WO2016048048A1 - Method for preparing ε-caprolactam by using novel caprolactam converting enzyme - Google Patents

Abstract

The present invention relates to a novel ε-caprolactam converting enzyme and, more specifically, to: a novel ε-caprolactam converting enzyme for converting aminocaproic acid into ε-caprolactam; and a method for preparing ε-caprolactam by using the ε-caprolactam converting enzyme or a microorganism having been transformed by a gene encoding the ε-caprolactam converting enzyme. According to the present invention, aminocaproic acid can be efficiently converted into ε-caprolactam through a biosynthetic process without using an organic solvent, and thus ε-caprolactam can be prepared in an environmentally friendly manner without producing a toxic material or an environmentally pollutive byproduct.

Description

Method for preparing ε-caprolactam using a novel caprolactam converting enzyme

The present invention relates to a novel caprolactam converting enzyme, and more particularly, to a novel caprolactam converting enzyme for converting aminocapronic acid into ε-caprolactam and to a gene encoding the caprolactam converting enzyme or the caprolactam converting enzyme. It relates to a method for producing caprolactam using the microorganisms.

ε-caprolactam is a precursor for synthetic polymer nylon 6, synthetic leather and polyurethane linkers, and is a monomer that is in the spotlight in the world market for $ 2,700 to 3,300. (epsilon) -caprolactam is a lactam type organic compound of 6-aminohexanoic acid (ε-aminohexanoic acid, 6-aminocapronic acid), and nylon-6 is produced by a ring-opening polymerization reaction of an ε-caprolactam monomer. The starting compound for producing ε-caprolactam is benzene, benzene is converted to cyclohexane or phenol, and the cyclohexane or phenol is subjected to cyclohexanone to cyclohexanone oxime), which intermediate is heated under sulfuric acid. This chemical reaction is known as the Beckman rearrangement. The starting compound benzene is produced through the purification of petroleum compounds. However, the process of producing ε-caprolactam through a chemical reaction is increasing the need for bioprocessing technology research due to problems such as the use of benzene toxic substances, the generation of environmental pollution by-products and the use of strong oxidizing agents.

Currently, as another method, a method of converting 6-aminohexaenoic acid, which is a precursor of ε-caprolactam, to an enzymatic method through a bio biosynthesis process (US Patent Publication No. 2009-0137759), 6-aminocaproic acid, non-naturally occurring microbial organisms having ε-caprolactam, hexamethylenediamine or levulinic acid pathways (US Patent Publication No. 2010-0317069), adipates, 6-aminocapronic acid or non-having ε-caprolactam pathways Research is being done on natural microbial organisms (US Pat. No. 7799545). Although studies have recently been reported on the conversion of ε-caprolactam from aminocaproic acid using enzymes in organic solvents (Tetrahedron Letters, 54: 370-372, 2013), the use of enzymes or microorganisms in water No results have been reported for ε-caprolactam conversion from capronic acid.

Recently, a study on a gene circuit using a regulator capable of detecting ε-caprolactam in a cell has been reported (Korea Patent Publication No. 2015-0056072). Through this, a small amount of ε-caprolactam can be detected and quantified in microorganisms, and a new enzyme search method has been studied. In addition, by using the enzyme search method using a gene circuit, it is possible to detect useful genes in various gene populations including the environment or metagenomic library, and to quickly detect various enzyme activities with sensitivity and expression control of compounds with high sensitivity. And quantification.

Accordingly, the present inventors have made intensive efforts to develop a new enzyme for generating ε-caprolactam from aminocapronic acid. As a result, a novel enzyme screened from metagenome is prepared using aminosenmic acid. It confirmed that the activity which converts into (epsilon) -caprolactam is excellent, and completed this invention.

The present invention provides a novel enzyme having an activity of converting aminocapronic acid into ε-caprolactam.

Another object of the present invention is to provide a method for preparing ε-caprolactam using an enzyme having an activity of converting the aminocapronic acid into ε-caprolactam.

In order to achieve the above object, the present invention provides a caprolactam converting enzyme having an amino acid sequence having at least 90% homology with SEQ ID NO: 1.

The present invention also provides a gene encoding the caprolactam converting enzyme and a recombinant vector containing the gene.

The present invention also provides a recombinant microorganism into which the gene or the recombinant vector is introduced.

The present invention also comprises the steps of converting aminocapronic acid into ε-caprolactam using the caprolactam converting enzyme; And it provides a method for producing a method for producing ε-caprolactam comprising the step of recovering the generated ε-caprolactam.

