be described with reference to Figures 2 to 6.

[0091] Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention may be a strain specialized in producing butyrate by decomposing tributyrin.

[0092] More specifically, referring to FIG. 1, Bacillus subtilis including an esterase coding gene according to one embodiment of the present invention is a strain identified by a process including a step of preparing a plate medium containing tributyrin (S110), a step of spreading and culturing a fecal suspension on a plate medium containing tributyrin (S120), a step of selecting a colony (S130), a step of subculturing the selected colony (S140), a step of identifying a strain (S150), and a step of selecting a strain (S160).

[0093] First, in the step of manufacturing a flat plate medium (S110), the flat plate medium containing tributyrin may be a medium containing tributyrin at a concentration of about 1%, and due to tributyrin, the flat plate medium has a visually opaque appearance.

[0094] Next, in the smearing and culturing step (S120), the fecal suspension may mean a suspension in which feces obtained from a human are dispersed in a solvent, and at this time, the solvent may be a liquid medium or phosphate buffer solution (PBS) used in a microbial or bacterial culture experiment.

[0095] Furthermore, the fecal suspension may be a diluted solution that is first suspended and then secondarily dissolved and diluted. More specifically, the diluted solution may be a diluted solution that is obtained by serially diluting the first suspended suspension with the aforementioned solvent at a ratio of 1:9, 1 / 10 at a time.

[0096] Next, in the smearing and culturing step (S120), the fecal suspension prepared and diluted by the aforementioned process is smeared on a plate medium containing tributyrin at a concentration of about 1%, and the plate medium on which the fecal suspension is smeared can be cultured at a condition of 37°C for about 48 hours.

[0097] Next, in the colony selection step (S130), colonies with a clear zone formed around the plate medium after about 48 hours of culture can be selected. The clear zone can be formed when tributyrin in the plate medium is decomposed by the ester bond decomposition enzyme, i.e., esterase, of bacteria contained in feces.

[0098] More specifically, referring to FIG. 2, a schematic diagram of a tributyrin degradation process of Bacillus subtilis comprising an esterase-encoding gene according to one embodiment of the present invention is illustrated. Tributyrin in a plate medium can be decomposed into three butyrates by the esterase of the bacteria. Consequently, when tributyrin is decomposed by bacteria in an opaque plate medium due to the inclusion of tributyrin, a transparent clear zone can be formed.

[0099] At this time, the bacteria having esterase activity for decomposing tributyrin, i.e., the bacteria capable of producing esterase, may preferably be Bacillus subtilis, and the most preferred Bacillus subtilis used in one embodiment of the present invention is based on the accession number KCTC 15208 BP (BM107 strain) described below, and may include all bacteria whose intrinsic activity for esterase is enhanced by a specific gene, i.e., the estB gene, which is an esterase coding gene according to one embodiment of the present invention.

[0100] Ultimately, the bacteria selected through the above colony selection (S130) process may be strains specialized in decomposing tributyrin to produce butyrate.

[0101] Next, in the subculturing step (S140), the colonies selected based on whether a clear zone is formed can be subcultured on a new plate medium.

[0102] Next, in the strain identification step (S150), the subcultured colonies can be identified based on the 16S rRNA sequence, and Bacillus subtilis, Lactobacillus sp., and Weissella sp. were identified.

[0103] Next, in the strain selection step (S160), the strains were selected based on hemolytic activity. More specifically, the hemolytic activity of bacteria is a characteristic that allows bacteria to acquire the iron necessary for survival within the host, and from a functional perspective, it can be considered an important virulence factor for the host. Therefore, since the strains must be free of this hemolytic activity for use in the body, the strains were selected based on the virulence factor for hemolytic activity, i.e., beta-hemolysin activity.

[0104] In relation to this, Fig. 3 is a result of confirming hemolysis for Bacillus subtilis, a host cell containing an esterase coding gene according to one embodiment of the present invention. At this time, in order to confirm the hemolysis reaction, each strain was spread on a plate medium (Tryptic soy agar, TSA) containing 5 to 7% sheep blood, and then cultured for about 24 hours under conditions of 36 to 37°C, and the hemolysis activity according to β-hemolysin activity was confirmed depending on the presence or absence of a hemolysis zone, i.e., a clear zone, in the medium.

[0105] Referring to FIG. 3, Example 1, which is Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention, shows no change in the medium.

[0106] In contrast, the control strain, Staphylococcus aureus (S. aureus), was shown to have clear zones formed around the colonies.

