Method for producing dipeptide using bioconversion process

Abstract

The present invention relates to a method for producing a dipeptide using a bioconversion process and, more specifically, to a method for producing L-alanyl-L-glutamine, the method comprising the steps of: (a) culturing a recombinant strain expressing the protein consisting of SEQ ID NO: 1; (b) mixing L-alanine methyl ester hydrochloride and glutamine with the culture; and (c) obtaining L-alanyl-L-glutamine.

Description

Method for producing dipeptides using a bioconversion process

[0001] This application claims the benefit of Republic of Korea Patent Application No. 10-2024-0008937, filed on January 19, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a method for producing a dipeptide using a bioconversion process, and more particularly, to a method for producing L-alanyl-L-glutamine, comprising the steps of: (a) culturing a recombinant strain expressing a protein consisting of sequence number 1; (b) mixing alanine methyl ester hydrochloride and glutamine with the culture; and (c) obtaining L-alanyl-L-glutamine.

[0003]

[0004] L-alanyl-L-glutamine (Ala-Gln, Cas No. 39537-23-0) is a dipeptide composed of the amino acids alanine and glutamine, C8H 15 It has the chemical formula N3O4. L-Alanyl-L-glutamine has high solubility, water solubility, and heat stability, and it is a stable compound in humans and animals, so it is widely used in various fields. In the food industry, it is used as a lactic acid bacteria growth promoter to help improve the taste, texture, and shelf life of foods. In the medical field, it is used as a nutritional supplement to enhance immune function, maintain intestinal function, and improve recovery after surgery or illness. It is also used in sports nutrition products to enhance athletic performance and reduce fatigue.

[0005] L-alanyl-L-glutamine is synthesized primarily through chemical synthesis and enzymatic biosynthesis. However, chemical synthesis is not industrially feasible due to its numerous reaction steps, numerous byproducts, and high toxicity. Therefore, enzymatic biosynthesis has recently become widely used. However, enzymatic biosynthesis also has drawbacks such as high enzyme usage and low peptide productivity.

[0006] Korean Patent Nos. 10-0762730 and 10-2183558 disclose novel enzymes derived from Sphingobacterium strains that efficiently synthesize a dipeptide, namely L-alanyl-L-glutamine. However, compared to known enzymes, there is a need to discover new enzymes that are more efficient and, in particular, capable of producing L-alanyl-L-glutamine even at high temperatures.

[0007]

[0008] Accordingly, the inventors of the present invention, in order to discover a novel enzyme other than the previously known L-alanyl-L-glutamine biosynthetic enzyme, searched for a novel enzyme based on homologs of the previously known enzymes, and confirmed that the protein consisting of sequence number 1 has the characteristic of having high L-alanyl-L-glutamine biosynthetic efficiency while maintaining its activity even at high temperatures, thereby completing the present invention.

[0009]

[0010] Accordingly, an object of the present invention is to provide a method for producing L-alanyl-L-glutamine, comprising the steps of: (a) culturing a recombinant strain expressing a protein consisting of sequence number 1 in a medium under conditions and for a time suitable for producing the protein; (b) mixing alanine methyl ester hydrochloride and glutamine with the culture; and (c) obtaining L-alanyl-L-glutamine.

[0011]

[0012] Another object of the present invention is to provide a composition for producing L-alanyl-L-glutamine, comprising at least one of a protein comprising sequence number 1 or a recombinant strain expressing a protein comprising sequence number 1.

[0013]

[0014] In order to achieve the above object, the present invention provides a method for producing L-alanyl-L-glutamine, comprising the steps of: (a) culturing a recombinant strain expressing a protein consisting of sequence number 1 in a medium under conditions and for a time suitable for producing the protein; (b) mixing alanine methyl ester hydrochloride and glutamine with the culture; and (c) obtaining L-alanyl-L-glutamine.

[0015]

[0016] In order to achieve another object of the present invention, the present invention provides a composition for producing L-alanyl-L-glutamine, comprising at least one of a protein consisting of SEQ ID NO: 1 or a recombinant strain expressing a protein consisting of SEQ ID NO: 1.

[0017]

[0018] The present invention is described in detail below.

