KR20090128767A - Method for preparing nylon 4 using the enzymatic and chemical reactions - Google Patents

Abstract

PURPOSE: A novel method for preparing nylon 4 combining enzyme reaction and chemical reaction is provided to produce from biomass-derived raw material and reduce production cost. CONSTITUTION: A method for preparing nylon 4 from biomass through enzyme reaction and chemical reaction comprises: a first step of using glutamic acid or glutamic acid dicarboxylase(GAD) to obtain 4-aminobutylic acid; a second step of producing 2-pyrrolidone from 4-aminobutylic acid using catalyst or dehydrating agent; and a third step of adding initiator and catalyst from 2-pyrrolidone to obtain the nylon 4. The glutamic acid dicarboxylase is gadA or gadB enyme from E.coli or gad1, gad2 or gad3 enzyme from lactobacillus. The E.coli is Escherichia coli W3110. The catalyst is alumina(Al2O3), chromium oxide (Cr2O3), or titanium dioxide (TiO2) or tungsten oxide (WO3).

Description

Method for preparing Nylon 4 using the enzymatic and chemical reactions from biomass

The present invention relates to a method for producing nylon 4 using enzymatic and chemical reactions from biomass, and more particularly, 4-aminobutyl acid and 2-pyri, based on glutamic acid or sodium glutamate produced by microbial fermentation. The present invention relates to a novel method for producing nylon 4, which combines enzymatic reactions and chemical reactions with rolidone as an intermediate.

Recently, global oil prices continue to rise due to global oil supply and unstable crisis, and basic oils such as naphtha produced in dependence on crude oil sharply increase the production cost of secondary petroleum compounds. The price of products in the industry is rising rapidly. As an alternative to the petrochemical industry that relies on crude oil, all human efforts to produce alternative production methods or alternative compounds derived from biomass using industrial biotechnology have recently been developed such as bioenergy, bioplastic, and bio compounds. It is visible in various fields.

In the bioenergy sector, bioethanol to replace gasoline has grown rapidly to 100 trillion gallons by the end of 2007, mainly in the US and Brazil, and biodiesel to replace diesel is rapidly growing in Europe. In the bioplastics market produced using biomass, the market is rapidly expanding to replace polylactic acid (Poly Lactic acid), commercialized by Cargill-Dausa in 2002, to replace existing PET and PS with new compounds. In addition, PHA-based bioplastics, which were previously developed and commercialized by Metabolix, are also in the process of constructing a factory to enter the general-purpose polymer market beyond the conventional medical polymer market. In addition, PBS using succinic acid has been widely commercialized and used. In the biochemical market, 1,3-propanediol, commercialized by DuPont at the end of 2006, is a successful example of replacing the existing petrochemical process with biomass-based industrial biotechnologies. It is expected to be replaced by sustainable industrial biotechnology.

In the case of nylon 4, it is common to prepare 2-pyrrolidone using 1,4-butanediol using C4 fraction of petrochemical as a raw material and to prepare nylon 4 using the same. The development of an economical alternative production technology for nylon 4 is expected to accelerate its commercialization. In addition, nylon 4 has excellent organic solvent resistance, abrasion resistance, high temperature, flame retardancy, and workability, which are characteristics of general nylon, and can be a new bio nylon that can substantially compensate for the shortcomings of bio plastics such as PLA, which has recently been commercialized. It seems to be.

In the case of nylon in the field of bioplastics related to the present invention, there have been existing attempts to replace the existing petrochemical method with biomass-derived methods in nylon 6 and nylon 6,6, which form the representative large nylon market. . In other words, in case of nylon 6, Frost 6 (PCT / US2005 / 020326) of the University of Michigan in 2005 (PCT / US2005 / 020326) uses nylon 6 through cyclization and deamine reaction using lysine, an amino acid for feed additive, which is mass-produced by conventional microbial fermentation. The technology for producing caprolactam, which is a monomer, has been developed. In the case of nylon 6,6, in 1994, Dr. et al. (J. Am. Chem. Soc. 116: 399-400 (1994)) produced cis, cis-muconic acid by microbial fermentation based on glucose, He has developed a technique to convert to adipic acid by conversion. However, other technical developments have not been reported for other nylons.