[0107] That is, Example 1, which is Bacillus subtilis including an esterase coding gene according to an embodiment of the present invention, may mean that it is a strain that does not cause hemolysis and has secured stability, as the β-hemolysin activity is negative. Therefore, as a host cell including an esterase coding gene according to an embodiment of the present invention, Bacillus subtilis may be preferable, and thus, Example 1 was selected.

[0108] Furthermore, Example 1, which includes an esterase coding gene according to one embodiment of the present invention, was confirmed to be a novel strain belonging to Bacillus subtilis, and was deposited with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and assigned the deposit number KCTC 15208 BP.

[0109] Referring back to FIG. 1, the strain selected in the strain selection step (S160) of Example 1 (strain deposited with accession number KCTC 15208 BP) may be a strain that exhibits activity in esterase capable of decomposing tributyrin into butyrate through the colony selection step (S130).

[0110] More specifically, referring to FIG. 4, the results of evaluating the tributyrin degradation ability of a host cell including an esterase coding gene according to one embodiment of the present invention are shown. At this time, Bacillus subtilis, Lactobacillus sp., and Weissella sp. strains were identified based on the strain identification based on the 16S rRNA gene base sequence, and thus, for the comparative evaluation of the tributyrin degradation ability for Example 1, standard strains BS10A1 (Bacillus subtilis), LF10 (Lactobacillus sp.), LF71 (Lactobacillus sp.), and WC68 (Weissella sp.) were used in an in vitro comparative experiment. Furthermore, for in vitro experiments to evaluate tributyrin degradation ability, each strain was inoculated into a liquid medium containing 10 mM tributyrin, and then shaken and cultured for approximately 24 hours at 37°C and 200 rpm. Next, to measure the butyrate content in the culture medium, the cultured medium was quantitatively analyzed using high-performance liquid chromatography (HPLC), and the degradation rate for each strain was evaluated by comparing the amount of butyrate in the culture medium with the theoretical maximum butyrate level.

[0111] The tributyrin degradation rate of Example 1, which is Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention, was found to be about 69%, which is higher than that of BS10A1 (Bacillus subtilis), LF10 (Lactobacillus species), LF71 (Lactobacillus species) and WC68 (Weissella species).

[0112] More specifically, the decomposition rate of BS10A1, which is the same species as Example 1, which is Bacillus subtilis including an esterase coding gene according to one embodiment of the present invention, was found to be about 56%, which is about 1.2 times higher than the decomposition rate of Example 1.

[0113] Furthermore, the decomposition rates of Lactobacillus species LF10 and LF71 were found to be approximately 4%, which is approximately 17 times higher than the decomposition rate of Example 1.

[0114] Furthermore, the decomposition rate of WC68, a species of Weissella, was shown to be approximately 2%, which is approximately 34.5 times higher than the decomposition rate of Example 1.

[0115] That is, among the identified strains, Example 1 does not have toxic activity to the host and may be a strain with enhanced esterase activity (production).

[0116] Finally, a host cell comprising an esterase coding gene according to one embodiment of the present invention may include a Bacillus subtilis strain having the accession number KCTC 15208 BP of Example 1.

[0117] Meanwhile, a host cell including an esterase coding gene according to one embodiment of the present invention may include, but is not limited to, the Bacillus subtilis strain of Accession No. KCTC 15208 BP of Example 1, and may include both Bacillus subtilis and various host cells including a specific gene derived based on Example 1.

[0118] In relation to this, referring to FIG. 5A, the results of strain selection based on the tributyrin decomposition ability of Bacillus subtilis comprising an esterase coding gene according to an embodiment of the present invention are illustrated. At this time, a total of 9 strains of Bacillus subtilis isolated from the feces of healthy people were used to evaluate the tributyrin decomposition ability of Bacillus subtilis according to an embodiment of the present invention. Furthermore, for an in vitro experiment to evaluate the tributyrin decomposition ability, a 1% BCP solution based on 10 ml of EtOH (absolute) and 0.1 g of BCP (Bromocresol purple) was added to a TB-LB agar plate, and a 0.01% TB-BCP agar plate was prepared, and then each strain was adjusted to an OD=1 and 10 ul was spotted. At this time, each strain was inoculated at 2% in 2 ml of LB (LB + 1% TB supplement) and cultured for 18 hours (overnight). Furthermore, the strains inoculated (spotted) at 10 μl each were cultured at 37 °C for 48 hours and then observed.

