KR102495306B1 - Novel esterase, nucleotides encoding the esterase and microorganism containing the same - Google Patents

Abstract

In the present specification, a polynucleotide having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1 and encoding an esterase, and uses thereof are provided.

Description

Novel esterases, polynucleotides encoding them and microorganisms containing them

The present invention relates to novel esterase-encoding polynucleotides and uses thereof, and more particularly to novel esterase-encoding polynucleotides configured to produce butyric acid using tributyrin as a substrate and pharmaceutical compositions based thereon.

Butyric acid is a type of short-chain fatty acid, and can be produced by butyric acid-producing bacteria present in the intestines of humans or animals. In recent studies, it has been reported that butyric acid exhibits various physiological actions such as anti-inflammatory action and colorectal cancer preventive action, and that various diseases can be prevented or improved by butyric acid.

For this reason, various attempts are being made to use butyric acid for the prevention and treatment of diseases or health promotion. On the other hand, butyric acid is a substance that emits a strong odor, so it can be difficult to add butyric acid itself to medicines or foods. Accordingly, various treatments for increasing the amount of butyric acid in the intestines of humans or animals have been performed.

Therefore, there is a continuous demand for the development of a new lactic acid enhancement system that effectively increases the level of lactic acid in the intestine.

The background description of the invention has been prepared to facilitate understanding of the present invention. It should not be construed as an admission that matters described in the background art of the invention exist as prior art.

As a method of increasing the level of butyric acid in the intestine, an intestinal butyrate-producing agent containing as an active ingredient a strain of Clostridium butyricum that produces butyric acid during metabolism has been proposed.

Such a probiotics composition can further enhance the level of butyric acid in the intestine, but it may be difficult to set a target concentration of butyric acid in the intestine, and the intestinal colonization rate of Clostridium butyricum strains may vary from person to person. That is, the efficiency of butyric acid production in the intestine by lactic acid-producing bacteria-based enhancement may differ from person to person. Moreover, in producing the enhancer, the Clostridium butyricum strain may have more difficult culture conditions than a fast-digesting or anaerobic strain such as Escherichia coli.

The inventors of the present invention based on the above-mentioned Clostridium butyricum As a way to overcome the limitations of intestinal lactic acid-producing bacteria enhancers, a new system for enhancing intestinal lactate levels was attempted.

As a result, the inventors of the present invention have discovered a novel butyric acid production gene that produces butyric acid using tributyrin as a substrate.

More specifically, the inventors of the present invention were able to discover a novel tributyrin esterase capable of producing butyric acid by hydrolyzing the ester bond of tributyrin and a novel polynucleotide derived from intestinal microorganisms encoding the same. .

At this time, the inventors of the present invention provide the novel esterase together with tributyrin, or provide the strain producing it together with tributyrin in the form of a prodrug, the level of butyrate in the intestine can be enhanced was able to recognize

In particular, the inventors of the present invention found that when an intestinal microorganism such as Escherichia coli transformed with a recombinant vector containing a novel tributyrin esterase-encoding polynucleotide is provided together with tributyrin, butyric acid production such as Clostridium butyricum It was expected that the intestinal colonization rate would be higher than when the bacteria were taken directly.

Furthermore, the inventors of the present invention found that the new system for enhancing intestinal lactate levels can easily set the target lactate level by controlling the level of tributyrin, a substrate, and the efficiency of lactate production in the intestine is higher than that of the prior art. It could be expected that it could be promoted more than lactic acid-producing bacteria.

In addition, the inventors of the present invention, when transforming E. coli under relatively simple culture conditions with a recombinant vector containing a novel tributyrin esterase coding gene, a strain capable of hydrolysing a large amount of ester bonds and producing butyric acid It could be expected that it could be obtained easily.

Furthermore, the inventors of the present invention found that the novel intestinal lactic acid level enhancing system as described above can be used to treat inflammatory bowel disease, Clostridium difficile infection, Crohn's disease, irritable bowel disease, ulcerative colitis, It can be recognized that it can be applied to the treatment of intestinal diseases such as Behcet's disease, tuberculous enteritis, and ischemic bowel disease.

Accordingly, the problem to be solved by the present invention is to provide a polynucleotide encoding a novel tributyrin esterase capable of effectively increasing butyric acid in the intestine, a novel tributyrin esterase, and a composition for treating and preventing intestinal diseases using the same. is to provide

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 following description.