On the other hand, there are some research and development examples for the intermediates 4-aminobutyl acid, 2-pyrrolidone, etc. in the production of nylon 4 of the present invention, first, in the case of 4-aminobutyl acid, gamma aminobutyl acid (GABA, r-aminobutyric) acid), which is known to have hypotension and analgesic effects due to neurotransmitter inhibition and is currently used as a food and pharmaceutical material (Erlander et al., Neurochem Res. 16: 215-226 (1991)). Such 4-aminobutyl acid can be prepared by glutamate decarboxylase based on glutamic acid, and various glutamic acid dicarboxylase have been reported to date (Proc. Natl. Acad. Sci. USA, 87: 8491 -8495 (1990), Protein Expression and Purification 8: 430-438 (1996), Biosci. Biotechnol. Biochem. 66 (12): 2600-2605 (2002)). However, most of them use 4-aminobutyl acid as a final product, and there is no example of producing nylon 4 through 2-pyrrolidone as a raw material. In the case of 2-pyrrolidone in 1990, Pathak et al. (Tetrahedron 46 (5): 1733-1744 (1990)) reported a method synthesized from 4-aminobutyl acid in the middle of the precursor synthesis of carcinogenic substances, 2003 BASF filed a patent for a method for continuously producing 2-pyrrolidone by reacting gamma-butylolactone, an existing petroleum compound, in a liquid phase with ammonia (PCT / EP2002 / 009721). However, these previous studies are related to the production of unit chemicals that are not related to the production of nylon 4.

As a result, the present inventors have made great efforts to develop nylon 4 having low dependence on petrochemical raw materials. As a result, the present inventors have led to the present invention for producing nylon 4 in three steps from glutamic acid or sodium glutamate.

An object of the present invention is to provide a method for producing nylon 4 using an enzyme reaction and a chemical reaction from biomass to replace the conventional method for producing petroleum-dependent nylon 4.

In order to achieve the above object, the present invention provides a method for preparing nylon 4 using an enzyme reaction and a chemical reaction from biomass.

The present invention relates to a novel production method for producing nylon 4 using sodium glutamate or glutamic acid produced by microbial fermentation from biomass as a starting material. By providing a source technology that can be produced from the raw materials can be reduced the dependence of petroleum compounds in the high oil price environment, and provides an effect that can significantly reduce the manufacturing cost, it will be a very useful invention in the petrochemical field.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for preparing nylon 4 using enzyme reactions and chemical reactions from biomass.

The method for preparing nylon 4 may include preparing 4-aminobutyl acid from glutamic acid or sodium glutamate using glutamic acid dicarboxylase (GAD) (step 1);

Preparing 2-pyrrolidone from the 4-aminobutyl acid prepared in step 1 using a catalyst or a dehydrating agent (step 2); And

Nylon 4 is prepared by adding an initiator and a catalyst from 2-pyrrolidone prepared in Step 2 to polymerize (Step 3).

Step 1 is a step of preparing 4-aminobutyl acid from glutamic acid or sodium glutamate produced by fermentation of microorganisms in biomass as in Scheme 1 below.

<Scheme 1>

The preparation of 4-aminobutyl acid in step 1 is carried out by producing a recombinant E. coli overproducing glutamic acid decarboxylase (GAD) having an activity of removing alpha-carboxyl groups of glutamic acid or sodium glutamate as a biocatalyst. Used for Preferably, the glutamic acid dicarboxylase of step 1 may be a gadA or gadB enzyme isolated from E. coli or a gad1, gad2 or gad3 enzyme isolated from the genus Lactobacillus.