[0119] Examples 1 to 9, which are Bacillus subtilis comprising an esterase-encoding gene according to an embodiment of the present invention, all exhibited the formation of a clear zone around the colony within the plate (plate medium). This may indicate that Examples 1 to 9, which are Bacillus subtilis comprising an esterase-encoding gene according to an embodiment of the present invention, all possess tributyrin decomposition ability.

[0120] Furthermore, tributyrin can be decomposed by the activity of esterase in bacteria, and this esterase activity can be expressed by a specific gene. More specifically, Example 1 was confirmed to include the estB gene, which is an esterase coding gene represented by the base sequence of SEQ ID NO: 1, in relation to the activity of esterase, as a result of the identification based on the previous 16S rRNA base sequence, and its base sequence is as follows. At this time, the amino acid composition of the estB protein (esterase) formed from the estB gene, which is an esterase coding gene represented by the base sequence of SEQ ID NO: 1 according to one embodiment of the present invention, is as shown in SEQ ID NO: 2 below, and the estB protein (esterase) mainly decomposes neutral fat composed of short-chain fatty acids of C3 to C10.

[0121] estB 유전자 : 5'-GTGAAAAAAGTACTTATGGCATTCATTATTTGTTTATCGCTGATTCTATCTGTTTTAGCCGCTCCGCCGTCTGGCGCAAAAGCTGAGTCAGTACATAATCCTGTCGTTCTTGTTCATGGAATAAGTGGTGCATCATACAACTTTTTCGCTATTAAAAACTACTTAATTTCTCAAGGCTGGCAAAGCAACAAACTGTACGCAATTGATTTTTATGATAAAACAGGAAACAACCTAAATAACGGCCCGCAGCTTGCTTCATATGTTGACCGTGTTTTAAAAGAGACTGGGGCAAAAAAAGTAGATATTGTGGCTCATAGTATGGGAGGCGCCAATACGCTGTACTATATTAAATATTTAGGCGGGGGCAATAAGATTCAAAATGTCGTAACGCTTGGAGGGGCTAATGGTTTAGTGTCATCAACCGCGCTGCCGGGCACAGACCCTAATCAAAAGATCCTCTATACATCTATTTACAGTCTCAATGATCAAATTGTCATCAATAGCTTGTCTCGGTTACAAGGAGCGCGAAACATCCAGCTTTATGGCATCGGTCATATTGGCTTGCTTTCTAATAGCCAAGTGAACGGCTATATCAAAGAAGGGCTGAATGGCGGAGGCCTCAATACAAATTAA-3'(서열번호 1)

[0122] estB 단백질 : MKKVLMAFIICLSLILSVLAAPPSGAKAESVHNPVVLVHGISGASYNFFAIKNYLISQGWQSNKLYAIDFYDKTGNNLNNGPQLASYVDRVLKETGAKKVDIVAHSMGGANTLYYIKYLGGGNKIQNVVTLGGANGLVSSTALPGTDPNQKILYTSIYSLNDQIVINSLSRLQGARNIQLYGIGHIGLLSNSQVNGYIKEGLNGGGLNTN(서열번호 2)

[0123] Furthermore, the estB gene represented by the base sequence of sequence number 1 appears to include not only Example 1 but also Examples 2 to 9 having tributyrin decomposition ability.

[0124] More specifically, referring to FIG. 5b, the PCR results of Bacillus subtilis including an esterase coding gene according to one embodiment of the present invention are shown. At this time, in order to confirm whether the estB gene represented by the base sequence of SEQ ID NO. 1 exists, primer sets of SEQ ID NO. 3 (estB inner F: TCA AGG CTG GCA AAG CAA CA) and SEQ ID NO. 4 (estB inner R: TGG ATG TTT CGC GCT CCT TGT A) were used based on the estB gene represented by the base sequence of SEQ ID NO. 1 described above. Furthermore, for a PCR experiment to confirm the estB gene represented by the base sequence of sequence number 1, a 20 ul sample containing 14.5 ul of DW, 2 ul of 10X Ex Taq buffer, 1 ul of dNTP Mix, 0.5 ul of Ex Taq, 0.5 ul of primer forward, 0.5 ul of primer reverse, and 1 ul of gDNA was prepared, and the PCR conditions were set to 94.0 ℃ 10 min, 94.0 ℃ 30 sec, 60.0 ℃ 30 sec, 72.0 ℃ 50 sec, 72.0 ℃ 5 min, and 4.0 ℃, and PCR was performed.