The present invention also comprises the steps of culturing the recombinant microorganisms in aminocapronic acid-containing medium to produce caprolactam; And it provides a method for producing ε-caprolactam comprising the step of obtaining the produced caprolactam.

According to the present invention, aminocapronic acid can be efficiently converted into ε-caprolactam through a bio biosynthesis process without using an organic solvent, thereby producing ε-caprolactam in an environmentally friendly manner without generating toxic substances or byproducts of environmental pollution. can do.

1 shows an SDS PAGE gel of caprolactam converting enzyme according to the present invention.

2 and 3 show the results of LC-MS analysis and NMR analysis demonstrating the conversion of caprolactam from aminocapronic acid using the caprolactam converting enzyme according to the present invention.

Figure 4 shows the comparison of the enzyme activity according to the temperature of the caprolactam converting enzyme according to the present invention.

Figure 5 shows a comparison of the enzyme activity according to the pH of the caprolactam converting enzyme according to the present invention. In Figure 5 according to the pH indicates the activity in PIPES buffer solution, ○ indicates enzyme activity in the HEPPS buffer solution.

6 is a schematic diagram of a CL-GESS gene circuit capable of detecting ε-caprolactam.

Although ε-caprolactam has been synthesized from benzene through conventional chemical reactions, development of synthetic methods through bio biosynthesis has been required due to problems such as the use of benzene, a toxic substance, the generation of environmental pollution by-products, and the use of strong oxidizers. The present inventors developed a genetic circuit using a regulator capable of detecting ε-caprolactam (Korean Patent Publication No. 2015-0056072), and discovered a caprolactam converting enzyme from metagenome using the gene circuit.

Hereinafter, the present invention will be described in detail.

1. ε-Caprolactam Converting Enzyme and Its Genes

One aspect of the invention relates to a caprolactam converting enzyme having an amino acid sequence having at least 90% homology with SEQ ID NO: 1 and a gene encoding the caprolactam converting enzyme. The gene encoding the caprolactam converting enzyme has a nucleotide sequence of SEQ ID NO: 2.

The epsilon caprolactam converting enzyme of the present invention comprises the amino acid sequence of SEQ ID NO: 1, wherein the epsilon caprolactam converting enzyme deletes, inserts, substitutes, or substitutes for amino acid residues within a range that does not affect the function of the protein. May be variants, or fragments of amino acids having different sequences. Amino acid exchange at the protein and peptide levels that does not alter the activity of the ε-caprolactam converting enzyme as a whole is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like. Accordingly, the present invention includes a protein having an amino acid sequence substantially identical to a protein comprising the amino acid sequence of SEQ ID NO: 1, and a variant thereof or an active fragment thereof. The substantially identical protein means those having homology of at least 80%, preferably at least 90%, most preferably at least 95% of amino acid sequences, but are not limited thereto, and have homology of at least 80% amino acid sequence and the same. If it has enzyme activity, it is included in the scope of the present invention.

Gene of the epsilon caprolactam converting enzyme is preferably composed of the nucleotide sequence of SEQ ID NO: 2. However, the gene encoding the ε-caprolactam converting enzyme and variants or active fragments thereof of the present invention may be modified in the coding region within a range not changing the amino acid sequence of the enzyme and its variants or active fragments expressed from the coding region. Modifications may be made, and various mutations may be made within a range that does not affect the expression of genes in parts other than coding regions, and such mutation genes are also included in the scope of the present invention. Therefore, the present invention includes a gene consisting of a base sequence substantially the same as the gene of SEQ ID NO: 2 and fragments of the gene. Genes consisting of the same base sequence means those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95%, but are not limited thereto, and at least 80% sequence homology. And encoded proteins are included in the present invention if they have the same enzymatic activity. As described above, as long as the gene of the ε-caprolactam converting enzyme of the present invention encodes a protein having equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof. It is included in the scope of the present invention.

The amino acid sequence of SEQ ID NO: 1 contained in the ε-caprolactam converting enzyme is preferably encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 2, but the present invention is not limited thereto, and the present invention has the same amino acid sequence. As long as the protein can be encoded, it may be encoded by a gene consisting of another base sequence which is substantially the same as the base sequence of SEQ ID NO: 2. Such base sequences may be single stranded or double stranded, and may be DNA molecules or RNA molecules.

