WO2015037823A1

WO2015037823A1 - Method for producing 6-aminohexanoic acid or caprolactam from lysine

Info

Publication number: WO2015037823A1
Application number: PCT/KR2014/006485
Authority: WO
Inventors: 변성민; 이수미; 최현준; 김용환
Original assignee: 광운대학교 산학협력단
Priority date: 2013-09-10
Filing date: 2014-07-17
Publication date: 2015-03-19
Also published as: KR20150029526A; KR101565253B1

Abstract

The present invention relates to a method for producing caprolactam or 6-aminohexanoic acid from lysine; and the production method of the present invention can create 6-aminohexanoic acid or caprolactam, having physical properties that are the same as those of existing nylon 6, by using lysine which is mass produced as an additive for feeds, thus improving economic viability, and without involving the use of fossil starting materials or toxic chemical catalysts, thus lessening the generation of environmentally polluting substances such as carbon dioxide.

Description

【Specification】

[Name of invention]

Method for preparing 6-aminonucleoanoic acid or caprolactam from lysine

Technical Field

The present invention relates to a process for preparing 6-aminonucleoanoic acid or caprolactam from lysine.

The present invention also relates to a process for producing nylon 6 from the caprolactam.

Background Art

Caprolactam is an aromatic hydrocarbon compound compound which can be used as a raw material for producing nylon 6 or as a raw material for the production of engineering resins and films. Caprolactam is particularly high in economic value due to increasing demand for engineering resin applications. Nylon 6, produced from caprolactam as a raw material, is usually produced by ring opening synthesis reaction of ε-caprolactam monomer.

There are three major processes for the chemical production of caprolactam. These processes are all using petroleum-derived cyclonucleic acid or benzoic acid, one of which is a cyclonucleanone oxime HC1 process produced by cyclonucleic acid oxidation, the other is a cyclonucleanone process produced by cyclonucleic acid oxidation, Another is a cyclonucleic acid carboxylic acid process with benzoic acid.

The most widely used of these is the cyclonucleanone process. The cyclonucleanone process converts benzene as a starting material to cyclohexane or phenol, and converts the cyclonucleic acid or phenol through cyclohexanone. Synthesis with cyclohexanone oxime and heating the intermediate under sulfuric acid. This chemical reaction is known as the Beckman rearrangement. Benzene, a starting material in the process, is produced through the purification of petroleum compounds.

The biggest disadvantage of this existing chemical process is that excess ammonia is produced (4.5 tonnes / ton) as a byproduct. Multinational petrochemical companies have developed processes to reduce ammonia over a long period of time.

In addition, these caprolactam synthesis processes are all based on fossil raw materials, and not only emit a lot of environmental pollutants during the conversion process, but also have a problem of consuming a large amount of energy since the process is performed at high temperature and high pressure. In order to solve this problem, attempts to produce caprolactam from biomass-derived materials, that is, attempts to produce biocaprolactam have been steadily progressing.

When lysine produced from sugar is used to produce caprolactam or 6-aminohexanoic acid, the precursor of caprolactam, these materials can be dehydrated and synthesized to result in nylon 6. Nylon 6 produced in this way would be referred to as bioplastic because it is based on biomass and not on fossil raw materials, and may be classified as bionylon 6. Bionylon 6 has the same physical properties as nylon 6, but is produced without the use of fossil raw materials, resulting in less emission of environmental pollutants during the production process. Can be.

A number of rudimentary chemical processes for producing caprolactam or its precursors through the chemical process of amino acid lysine are known as follows. U. S. Patent No. 6,300, 496 discloses a process for producing caprolactam from lysine via 6-amino carpric acid via a chemical process. US Pat. No. 7,399,855 discloses a technique for chemically converting lysine to caprolactam in an alcoholic organic solvent environment. In addition, US Patent Publication No. 2010/014003 A technique for chemically converting lysine caprolactam under an organic solvent in which a chemical catalyst and hydrogen gas is present is disclosed. However, all of these processes have the disadvantage that the reaction conditions are high temperature and high pressure conditions and toxic chemical catalysts are used.

U.S. Pat.No. 7,799,545 describes a technique for producing caprolactam via 6-aminocapric acid through a biological process or a chemical fusion process. And caprolactam are disclosed. However, the technology does not use lysine, so it seems to be less economical. [Detailed Description of the Invention]

[Technical problem]

The present inventors can produce 6-aminonucleoanoic acid without the above problems by treating lysine with cyclotalamino enzyme to produce homoproline and treating reductase to produce 6-aminonucleoanoic acid. By reducing the 6-aminonucleosanic acid and treating it with hydrolase, it is possible to produce caprolactam by proceeding with dehydration reaction, thereby completing the present invention by elucidating that nylon 6 can be produced.