[0125] As a result of PCR, all of Examples 1 to 9 were found to have a band at 366 bp corresponding to the estB gene. That is, Examples 1 to 9 for Bacillus subtilis including an esterase coding gene according to an embodiment of the present invention all include the estB gene represented by the base sequence of SEQ ID NO: 1, and this may mean that the esterase is active due to the expression of the estB gene represented by the base sequence of SEQ ID NO: 1.

[0126] Furthermore, in order to confirm the tributyrin decomposition ability of the enzyme for the estB gene represented by the base sequence of SEQ ID NO: 1, which contributes to butyrate production activity in Bacillus subtilis comprising an esterase coding gene according to an embodiment of the present invention, a recombinant E. coli strain was prepared and verified. Referring to FIGS. 6A and 6B , transformed E. coli comprising an esterase coding gene according to an embodiment of the present invention and the results of the tributyrin decomposition ability thereof are illustrated.

[0127] First, FIG. 6a is a vector image including an esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention, and referring to FIG. 6a, for the production of a recombinant strain, E. coliDH5α was used as a host, and a pCDF vector was used as a gene delivery vehicle. The pCDF vector for the transformed E. coli including the esterase coding gene according to an embodiment of the present invention originates from CloDF13, includes a spectinomycin resistance gene, a small multidrug resistance (SMR) gene for an active efflux mechanism as a selection marker in relation to resistance, and includes J230106 as a promoter.

[0128] Next, by using GA_estB_F and GA_estB_R primers and PCR (annealing temperature 59℃ elongation time 20 seconds), an amplicon for the esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to one embodiment of the present invention was obtained, and then the backbone of the vector having CloDF13 and spectinomycin resistance genes was amplified.

[0129] The estB amplicon (insert) and vector for the esterase coding gene represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention were gel-purified, and then the concentrations were confirmed and mixed at a ratio of 3:1. E. coli DH5α was used as the recombinant host, and Gibson assembly was performed at 50°C for 30 minutes based on NEBuilder®HiFi DNA Assembly Master Mix (Cat no: E2621S). The detailed conditions for the Gibson assembly are as shown in Table 1 below.

[0130] Vector(60ng)1.63μlInsert(32.81ng)0.74μl2x Master mix(0.5x)2.5μlD.W(up to 10μl)5.13μlTotal10μl

[0131] FIG. 6b is a result of confirming whether an amplicon was generated for an esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention in the recombinant E. coli (E. coliharboring pCDF estB) generated in the aforementioned FIG. 6a. At this time, in order to confirm whether an amplicon was generated, the cells were streaked on an LB plate medium (Spec50) containing spectinomycin (50 ug / ml), and then cultured at 37°C for about 16 hours, and then 8 typical colonies were randomly selected. Furthermore, PCR was performed with Forward primer: IVT_T7 and Reverse primer: pET21b_Terminator to confirm whether an amplicon was generated for an esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention in a 1108 bp region. Referring to (a) of FIG. 6b, all of the randomly selected colonies 1 to 8 appear to contain an esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention. Furthermore, referring to (b) of FIG. 6b, since colonies 1, 3, 5, and 7 did not show a light green color, they were selected as spectinomycin-resistant colonies. Accordingly, referring to FIG. 6c, the results of Sanger base sequence analysis for colonies 1, 3, 5, and 7 confirmed in the aforementioned FIG. 6b are illustrated, and it appears that colonies 3 and 7 among the recombinant E. coli generated by the aforementioned process included an esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention.

[0132] Furthermore, referring to 6d, in order to confirm the tributyrin decomposition ability of the transformed host cell, the results of the clear zone confirmation for the recombinant E. coli (E. coli harboring pCDF estB) colonies of No. 3 and No. 7, into which the esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to one embodiment of the present invention selected from FIG. 6c was inserted are shown. At this time, after preparing a plate medium (LB + TB supplement 1%) containing tributyrin (tributyrin, TB) at 1%, the recombinant E. coli (#3, #7) and the control E. coli (E. coli harboring pCDF GFP / #2, #6) colonies were smeared, and the tributyrin decomposition ability was evaluated by confirming the clear zone formed around the smear area. Furthermore, the control E. coli (E. coli harboring pCDF GFP / #2, #6) is a recombinant E. coli in which a gene including a sequence including a mutation in a part of the sequence of the esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention has been inserted, and the functional effect on the esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to an embodiment of the present invention can be compared and confirmed.