In order to solve the above problems, a polynucleotide having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1 and encoding esterase is provided according to an embodiment of the present invention.

In order to solve the above problems, an esterase having 80% or more homology with the amino acid sequence of SEQ ID NO: 2 according to another embodiment of the present invention is provided.

According to a feature of the present invention, the esterase can be configured to take tributirin as a substrate and hydrolyze triburin to produce butyrate.

In order to solve the above problems, E. coli (accession number: accession number KCTC 14204BP) producing esterases according to another embodiment of the present invention is provided.

According to a feature of the present invention, the E. coli is an E. coli whose esterase activity is enhanced compared to the intrinsic activity, and its esterase activity is not enhanced compared to the intrinsic activity. there is.

In order to solve the problems as described above, a composition for treating or preventing intestinal diseases containing esterase and tributirin according to another embodiment of the present invention is provided.

According to a feature of the present invention, the esterase may have 80% or more homology with the amino acid sequence of SEQ ID NO: 2.

According to another feature of the present invention, the intestinal disease is inflammatory bowel disease, Clostridium difficile infection, Crohn's disease, irritable bowel disease, ulcerative colitis, Behcet's disease, tuberculous enteritis, and ischemic It may be at least one of intestinal disorders.

In order to solve the above problems, according to another embodiment of the present invention, E. coli producing esterase (Accession No.: Accession No. KCTC 14204BP) and tributyrin (trybutirin), including intestinal disease treatment A prodrug composition for use or prevention is provided.

In order to solve the above problems, according to another embodiment of the present invention, a recombinant vector comprising a nucleotide sequence having 80% or more homology with the nucleotide sequence of SEQ ID NO: 1 is provided.

In order to solve the above problems, according to another embodiment of the present invention, a microorganism transformed with a recombinant vector containing a nucleotide sequence having 80% or more homology with the nucleotide sequence of SEQ ID NO: 1 is provided. .

In order to solve the problem as described above, according to another embodiment of the present invention, transforming a recombinant vector containing a nucleotide sequence having 80% or more homology with the nucleotide sequence of SEQ ID NO: 1 into a host cell , culturing the transformed host cell to obtain esterase; and separating and purifying the esterase.

According to the features of the present invention, Host cells are Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus reuteri And Bacillus subtilis ( Bacillus subtilis ) It may be at least one.

Hereinafter, the present invention will be described in more detail through examples. However, since these examples are only for illustrative purposes, the scope of the present invention should not be construed as being limited by these examples.

The present invention can provide an intestinal butyric acid enhancement system based on polynucleotides encoding novel esterases.

More specifically, the present invention not only provides a polynucleotide encoding the novel esterase, but also provides a strain producing the novel esterase transformed with a vector containing the polynucleotide in the form of a prodrug together with tributyrin. , which can increase the level of butyric acid in the intestine.

In particular, since the present invention provides an intestinal microorganism transformed with a recombinant vector having a novel tributyrin esterase-encoding polynucleotide together with tributyrin, the colonization rate in the intestine can be higher than when lactic acid-producing bacteria are directly ingested. .

According to the present invention, by controlling the level of tributyrin, a substrate for novel esterase, it is possible to easily set a target concentration of butyric acid in the intestine, so that the efficiency of butyric acid production in the intestine can be improved compared to conventional butyrate-producing bacteria.

In addition, according to the present invention, it is possible to transform Escherichia coli under relatively simple culture conditions with a vector containing a novel tributyrin esterase-encoding gene, thereby easily obtaining a strain capable of hydrolysing a large amount of ester bonds.

Furthermore, the present invention is directed to treating inflammatory bowel disease, Clostridium difficile infection, Crohn's disease, irritable bowel disease, ulcerative colitis, Behçet's disease, tuberculous enteritis, and ischemic bowel disease. It can be applied to the treatment of intestinal diseases.

Effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

1 exemplarily illustrates the procedure of a method for preparing an esterase used in various embodiments of the present invention.
2 to 4 show the tributyrin hydrolysis activity of Escherichia coli (Accession Number: Accession Number KCTC 14204BP) producing esterases used in various embodiments of the present invention.
5A and 5B show the results of blast analysis of polynucleotides encoding esterases with enhanced activity compared to intrinsic activity.