In one aspect of the present invention, a meta-genome library derived from a tidal flat and a transformant co-transformed into E. coli were cultured in an aminocaproic acid-containing solid medium using a CL-GESS gene circuit using a regulator capable of detecting ε-caprolactam. By using the generated epsilon caprolactam, a colony expressing a fluorescent gene, which is a reporter gene, was selected, and the colonies were cultured to separate and amplify the separated gene to obtain a caprolactam converting enzyme gene.

2. Recombinant Vectors and Transformants of ε-Caprolactam Converting Enzymes

Another aspect of the invention relates to a recombinant vector containing a gene encoding said caprolactam converting enzyme and a recombinant microorganism into which said gene or said recombinant vector is introduced.

The recombinant vector includes, but is not limited to, plasmid vector, cosmid vector, bacteriophage vector, viral vector, and the like.

The recombinant vector is an expression control sequence such as a promoter, a terminator, an enhancer, or a sequence for secretion, etc., depending on the type of host cell to produce the ε-caprolactam converting enzyme of the present invention. Can be combined as appropriate for the purpose.

The recombinant vector may further include a selection marker for selecting a host cell into which the vector has been introduced, and, in the case of a replicable expression vector, may include a replication origin.

In addition, the recombinant vector may include a sequence for facilitating the purification of the expression protein, specifically, the gene encoding the separation and purification tag to be operable to the gene encoding the ε-caprolactam converting enzyme of the present invention Can be connected. In this case, the separation and purification tag may be used alone, or GST, poly-Arg, FLAG, histidine-tag (His-tag) and c-myc, or two or more of them may be sequentially connected.

The gene encoding the epsilon caprolactam converting enzyme may be cloned through a restriction enzyme cleavage site. When a gene encoding a protein cleavage recognition site is used in the vector, a gene of the epsilon caprolactam converting enzyme may be used. Linked in frame, the enzyme can be obtained and then cleaved with protein cleavage enzymes to produce the original form of ε-caprolactam converting enzyme.

In addition, a recombinant expression vector comprising a gene encoding an epsilon caprolactam converting enzyme is introduced into the recombinant microorganism of the present invention.

A transformant may be prepared by transforming the recombinant vector according to the present invention into any one suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells and animal cells according to expression purposes. For example, the host cell may be Escherichia coli ( E. coli BL21 (DE3), DH5α, etc.) or yeast cells (Saccharomyces genus, Pichia genus, etc.). In this case, appropriate culture methods, media conditions and the like can be easily selected by those skilled in the art according to the type of host cell.

As a method of introducing a recombinant expression vector for producing a transformant of the present invention, a known technique, that is, a heat shock method, an electric shock method, or the like may be used.

In a specific embodiment of the present invention, a caprolactam converting enzyme gene screened in a metagenome library is inserted into a pET 28 (+) (Novagen, USA) vector to prepare a recombinant vector containing a caprolactam converting enzyme gene and the vector is expressed in E. coli ER2566. Strain (Novagen, USA) was transformed to obtain a transformant.

3. Production method of ε-caprolactam converting enzyme

Another aspect of the present invention relates to a method for producing caprolactam converting enzyme by culturing and purifying recombinant E. coli introduced with a recombinant vector containing a caprolactam converting enzyme gene.

The method for producing ε-caprolactam converting enzyme of the present invention comprises the steps of: 1) culturing a transformant into which a recombinant expression vector containing a gene encoding the ε-caprolactam converting enzyme of the present invention is introduced; 2) inducing expression of a gene encoding the ε-caprolactam converting enzyme in the transformant cultured in step 1); And 3) separating the epsilon caprolactam converting enzyme from the culture of the transformant in which the expression of the epsilon caprolactam converting enzyme gene is induced in step 2).

The N-terminus of the gene encoding the epsilon caprolactam converting enzyme of step 1) may be further linked to a gene or protein cleavage cleavage site encoding the tag for separation and purification, thereby resulting in a recombinant epsilon caprolactam converting enzyme. It may be possible to purify or to obtain the ε-caprolactam converting enzyme in its original form.

Cultivation of the transformant of step 1) may be performed according to a known method, and conditions such as culture temperature, incubation time and pH of the medium may be appropriately adjusted. In addition, the culture method may include batch culture, continuous culture and fed-batch culture. The culture medium used should suitably meet the requirements of the particular strain.