[0019]

[0020] The present invention relates to an L-alanyl-L-glutamine biosynthetic enzyme, wherein the enzyme refers to a protein having the following amino acid sequence.

[0021] Sequence number 1:

[0022] MSGLSFAQDVKADSLYVRQHYDKIEQLIPMRDGTKLFTAIYMPKDKSKNYPVLLNRTPYTVAPYGADAYKKSLGNFPAEMREGFIFVYQDVRGRWMSEGEFEDVRPVNKSKNKKAIDETTDTYDTLEWLSKNMKNYNQKAGIYGISYPGF YSTMSLINSHPTLKAVSPQAPVTNWYLGDDFHHNGVLFLNDSFKFMSSFGVKRPQPITPDKGPKSFEYPIKDNYRFYLEGGSVKELKNTYFQDNIKFYNDLFAHPDYDQFWQDRNPLPHLTHVKPAVMTVGGFFDAEDAYGAFETYKAIEK QNPNATNILVAGPWFHGGWVRAKGDTFGDMQFGSSTGEYYQQQIELPFFNYYLKDKGDFKPTEARIFITGSNEWKQFETWPPKNSSTKKMFLQANGKIAFNQSNTATFDEYVSDPNHPVPYQEGVLETRSREYMVDDQRFASTRPDVMVY QTDVLTEDITVTGPVINHLFVSSTGTDADYVVKLIDVYPEDTPKFNEKLMAGYQNLIRAEIMRAKYRNSFEKPEAMIPNQKTSVMYTMPDVGHTFKKGHRIMIQVQNTWFPLADRNPQQFMNVYEATAKDFLKQTQRIYHDSFIEIPVLNK

[0023]

[0024] The protein consisting of the above sequence number 1 is a protein known as a CocE / NonD family hydrolase derived from Empedobacter brevis, and the fact that the protein is an α-amino acid ester acyltransferase (AET) having L-alanyl-L-glutamine biosynthetic activity was first confirmed by the present inventor. In particular, as confirmed by the present inventor, the protein showed a 67% higher L-alanyl-L-glutamine conversion rate when reacted at 25°C and a 179% higher L-alanyl-L-glutamine conversion rate when reacted at 37°C compared to the Sphingobacterium-derived enzyme disclosed in the above-mentioned prior patent document. In particular, at 50°C, the enzyme of the prior patent document did not show any activity at all, but the enzyme according to the present invention showed a similar degree of L-alanyl-L-glutamine at 25°C and 37°C.

[0025]

[0026] Accordingly, the present invention provides a method for producing L-alanyl-L-glutamine, comprising the steps of: (a) culturing a recombinant strain expressing a protein consisting of sequence number 1 in a medium for a time and under conditions suitable for producing the protein; (b) mixing alanine methyl ester hydrochloride and glutamine with the culture; and (c) obtaining L-alanyl-L-glutamine.

[0027]

[0028] Below, each step of the method according to the present invention is described in detail.

[0029]

[0030] (a) a step of culturing a recombinant strain expressing a protein consisting of sequence number 1 in a medium under conditions and for a time suitable for producing the protein;

[0031]

[0032] In the present invention, the recombinant strain refers to a strain comprising a vector including a gene encoding a protein consisting of SEQ ID NO: 1, preferably a pET28a vector. In addition, the recombinant strain may be a strain transformed with a vector including a gene encoding a protein consisting of SEQ ID NO: 1. The 'vector' is used for the purpose of replication or expression of the protein of the present invention for the recombinant production of the protein consisting of SEQ ID NO: 1 of the present invention, and generally includes at least one of a signal sequence, a replication origin, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably, it may be a vector including a gene encoding a protein consisting of SEQ ID NO: 1 of the present invention operably linked to a regulatory sequence, for example, a promoter. Accordingly, the recombinant strain may be a strain transformed with a vector including a gene encoding a protein consisting of SEQ ID NO: 1 operably linked to a promoter.

[0033]

[0034] A plasmid, a type of vector, is a linear or circular double-stranded DNA molecule into which foreign polynucleotide fragments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA fragments can be incorporated into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors, including those of bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell by introduction into the host cell and are thereby replicated together with the host genome.