When it is referred to as being "directly connected" or "directly connected" to a component (e.g., a second component), another component (e.g., a third component) between the certain component and the other component is referred to. It can be understood that does not exist.

As used in this document, the expression "configured to" means "suitable for," "having the capacity to," depending on the circumstances. ," "designed to," "adapted to," "made to," or "capable of." The term "configured (or set) to" may not necessarily mean only "specifically designed to" hardware. Instead, in some contexts, the expression "a device configured to" may mean that the device "is capable of" in conjunction with other devices or components. For example, the phrase "a processor configured (or configured) to perform A, B, and C" may include a dedicated processor (eg, an embedded processor) to perform those operations, or by executing one or more software programs stored on a memory device. , may mean a general-purpose processor (eg, CPU or application processor) capable of performing corresponding operations.

Terms used in this document are only used to describe a specific embodiment, and may not be intended to limit the scope of other embodiments. Singular expressions may include plural expressions unless the context clearly dictates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as commonly understood by a person of ordinary skill in the art described in this document. Among the terms used in this document, terms defined in a general dictionary may be interpreted as having the same or similar meaning as the meaning in the context of the related art, and unless explicitly defined in this document, an ideal or excessively formal meaning. not be interpreted as In some cases, even terms defined in this document cannot be interpreted to exclude the embodiments of this document.

Each feature of the various embodiments of the present invention can be partially or entirely combined or combined with each other, and as those skilled in the art can fully understand, various interlocking and driving operations are possible, and each embodiment can be implemented independently of each other. It may be possible to implement together in an association relationship.

Hereinafter, terms used within the present specification will be described for clarity of explanation.

As used herein, the term “esterase” refers to an enzyme that hydrolyzes ester bonds, and may be used interchangeably with esterase, esterase, and the like. Preferably, esterase within 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 the hydrolysis.

As used herein, the term "butyric acid" is one of short-chain fatty acids, and may be interpreted as the same as butyric acid and butyric acid. In this case, lactic acid may be a natural product in vivo in which carbohydrates (cellulose, hemicellulose, starch) are mainly produced in the large intestine or rumen by microbial fermentation. On the other hand, butyric acid can act as an important fuel for intestinal endothelial cells in the intestine, so it is helpful for intestinal health, and can lower inflammation in the intestine, so it can be effective against various intestinal diseases including inflammatory bowel disease. Furthermore, butyric acid may be effective in anticancer effects, neuroprotective effects, immunity enhancement and anti-inflammatory effects, and further reducing obesity and preventing diabetes.

As used herein, the term "intrinsic activity" may refer to an active state naturally possessed by microorganisms. At this time, "E. coli, enhanced compared to the intrinsic activity" may mean E. coli having more improved hydrolytic activity compared to the esterase activity originally possessed by E. coli.

For example, E. coli producing the esterase of accession number KCTC 14204BP used in various embodiments of the present invention is an E. coli whose tributyrin esterase activity is enhanced compared to the intrinsic activity, and tributyrin The hydrolysis ability for may be high. That is, E. coli producing the esterase of Accession No. KCTC 14204BP can also produce butyric acid at a higher level than E. coli that is not enhanced with respect to its intrinsic activity.

Meanwhile, E. coli producing the esterase of accession number KCTC 14204BP may have a polynucleotide having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1 and encoding the esterase.

However, it is not limited thereto, and a strain transformed with a recombinant vector having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1 and having a polynucleotide encoding esterase has an esterase activity in intrinsic activity It may be an enhanced strain compared to. That is, the transformed strain has a higher ability to hydrolyze tributyrin than the non-transformed strain, and thus can produce butyric acid at a high level.

At this time, the strain enhanced compared to the intrinsic activity can produce an esterase consisting of an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 2.

As used herein, the term "homology" may indicate the same degree as the nucleotide sequence or amino acid sequence disclosed herein. Specifically, the comparison of homology can express the homology between two or more sequences as a percentage (%). For example, through the comparison of the homology, a polypeptide consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of the esterase according to various embodiments of the present invention can be regarded as substantially retaining the biological activity of the esterase. .

For example, through homology comparison, a gene consisting of a nucleotide sequence that is 80% or more identical to the nucleotide sequence of the polynucleotide encoding the esterase of SEQ ID NO: 1 is substantially biologically active of the esterase according to an embodiment of the present invention. The same activity as can be seen as being able to encode esterases.

