JP2008061501A - Method for designing enzyme for catalyzing direct condensation reaction of free carboxylic acid amine, and method for enzymatically producing nylon oligomer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme for catalyzing a direct condensation between a free carboxyl group and a free amino group, and to provide a method for enzymatically synthesizing a non-natural amide that uses such an enzyme. <P>SOLUTION: A nylon oligomer is synthesized from 6-aminocaproic acid as the substrate, in the presence of a nylon oligomer-synthesizing enzyme having a specific sequence. This enzyme can catalyze direct condensation between a free carboxyl group and a free amino group under a mild condition of about 30°C, in a very short time and can synthesize nylon oligomer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for enzymatically producing a nylon oligomer using an enzyme capable of synthesizing a nylon oligomer in a fine water system. The present invention also relates to a method for designing a non-natural amide compound synthase based on protein engineering / molecular evolution engineering techniques.

  6-Nylon is a polymer in which 100-mer or more of 6-aminocaproic acid is polymerized. Add a small amount of water, nylon salt, aminocaproic acid, base, etc. to caprolactam and heat at 220 ° C to 300 ° C for several hours to several tens of hours. And then polymerized. In addition, by using a condensing agent, an amide bond can be generated at room temperature.

  Generally, since the carbonyl group of carboxylic acid itself has low electrophilicity, it can be derived into a highly reactive active ester, or an active intermediate can be prepared using various activators such as N, N-dicyclohexylcarbodiimide (DCC). After generation, it is reacted with alcohol or amine. This method requires equimolar amounts of condensing agent with the raw material, but the condensing agent is generally expensive. In addition, after the reaction, a by-product separation / purification step is required, resulting in a further increase in production cost.

  On the other hand, the enzymatic synthesis method of synthesizing a target product from a substrate using an enzyme as a catalyst has the characteristics that it has high substrate specificity and stereospecificity and can be carried out at normal temperature and pressure. As a method for synthesizing an organic substance using such an enzyme, Patent Document 1 discloses a method for synthesizing an amide using a hydrolase derived from a microorganism belonging to the genus Streptomyces.

  Further, Patent Document 2 discloses a method of synthesizing an oligosaccharide chain up to a polysaccharide derivative monomer using an enzyme (phosphorylase in the case of maltopentaose) using a monomer chemically bonded to an oligosaccharide as a primer.

Here, the present inventors have studied non-natural amide compounds (nylon oligomers) synthesized as a by-product of the 6-nylon industry as a model system, and have conducted research on microbial adaptation mechanisms for non-natural substances. 3D structure analysis of 6-aminocaproic acid linear dimer hydrolase (EII / EII ') and mutant enzyme Hyb-24 of Arthrobacter genus KI72 has been completed with a high resolution of 1.4 to 1.8 mm. (Non-Patent Document 1).
JP 2004-81107 A JP-A-6-199883 Negoro et al. J. Biol. Chem. 280, 39644-39652 (2005).

  Examples of using non-aqueous or fine water (mixed solvent system of organic solvent and small amount of water) and reverse hydrolysis reaction, synthesis of fatty acid esters using lipase, peptide synthesis using protease, etc. However, since by-product water molecules are involved in the hydrolysis of the reverse reaction, it is usually necessary to convert them into carboxylic acid esters in advance. However, even under such conditions, the peptide synthesis in the fine water system is slower than the forward hydrolysis rate, and dehydration condensation requires several hours to 10 days (usually about 10 to 20 hours).

  If a direct amide bond can be formed from a free carboxyl group and an amino group, the protection process can be omitted, so that various amide compounds can be easily synthesized. As described above, both chemical and enzymatic synthesis methods are free. Direct condensation between carboxyl and amino groups is very unlikely. In addition, ester peptides using lipase / protease are all directed to physiological substrates and are difficult to apply to non-natural compounds.

  An object of the present invention is to provide a method for enzymatic synthesis of an unnatural amide using an enzyme that catalyzes a direct condensation reaction between a free carboxyl group and a free amino group. Another object of the present invention is to provide a protein engineering / molecular evolutionary technique for designing the enzyme.