Specifically, the method using gadA or gadB isolated from Escherichia coli isolates the chromosome of wild type E. coli W3110 and performs a PCR reaction using this as a template to clone the glutamic acid dicarboxylase (gadA or gadB) of Escherichia coli and then, in Escherichia coli The recombinant E. coli biocatalyst is produced by overexpressing a gene and introducing the enzyme into an expression vector capable of mass production, thereby overproducing glutamic acid dicarboxylase. The recombinant Escherichia coli thus prepared is cultured under optimal conditions for enzyme production to obtain enzyme strains, and then ultrasonically pulverized or treated with organic solvents such as chloroform or hexane so that the substrate can react with sodium glutamate or glutamic acid. For optimal production of 4-aminobutyl acid, pH was maintained at an optimal condition using an appropriate buffer solution, and PLP (Pyrridoxal 5-phosphate), a coenzyme of enzyme reaction, was added, followed by reaction under various substrate conditions. Butyl acid is obtained. The 4-aminobutyl acid thus obtained is first separated by chromatography, concentrated and crystallized, and used for the next step of reaction.

On the other hand, the method using gad1, gad2 or gad3 isolated from Lactobacillus is isolated from the chromosome of Lactobacillus brevis ( Lactobacillus brevis ) and then subjected to a PCR reaction using the template as a glutamic acid dicarboxylase (gad1, gad2) of Lactobacillus Alternatively, after cloning gad3), E. coli overexpresses the gene and introduces it into an expression vector capable of mass-producing the enzyme, thereby producing a recombinant E. coli biocatalyst that overproduces glutamic acid dicarboxylase. Subsequent methods are carried out in the same manner as using the enzyme isolated from E. coli.

E. coli strain may be used to obtain the gad enzyme E. coli, including the gad A or B enzyme gene, preferably E. coli ( E. coli ) W3110 (KCTC 223) strain.

In addition, Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus casi ( Lactobacillus casei ), Lactobacillus ashdophyllus ( Lactobacillus acidophilus ), Lactobacillus vulgaricus ( Lactobacillus bulgaricus ) The Bacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus spokes to jeneseu (Lactobacillus sporogenes), Lactobacillus buffer lactofermentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus) and Lactobacillus ruteri selected from the group consisting of (Lactobacillus reuteri) Any one may be used, but preferably, Lactobacillus brevis ATCC 367 may be used.

Step 2 of the preparation method of the present invention is to prepare 2-pyrrolidone from 4-aminobutyl acid obtained in step 1 as shown in Scheme 2.

<Scheme 2>

Specifically, in the step, a toluene solution of 4-aminobutyl acid and neutral alumina dissolved in a reactor to which a Dean-Stark device is attached is heated and reacted. After the reaction was terminated, cooled to room temperature and filtered using a glass filter. The reaction is washed several times with methanol / chloroform mixed solvent during the filtration. After filtration, the solvent is removed by vacuum distillation using a rotary evaporator to prepare 2-pyrrolidone.

Instead of neutral alumina, chromium oxide (Cr ₂ O ₃ ), titanium dioxide (TiO ₂ ) or tungsten oxide (WO ₃ ) may be used in step ₂ , and phosphorus oxide (P ₂ O ₃ ) and iodine ( I ₂ ), zinc chloride (ZnCl ₂ ), BF3-etherate (BF ₃ -etherate), dimethyl sulfoxide, dimethyl sulfoxide, phosphorus oxychloride-pyridine compound (POCl ₃ -pyridine), potassium hydrogen sulfate (KHSO ₄ ) Or potassium hydroxide (KOH) may be used, but is not limited thereto.

Step 3 of the preparation method of the present invention is to prepare nylon 4 from 2-pyrrolidone obtained in step 2 as shown in Scheme 3.

<Scheme 3>

Specifically, the nylon 4 polymerization of step 3 is put 2-pyrrolidone in a reactor with a stirrer, and heated to 80 ^o C under a nitrogen stream after heating. Then, 3.4 g of potassium hydroxide is added to cause a reaction between the monomer and potassium hydroxide to generate water. At this time, the generated water is removed from the reactor in a vacuum of about 1 Torr, and the reaction is continued until an appropriate amount of water comes out. At this time, the reaction time is preferably about 15 minutes. Then, the reactants in the hot state are transferred to a polyethylene bottle filled with nitrogen under nitrogen atmosphere, and then polymerized by adding an appropriate amount of silicon tetrachloride together. After about 10 minutes, a white powdery precipitate began to form at a reaction temperature of 50 ^° C., and nylon 4 began to be produced. After about 24 hours, the reaction was terminated. The nylon 4 thus prepared is present in a very hard solid state, and the nylon 4 in the solid state is crushed with a hammer and then mixed with 0.1% formic acid to remove unreacted monomer. During the filtration, it is first washed with 0.1% formic acid, then with a sufficient amount of distilled water, and finally with alcohol. After washing, drying in vacuo may yield nylon 4 in a pure state.