In addition, the esterase may be performed by adding, deleting, or replacing the amino acid sequence of SEQ ID NO: 2, or a combination thereof. Specifically, if the polypeptide substantially retains the biological activity with the esterase according to various embodiments of the present invention even with deletion or addition such as deletion mutation in the amino acid of SEQ ID NO: 2, addition and swapping of functional sequences, the intrinsic activity is It can be seen as an enhanced esterase.

As used herein, the term "vector" contains DNA sequences operably linked to regulatory sequences capable of controlling expression of a protein in a host cell and other sequences introduced to facilitate genetic manipulation or to optimize expression of a protein. It means a DNA construct that The control sequence may include a promoter for controlling transcription, an operator optionally added to control transcription, an appropriate mRNA ribosome binding site, and sequences for controlling termination of transcription and translation.

Specifically, the vector is a vector whose promoter is a CAT promoter, and may include a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector such as an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector. The vector may be integrated into the genome of a host cell.

As used herein, the term "recombination" may be genetic recombination, and the genetic recombination may mean the joining of genetic DNA or synthesized genes separated from heterologous organisms. "Recombinant vector" may refer to a vector recombined to include the polynucleotide of SEQ ID NO: 1.

As used herein, the term "transformation" may mean that the genetic properties of an organism are changed by accepting DNA from the outside. The transformation method may include, but is not limited to, methods using ribosomes, electroporation, chemicals that increase free DNA uptake, DNA direct injection into host cells, particle gun bombardment, and microfiring. The method can be used as a transformation method.

As used herein, the term "host cell" may refer to a cell used for viral infection or a bacterium used for propagation of a vector such as a plasmid or phage DNA and an inserted gene.

For example, the host cell may be a host cell transformed with the recombinant vector. Transformable host cells include prokaryotic host cells such as Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis or Staphylococcus. but is not limited thereto. In addition, fungi (e.g., Aspergillus), yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae), Shizosaccharomaces ( Schizosaccharomyces), Neurospora crassa), and the like can be used as host cells.

Thus, within the present specification, transformed host cells may be used interchangeably with transformed microorganisms.

Meanwhile, cells derived from higher eukaryotes including insect cells, plant cells, and mammals may be used as host cells. However, it is not limited thereto, and various cells may be used as host cells transformed with the recombinant vector.

Preferably, the host cell is Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus reuteri And Bacillus subtilis ( Bacillus subtilis ) It may be at least one intestinal microorganism. These intestinal microorganisms may have a higher colonization rate in the intestine than other host cells, and thus may be more effective in promoting butyric acid in the intestine.

As used herein, the term "expression" may mean expression or production of a polypeptide from a gene encoding an esterase of the transformed host cell.

Isolation and purification means separating and purifying a polypeptide as an active ingredient. For example, in the process of separating and purifying a polypeptide according to one embodiment of the present invention, methods known in the art may be used without limitation, and ion exchange chromatography may be preferably used. In this case, the polypeptide may be an esterase according to various embodiments of the present invention.

As used herein, the term "composition for treating or preventing intestinal disease" may refer to a pharmaceutical composition having a therapeutic effect on intestinal disease.

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 organisms and does not inhibit the biological activity and properties of the administered compound. For example, acceptable pharmaceutical carriers for pharmaceutical compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, and maltodextrin. It may be a solution, glycerol, ethanol, and a mixture in which one or more of these components are mixed. In addition, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to treat or prevent intestinal diseases, and may be formulated into injection formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. can

The composition for treating or preventing intestinal diseases may be administered through oral administration or parenteral administration, and may be administered by coating or spraying the diseased area. The composition for treating or preventing intestinal disorders may also be parenterally administered by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or local administration.

Appropriate application, spraying, and dosage of the composition for treating or preventing intestinal disorders include formulation method, administration method, age, weight, sex, severity of disease symptoms, food, administration time, administration route, and excretion of target animals and patients. It can vary by factors such as speed and response sensitivity. In addition, a skilled doctor or veterinarian can easily determine and prescribe the dosage of the composition for treating or preventing intestinal diseases that is effective for the desired treatment.

Formulations for oral administration containing a composition for treating or preventing intestinal disorders 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. there is. At this time, in order to formulate formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearate A lubricating oil such as magnesium acid, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.