[0133] E. coli (#3 / #7) containing an esterase coding gene according to one embodiment of the present invention is shown to have a clear zone formed in the peripheral region, while the control E. coli (#2 / #6) is shown to have no clear zone.

[0134] That is, it can mean that when the esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to one embodiment of the present invention is introduced into E. coli, the esterase protein is expressed to decompose tributyrin and produce butyrate therefrom. Therefore, it can be suggested that the esterase coding gene (estB gene) represented by the base sequence of SEQ ID NO: 1 according to one embodiment of the present invention contributes to improving the intrinsic activity of esterase for butyrate production in various microorganisms.

[0135] Ultimately, the host cell comprising the esterase coding gene according to one embodiment of the present invention may include not only the strain of Example 1 deposited under accession number KCTC 15208 BP, but also all bacteria comprising the estB gene. For example, the host cell comprising the esterase coding gene according to one embodiment of the present invention may include, but is not limited to, Bacillus subtilis or Escherichia coli transformed to include the estB gene.

[0136] According to the above process, Bacillus subtilis according to one embodiment of the present invention, which decomposes tributyrin and has enhanced butyrate production activity, can be selected, and further, an esterase coding gene represented by the base sequence of SEQ ID NO: 1 derived from Bacillus subtilis, which can enhance the intrinsic activity for esterase, can be derived.

[0137] Furthermore, Bacillus subtilis containing an esterase coding gene according to one embodiment of the present invention is a safe bacterium that does not have hemolytic activity in a host, and can be used as a food or medicine in the host's body.

[0138] For example, Bacillus subtilis, a host cell comprising an esterase-encoding gene according to an embodiment of the present invention, can be used as a prodrug composition for preventing or treating inflammatory bowel disease. More specifically, a prodrug refers to a drug that has been chemically modified to have its physical and chemical properties adjusted, and although it does not exhibit physiological activity itself, it can be chemically or enzymatically converted into the original drug in the body after administration to exhibit a pharmaceutical effect. Accordingly, Bacillus subtilis, a host cell comprising an esterase-encoding gene according to an embodiment of the present invention, can be used as a prodrug composition for preventing or treating inflammatory bowel disease, and can include Bacillus subtilis comprising an esterase-encoding gene according to an embodiment of the present invention described above.

[0139] Meanwhile, since the Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention is ultimately intended to produce butyrate from tributyrin, a prodrug composition for preventing or treating inflammatory bowel disease comprising the Bacillus subtilis comprising an esterase coding gene according to one embodiment of the present invention may further include tributyrin in addition to the bacteria in order to improve butyrate production efficiency.

[0140] Furthermore, the host cell Bacillus subtilis comprising the esterase coding gene according to an embodiment of the present invention can have improved butyrate production efficiency by activating esterase since it comprises the estB gene. In addition, when improved butyrate production occurs in the host body, the host cell Bacillus subtilis comprising the esterase coding gene according to an embodiment of the present invention can treat or prevent an intestinal disease of the host. At this time, the host cell comprising the esterase coding gene according to an embodiment of the present invention is not limited to Bacillus subtilis, and may include all host cells comprising the estB gene. For example, E. coli transformed to include the estB gene represented by the base sequence of SEQ ID NO: 1 can also have the same effect.

[0141]

[0142] Verification of an esterase coding gene and a host cell containing the same according to one embodiment of the present invention

[0143] Hereinafter, with reference to FIGS. 7a to 8c, the antibiotic resistance and therapeutic efficacy of a host cell including an esterase coding gene according to one embodiment of the present invention will be verified.

[0144] Figures 7a and 7b show the results of an experiment on antibiotic resistance of a host cell including an esterase-encoding gene according to an embodiment of the present invention. At this time, the antibiotic susceptibility experiment was performed using an E-TEST strip (bioMerieux). More specifically, a Bacillus subtilis strain, Example 1, deposited under the accession number KCTC 15208 BP, as a host cell including an esterase-encoding gene according to an embodiment of the present invention, was cultured overnight, and the cultured Example 1 strain was diluted with 1X PBS according to the 0.5 McFarland standard. The diluted strain was streaked on a Tryptic soy agar medium, and an E-test strip was placed on the medium on which the strain was streaked, and then cultured for 48 hours under anaerobic conditions. Furthermore, the cut-off value for antibiotic resistance was based on the EFSA (European Food Safety Authority) standard.