It is therefore an object of the present invention to provide a method for producing 6-aminonucleoanoic acid or caprolactam from lysine and a method for producing nylon 6 using the same without environmentally friendly energy consumption.

Technical Solution

One aspect of the present invention comprises the steps of (a) treating L- lysine with a cyclotalamino enzyme to produce homoproline; And (b) treating the homoproline with a reductase. According to one aspect of the invention, the step (a) is carried out at a temperature of 25 ° C to 80 ° C It may be made, the step (a) may be made at a condition of pH 6 to 7. Another aspect of the present invention provides a method of producing a homoproline comprising the steps of: (a) treating L-lysine with a cyclotalamino enzyme to produce homoproline; (b) treating the homoproline with a reductase to produce 6-aminonucleosanic acid; (c) treating the 6-aminonucleoanoic acid to produce a reduced 6-aminonucleoanoic acid; And (d) treating the reduced 6-aminonucleosanic acid with a hydrolase to proceed with dehydration reaction.

Here, as the reducing agent, NADH, lipoate, or dithiothritol may be used.

As the hydrolase, lactamase may be used. When caprolactam is synthesized outside of the body, dehydration reaction may be caused by using an alkaline catalyst in addition to hydrolase.

Another aspect of the present invention provides a method for producing nylon 6 comprising the step of condensing caprolactam produced by the above method with nylon 6.

Advantageous Effects

While providing a nylon of the same physical properties as the existing nylon 6 through the production method of the present invention, it is possible to reduce the generation of environmental pollutants such as carbon dioxide without using fossil raw materials or toxic chemical catalysts.

In addition, by utilizing lysine, which is produced in large quantities as feed additives, 6-aminonucleoanoic acid or caprolactam can be more economically improved.

[Brief Description of Drawings]

1 is a diagram showing a biosynthetic pathway for synthesizing 6-aminonucleoanoic acid from lysine.

2 shows the biosynthetic pathway for synthesizing caprolactam from lysine Picture.

3 is a flowchart showing the steps of producing nylon 6 from lysine.

Figure 4 shows the pET-22b (+)-spLCD recombinant gene sequence.

5 is a photograph showing the results of SDS-PAGE analysis of the target protein (lysine cycle deamination enzyme) and the purity analysis using the same.

FIG. 6 is a graph showing the reaction specificity of lysine cycles and diamines for various substrates.

7 is a graph comparing relative L-lysine conversion according to the type of buffer. Figure 8 shows the reaction rate of the fluctuations—degrees change with temperature—extra note- ^ Graf.

9 is a graph showing a change in conversion rate with a change in pH condition.

10 is a graph showing the conversion of L-lysine and L-pipecolic acid production reaction using lysine cyclodiaminase under optimal conditions (200 mM PIPES buffer, pH 7.0, temperature 60 ^° C., L-lysine). .

Figure 11 is a diagram showing the pET-22b (+ Hc plinase racemized recombinant gene sequence.

12 is a graph showing the results of conversion of substrates (proline, homoproline) reaction by proline racemase.

Figure 13 is a diagram showing the synthetic gene and amino acid sequence of Clostridium sticklandii-derived D-plin reductase A.

Figure 14 is a diagram showing the synthetic gene and amino acid sequence of D-proline reductase A derived from Bifidobacterium thermophilum RBL67.

15 is a photograph showing the results of Western blot analysis for the expression of the target protein (D-proline reductase A), post-expression splitting, and purity analysis.

[Form for implementation of invention] One aspect of the present invention comprises the steps of (a) treating L- lysine with a cyclotalamino enzyme to produce homoproline; And (W treating the homoproline with a reductase, a method for preparing 6-aminonucleosanoic acid (see Fig. 1). Homoproline is produced through the process of deamination, and the resulting homoproline is treated with reductase to produce 6-aminonucleosanic acid.

Lysines used in the present invention include commercially available L-lysine, such as L-lysine dihydrochloride, L-lysine hydrochloride, L-lysine phosphate, L-lysine diphosphate, L-lysine acetate and L-lysine. And L-lysine sources, and the steps necessary to bring L-lysine into proper condition for subsequent reaction are known to those of ordinary skill in the art. It is also well known in the art that commercially available lysine materials may be used and the step of separating L-lysine from D-lysine, for example, through separation of optical isomers and other separation and purification techniques. It will be self-evident to those who have it.