As used herein, the term "bowel disease" refers to inflammatory bowel disease, Clostridium difficile infection, Crohn's disease, irritable bowel disease, ulcerative colitis, Behçet's disease, tuberculous enteritis, and at least one of ischemic bowel disease. Preferably, bowel disease within the present specification may be inflammatory bowel disease, but is not limited thereto.

As used herein, the term "prodrugs" refers to a drug whose physical and chemical properties are controlled by chemically changing a drug, and does not itself exhibit physiological activity, but chemically in the body after administration, Or, by the action of an enzyme, it can be changed into the original drug and exert its medicinal effect.

For example, a prodrug composition for treating or preventing intestinal diseases, including E. coli producing an esterase of Accession No. KCTC 14204BP and trybutirin, can reach the intestine when administered orally. can At this time, esterase produced by metabolism of Escherichia coli settled in the intestine can increase the level of butyrate in the intestine by hydrolyzing tributyrin. At this time, the level of lactic acid in the intestine to be achieved can be easily set by adjusting the level of tributyrin in the prodrug.

On the other hand, the prodrug consisting of Escherichia coli and tributyrin is liquid and solid pharmaceutical preparations, such as injectable solutions, suspensions, emulsions, tablets, coated tablets, coated tablet cores, capsules, solutions, troches, dispersions, It can be formulated as pellets, granules, suppositories, hard or soft gelatin capsules, and the like.

According to the features of the present invention, the prodrug composition for treating or preventing intestinal disease may have various compositions as long as it reaches the intestine and hydrolyzes tributyrin and increases the level of butyric acid. For example, a prodrug composition for treating or preventing intestinal disorders may consist of esterase having 80% or more homology to the amino acid sequence of SEQ ID NO: 2 and tributyrin. Alternatively, the prodrug composition for treating or preventing intestinal diseases is a strain transformed with a recombinant vector containing a polynucleotide encoding an esterase having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1 and tributyrin. may be done At this time, the prodrug may be encapsulated so as not to be decomposed by gastric acid, antibiotics, bile acids, digestive enzymes, etc. until it reaches the intestine. Furthermore, the transformed strain may itself be homogenous, capable of generating spores and germination in the intestine.

Hereinafter, with reference to FIG. 1, an esterase production procedure according to an embodiment of the present invention will be described in detail.

1 exemplarily illustrates the procedure of a method for preparing an esterase used in various embodiments of the present invention.

Referring to FIG. 1, first, a recombinant vector containing a nucleotide sequence having 80% or more homology with the nucleotide sequence of SEQ ID NO: 1 is transformed into a host cell for the production of esterase (S110). Then, the esterase is obtained by culturing the transformed host cell (S120). Finally, the esterase is separated and purified (S130)

More specifically, in the step of transforming the recombinant vector into a host cell (S110), a polynucleotide having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1 according to various embodiments of the present invention and configured to encode an esterase A recombinant vector containing is prepared. In this case, the vector may be pBAD24, but is not limited thereto. Then, the recombinant vector is Escherichia coli, and further Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus reuteri ( Lactobacillus Reuteri ) And Bacillus subtilis ( Bacillus subtilis ) It can be transformed by using an intestinal strain containing at least one of the host cells.

Then, in the step of obtaining the esterase (S120), the esterase may be produced by culturing the transformed host cell. In this case, the esterase may have 80% or more homology with the amino acid sequence of SEQ ID NO: 2, and may be tributyrin esterase that produces butyric acid using tributyrin as a substrate.

Finally, in the step of separating and purifying the esterase (S130), the tributyrin esterase can be purified through various filtering operations.

Tributyrin esterase used in various embodiments of the present invention can be produced with high efficiency by the esterase production method according to an embodiment of the present invention according to the above procedure.

Evaluation 1: Esterase activity of E. coli according to various embodiments of the present invention

Hereinafter, the evaluation results of the esterase activity of E. coli according to various embodiments of the present invention will be described in detail with reference to FIGS. 2 to 4.

In this evaluation, Escherichia coli transformed with the recombinant vector containing the nucleotide sequence of SEQ ID NO: 1, Accession No. KCTC 14204BP, was set as an experimental group, and E. coli without it was set as a control group.