  The present inventors are a nylon oligomer-degrading bacterium, Arthrobacter sp. KI72 strain 6-aminohexanoate-oligomer hydrolase (aminocaproic acid linear dimer hydrolase) (highly active EII, low activity EII ', mutation Focusing on the enzyme Hyb-24), the three-dimensional structure was analyzed. Various mutant enzymes with greatly different functions were obtained. Among them, an enzyme that catalyzes the synthesis reaction of nylon oligomer from 6-aminocaproic acid in a 90% tert-butyl alcohol / 10% water mixed system was found, and the present invention was completed.

Specifically, the present invention
The present invention relates to a method for enzymatically producing a nylon oligomer, comprising synthesizing a nylon oligomer in an organic solvent / water mixed solvent using 6-aminocaproic acid as a substrate and the nylon oligomer synthetase described in SEQ ID NO: 2 (claim) Item 1).

  Nylon oligomer synthase having the amino acid sequence shown in SEQ ID NO: 2 is a mutant enzyme having a structure in which two amino acid residues of Hyb-24 (having the amino acid sequence shown in SEQ ID NO: 1) are substituted. A nylon oligomer (2-6 mer) can be synthesized from 6-aminocaproic acid in an aqueous system.

The present invention also provides:
Protein engineering and molecular evolution engineering based on the principle of changing the interaction between the substrate and the enzyme by selecting the length of the loop, the type of amino acid residue in the loop, and the type of amino acid involved in substrate binding in the cleft The present invention relates to a method for designing a synthetic enzyme for an unnatural amide compound.

  The nylon oligomer synthase of the present invention can synthesize a nylon oligomer by catalyzing the direct condensation between a free carboxyl group and a free amino group in a very short time under mild conditions of about 30 ° C.

  Embodiments of the present invention will be described below with reference to the drawings as appropriate. The present invention is not limited to these.

<Production of nylon oligomer synthase Hyb-24DN>
First, a method for producing Hyb-24DN, which is a mutant enzyme of 6-aminohexanoate-oligomer hydrolase (aminocaproic acid linear dimer hydrolase) of Arthrobacter sp. KI72 strain, will be described. In addition, the outline of the manufacturing process of Hyb-24DN is shown in FIG.

(Preparation of crude Hyb-24DN solution)
Hyb-24DN (SEQ ID NO: 2) is a mutant enzyme containing 2 substitutions of G181D / H266N in Hyb-24 (SEQ ID NO: 1) (Gly → Asp at position 181 of SEQ ID NO: 1, and His → Asn at position 266, respectively) Replacement). A recombinant plasmid (pHY3DN1) in which the gene encoding the Hyb-24DN enzyme was incorporated into an E. coli vector (pKP1500) was introduced into E. coli KP3998 by transformation. 300 mL of TBA medium (composition: bactotryptone 20 g / L, yeast extract 5 g / L, NaCl 0.5 g / L, MgCl ₂ 0.19 g / L, glucose 0.9 g / L, ampicillin 100 mg / L, pH 7.0) at 37 ° C. until the logarithmic growth phase (about 4 hours)

  Next, IPTG (isopropyl β-D-thiogalactoside) was added to a final concentration of 1 mM, and the culture was further continued for 20 hours. Bacteria are collected by centrifugation (4 ° C, 8000 rpm, 5 minutes), washed twice with buffer A (20 mM phosphate buffer pH 7.3 containing 10% glycerol), and then reconstituted in 20 mL of buffer A. Suspended. This cell suspension was subjected to ultrasonic disruption treatment (20 kHz, 5 minutes × 4 times) and then centrifuged (4 ° C., 17000 rpm, 30 minutes), and the supernatant was used as a cell extract.

(Purification of mutant enzyme by ion exchange column chromatography)
The cell extract obtained above was charged onto a Hi-Trap Q Sepharose (registered trademark) column (column volume 5 mL) that had been equilibrated with buffer A in advance. After washing with 15 mL of buffer A, the sodium chloride concentration was linearly changed from 0 M to 0.5 M and eluted in a total volume of 300 mL. The flow rate was 3 mL / min, and 6 mL each was collected.