As the polymerization initiator of step 3, trichlorosilane (SiHCl ₃ ) or (C ₅ H ₅ ) ₂ TiCl ₂ may be used instead of silicon tetrachloride, and sodium hydroxide (NaOH) or calcium hydride (CaH) instead of potassium hydroxide as a catalyst. ₂ ) may be used, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to these examples.

Example 1 Preparation of Nylon 4 (1)

Step 1: Preparation of 4-Aminobutyl Acid Using gadA Enzyme Isolated from Escherichia Coli

4-Aminobutyl acid was prepared from glutamic acid or glutamic acid using glutamic acid dicarboxylase (GAD) enzyme by the method shown in the following scheme.

<Scheme 1>

In order to clone the gad A gene from the whole E. coli nucleotide sequence, E. coli strain W3110 (KCTC 223) was cultured and chromosomal DNA was isolated using a bacterial chromosome recovery system (Promega).

Using the isolated chromosomal DNA was prepared a primer for the expression of E. coli trc promoter and the specific sequence is as follows.

<Primer 1>

Forward primer 5'-gctagcaatggataagaagc-3 '(SEQ ID NO: 1)

Reverse primer 5’-gagctcagtccacacaaatt-3 ’(SEQ ID NO: 2)

Using the primers, the PCR reaction was performed in the form of repeating 30 seconds at 1 step 95 ° C., 30 seconds at 2 steps 55 ° C., 30 minutes at 3 steps 72 ° C., and securing gad A gene 30 times. Cloning was performed using Topo cloning system (Invitrogen). By subcloning the cloned gad A gene into the pSE380 vector (Invitrogen), a final vector capable of finally overexpressing the gad A gene in E. coli was produced.

By transforming the vector into Escherichia coli, an enzyme strain capable of overexpressing a ga dA protein was prepared. The strain was inoculated in LB medium containing ampicillin, incubated at 30 ° C. for 5 hours, and then gadA was added by adding 0.5 mM IPTG. Enzyme overexpression was induced and further cultured for 6 hours, and then the enzyme strain was recovered by centrifugation. In order to obtain gadA enzyme from the recovered enzyme strain, the strain was crushed using an ultrasonic mill. The crushing conditions were added 0.1 mM EDTA, 0.2 mM PLP, 5 mM DTT in 0.1 M Tris-HCl (pH 7.4), and then crushed in an ultrasonic mill for 5 minutes, and then precipitated at 14,000 rpm for 20 minutes using a centrifuge. The supernatant was recovered and used as the enzyme addition solution for the reaction.

For reaction, sodium glutamate or glutamic acid was added to 0.2 M sodium acetate buffer (pH 5.0) as a substrate at a concentration of 1 M, followed by 1/10 of the enzyme by volume, 1 mM PLP, and 37 ° C. The reaction was performed while shaking at 150 rpm. After 1 hour of reaction, it was confirmed that sodium glutamate or glutamic acid was converted to 100% 4-aminobutyl acid based on the molar conversion rate. This is a better result than any conventional invention or literature.

In order to recover 4-aminobutyl acid produced by the reaction, 1 mL of 5N sodium hydroxide was added to 100 mL of the enzyme reaction solution to adjust the pH to 10.0 or more, and then passed through a column in which a cation exchange resin was deposited. After attaching butyric acid, it was recovered by desorbing 4-aminobutyl acid using an eluent composed of 50% methanol and 0.1 N hydrochloric acid, concentrated using a vacuum evaporator, and then produced 92% by crystallization. Aminobutyl acid could be recovered.

Step 2: 2- Pyrrolidone  Produce

By the method shown in Scheme 2 below, 2-pyrrolidone was prepared from 4-aminobutyl acid obtained in step 1.