With respect to the temperature condition, the de-amination process may be 25 ⁰ C to 80 ° C, preferably from 40 ° C to 65 ⁰ C, more preferably from 55 ° C to 60 ^o C. If the reaction temperature increases by 10 ^o C, the enzyme reaction rate increases approximately 2 times, but when the reaction temperature exceeds 80 ^o C, the enzyme instability increases and the reaction rate and conversion rate decrease drastically.

With regard to the H conditions, the deamination process is neutral condition, and may preferably be pH 6-7. This is because the protein is inactivated in the acidic pH range.

Another aspect of the present invention provides a method for producing a homoproline comprising the steps of: (a) treating L-lysine with a cyclotalamino enzyme to produce homoproline; (b) treating the homoproline with a reductase to produce 6-aminonucleosanic acid; (c) above 6- Treating the aminonucleosanic acid with a reducing agent to produce reduced 6-aminonucleosanool; And (d) treating the reduced 6-aminonucleosanic acid with a hydrolase or an alkali catalyst to provide dehydration reactions (see FIG. 2).

Here, as a reducing agent, NADH or lipoate may be used when synthesized in vivo, and dithiotrattle or NADH may be used when synthesized in vivo.

The hydrolase may be used to synthesize caprolactam in vivo, and may be performed by causing dehydration by using an alkaline catalyst in addition to the hydrolase when synthesizing caprolactam in vivo.

According to another aspect of the invention, the invention provides a process for producing nylon 6 comprising the step of polymerizing caprolactam produced by the process to nylon 6 (see FIG. 3). <Example 1>

The C-terminus of the lysine cyclodeaminse enzyme gene (EFH32217.1,297153222) derived from Streptomyces pristinaespiralis present in the NCBI gene bank can be easily isolated. A histidine tag was added, and a gene having a new nucleotide sequence that could be selectively cleaved by a restriction enzyme of Nciel at the N-terminus and Xhol at the C-terminus was synthesized (FIG. 4). The synthesized gene was specifically cleaved using Ndel / Xhol restriction enzyme and then attached to pET-22b (+) plasmid vector prepared by cleavage by the same restriction enzyme using lagease enzyme. As a result, pET-22b ( +)-spLCD recombinant gene was constructed. The produced recombinant gene has a T7 / lac gene moiety controlled by IPTG for expression, so that expression can be selectively induced by IPTG addition in the future. <Example 2>

The recombinant gene prepared in Example 1 was transformed into E. colistrain BL21 (DE3) for expression, and then cultured at 37 ° C. in LB medium containing ampicillin (lmg / ml). 0.1 M IPTG was administered to induce the expression of the target protein, followed by further incubation for 16 hours in a shaker at 200 rpm. The obtained E. coli cells were harvested using a centrifuge, and then cells were disrupted using an ultrasonic grinder under crushing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCUOmM imidazole). The crushed cell solution was added to the Ni-NTA column to selectively separate only lysine cyclodeaminase, a target protein to which a histidine tag was attached. In order to measure the activity of the isolated enzyme, the enzyme solution was added to the reaction solution (10mM L-lysine, 0.4mM NAD ⁺ , 200mM pH6.9 PIPES buffers) and sampled at regular intervals. 10 μΐ of H ₂ SO ₄ (v / v) was added. Concentrations of L-lysine and L-pipecolic acid (L-homoproline) were measured using HPLC with Chirobiotic ™ T chiral column (5μπι, 25 cm x 4.6 mm, Astec comp) and UV spectrometer. Quantification

As a result of measuring the concentration of the target enzyme protein purely purified by Ni-NTA column, the concentration was 6 mg / ml. In FIG. 5, SDS-PAGE analysis was performed to confirm the purity of the target protein. As a result, it was confirmed that the target protein was obtained in a dissolved state at 40.3 kDa. In addition, the ratio of the insoluble protein was considerably low. It was found that the production of E. coli in the form of recombinant protein is very effective. <Example 4>

Banung experiment was carried out using the enzyme obtained in Example 3 for a substrate of various lengths similar to lysine. Substrates include L-2,3-diaminopropionic acid (L-2,3-diaminoprapiomc, L-C3), L-2,4-diaminopropionic acid, including L-lysine (L-C6). (L-2,4-diaminot) ii1; yricacicl, L-C4), L-ornithine (L-C5), D-ornithine (D— ornithine, D-C5), D-lysine ( D-lysine, D-C6), L-2,7 heptanoic acid (L-2,7-diaminoheptanoic acid, L-C7) and the like were used.