  Thereafter, each fraction was subjected to SDS-PAGE analysis, the fraction from which the target protein was eluted was identified, and concentrated with a centrifugal concentrator (MILLIPORE, YM-10). This Hyb-24DN solution (5 mg / mL) was used for the nylon oligomer synthesis reaction described later.

(Selection of reaction conditions suitable for synthesis of nylon basic unit)
To 450 μL of each organic solvent shown in Table 1, 50 μL of 100 mM 6-aminohexanoic acid (Ahx) and 10 μL of Hyb-24DN solution (5 mg / mL) were added and reacted at 30 ° C. for 24 hours.

  1 μL of the reaction solution is loaded onto a silica thin layer plate (Merck) and developed with a developing solvent (1-propanol: water: ethyl acetate: ammonia water = 24: 12: 4: 1.5 (volume ratio)), then 0.2% A ninhydrin solution was sprayed to confirm the reaction product.

  As a result, as shown in FIG. 2, when n-propyl alcohol (n-Propanol), n-butyl alcohol (n-Butanol) and tert-butyl alcohol (tert-Butanol) were used, Ahx dimer Synthesis was confirmed. When tert-butyl alcohol was used, the synthesis of Ahx trimer and Ahx tetramer was also confirmed. In FIG. 2, Al2 represents an Ahx dimer, Al3 represents an Ahx trimer, and Al4 represents an Ahx tetramer.

  On the other hand, when organic solvents other than these three types were used, the synthesis of Ahx dimer was not confirmed.

[Example 1]
Hyb-24DN solution (5 mg / mL) was diluted to an enzyme concentration of 1.25 mg / mL. To 450 μL of a mixed solvent of 90% by volume of tert-butyl alcohol and 10% by volume of water, add 50 μL of 100 mM 6-aminohexanoic acid (Ahx) and 10 μL of Hyb-24DN diluted solution (1.25 mg / mL). The reaction was carried out at 180 ° C. for 180 minutes.

  During this time, 1 μL of the reaction solution was sampled over time, and the reaction product was confirmed by the method using the 0.2% ninhydrin solution described above. The result is shown in FIG.

  It was revealed that about 50% of the substrate was converted to Al2 in a reaction time of 5 minutes, and then the synthesis from dimer to tetramer proceeded slowly until 180 minutes.

  Next, whether about 60 to 70% of the raw material was converted to Al2 and then the reaction was stopped due to enzyme instability or whether the reaction reached equilibrium at that time.

  Collect 450 μL of tert-butyl alcohol and 5 μL of Hyb-24DN solution (1.25 mg / mL) in 10 microtubes, respectively, after 0, 5, 10, 15, 30, 45, 60, 90, 120 and 180 minutes 50 μL of 100 mM Ahx was added, and the enzyme reaction was performed at 30 ° C. As a result, as shown in FIG. 4, since no significant decrease in synthetic activity was observed after 180 minutes, the synthesis reaction was stopped because synthesis and decomposition were in equilibrium. it was thought.

[Example 2]
Add 100 μM 4-aminobutanoic acid 50 μL and Hyb-24DN diluted solution (1.25 mg / mL) 10 μL to 450 μL of a mixed solvent of tert-butyl alcohol 90% by volume and water 10% by volume. Reacted.

[Example 3]
To 450 μL of a mixed solvent of 90% by volume of tert-butyl alcohol and 10% by volume of water, add 50 μL of 100 mM 5-aminoheptanoic acid and 10 μL of Hyb-24DN diluted solution (1.25 mg / mL). Reacted for 1 minute.

  About Example 2 and Example 3, 1 microliter of reaction solutions 180 minutes after were sampled, and the reaction product was confirmed by the method using the 0.2% ninhydrin solution mentioned above. As a result, as shown in FIG. 5, dimer synthesis was observed from 5-aminopentanoic acid and 4-aminobutyric acid as well as 6-aminohexanoic acid.