2 to 4 show the tributyrin hydrolysis activity of Escherichia coli (Accession Number: Accession Number KCTC 14204BP) producing esterases used in various embodiments of the present invention.

First, referring to FIG. 2, the result of inoculating Escherichia coli of Accession No. KCTC 14204BP and E. coli of the control group on a nutrient agar medium containing tributyrin (Nutrition agar+Tributyrin 0.8%) is shown.

More specifically, when Escherichia coli of accession number KCTC 14204BP used in various embodiments of the present invention is inoculated, it appears that a transparent ring is generated according to the decomposition of tributyrin. At this time, the diameter of the ring appears larger than that of the control group.

These results may mean that the Escherichia coli of accession number KCTC 14204BP used in various embodiments of the present invention hydrolyzes tributyrin and produces butyric acid, unlike the control Escherichia coli.

Next, referring to FIGS. 3 and 4, NB (peptone 2.5g + yeast extract 1.5g + NaCl 1.5g / 500ml) medium containing 5mM tributyrin and 0.02% TritonX100 After culturing E. coli of accession number KCTC 14204BP and E. coli of the control group for 24 hours, the supernatant was taken and filtered, and the concentration of butyric acid was analyzed through HPLC analysis. The result is shown. At this time, the analysis of lactic acid concentration was performed three times.

More specifically, in the case of the control group, the average level of lactate, a product of tributyrin hydrolysis, was 81.57333 μg/ml during cultivation. However, the level of butyric acid by Escherichia coli of accession number KCTC 14204BP used in various examples of the present invention appears to be 1314.787 μg/ml on average.

These results mean that the butyric acid production of Escherichia coli of accession number KCTC 14204BP used in various embodiments of the present invention is about 16 times higher than that of general Escherichia coli, and the hydrolysis ability for tributyrin is 10 times higher than that of the control Escherichia coli. that can mean

As a result of the above evaluation 1, E. coli of Accession No. KCTC 14204BP, transformed with the recombinant vector containing the nucleotide sequence of SEQ ID NO: 1 according to various embodiments of the present invention, esterase consisting of the amino acid sequence of SEQ ID NO: 2 It can be produced to hydrolyze tributyrin and increase the level of butyric acid in the intestine. Accordingly, the prodrug composition containing Escherichia coli and tributyrin, or the pharmaceutical composition containing tributyrin and esterase consisting of the amino acid sequence of SEQ ID NO: 2, promotes intestinal health by increasing the level of butyric acid in the intestine, May reduce intestinal inflammation. Accordingly, the compositions may be effective against various intestinal diseases including inflammatory bowel disease. Furthermore, the compositions may be effective for anti-cancer effects, neuroprotective effects, immunity enhancement and anti-inflammatory effects, and further reducing obesity and preventing diabetes. More specifically, the compositions are useful for treating inflammatory bowel disease, Clostridium difficile infection, Crohn's disease, irritable bowel disease, ulcerative colitis, Behçet's disease, tuberculous enteritis, and ischemic bowel disease. It can be applied to the treatment of the same intestinal disease.

On the other hand, the effect of the present invention is not limited thereto.

For example, a strain transformed with a recombinant vector containing a nucleotide sequence having 80% or more homology with the nucleotide sequence of SEQ ID NO: 1 may have the same activity as Escherichia coli of Accession No. KCTC 14204BP. More specifically, the transformed strain hydrolyzes tributyrin to produce an esterase that enhances the level of butyric acid in the intestine, thereby contributing to increasing the level of butyric acid.

In particular, since the present invention provides an intestinal microorganism such as Escherichia coli transformed with a novel tributyrin esterase-encoded polynucleotide together with tributyrin, the colonization rate in the intestine may be higher than when the lactic acid-producing bacteria are directly ingested.

In addition, according to the present invention, by controlling the level of tributyrin, which is a substrate for novel esterase, it is possible to easily set a target concentration of butyric acid in the intestine, so that the efficiency of butyric acid production in the intestine can be improved compared to conventional butyrate-producing bacteria.

In addition, according to the present invention, it is possible to transform Escherichia coli under relatively simple culture conditions with a vector containing a novel tributyrin esterase-encoding gene, thereby easily obtaining a strain capable of hydrolysing a large amount of ester bonds.