Separation of ε-caprolactam converting enzyme from the culture produced by the culture of step 3) can be carried out in a conventional method in the art, such as centrifugation, filtration. In addition, the ε-caprolactam converting enzyme isolated by the above method can be purified in a conventional manner, for example, using salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (acetone, ethanol, etc.). Protein fraction precipitation), dialysis, gel filtration, ion exchange, chromatography such as reversed phase column chromatography, and ultrafiltration and the like can be used alone or in combination to purify the enzyme of the present invention.

In a specific embodiment of the present invention, it was confirmed that the protein purified as described above exhibits the activity of the ε-caprolactam converting enzyme (see FIGS. 4 and 5). From the above results, it can be seen that the mass production of ε-caprolactam converting enzyme is possible according to the production method of the present invention.

4. Preparation method of epsilon caprolactam and composition for preparation of epsilon caprolactam

Another aspect of the invention is the step of converting aminocapronic acid to ε-caprolactam using a caprolactam converting enzyme; And it relates to a method for producing ε-caprolactam comprising the step of recovering the generated ε-caprolactam. The production method may include the product for producing ε-caprolactam used in the production of ε-caprolactam.

In addition, the method for producing ε-caprolactam of the present invention comprises the steps of 1) reacting the ε-caprolactam converting enzyme of the present invention with aminocapronic acid; And 2) recovering ε-caprolactam from the reaction product resulting from the reaction in step 1). The method for producing ε-caprolactam and the composition for preparing ε-caprolactam is to use the enzymatic activity of the ε-caprolactam converting enzyme of the present invention in vitro . In other words, [epsilon] -caprolactam conversion enzyme is used to generate [epsilon] -caprolactam from its substrate, aminocapronic acid.

The reaction of step 1) may be performed at a temperature range of 30 ° C. to 50 ° C., preferably at a temperature range of 35 ° C. to 45 ° C., more preferably at a temperature of about 37 ° C., but is not limited thereto. In addition, the reaction of step 1) may be performed at a pH range of 6.5 to 8.5, preferably at a pH range of 7 to 8, more preferably at a pH of 7.5, but is not limited thereto.

In addition, the reaction of step 1) may include coenzymes such as NAD, NADH, and FDH together with the ε-caprolactam converting enzyme in order to enhance the activity of the ε-caprolactam converting enzyme, in particular FDH together. It is preferred to be included.

In the reaction of step 1), the aminocapronic acid is preferably injected from the outside, the aminocapronic acid should be injected in a sufficient amount in accordance with the desired amount of ε-caprolactam. In addition, the injection of the aminocaproic acid may be performed continuously. In particular, when the aminocapronic acid is continuously injected, it is possible to continuously produce ε-caprolactam by the ε-caprolactam converting enzyme.

In a specific embodiment of the present invention, the purified caprolactam converting enzyme was reacted in an aminocapronic acid containing solution to obtain ε-caprolactam (FIG. 2).

In another aspect, the present invention comprises the steps of culturing the recombinant microorganism in an aminocapronic acid containing medium to produce caprolactam; And it relates to a method for producing ε-caprolactam comprising the step of obtaining the produced caprolactam.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[ Example  One]

Redesigned Genetic Circuits for Caprolactam Detection (CL- GESS Manufacturing

In accordance with the contents disclosed in Korean Patent Laid-Open No. 2015-0056072, a CL-GESS gene circuit capable of detecting ε-caprolactam was prepared as shown in FIG. 6.

In the CL-GESS gene circuit prepared as described above, since the NitR protein binds with ε-caprolactam to activate the nitA promoter, expression of fluorescent protein is induced in the presence of ε-caprolactam. Thus the CL-GESS can be used to confirm the presence of caprolactam.

[ Example  2]

CL- GESS  Discovery of Caprolactam Converting Enzyme

In order to discover caprolactam converting enzyme using CL-GESS prepared in Example 1, DE3 was inserted into chromosomal DNA of E. coli EPI300 (EPicentre, USA) using a DE3 Lysogenization Kit (Novagen, USA). EPI300 (DE3) was prepared.

E. coli EPI300 (DE3) prepared as described above, co-transformed the metagenome library derived from the mud flat and CL-GESS prepared in Example 1, transformants were 50 mM aminocapronic acid, 100 ㎍ / ㎖ ampicillin , LB solid medium containing 34 μg / ml chloramphenicol was plated and incubated at 37 ° C. for 24 hours to induce fluorescence. The degree of fluorescence of the single colonies on the solid medium was observed with a fluorescence microscope (Nicon AZ100-M, Japan) to select single colonies with the fluorescence expression within the top 5%.