<Comparison of activity with similar enzymes>
As a similar enzyme to Hyb-24DN, a mutant enzyme containing 2 substitutions of G181E / H266N in Hyb-24 (SEQ ID NO: 1) (Gly → Glu at 181th in SEQ ID NO: 1, and His → Asn at 266th, respectively) Hyb-24EN (SEQ ID NO: 3) was used. The manufacturing method of Hyb-24EN is the same as Hyb-24DN.

  FIG. 6 shows a comparison of the degradation activity and synthesis activity of Hyb-24DN and Hyb-24EN. First, the degradation activity of Hyb-24EN was very low compared to Hyb-24DN. As for the decomposition reaction, Hyb-24EN only catalyzed the synthesis reaction of 6-aminohexane dimer.

(Hyb-24DN 3D structure features)
Hyb-24DN belongs to the group of enzymes having β-lactamase folds, like EII (SEQ ID NO: 4), EII ′ (SEQ ID NO: 5) and Hyb-24. The enzyme group having β-lactamase fold has various functions such as β-lactam hydrolysis, carboxylic acid ester hydrolysis, DD-peptide hydrolysis and the like. As a result of analyzing the three-dimensional structure of Hyb-24DN, It was found that the position of the ˜177th amino acid residue has a unique property that it moves greatly with the binding of the substrate (inductive fitting with loop movement).

  That is, in Hyb-24DN, most of the water molecules (7 molecules) in the catalyst cleft are eliminated from the binding stage of Hyb-24DN and substrate binding (FIG. 7) to the stage of acylation of the substrate (FIG. 8). The And in reverse reaction (Al2 synthesis reaction), it becomes an efficient reaction field for amide bond production | generation. For this reason, it is possible to generate an amide bond from a free carboxyl group and a free amino group in a very short reaction under a mild condition of about 30 ° C.

  Such a non-aqueous environment in the cleft is due to the movement of the 167th to 177th flexible loops. Inductive adaptation involving loop movement is known for enzyme groups related to sugar metabolism such as hexokinase, but there are no reports of related enzymes such as various proteases having Ser as an active center and β-lactamase.

  After loop movement, the same residue is coordinated to the catalytic center by rotation of the Tyr170 side chain. Tyr170 has a distance from the substrate amide nitrogen at a position approximately equivalent to the catalyst center Tyr215. That is, a new catalyst center involved in the amide hydrolysis / synthesis reaction is formed.

  The interaction between the substrate and the enzyme can be changed by selecting the length of the loop, the type of amino acid residue in the loop, and the type of amino acid involved in substrate binding in the cleft. Therefore, direct condensation of various carboxylic acids and amines is possible by utilizing the non-aqueous space in the enzyme cleft.

  By constructing a gene library in which mutations are introduced into the cleft of nylon oligomer degrading enzyme based on the three-dimensional structure information, it can be expected to produce various useful amide compounds using these.

It is a figure which shows the outline of the manufacturing process of Hyb-24DN. It is the photograph of the thin layer chromatography at the time of using 6 types of organic solvents. 2 is a photograph of thin layer chromatography showing the reaction time and the change of the reaction product in Example 1. FIG. 2 is a photograph of a thin layer chromatography showing the contact time of Hyb-24DN / tert-butyl alcohol and the change of the reaction product in Example 1. FIG. It is a photograph of the thin layer chromatography which shows the reaction product in Example 1- Example 3. It is a figure showing the comparison of the decomposition activity and synthetic | combination activity of Hyb-24DN and Hyb-24EN. It is a figure showing the structure in the cleft of Hyb-24DN. It is a figure showing the structure in the cleft in the coupling | bonding step of Hyb-24DN and a substrate coupling | bonding.

Claims (2)

  A method for producing a nylon oligomer, comprising synthesizing a nylon oligomer in an organic solvent / water mixed solvent using 6-aminocaproic acid as a substrate and the nylon oligomer synthetase described in SEQ ID NO: 2.   By modifying the length of the flexible loop in the nylon oligomer degrading enzyme, the type of amino acid residue in the loop, and the type of amino acid involved in substrate binding in the cleft using protein engineering / molecular evolution engineering techniques, A method for designing a synthetic enzyme for a non-natural amide compound based on the principle of changing the interaction.