Evaluation 2: Identification of esterase coding genes and esterases according to various embodiments of the present invention

Hereinafter, with reference to FIG. 5 , results of identification of esterase coding genes according to various embodiments of the present invention and esterases according to various embodiments of the present invention will be described.

5A and 5B show the results of blast analysis of polynucleotides encoding esterases with enhanced activity compared to intrinsic activity.

First, referring to FIG. 5A , the result of blast n analysis for a polynucleotide encoding an esterase having the nucleotide sequence of SEQ ID NO: 1 and having an enhanced activity compared to the endogenous activity is shown. At this time, the target genomes are listed in the order of high similarity to the polynucleotide according to various embodiments of the present invention according to E value (E valude).

More specifically, the complete genome of Clostridium lentocellum DSM 5427, which was analyzed to have the highest similarity with the polynucleotide according to various embodiments of the present invention, corresponds to only 72% The region appears to have 67.41% homology with polynucleotides according to various embodiments of the present invention.

Furthermore, in the herbinix species, SD1D genome, chromosome: I, which was analyzed to have the second highest degree of similarity with the polynucleotide according to various embodiments of the present invention, only 71% of the region corresponds to that of the present invention. It is shown to have 67.9% homology with polynucleotides according to various examples.

Such an analysis result may mean that the polynucleotide according to various embodiments of the present invention is a genome different from conventionally known genomes.

In this regard, further referring to FIG. 5B , a blast x result obtained by aminolating and analyzing a polynucleotide encoding an esterase having the nucleotide sequence of SEQ ID NO: 1 and having enhanced activity compared to intrinsic activity is shown. At this time, the target proteins are listed in order of high similarity to the polynucleotide according to various embodiments of the present invention according to E value (E valude).

More specifically, the alpha / beta hydrolase fold domain-containing protein (alpha / beta hydrolase fold domain-containing protein) is shown to have only 60.46% homology with proteins based on polynucleotides according to various embodiments of the present invention.

Furthermore, the hypothetical protein derived from Herbinix species, which was analyzed to have the third highest similarity with the protein based on the polynucleotide according to various embodiments of the present invention, is a polynucleotide according to various embodiments of the present invention It appears to have only 59.09% homology with the based protein.

These analysis results indicate that the polynucleotide of SEQ ID NO: 1 according to various embodiments of the present invention is a novel genome different from previously known genomes, and the esterase of SEQ ID NO: 2 is a novel protein different from previously known proteins. can do.

In addition, it may be suggested that such genetic differences contribute to enhancing the intrinsic activity of esterases according to various embodiments of the present invention.

Accordingly, a strain transformed with a recombinant vector containing a polynucleotide having 80% or more homology to the nucleotide sequence of SEQ ID NO: 1, or an esterase consisting of an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 2 can be provided as a variety of compositions for promoting intestinal health.

Each feature of the various embodiments of the present invention can be partially or entirely combined or combined with each other, and as a person skilled in the art can fully understand, various interlocking and driving operations are possible, and each embodiment can be implemented independently of each other. and may be implemented together in a related relationship.