[0035]

[0036] In the present invention, the recombinant strain may be E. coli, and in one embodiment of the present invention, E. coliBL21(DE3) was used, but is not limited thereto, and includes all bacteria and E. coli that can be used as a transformation host as is well known to those skilled in the art.

[0037] The above 'transformation' refers to a change in the genotype of a host cell by introduction of a foreign polynucleotide (a polynucleotide encoding the protein of the present invention or a fragment thereof), and refers to introduction of the foreign polynucleotide into the host cell regardless of the method used for the transformation. The foreign polynucleotide introduced into the host cell may be maintained by integration into the genome of the host cell or may be maintained without integration, and the present invention includes both.

[0038]

[0039] In one embodiment of the present invention, the E. coli BL21 (DE3) used as a recombinant strain refers to a strain genetically engineered to easily produce L-alanyl-L-glutamine. That is, the wild-type BL21 (DE3) uses a dipeptide transporter (dppA) to introduce a dipeptide into the cell, and uses a peptidase (pepABDN) to break down the dipeptide introduced into the cell into a single amino acid. In addition, the wild-type BL21 (DE3) uses glutaminase (glsAB) to convert L-glutamine into L-glutamate. Therefore, in order to prevent the decomposition of L-alanyl-L-glutamine produced by the protein according to the present invention and to prevent L-glutamine used as a substrate of L-alanyl-L-glutamine from being converted into L-glutamic acid, the dppA, pepA, pepB, pepD, pepN, glsA and glsB genes of wild type BL21 (DE3) are deleted.

[0040]

[0041] In the present invention, the time, conditions, and medium suitable for producing the protein may vary depending on the type of host strain used as the recombinant strain.

[0042] The above "cultivation" refers to growing the recombinant strain under appropriately controlled environmental conditions. The cultivation process can be performed using appropriate media and culture conditions known in the art. This cultivation process can be easily adjusted and used by a skilled practitioner depending on the selected microorganism. Specifically, the cultivation may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0043] The above 'medium' refers to a material containing nutrients as main components necessary for culturing the recombinant strain, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, any medium and other culture conditions used for culturing the recombinant strain may be used without special restrictions as long as it is a medium used for culturing general microorganisms, and the recombinant strain may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and / or vitamin.

[0044] The carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0045] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0046] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in a batch or continuous manner, but are not limited thereto.

[0047] Additionally, during the above culturing, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. Additionally, during the culturing, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Additionally, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or to maintain anaerobic and microaerobic states, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0048] In the above culture, the culture temperature can be maintained at 20°C to 45°C, specifically 25°C to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0049] In one embodiment of the present invention, E. coli BL21 (DE3) was used as a host strain and cultured in LB medium (Luria-Bertani broth) at 25°C for 18 hours with shaking at 200 rpm. However, this is not a limitation, and a person skilled in the art can appropriately change and apply culture conditions according to what is well known in the art. It is obvious to a person skilled in the art that any of the above-described conditions can be applied as long as it is a culture condition for a host strain capable of expressing a protein consisting of the sequence number 1 of the present invention.

[0050]

[0051] (b) a step of mixing L-alanine methyl ester hydrochloride and glutamine into the culture;

[0052]

[0053] The step (b) according to the present invention refers to a step of substantially synthesizing L-alanyl-L-glutamine by administering a substrate for biosynthesis of L-alanyl-L-glutamine.

[0054] The above substrate refers to a substrate containing a carboxyl group component and an amine group component. In the present invention, the carboxyl group component may be selected from an amino acid ester or an amino acid amide, and preferably may be L-alanine methyl ester hydrochloride. The amine group component may be selected from an amino acid, an amino acid with a C-terminal protected, or an amine, and preferably may be L-glutamine.

[0055] In the step (b) according to the present invention, it is advantageous for production to set the concentration of the substrate according to the proportional reaction relationship of the reactants, and there is no upper / lower limit restriction on the concentration of the reaction substrate, but the carboxyl group component and the amine component can be administered at a concentration of 50 to 600 mM.

[0056] When administering the above substrate, the carboxyl group component and the amine component may be dissolved in a buffer solution and administered. The buffer solution is not limited thereto, but may include a borate buffer solution, and the pH may be adjusted to a range of pH 8 to pH 10, preferably pH 8 to pH 9, and then administered.