Single colonies selected as described above were inoculated in LB liquid medium containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol, followed by shaking culture at 37 ° C. for 12 hours, followed by 50 mM aminocaproic acid, 100 μg. Inoculated in LB liquid medium containing a / ml ampicillin, 34 μg / ml chloramphenicol (experimental group) and LB liquid medium containing 100 μg / ml ampicillin, 34 μg / ml chloramphenicol (control), for 24 hours. Fluorescence was induced by shaking culture at 37 ° C., and fluorescence expression in the cells was analyzed using the FACS Calibur system (Becton Dickinson, USA). The detector was set to detect FSC, SSC, and FL1-H (excitation = 488 nm, emission = 530/30 nm), and the data of 10,000 sample cells were analyzed by FlowJo (Tree Star, Inc., USA). .

As a result, it was confirmed that the fluorescence was increased in one single colony compared to the control group, the metagenomic gene was isolated from the single colony, and the nucleotide sequence was analyzed. As a result, the metagenomic gene introduced into the single colony was sequenced. It was confirmed that it has the nucleotide sequence of No. 3 (Macrogen, Korea).

As a result of analyzing the nucleotide sequence of SEQ ID NO: 3 with an ORF finder, it was confirmed that 13 ORFs were present in the nucleotide sequence of SEQ ID NO: 3, and the aminocapronic acid was cloned and expressed by the 13 ORFs, respectively. The ORF of the caprolactam converting enzyme showing the activity of converting to epsilon -caprolactam was derived. The ORF of the caprolactam converting enzyme as described above was isolated and the nucleotide sequence was analyzed. As a result, it was confirmed that the ORF of the caprolactam converting enzyme had the nucleotide sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 1.

[ Example  3]

Preparation of Recombinant Expression Vectors and Transformants Comprising Caprolactam Converting Enzyme Genes

To prepare a caprolactam converting enzyme having a nucleotide sequence of SEQ ID NO: 2 identified in Example 2, primer pairs of SEQ ID NOs: 4 and 5 were designed.

SEQ ID NO:	order
4 (forward primer)	5'-TTTCATATGAACTTAACGGGAAAA-3 '
5 (reverse primer)	5'-TTTCTCGAGTTATTGCGCTACCCAAC-3 '

The primers were designed with NdeI and XhoI restriction enzyme cleavage portions, respectively, and amplified the nucleotide sequence of the gene by PCR using the primers.

The PCR product containing the amplified caprolactam converting enzyme gene was inserted into the plasmid vector pET28 (+) (Novagen, USA) using restriction enzymes NdeI and XhoI to construct a pET28 (+) / caprolactam converting enzyme expression vector.

The recombinant expression vector thus obtained was transformed into E. coli ER2566 strain (Novagen, USA) by a conventional transformation method to prepare a 'recombinant ER2566 strain'.

In addition, the transformants were stored frozen before incubation for the production of caprolactam by addition of 20% glycerin solution.

[ Example  4]

Production of Caprolactam Converting Enzyme

The recombinant ER2566 strain prepared in Example 3 was inoculated into a test tube containing 3 ml of LB medium, followed by incubation with a shake incubator at 37 ° C. until absorbance was 2.0 at 600 nm. The culture was carried out by adding the culture medium to a 2,000 ml flask containing 500 ml of LB medium. When the absorbance at 600 nm was 0.6 in the above culturing, IPTG was added to a final concentration of 0.1 mM to induce overexpression of caprolactam converting enzyme. Stirring rate was adjusted to 200rpm, the culture temperature was maintained at 37 ℃, after the addition of IPTG was incubated by adjusting the stirring speed to 150rpm, the culture temperature to 16 ℃.

As described above, the culture medium of the recombinant ER2566 strain induced overexpression of caprolactam converting enzyme was centrifuged at 6,000 g for 30 minutes at 4 ° C., washed twice with 0.85% sodium chloride (NaCl), and then 50 mM sodium phosphate and 300 mM Sodium chloride, 10 mM immidazole and 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride) were added to disrupt the cell solution with a sonicator.

The cell lysate was again centrifuged at 13,000 × g, 4 ° C. for 20 minutes to take only the supernatant, followed by His-tag in a fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA, USA). An IMAC adsorption column was used to isolate only the caprolactam converting enzyme, and the enzyme isolated as described above was confirmed by SDS-PAGE.