<Scheme 2>

In a 500 mL reactor equipped with a Dean-Stark apparatus, 250 mL of a toluene solution dissolved in 5.2 g (50 mmol) of 4-aminobutyl acid and 15 g (150 mmol) of neutral alumina was added and heated to reflux for 10 hours. To react. In the reaction, a metal oxide such as Cr ₂ O ₃ , TiO _2, or WO ₃ may be used instead of alumina, and P ₂ O ₅ , I ₂ , ZnCl ₂ , BF ₃ -etherate, Dimethyl sulfoxide, POCl ₃ -Pyridine Dehydrating agents such as KHSO ₄ or KOH can also be used.

After completion of the reaction, the mixture was cooled to room temperature and then filtered using a glass filter. The reaction was washed several times using a methanol / chloroform (1: 1) mixed solvent during filtration. After filtration, the solvent contained in the filtrate was removed by vacuum distillation using a rotary evaporator. After removing the solvent, 4.1 g of 2-pyrrolidone was obtained as a yellow oily liquid sample, and the yield was about 96%.

Step 3: Preparation of Nylon 4

Nylon 4 was prepared from 2-pyrrolidone obtained in step 2 in the manner shown in Scheme 3 below.

<Scheme 3>

Into a 250 mL reactor equipped with a Claisen head, a thermometer and a stirrer for vacuum distillation, 120 g of 2-pyrrolidone prepared in Step 2 was used, followed by a heating mantle. And heated to 80 ^° C. under a nitrogen stream. Then, 3.4 g of potassium hydroxide (97% purity) was added to cause a reaction between the pyrrolidone monomer and potassium hydroxide, in which water was incidentally generated. The produced water was removed from the reactor in a vacuum of about 1 Torr, and the reaction was continued until about 20 mL of water came out. The reaction time was about 15 minutes. The hot reactant was transferred to a polyethylene bottle filled with nitrogen under a nitrogen atmosphere, and then 0.5 g of silicon tetrachloride (SiCl ₄ ; Silicone tetrachloride) was added as a reaction initiator to proceed with polymer polymerization. After about 10 minutes, a white powdery precipitate of nylon 4 began to form at a reaction temperature of 50 ^° C., and after about 24 hours, the reaction was terminated.

The nylon 4 in a very hard solid state prepared above was crushed using a hammer and then mixed with 0.1% formic acid and filtered. The unreacted monomer was removed by the filtration, and during filtration, washing with 0.1% formic acid first, followed by washing with a sufficient amount of distilled water, and finally with alcohol. After washing, dried in a vacuum to prepare a nylon 4 in a pure state. The amount of nylon 4 produced was about 74 g. (Monomer conversion: 74%)

Instead of potassium hydroxide, metal hydroxides or metal hydrides such as sodium hydroxide and CaH ₂ may be used as the catalyst during the reaction, and metal hydrides such as SiHCl ₃ and (C ₅ H ₅ ) ₂ TiCl ₂ may be used instead of silicon tetrachloride as an initiator. Can be used.

Example  2: Manufacture of Nylon 4 (2)

Nylon 4 was prepared in the same manner as in Example 1, except that 4-aminobutyl acid was prepared as follows using gadB enzyme isolated from Escherichia coli.

In order to clone the gad B gene from the entire E. coli nucleotide sequence, a primer was prepared to express the trc promoter of E. coli using the chromosomal DNA obtained in Example 1, and the specific nucleotide sequence is as follows.

<Primer 2>

Forward primer 5’-gctagcaatggataagaagc-3 ’(SEQ ID NO: 3)

Reverse primer 5’-gagctcagtccatacaaatt-3 ’(SEQ ID NO: 4)

PCR was carried out in the same manner as in Example 1 using the primers, subcloning the E. coli expression vector, E. coli was transformed, and the strain was ultrasonically ground to obtain 4-aminobutyl acid in 90% yield. The subsequent steps were carried out in the same manner as in Example 1 to prepare nylon 4.