[0057] Meanwhile, in the step (b) according to the present invention, when administering the substrate, the density of the recombinant strain may be OD600 = 0.1 to 4.0, but is not limited thereto.

[0058]

[0059] As described above, according to the experiments conducted by the inventor of the present invention, the protein comprising sequence number 1 according to the present invention has a constant level of L-alanyl-L-glutamine conversion rate within the range of 25°C to 50°C. In consideration of this, step (b) according to the present invention can be performed at any temperature within 15°C to 55°C, preferably within 25°C to 50°C, and a person skilled in the art can determine an appropriate temperature within the above range as needed to perform step (b).

[0060]

[0061] (c) a step of obtaining L-alanyl-L-glutamine;

[0062]

[0063] The step (c) according to the present invention refers to a step of terminating the reaction and recovering the finally produced L-alanyl-L-glutamine.

[0064] In the method for producing L-alanyl-L-glutamine according to the present invention, when the recombinant strain proceeds with production and the reaction progresses to a required degree, the reaction can be terminated by physically separating the recombinant strain from the newly synthesized L-alanyl-L-glutamine, for example, by using centrifugation, and the recombinant strain thus separated can be introduced into a new reaction system as a process strain and used repeatedly.

[0065] The above 'recovery' may be collecting the target L-alanyl-L-glutamine using a suitable method known in the art according to the culture method of the above-described recombinant strain, i.e., a culture method such as batch, continuous or fed-batch. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the target L-alanyl-L-glutamine may be recovered from the medium or the recombinant strain using a suitable method known in the art.

[0066]

[0067] In addition, the present invention provides a composition for producing L-alanyl-L-glutamine, comprising at least one of a protein comprising sequence number 1 or a recombinant strain expressing a protein comprising sequence number 1.

[0068]

[0069] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the production composition according to the present invention may further include additives such as acceptable carriers, excipients, or diluents in addition to the recombinant strain. The production composition of the present invention includes the recombinant strain produced by the method of the present invention, and may include 99.9% to 0.1 wt% of the carrier.

[0070]

[0071] In the present invention, the protein consisting of the sequence number 1 and the recombinant strain expressing the protein consisting of the sequence number 1 are as described above.

[0072]

[0073] The method of the present invention can efficiently synthesize and / or produce L-alanyl-L-glutamine, and in particular, the protein according to the present invention has the characteristic of being able to synthesize L-alanyl-L-glutamine even at high temperatures, and thus can be usefully used for producing L-alanyl-L-glutamine.

[0074]

[0075] Figure 1 is a schematic diagram showing the process by which a recombinant strain according to the present invention produces L-alanyl-L-glutamine.

[0076] Figure 2 shows the conversion rate of L-alanyl-L-glutamine at a reaction temperature of 25°C compared to that of an enzyme in a prior literature.

[0077] Figure 3 shows the conversion rate of L-alanyl-L-glutamine at a reaction temperature of 37°C compared to that of an enzyme in a prior literature.

[0078] Figure 4 shows the conversion rate of L-alanyl-L-glutamine at a reaction temperature of 50°C compared to that of an enzyme in a prior literature.

[0079]

[0080] The present invention is described in detail below. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0081]

[0082] Example 1. Production of L-alanyl-L-glutamine producing strain

[0083] 1-1. Disruption of dipeptide transporter and peptidase genes

[0084] Wild-type BL21 (DE3) can import dipeptides into cells using the dipeptide transporter (dppA) and break down dipeptides into single amino acids using peptidases (pepABDN). Furthermore, it can convert L-glutamine into L-glutamate using glutaminase (glsAB).

[0085] Therefore, in order to prevent the degradation of alanyl-glutamine (L-alanyl-L-glutamine, Ala-Gln) and to prevent the conversion of L-glutamine, which is used as a substrate for alanyl-glutamine, into L-glutamic acid, deletion of dppA, pepA, pepB, pepD, pepN, glsA, and glsB in wild-type BL21 (DE3) was performed.