6 is a graph showing the relative conversion of reactions with various substrates. As expected, the most converting substrate was L-lysine. In addition, it was confirmed that the C5 and C7 compounds also reacted. The enzyme obtained in Example 3 exhibited broad substrate specificity with respect to carbon length while exhibiting strict optical specificity. In other words, the D-type substrate hardly reacted.

Several buffers were tested such as HEPES, Tris-HCl, MOPS, PIPES and potassium phosphate (KPi) to optimize reaction. Figure 7 shows the relative L- lysine conversion according to the type of buffer. As can be seen in Figure 7, the PIPES buffer showed the best activity. The PIPES buffer is presumed to be due to the small degradation activity for NADH used as a cof actor of the enzyme.

In order to determine the optimum temperature, the reaction rate was measured while changing the reaction temperature as shown in FIG. 8. The initial reaction rate at each temperature is 0.71xlO-7mol / Ls s (25 ° C), 1.57 lO-7mol / Ls s (37 ° C) and 4.65xlO-7mol / L-s (60 ^° C), respectively. It was estimated. this is Increasing the reaction temperature by 10 ° C is consistent with the general rule that the reaction reaction doubles. Up to 60 ^o C, the reaction rate increased relatively, after which the instability of the enzyme increased, resulting in a rapid decrease in reaction rate and conversion rate.

In order to determine the optimum reaction pH, the conversion rate of L-lysine was measured by using different buffers for each pH region (pH4-5: sodium acetate buffer, pH 6-8: sodium phosphate, pH 9-10: sodium carbonate). buffer). As shown in FIG. 9, the highest conversion was shown in the neutral pH condition, and the conversion was less than 10% under the acidic pH condition of pH 5. This is presumably due to the inactivation of proteins in the acidic pH region.

As a result of reaction in the optimum reaction conditions (200 mM PIPES buffer, pH 7.0, temperature 60 ^° C, L-lysine) as shown in Figure 10, a conversion of more than 90% was achieved. It is assumed that this high conversion can be achieved because the deaminolated cyclization reaction is an irreversible reaction. It is believed that lysine cyclodiamines can effectively synthesize L-pipecolic acid (L-homoproline) from L-lysine in in-vitw and in-vivo states.

Example 9

Using a nucleotide sequence of the proline racemase enzyme gene (EAN89436, 70875934) from the Trypanosoma cruzi gene present in the NCBI gene bank, a histidine tag is added to the N-terminus for easy separation. A gene having a new base sequence that can be selectively cleaved by Ndel at the end and Xhol restriction enzyme at the C-terminal end was synthesized (FIG. 11). The synthesized gene was specifically cleaved using Ndel / Xhol restriction enzyme and then attached to pET-22b (+) plasmid vector prepared by cleavage by the same restriction enzyme using T4 ligase enzyme. ) -tc plinase racemized recombinant gene was constructed. The recombinant gene produced has a part of the T7 / lac gene controlled by IPTG for expression, so that expression can be selectively induced by IPTG addition in the future.

E. coli for expressing the recombinant gene prepared in Example 9

(E olistrainBL21 (DE3)) was transformed and then cultured at 20 ° C. in LB medium containing ampicillin (lmg / ml). After administration of 0.1 M IPTG to induce the expression of the target protein, 48 hours were further incubated in a shaker at 200 rpm. The obtained E. coli cells were harvested using a centrifuge, and then cells were disrupted using an ultrasonic grinder under a crushing buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCUOmM imidazole). The crushed cell solution was added to the Ni-NTA column to selectively separate only the histidine-tagged target protein racenase.

In order to measure the activity of the isolated enzyme, 0.97 mg / ml of enzyme solution was added to the reaction solution (25 mM L—proline, 200 mM sodium acetate buffer, pH 6.0), sampled at regular intervals, and the reaction was stopped. 10 μl of 10% H ₂ SO ₄ (v / v) was added thereto. The concentration of semi-aqueous L-proline and product D-proline were quantified using Chirobiotic ™ T chiral column (5μπι, 25cmX4.6mm, Astec comp) and HPLC equipped with UV spectrometer.

<Example 11> Expression optimization was performed to express the enzyme under optimal expression conditions (48hr after 20 ^o C incubation, 20 ° C induction) and reacted under the following conditions (200 mM, sodium acetate buffer, pH 6.0, temperature 37 ° C, L-proline). As a result, as can be seen in Figure 12, it can be seen that 90% racemization reaction in 1 hour. The reaction results of L-type and D-type resulted in equilibrium of reaction at the concentration of 50:50.