Although the embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and may be variously modified and 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 idea of the present invention, but to explain, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of the present invention should be construed according to the claims below, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY <120> NOVEL NUCLEOTIDES ENCODING ESTERASE AND USE THEREOF <130> 20PD5341EN <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 795 <212> DNA <213> artificial sequence <220> <223> DNA sequence for novel esterase encoding gene <400> 1 atggcattat taacacttca atattacagt acggccttat gcaggcccac cagagtacaa 60 ttgcttctgc cgaatgatgt ccaggagatg gctgtgcagg acaacgaaaa ttataataga 120 ccgatgaaga cgcttgtctt gctgcatggc tacagcggca gctgtgacga ctggctgact 180 ggcagcagag tccaggaaat atcgggtatg tataacctgg caattgcaat gccgtcaggg 240 gaaaacagtt tttacctgga tcagaaggga accggaaggg catatgccac atttgtgggt 300 aaggagctta ttgcgtatct gcaaaagact ttcggcattg ccatgaaggc tgaggatacc 360 ttcatagggg gcttatccat gggaggattt ggcgccctgc acacggggct tttgtatccg 420 gagaattact caaagattgc tgctctttcg tccgcactga tcgtgcatga ggttatgggaa 480 atgaaagaag gcatcggcaa tgctgtggct gactatgatt actataaagc cacattcgga 540 gagccgaaag agctggcaat gagcaaaaat aatccggaat atattatcag acagcgcaga 600 gagaggggag agtccattca gcctgttttt atggcctgtg gaactgagga ctttttactg 660 gaaaataacc ggagctttta tgagttcctg aaaaaggagg gcatagaggt agattatcac 720 gaaagtccgg gaactcatga ctggaaattc tggaatgaat atctggaacc ggcagttaaa 780 tggatgctgc aatga 795 <210> 2 <211> 264 <212> PRT <213> artificial sequence <220> <223> Protein sequence for novel esterase <400> 2 Met Ala Leu Leu Thr Leu Gln Tyr Tyr Ser Thr Ala Leu Cys Arg Pro 1 5 10 15 Thr Arg Val Gln Leu Leu Leu Pro Asn Asp Val Gln Glu Met Ala Val 20 25 30 Gln Asp Asn Glu Asn Tyr Asn Arg Pro Met Lys Thr Leu Val Leu Leu 35 40 45 His Gly Tyr Ser Gly Ser Cys Asp Asp Trp Leu Thr Gly Ser Arg Val 50 55 60 Gln Glu Ile Ser Gly Met Tyr Asn Leu Ala Ile Ala Met Pro Ser Gly 65 70 75 80 Glu Asn Ser Phe Tyr Leu Asp Gln Lys Gly Thr Gly Arg Ala Tyr Ala 85 90 95 Thr Phe Val Gly Lys Glu Leu Ile Ala Tyr Leu Gln Lys Thr Phe Gly 100 105 110 Ile Ala Met Lys Ala Glu Asp Thr Phe Ile Gly Gly Leu Ser Met Gly 115 120 125 Gly Phe Gly Ala Leu His Thr Gly Leu Leu Tyr Pro Glu Asn Tyr Ser 130 135 140 Lys Ile Ala Ala Leu Ser Ser Ala Leu Ile Val His Glu Val Met Glu 145 150 155 160 Met Lys Glu Gly Ile Gly Asn Ala Val Ala Asp Tyr Asp Tyr Tyr Lys 165 170 175 Ala Thr Phe Gly Glu Pro Lys Glu Leu Ala Met Ser Lys Asn Asn Pro 180 185 190 Glu Tyr Ile Ile Arg Gln Arg Arg Glu Arg Gly Glu Ser Ile Gln Pro 195 200 205 Val Phe Met Ala Cys Gly Thr Glu Asp Phe Leu Leu Glu Asn Asn Arg 210 215 220 Ser Phe Tyr Glu Phe Leu Lys Lys Glu Gly Ile Glu Val Asp Tyr His 225 230 235 240 Glu Ser Pro Gly Thr His Asp Trp Lys Phe Trp Asn Glu Tyr Leu Glu 245 250 255 Pro Ala Val Lys Trp Met Leu Gln 260

Claims (13)

A polynucleotide having the nucleotide sequence of SEQ ID NO: 1 and encoding esterase. Esterase having the amino acid sequence of SEQ ID NO: 2. According to claim 2,
The esterase,
Tributirin as a substrate,
Esterase, configured to hydrolyze the tributyrin to produce butyrate. E. coli producing an esterase having the amino acid sequence of SEQ ID NO: 2 (Accession Number: KCTC 14204BP). According to claim 4,
The Escherichia coli,
E. coli with enhanced esterase activity compared to intrinsic activity,
Escherichia coli having a hydrolysis capacity for tributyrin that is 10 times higher than that of E. coli in which the esterase activity is not enhanced relative to the intrinsic activity. A composition for treating or preventing inflammatory bowel disease, comprising esterase having the amino acid sequence of SEQ ID NO: 2 and tributyrin. delete delete A prodrug composition for treating or preventing inflammatory bowel disease, comprising E. coli according to claim 4 and tributirin. A recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1. A microorganism transformed with the recombinant vector according to claim 10. Transforming the recombinant vector according to claim 10 into a host cell;
culturing the transformed host cell to obtain esterase; and
Esterase production method comprising the step of separating and purifying the esterase. According to claim 12,
The host cell is Lactobacillus rhamnosus , Lactobacillus acidophilus , Lactobacillus reuteri And Bacillus subtilis ( Bacillus subtilis ) At least one, the esterase production method.

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