As a result, as shown in Figure 1, it was confirmed that the caprolactam converting enzyme was isolated (Fig. 1).

[ Example  5]

Confirmation of Caprolactam Converting Enzyme Activity

In order to measure the activity of the caprolactam converting enzyme isolated in Example 4, the reaction was performed for 10 minutes using 50 mM pH 7.5 HEPES buffer solution containing 1 mM aminocapronic acid and 10 units / ml caprolactam converting enzyme. After the reaction, the reaction was stopped by adding hydrogen chloride (HCl) to a final concentration of 200 mM.

In addition, in the measurement of enzyme activity, the concentration of precursor aminocapronic acid and product caprolactam were measured by high pressure liquid chromatography equipped with LC-Mass detector and ZORBAX ECLIPSE XDB-C18 (Agilent, USA) column. The ZORBAX ECLIPSE XDB-C18 (Agilent, USA) column was used at 10 ° C. at 0.4 mL / min, using solvent A (0.1% formic acid; formic acid) and solvent B (100% acetonitrile; acetonitrile). -90% acetonitrile was allowed to pass sequentially. In addition, NMR analysis of the reaction solution in which caprolactam converting enzyme and aminocaproic acid reacted was requested to the Basic Science Research Institute (Ochang) to confirm the degree of conversion of aminocapronic acid to caprolactam.

As a result, it was confirmed that aminocapronic acid is converted to caprolactam by caprolactam converting enzyme (FIGS. 2 and 3), and the specific activity of caprolactam converting enzyme is 20 ± 1.2 nmole / min as shown in Table 2. / mg.

	Specific activity (nmole / min / mg)
Caprolactam Converting Enzyme Isolated from Example 4	20 ± 1.2

[ Example  6]

Investigation of pH and Temperature Effects on Caprolactam Converting Enzyme Activity

With respect to the caprolactam converting enzyme isolated in Example 4, activity according to pH and temperature changes was confirmed.

6-1. Confirmation of Caprolactam Converting Enzyme Activity According to Temperature

Caprolactam converting enzyme was reacted with 50 mM pH 7.5 HEPES buffer solution containing 1 mM aminocapronic acid and 10 units / ml caprolactam converting enzyme at a temperature ranging from 25 to 50 ° C. for 10 minutes, followed by a final concentration of 200 mM. Hydrogen chloride (HCl) was added to stop the reaction.

As a result, the optimum temperature of the caprolactam converting enzyme was confirmed to be 40 ℃ (Fig. 4).

6-2. Confirmation of Caprolactam Converting Enzyme Activity According to pH

PH ranges from 6.5 to 7.5 using 50 mM piperazine-N, N'-bis (2-ethane sulfonic acid) (PIPES) buffer containing 1 mM aminocapronic acid (substrate) and 10 units / ml caprolactam converting enzyme The enzyme reaction was carried out until, and the enzyme reaction was carried out using a 50 mM HEPES buffer containing 1 mM aminocapronic acid and 10 units / ml caprolactam converting enzyme from pH 7.5 to 8.5. Specifically, the enzymatic reaction was performed at 40 ° C. for 10 minutes, and the reaction was stopped again by adding a final concentration of 200 mM hydrogen chloride (HCl).

As a result, it was confirmed that the optimum pH of the epsilon caprolactam converting enzyme was 7.5 (FIG. 5).

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Since the novel ε-caprolactam converting enzyme according to the present invention has excellent activity for converting aminocapronic acid into ε-caprolactam, the ε-caprolactam converting enzyme of the present invention is useful in a field requiring ε-caprolactam. Can be used.

Claims (7)

1. Ε-caprolactam converting enzyme comprising an amino acid sequence having at least 80% homology with SEQ ID NO: 1.
2. Gene encoding the epsilon -caprolactam converting enzyme of claim 1.
3. The method according to claim 2,

   The gene is a gene consisting of the nucleotide sequence of SEQ ID NO: 2.
4. Recombinant expression vector containing the gene of claim 2.
5. A transformant wherein the recombinant expression vector of claim 4 is introduced into a host cell.
6. Converting aminocaproic acid to ε-caprolactam using the ε-caprolactam converting enzyme of claim 1; And

   Recovering the generated ε-caprolactam;

   Method for producing ε-caprolactam comprising a.
7. Culturing the transformant of claim 5; And

   Recovering ε-caprolactam from the culture of the transformant;

   Method for producing ε-caprolactam comprising a.

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