Example  3: Manufacture of Nylon 4 (3)

Nylon 4 was prepared in the same manner as in Example 1, except that 4-aminobutyl acid was prepared as follows using gad1 enzyme isolated from Lactobacillus.

In order to clone the gad gene in the Lactobacillus strain known to have excellent activity of glutamic acid dicarboxylase, the gad 1 gene was cloned from Lactobacillus brevis , whose whole nucleotide sequence is known. That is, in order to first obtain Lactobacillus brevis chromosomal DNA, the strain was incubated in MRS medium at 37 ° C. for 16 hours, and then chromosomal DNA was obtained using the same promegasa kit used in Escherichia coli. From the obtained chromosomal DNA, gad 1 genes were prepared for fusion to the trc promoter that can be expressed in Escherichia coli, respectively, and specific nucleotide sequences are as follows.

<Primer 3>

Forward primer 5’-gagctcgttagtgagtgcctttgtgg-3 ’(SEQ ID NO: 5)

Reverse primer 5’-ggatccaaaaccatggttccgtgata-3 ’(SEQ ID NO: 6)

PCR was carried out in the same manner as in Example 1 using the primers, subcloning the E. coli expression vector, E. coli was transformed, and the strain was ultrasonically ground to obtain 4-aminobutyl acid in 90% yield. The subsequent steps were carried out in the same manner as in Example 1 to prepare nylon 4.

Example  4: Manufacture of Nylon 4 (4)

Nylon 4 was prepared in the same manner as in Example 1, except that 4-aminobutyl acid was prepared as follows using gad2 enzyme isolated from Lactobacillus.

In order to clone the gad 2 gene from the entire nucleotide sequence of Lactobacillus brevis, a primer for the expression of the trc promoter of E. coli was prepared using the chromosomal DNA obtained in Example 3, and the specific nucleotide sequence is as follows.

<Primer 4>

Forward primer 5'-ctgcaggtacaccgatcttactccgg-3 '(SEQ ID NO: 7)

Reverse primer 5’-ggatcctgcgtaaactcaagcttgaa-3 ’(SEQ ID NO: 8)

PCR was carried out in the same manner as in Example 1 using the primers, subcloning the E. coli expression vector, E. coli was transformed, and the strain was sonicated to obtain 4-aminobutyl acid in 94% yield. The subsequent steps were carried out in the same manner as in Example 1 to prepare nylon 4.

Example  5: Preparation of Nylon 4 (5)

Nylon 4 was prepared in the same manner as in Example 1, except that 4-aminobutyl acid was prepared as follows using gad3 enzyme isolated from Lactobacillus.

In order to clone the gad 2 gene from the entire nucleotide sequence of Lactobacillus brevis, primers were prepared to express the trc promoter of Escherichia coli using the chromosomal DNA obtained in Example 3. Specific sequences are as follows.

<Primer 5>

Forward primer 5'-gagctcattaatgtaatacccgttag-3 '(SEQ ID NO: 9)

Reverse primer 5’-cgatcgctgtgtccaaccagttgcag-3 ’(SEQ ID NO: 10)

PCR was carried out in the same manner as in Example 1 using the primers, subcloning the E. coli expression vector, E. coli was transformed, and the strain was ultrasonically ground to obtain 4-aminobutyl acid in 85% yield. The subsequent steps were carried out in the same manner as in Example 1 to prepare nylon 4.

Example  6: Manufacture of nylon 4 (6)

Nylon 4 was prepared in the same manner as in Example 1, except that 4-aminobutyl acid was prepared in the first step of Example 1 using chloroform instead of the ultrasonic mill.

Since the use of an organic solvent such as chloroform can secrete the enzyme out of the cells, the yield of 4-aminobutyl acid was compared using the organic solvent.

The strain was treated with the addition of 1/10 of the volume of chloroform to obtain 95% of the converted 4-aminobutyl acid, which was used in the subsequent steps in the same manner as in Example 1 to prepare nylon 4.

Example  7: Preparation of nylon 4 (7)

Nylon 4 was prepared in the same manner as in Example 1, except that 4-aminobutyl acid was prepared in the first step of Example 1 using hexane instead of the ultrasonic mill.