[0086] To this end, the upstream and downstream regions of the target genes were obtained using the primers listed in Table 1, and cloned using the Gibson assembly method with the chromosomal transformation vector pCBT1 digested with EcoRV restriction enzyme to construct the recombinant plasmids pCBT1-ΔdppA, pCBT1-ΔpepA, pCBT1-ΔpepB, pCBT1-ΔpepD, pCBT1-ΔpepN, pCBT1-ΔglsA, and pCBT1-ΔglsB listed in Table 2.

[0087] Polymerase is KOD One TM Polymerase was used, and the PCR amplification conditions were denaturation at 98°C for 1 minute, followed by 29 cycles of denaturation at 98°C for 10 seconds, annealing at 55°C for 5 seconds, and polymerization at 68°C for 3 seconds, and then polymerization at 72°C for 1 minute. Cloning using Gibson assembly was performed by mixing the Gibson assembly reagent and each purified gene fragment and reacting at 50°C for 1 hour.

[0088] Each of the recombinant plasmids constructed above, pCBT1-ΔdppA, pCBT1-ΔpepA, pCBT1-ΔpepB, pCBT1-ΔpepD, pCBT1-ΔpepN, pCBT1-ΔglsA, pCBT1-ΔglsB vectors, was sequentially transformed into E. coliBL21 (DE3) strain by electroporation, and then a second crossing process was performed to produce a strain lacking dppA, pepA, pepB, pepD, pepN, glsA, and gllsb, which was named CE11EC007. The genetic manipulation was confirmed by PCR using the primers listed in Table 2.

[0089]

[0090]

[0091] Target gene primer direction sequence (5' -> 3') Sequence number dppA Forward GAAACCAGCATGATCAGCATC31 Reverse CACGAAGTGGCGAGTAATC32 pepA Forward CACGCGGTAGGCTTTATAG33 Reverse AATTTGCGCGAGGAACAC34 pepB Forward GATATCACCATCGGCTTCAATG35 Reverse CGATGATGTTGACGCCATC36 pepD Forward CCTTATCCGATGCGGTATTTAC37 Reverse GTAACCTGCCTGAAGTTAAAAAG38 pepN Forward GCTTAACGACGATGGTTTCTTGTACTTC39 Reverse GTATGAATACTGCTCGTCTGAAC40 glsA Forward GTTTCTTGCTGGCGCTC41 Reverse CGACGATCACATAACCCACATG42 glsB Forward GATAGCCAGTGAAACCAGTC43 Reverse CTGAAAAAATGCGTACTGGAAC44

[0092] 1-2. Cloning of an alpha-amino acid ester acyltransferase from Sphingobacterium sp. and a novel alpha-amino acid ester acyltransferase gene.

[0093] A gene encoding an alpha-amino acid ester acyltransferase derived from Sphingobacterium sp. used in a prior patent and a gene encoding a novel alpha-amino acid ester acyltransferase, F0358_12975, were synthesized and introduced into the pET28a vector to construct two types of plasmids, which were named pSAET and pCutisAET, respectively. Table 3 shows the amino acid sequences of SAET and F0358_12975 used in the prior patent (Korean Patent No. 10-0762730).

[0094]

[0095]

[0096]

[0097] 1-3. Construction of L-alanyl-L-glutamine producing strain

[0098] The vectors pSAET and pCutisAET constructed in the above Example 1-2 were transformed into the CE11EC007 strain constructed in the above Example 1-1 to obtain two strains producing L-alanyl-L-glutamine, and these strains were named Ec_SAET and Ec_CutisAET, respectively.

[0099]

[0100] Example 2. Evaluation of the productivity of L-alanyl-L-glutamine biosynthetic strains.

[0101] In order to confirm the L-alanyl-L-glutamine production ability of the Ec_SAET and Ec_CutisAET strains produced in Example 1 above, the strains were cultured using the following method.

[0102] First, 0.25 ml of seed culture was inoculated into a 250 ml corner-baffle flask containing 25 ml of LB medium (Luria-Bertani broth) supplemented with 50 μg / ml kanamycin, and when the OD600 value reached 0.4 to 0.6, 0.6 mM IPTG (Isopropyl-β-D-thio-galactoside) was added to induce the expression of the recombinant protein. The culture was shaken at 200 rpm for 18 h at 25°C, and after the culture was completed, 1 ml of the culture was centrifuged to recover the cells, washed with an equal volume of PBS (Phosphate-buffered saline (pH 7.0)), and used for the enzymatic reaction.