After optimizing codons in E. coli using the nucleotide sequences of the D-proline reductase A gene derived from Clostridium sticklandii and Bifidobacterium thennophilum RBL67 from the NCBI gene bank (YP_003937258.1, 310659537 I ΥΡ_007594109.1, 470203397), C-terminal 6χ-histidine tag is added to allow separation and purification even after expression and cleavage at Ν-terminal, and Nnucleotide at N-terminal and specific sequence that can be selectively cleaved by restriction enzyme of Xhol at C-terminal Branches synthesized genes (FIG. 14). The synthesized gene was specifically cleaved using Ndel / Xhol restriction enzyme and then attached to pET-22 (+) plasmid vector prepared by cleavage by the same restriction enzyme using T4 ligase enzyme, resulting in pET-22b. (+)-csPrdA I pET-22b (+)-MPrdA recombination gene was constructed. The produced recombinant gene has a part of the T7 / lac gene controlled by IPTG for protein expression, so that overexpression can be selectively induced by IPTG addition in the future.

The recombinant gene prepared in Example 12 was transformed into a water-soluble E. coli (£: coli strain BL21 (λ! Ε3)) for expression, and then 37 ° C and 200 rpm in LB medium containing ampicillin (0.1 mg / ml) Incubated with. 1 mM to induce overexpression of the target protein. IPTG was administered and further cultured for 16 hours in a shaker at 25 ° C, 200 rpm. The obtained E. coli cells were harvested using a centrifuge, and then cells were disrupted using an ultrasonic grinder under a crushing buffer (50 mM NaH ₂ P0, 300 mM NaCl, 10 mM imidazole). After disruption, the cell solution from which cell debris was removed was added to the Ni-NTA column to selectively separate only the 6 × -histidine-tagged target protein (D-Prinlin Reductase A).

In order to confirm the expression and purity of the isolated enzyme, we used Western blot method using antigen-antibody reaction against 6x-histidine tag after SDS-PAGE, and Clostridium sticklandu-derived D-prine reductase was In case of expression, 69.5 kDa, the alpha unit having a pyruvoyl (Pyruvoyl) group is 23.8kDa, the beta unit is 45.7kDa after the post-expression split process. B-fidobacterium thermophilum RBL67-derived D-plin reductase had 66.9 kDa when expressed for all enzymes, 19.8 kDa for alpha units having pyruboyl groups and 47.1 kDa for beta units after splitting after expression (Fig. 15). ).

Substrate conversion experiments were conducted on substrates (D-proline, D-homopropine) using the enzyme expressed in Example 13. The reaction was carried out under the following reaction conditions (200mM, sodium acetate buffer, H 6.0, temperature 37 ⁰ C, D-proline / D-homoproline), and as shown in Table 1 below, 90% reaction occurred in 10 hours. You can see the progress. These results show that D-plin is converted to 5-aminopentanoic acid, and D-homoplin is converted to 6-aminohexanoic acid. there was. 6-aminonucleoanoic acid can be readily converted to caprolactam by a simple dehydration reaction catalyst.

Table 1 Response time (hr) D-proline conversion (%) D-homoproline conversion (%)

1 10 5

5 60 20

10 90 40

Claims

【Scope of Claim】

[Claim 1]

(a) producing homoproline by treating L-lysine with cyclodeaminase; and

(b) treating the homoproline with reductase

Method for producing 6-aminohexanoic acid comprising.

[Claim 2]

The method of claim 1, wherein step (a) is performed at a temperature of 25°C to ^80oC .

[Claim 3]

The method of claim 1, wherein step (a) is performed under conditions of pH 6 to 7.

【Claim 4】

(a) treating L-lysine with cyclodeaminase to produce homoproline;

(b) producing 6-aminohexanoic acid by treating the homoproline with reductase;

(c) treating the 6-aminohexanoic acid with a reducing agent to produce reduced 6-aminohexanoic acid; and

(d) performing a dehydration reaction by treating the reduced 6-aminohexanoic acid with a hydrolytic enzyme or an alkaline catalyst.

A method for producing caprolactam comprising.

[Claim 5]

In clause 4,

The method of claim 1, wherein the reducing agent is NADH, thiothreitol, or lipoate.

【Claim 6】

According to claim 4,

The method of claim 1, wherein the hydrolytic enzyme is lactamase. [Claim 7]

A method for producing nylon 6, comprising the step of polymerizing caprolactam produced by the method of any one of claims 4 to 6 into nylon 6.