The strain was treated with the addition of 1/20 volume of hexane to obtain 90% of the converted 4-aminobutyl acid, which was then used in the same manner as in Example 1 to prepare nylon 4 .

Example  8: Manufacture of nylon 4 (8)

Nylon 4 was prepared in the same manner as in Example 2, except that 4-aminobutyl acid was prepared by using chloroform instead of the ultrasonic mill in the first step of Example 2 as follows.

The strain was treated with the addition of 1/10 of the volume of chloroform to obtain 85% of the converted 4-aminobutyl acid, which was followed by the same steps as in Example 2 to prepare nylon 4.

Example  9: Manufacture of Nylon 4 (9)

Nylon 4 was prepared in the same manner as in Example 2, except that 4-aminobutyl acid was prepared in the first step of Example 2 using hexane instead of the ultrasonic mill.

The strain was treated with the addition of 1/20 volume of hexane to obtain 87% of converted 4-aminobutyl acid, which was followed by the same steps as in Example 2 to prepare nylon 4.

Experimental Example  1: Identification of 4-Aminobutyl Acid of Step 1

Sodium glutamate and 4-aminobutyl acid can be qualitatively analyzed by thin layer chromatography analysis (TLC), which is normal butanol / acetic acid / water as developer on a 0.2 mm coated aluminum sheet (Merck type 60, Deamstadt, Germany) coated with silica gel. (5/3/2, v / v / v) with about 30 minutes and then sprayed with 60% ninhydrin solution and heated to visualize sodium glutamate and 4-aminobutyl acid to 4-amino from sodium glutamate The production of butyric acid was confirmed. For quantitative analysis, the HPLC method using an XTerra (Waters, RP 185m, 4.6mm X 150mm) column was used to derivatize the sample by OPA derivatization method, which is generally used for amino acid analysis, and then quantitated by HPLC. It was confirmed that 4-aminobutyric acid was prepared successfully.

Experimental Example  2: 2- of step 2 Pyrrolidone  Sympathy

1 H-NMR (BRUKER AMX-500), IR (Nicolet IR-Plan Advantage), GC / MS (Hewlett-Packard 5890A Gas Chromatography / Jeol JMS-DX303 Mass Spectrometer) for the material of step 2 in Examples 1-9 Structural analysis was performed using. The results are shown in Figures 2 to 4, it was confirmed as 2-pyrrolidone as follows.

bp ₇₆₀ : 245 ℃

NMR (CDCl ₃ ): δ 1.88 (m, 2H, H-4), 2.06 (t, 2H, H-3), 3.17 (t, 2H, H-5). 7.52 (s, 1H, NH)

IR (KBr): 3219 (NH), 2983 (cyclic alkane), 1673 (cyclic amide) cm ^-1

Mass (m / z): 85 (M ⁺ , 100), 42 (47), 41 (46), 30 (28)

Experimental Example  3: Characterization of Nylon 4 in Step 3

The thermal properties of the prepared nylon 4 of the step 3 in Examples 1 to 9 by using DSC to examine the thermal properties. The measurement results are as follows.

<Thermal analysis conditions>

Temperature range: 25 ~ 300 ^o C

Heating and cooling rate: 10 ^o C per minute

Atmosphere: Nitrogen atmosphere

<Thermal Analysis Results>

Tg: 110 ^o C (from 2nd heating curve)

Tm: 251 ^o C (from 2nd heating curve)

As a result of the thermal analysis, it was confirmed that nylon 4 having the same properties as that produced by the conventional petrochemical method was produced. Therefore, when using the present invention it is possible to secure a sustainable alternative production method in a high oil price environment, it will be possible to manufacture nylon 4 in an economical way.

1 is a simplified diagram of a method of making nylon 4 of the present invention.

2 is an NMR spectral result of 2-pyrrolidone synthesis result.

3 is an FT-IR spectral result of the 2-pyrrolidone synthesis result.

4 is a mass spectral result of the 2-pyrrolidone synthesis result.