[0145] First, referring to FIG. 7a, the MIC concentration of Example 1 according to one embodiment of the present invention is shown to be below the cut-off value for seven antibiotics (vancomycin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, chloramphenicol) excluding streptomycin. That is, it may mean that Bacillus subtilis, a host cell including an esterase coding gene according to one embodiment of the present invention, is not resistant to antibiotics.

[0146] Meanwhile, in the case of Streptomycin antibiotics, the MIC concentration of Example 1 was found to be higher than the cut-off value.

[0147] However, this appears to be at a similar level to the strain of a pharmaceutical product approved and sold on the market. More specifically, referring to FIG. 7b, the results of a Streptomycin resistance test for the B. subtilisR0179 strain (comparative example) isolated from Medilac-DS, a commercially available pharmaceutical, are shown, and the MIC of Example 1 according to an embodiment of the present invention appears to have formed a clear zone (indicated by an oval) similar to that of the comparative example. That is, it may mean that Bacillus subtilis, which is a host cell including an esterase coding gene according to an embodiment of the present invention, has a resistance to Streptomycin antibiotics similar to that of the strain approved and sold on the market.

[0148] Therefore, Bacillus subtilis, a host cell comprising an esterase coding gene according to one embodiment of the present invention, and a composition comprising the same may not cause antibiotic resistance, and thus may be safe from risks such as treatment failure due to antibiotic resistance, loss of therapeutic agent selectivity, and increased likelihood and severity of disease.

[0149] Figures 8a to 8c show the results of the verification of the therapeutic efficacy of Bacillus subtilis, a host cell containing an esterase-coding gene according to one embodiment of the present invention, and a prodrug composition containing the same for preventing or treating inflammatory bowel disease, on inflammatory bowel disease (IBD). At this time, 9-week-old male C57BL / 6 mice were used to verify the therapeutic efficacy on IBD. More specifically, the therapeutic efficacy verification experiment was performed by administering 2% DSS (dextran sodium sulfate) in drinking water for 5 days to induce IBD after a one-week adaptation period for 9-week-old mice, and then replacing the drinking water with 1% DSS and administering the drinking water for 5 days. Furthermore, after the DSS drinking water administration, regular drinking water without DSS was provided for 5 days. Comparative Example 1 (control + PBS), Comparative Example 2 (NC + PBS), and Example 1 (NC + strain deposited with accession number KCTC 15208 BP according to an embodiment of the present invention) were fed a normal chow diet (NC), and Comparative Example 3 (TB + PBS) and Example 2 (TB + strain deposited with accession number KCTC 15208 BP according to an embodiment of the present invention) were fed a normal diet for 5 days, and then fed a diet containing tributyrin (tributyrin diet, TB) from the 6th to the 9th day. Furthermore, Comparative Example 1 was a normal individual without IBD, and DSS was not included in the drinking water. The PBS and strains of Comparative Examples 1 to 3, Example 1, and Example 2 were administered orally at 100 ul / day. The experiments on mice described above were performed in accordance with the Yonsei Biomedical Research Institute and regulations within ARRIVE (Animal Research: Reporting of In Vivo Experiment) guidelines.

[0150] First, FIG. 8a shows the results of intestinal length according to treatment of a host cell, Bacillus subtilis, which includes an esterase coding gene according to one embodiment of the present invention, and a prodrug composition for preventing or treating inflammatory bowel disease comprising the same.

[0151] Referring to Fig. 8a(a), the experimental groups in which inflammatory bowel disease occurred, Comparative Example 2, Example 1, Comparative Example 3, and Example 2, were shown to have shorter colon lengths than the control group in which inflammatory bowel disease did not occur, Comparative Example 1. In other words, the length of the large intestine, including the colon, may be shortened due to inflammatory bowel disease.

[0152] However, referring to FIG. 8a(b), the colon length of Example 2 according to one embodiment of the present invention is about 50 to 70 mm, which is similar to the colon length of Comparative Example 1, which is a control group that does not include inflammatory bowel disease (IBD), of about 60 to 80 mm.

[0153] That is, by treating a host cell, Bacillus subtilis, which includes an esterase coding gene according to one embodiment of the present invention, and a prodrug composition for preventing or treating inflammatory bowel disease, which includes tributyrin, it may mean that inflammatory bowel disease can be recovered to a level similar to that of a normal individual.