[0103] The enzymatic reaction was initiated by adding 100 μl of the wet cells prepared by the above method to 900 μl of 0.2 M borate buffer (pH 8.5) containing 50 mM L-alanine methyl ester hydrochloride and 100 mM glutamine. The enzymatic reaction was carried out for 1 hour, and after completion of the reaction, the cells were removed using a centrifuge, and the enzyme reaction solution was analyzed by HPLC. The results of the enzymatic reaction are shown in Table 4.

[0104]

[0105] Strain light reaction temperature (℃) L-alanyl-L-glutamine conversion rate (%) * Ec_SAET2526.13714.9500 Ec_CutisAET2543.53741.55044.1

[0106] * The conversion rate (%) of L-alanyl-L-glutamine was calculated as follows.

[0107] (Molar concentration of L-alanyl-L-glutamine produced X 100) χ (Molar concentration of L-alanine methyl ester hydrochloride added to the reaction)

[0108]

[0109] As a result of the above experiment, CutisAET showed an L-alanyl-L-glutamine conversion rate (%) that was 67% and 179% higher than that of SAET at enzyme reaction temperatures of 25°C and 37°C, respectively (see Table 4, Figs. 2 and 3). In particular, at a reaction temperature of 50°C, CutisAET achieved an L-alanyl-L-glutamine conversion rate (%) of 44.1%, but SAET did not produce L-alanyl-L-glutamine (see Table 4 and Fig. 4).

[0110]

[0111] As described above, the method of the present invention can efficiently synthesize and / or produce L-alanyl-L-glutamine, and in particular, the protein according to the present invention has the characteristic of being able to synthesize L-alanyl-L-glutamine even at high temperatures, and thus can be usefully used in the production of L-alanyl-L-glutamine, and thus has high industrial applicability.

Claims

1. (a) A step of culturing a recombinant strain expressing a protein consisting of sequence number 1 in a medium under conditions and for a time suitable for producing the protein; (b) a step of mixing L-alanine methyl ester hydrochloride and glutamine into the culture; and (c) a step of obtaining L-alanyl-L-glutamine; A method for producing L-alanyl-L-glutamine comprising:

2. A method for producing L-alanyl-L-glutamine, characterized in that the protein in claim 1 is alpha-amino acid ester acyltransferase (AET).

3. A method for producing L-alanyl-L-glutamine, characterized in that in the first paragraph, step (b) is performed at a temperature of 25°C to 50°C.

4. A method for producing L-alanyl-L-glutamine, characterized in that in claim 1, the strain is Escherichia coli.

5. A method for producing L-alanyl-L-glutamine, characterized in that in the first paragraph, the step (b) is a step of synthesizing L-alanyl-L-glutamine by administering a substrate for the biosynthesis of L-alanyl-L-glutamine.

6. A method for producing L-alanyl-L-glutamine, characterized in that in paragraph 4, the E. coli has deletions of the dppA, pepA, pepB, pepD, pepN, glsA, and glsB genes.

7. A method for producing L-alanyl-L-glutamine, characterized in that in claim 4, the strain is transformed with a vector including a gene encoding a protein consisting of the sequence number 1 operably linked to a promoter.

8. A method for producing L-alanyl-L-glutamine, characterized in that in paragraph 5, the substrate comprises a carboxyl group component and an amine component.

9. A method for producing L-alanyl-L-glutamine, characterized in that in paragraph 8, the carboxyl group component and the amine component are administered at a concentration of 50 to 600 mM.

10. A method for producing L-alanyl-L-glutamine, characterized in that in paragraph 8, the carboxyl group component is selected from the group consisting of amino acid ester, amino acid amide, and L-alanine methyl ester hydrochloride.

11. A composition for producing L-alanyl-L-glutamine, comprising at least one of a protein comprising sequence number 1 or a recombinant strain expressing a protein comprising sequence number 1.

12. A composition for producing L-alanyl-L-glutamine, characterized in that the protein in claim 11 is α-amino acid ester acyltransferase (AET).

13. A composition for producing L-alanyl-L-glutamine, characterized in that the protein in claim 11 has enzymatic activity even at 50°C.

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