Claims (10)

Preparing 4-aminobutyl acid from glutamic acid or sodium glutamate using glutamic acid dicarboxylase (GAD) (step 1); Preparing 2-pyrrolidone from the 4-aminobutyl acid prepared in step 1 using a catalyst or a dehydrating agent (step 2); And A method for producing nylon 4 using enzymatic and chemical reactions from biomass comprising the step (step 3) of preparing nylon 4 by polymerizing by adding an initiator and a catalyst from 2-pyrrolidone prepared in step 2. The enzyme reaction from biomass according to claim 1, wherein the glutamic acid dicarboxylase of step 1 is a gadA or gadB enzyme isolated from E. coli or a gad1, gad2 or gad3 enzyme isolated from the genus Lactobacillus. Method for preparing nylon 4 using a chemical reaction. The method of claim 2, wherein the glutamic acid dicarboxylase is subjected to a polymerase chain reaction (PCR) from chromosomal DNA of E. coli or Lactobacillus to obtain gad A or gad B and gad 1, gad 2 or gad 3 genes, respectively. ; Subcloning said gad A, gad B, gad 1, gad 2 or gad 3 into an E. coli expression vector, respectively; Transforming E. coli into an expression vector subcloned into the gene; And Recovering glutamic acid decarboxylase expressed by E. coli transformed using ultrasonic pulverization or chloroform or hexane after culturing the E. coli; Method for producing nylon 4 using the enzyme reaction and chemical reaction from the biomass, characterized in that obtained by the method comprising a. The method of claim 1 or 2, wherein the E. coli is Escherichia coli ( E. coli ) W3110 strain from the biomass, characterized in that the production of nylon 4 using the enzyme reaction and chemical reaction. According to claim 1 or 2, wherein the genus Lactobacillus Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus casei ( Lactobacillus) casei ), Lactobacillus acidophilus , Lactobacillus vulgaris ( Lactobacillus) bulgaricus ), Lactobacillus rhamnosus ), Lactobacillus sporogenes ), Lactobacillus fermentum ), Lactobacillus helveticus ) and Lactobacillus reuteri ( Lactobacillus reuteri ) is a method for producing nylon 4 using an enzyme reaction and a chemical reaction, characterized in that any one selected from the group consisting of. The method of claim 1, wherein the 4-aminobutyl acid of Step 1 is obtained from glutamic acid or sodium glutamate using glutamic acid dicarboxylase to obtain 4-aminobutyl acid, which is purified using a column and a vacuum evaporator on which a cation exchange resin is deposited. Method for producing nylon 4 using the enzyme reaction and chemical reaction from the biomass, characterized in that. The method of claim 1, wherein the catalyst of step 2 is any one selected from the group consisting of alumina (Al ₂ O ₃ ), chromium oxide (Cr ₂ O ₃ ), titanium dioxide (TiO ₂ ) and tungsten oxide (WO ₃ ). Method for producing nylon 4 using the enzyme reaction and chemical reaction from the biomass, characterized in that the. The dehydrating agent of claim 2, wherein the dehydrating agent of step 2 is phosphorus oxide (P ₂ O ₃ ), iodine (I ₂ ), zinc chloride (ZnCl ₂ ), BF ₃ -ether compound (BF ₃ -etherate), dimethyl sulfoxide (Dimethyl) sulfoxide), phosphorus oxychloride-pyridine compound (POCl ₃ -pyridine), potassium hydrogen sulfate (KHSO ₄ ) and potassium hydroxide (KOH) from any one selected from the group consisting of enzymatic and chemical reaction from biomass Nylon 4 manufacturing method using. The biomass of claim 1, wherein the initiator of step 3 is any one selected from the group consisting of silicon tetrachloride (SiCl ₄ ), trichlorosilane (SiHCl ₃ ), and (C ₅ H ₅ ) ₂ TiCl ₂ . Method for producing nylon 4 using the enzyme reaction and chemical reaction. The method according to claim 1, wherein the catalyst of step 3 is any one selected from the group consisting of potassium hydroxide (KOH), sodium hydroxide (NaOH) and calcium hydride (CaH ₂ ) and the enzyme reaction from biomass and Method for preparing nylon 4 using a chemical reaction.

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