[0154] Furthermore, the colon length of Example 1, a composition comprising solely Bacillus subtilis, a host cell comprising an esterase-encoding gene according to an embodiment of the present invention, was found to be longer than that of Comparative Example 2, an intestinal disease experimental group to which the strain was not administered. In other words, this may mean that inflammatory bowel disease can be cured even with single treatment of Bacillus subtilis, a host cell comprising an esterase-encoding gene according to an embodiment of the present invention.

[0155] Furthermore, referring to FIG. 8b, the results of H&E staining images of colon tissues are illustrated, and it is shown that the colon tissues (epithelial cells) of Examples 1 and 2 have a histological morphology similar to that of Comparative Example 1, a healthy control group that does not include IBD. That is, as described above in FIG. 8b, it may mean that treatment with Bacillus subtilis, a host cell including an esterase coding gene according to an embodiment of the present invention, or a prodrug composition including the same can restore inflammatory bowel disease.

[0156] Furthermore, referring to FIG. 8c, the results of the intestinal butyrate concentration according to the treatment of Bacillus subtilis, a host cell including an esterase coding gene according to an embodiment of the present invention, and a prodrug composition for preventing or treating inflammatory bowel disease including the same are illustrated. At this time, for the sample collection for measuring the intestinal butyrate concentration, first, the cecum was removed from the mouse, and the contents of the cecum were recovered. After the contents of the cecum were weighed, 1 ml of PBS was added per 100 mg of the contents of the cecum, centrifugation was performed at 8,000 rpm for 15 minutes to obtain a supernatant, and methanol in an amount four times that of the obtained supernatant was added to the supernatant, and centrifugation was performed again at 8,000 rpm for 15 minutes to obtain a final analysis sample (supernatant). Butyrate in the final analytical sample was measured using LC-MS (Liquid chromatography - mass spectrometry / Vanquish Flex UHPLC / Thermo Fisher Scientific Inc.). A Waters Cortects C8 column (2.1x150, 1.6 μm) was used, and the column temperature was 50 °C. Furthermore, 2 mM ammonium acetate and MeOH for A / B were used as the mobile phases of LC-MS.

[0157] The butyrate (or butyric acid) concentrations of Comparative Example 2, Example 1, and Comparative Example 3, which include inflammatory bowel disease, were found to be lower than those of Comparative Example 1, which is a normal control group. That is, the butyrate concentration in the cecum may be lowered due to inflammatory bowel disease.

[0158] However, the butyrate concentration of Example 2 according to one embodiment of the present invention was found to have a higher value of about 330 μM than that of Comparative Example 1, which is a control group not including IBD, at about 320 μM. That is, this may mean that by treating a host cell, Bacillus subtilis, which includes an esterase-encoding gene according to one embodiment of the present invention, and a prodrug composition for preventing or treating inflammatory bowel disease, which includes tributyrin, the intestinal butyrate level can be restored to a normal individual level.

[0159] Furthermore, the butyrate concentration of Example 1, which is a composition solely comprising Bacillus subtilis, a host cell comprising an esterase coding gene according to an embodiment of the present invention, was found to be about 300 μM, which is higher than about 195 μM of Comparative Example 2, an intestinal disease experimental group to which the strain was not administered. In other words, this may mean that the intestinal butyrate level can be restored to a normal individual level even with a single treatment of Bacillus subtilis, a host cell comprising an esterase coding gene according to an embodiment of the present invention.

[0160] That is, Bacillus subtilis, which is a host cell including an esterase coding gene according to one embodiment of the present invention, and a prodrug composition for preventing or treating inflammatory bowel disease including the same can induce active production of butyrate in the colon, and various functional effects such as alleviating inflammation of colon epithelial cells, strengthening barrier function, suppressing colon polyps, and improving cognitive function can occur due to the produced butyrate.

[0161] Meanwhile, as described above, the host cell Bacillus subtilis including an esterase-encoding gene according to an embodiment of the present invention and the prodrug composition for preventing or treating inflammatory bowel disease including the same are intended to be active in the intestine of the host (subject), and therefore, a formulation and administration method that can reach the colon may be preferable. For example, as described above, the host cell Bacillus subtilis including an esterase-encoding gene according to an embodiment of the present invention and the prodrug composition for preventing or treating inflammatory bowel disease including the same may be formulated and used in a capsule form, but is not limited thereto, and may be formulated in a rectally administered form or an orally ingestible form.

[0162] According to the above results, the host cell Bacillus subtilis containing an esterase coding gene according to one embodiment of the present invention and the prodrug composition containing the same for preventing or treating inflammatory bowel disease can provide a high concentration of butyrate in the intestine of the host, and thus can treat or prevent various intestinal diseases including inflammatory bowel disease in the subject.

[0163] At this time, the high concentration of butyrate may be contributed by the high esterase activity of Bacillus subtilis according to one embodiment of the present invention. Furthermore, the esterase activity that can maximize the efficiency of tributhrin degradation may be determined by the inclusion of a specific gene, i.e., the estB gene represented by the base sequence of SEQ ID NO: 1 according to one embodiment of the present invention.

[0164] Although the embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be implemented without departing from the technical spirit of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain it, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and not restrictive. The protection scope of the present invention should be interpreted by the claims below, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

[0165]

[0166] [Accession number]

[0167] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0168] Accession number: KCTC15208BP

[0169] Date of acceptance: 202211128

[0170]

[0171]

[0172] [National Research and Development Project Supporting This Invention]

[0173] [Project ID] 1425177671

[0174] [Assignment Number] S3287890

[0175] [Ministry Name] Ministry of SMEs and Startups

[0176] [Name of Project Management (Specialist) Agency] Small and Medium Business Technology Information Promotion Agency

[0177] [Research Project Name] Small and Medium Enterprise Technology Development Support Project

[0178] [Research Project Name] Development of Microbiome-Based Microbial Therapeutics

[0179] [Name of the project performing organization] Biome Co., Ltd.

[0180] Research Period: May 1, 2022 - April 30, 2024

[0181]

[0182] [Project ID] 1711200956

[0183] [Assignment Number] 2022M3A9F3017506

[0184] [Ministry Name] Ministry of Science and ICT

[0185] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0186] [Research Project Name] Biomedical Technology Development

[0187] [Research Project Name] Establishment and Validation of a Biodelivery System for Gene Delivery with Therapeutic Effects Using CRISPR-Based Genome Editing Technology

[0188] [Name of the project performing organization] Yonsei University

[0189] Research Period: April 1, 2022 - December 31, 2026

Claims

1. A gene represented by the base sequence of sequence number 1 encoding an esterase derived from Bacillus subtilis.

2. An esterase represented by the amino acid sequence of sequence number 2.

3. In paragraph 2, The above esterase is, An esterase that breaks down the substrate tributyrin to produce butyrate.

4. In paragraph 2, The above esterase is, An esterase expressed from a gene represented by the base sequence of sequence number 1 derived from Bacillus subtilis 5. A recombinant vector comprising a gene encoding the esterase of clause 1.

6. A host cell transformed with the recombinant vector of Article 5.

7. In paragraph 6, The above host cell, A host cell characterized by producing an esterase represented by the amino acid sequence of sequence number 2.

8. In paragraph 7, The above host cell, A host cell characterized in that the activity of the above esterase is increased compared to the wild type.

9. In paragraph 6, The above host cell, Host cell, which is Escherichia coli or Bacillus subtilis.

10. Bacillus subtilis BM107, deposited under accession number KCTC 15208BP, has excellent butyrate production ability.

11. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, in which the gene represented by the base sequence of sequence number 1 is overexpressed.

12. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, characterized by producing butyrate by decomposing tributyrin.

13. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, which has negative β-hemolysin activity.

14. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, which is not resistant to at least one of the antibiotics vancomycin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.

15. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, which has a tributyrin degradation rate 1.2 times higher than that of Bacillus subtilis not containing the gene represented by the base sequence of the above sequence number 1.

16. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, which has a tributyrin degradation rate 17 times higher than that of Lactobacillus sp.

17. In paragraph 10, The above Bacillus subtilis BM107, Bacillus subtilis BM107, which has a tributyrin degradation rate 34.5 times higher than that of Weissella sp.

18. A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a host cell according to any one of claims 6 to 9 and tributyrin.

19. In paragraph 18, The above inflammatory bowel disease is, A composition for preventing or treating inflammatory bowel disease, comprising at least one of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, transitional colitis, and Behcet's syndrome.

20. As a method for promoting esterase activity, A step of transforming a host cell with a recombinant vector containing a gene represented by the base sequence of sequence number 1, and Comprising a step of overexpressing a gene represented by the base sequence of the above sequence number 1, A method for enhancing esterase activity, wherein the gene represented by the base sequence of the above sequence number 1 is a gene encoding an esterase represented by the amino acid sequence of the sequence number 2.

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