NITRILASE FROM ARABIS ALPINA, ITS ENCODING GENE, VECTOR, RECOMBINANT BACTERIAL STRAIN AND USES THEREOF

Abstract

The disclosure provides a nitrilase from Arabis alpina, which belongs to genus Arabis, family brassicaceae. The disclosure further provides the encoding gene, vector, recombinant bacterial strain, and the application in the manufacturing of (S)-3-cyano-5-methylhexanoic acid. The wet resting cells containing nitrilase Aa-Nit can kinetically resolve racemic IBSN at 1.2 M with a 42% conversion rate in 15 hr and >99% ee value. The disclosure provides a regio- and stereoselective method for the preparation of (S)-3-cyano-5-methylhexanoic acid. This method provides an atom economical, mild, environmental friendly industrial method to manufacture (S)-3-cyano-5-methylhexanoic acid.

Claims

1-4. (canceled)

5. A method of producing (S)-3-cyano-5-methylhexanoic acid comprising performing hydrolysis of racemic 2-isobutylsuccinimide Arabis alpina nitrilase comprising the amino acid sequence shown in SEQ ID NO. 1.

6. The method of claim 5, wherein a nitrilase gene-containing, recombinant bacterial strain is fermented and grown to provide wet cells comprising the nitrilase.

7. The method of claim 6, where the amount of nitrilase gene-containing, recombinant bacterial strain used for hydrolysis is 50 g/L based on the weight of the wet cells.

8. The method of claim 6, wherein (a) the nitrilase gene-containing, recombinant bacterial strain is inoculated into a liquid LB broth containing 50 g/mL of kanamycin and is grown for 12 hr at 37 C.; (b) the LB broth of step (a) is inoculated into fresh liquid LB broth containing 50 g/mL of kanamycin at 2% (v/v); (c) the nitrilase gene-containing, recombinant bacterial strain is grown to cell concentration (OD.sub.600) 0.4-0.8 at 37 C.; IPTG is added to the LB broth of step (b) until the concentration reaches 0.2 mM, (d) the nitrilase-gene containing, recombinant bacterial strain is induced to grow at 28 C. for 12 hr; the LB broth of step (c) is centrifuged at 4 C. at 12000 rpm for 5 min; and (e) the wet cells are collected.

9. The method of claim 6, wherein hydrolysis of racemic 2-isobutylsuccinimide takes place at a pH 5.0-10.0 buffer, at 25-45 C. while stirring at 150 rpm

10. The method of claim 6, wherein (S)-3-cyano-5-methylhexanoic acid is isolated and purified from the reaction mixture following hydrolysis.

11. The method of claim 7, wherein the concentration of racemic 2-isobutylsuccinimide is 0.15-1.5 mol/L.

Description

ILLUSTRATIONS

[0018] FIG. 1. Nitrilase Aa-Nit catalyzed kinetic resolution of IBSN.

[0019] FIG. 2. SDS-PAGE analysis of Nitrilase Aa-Nit. M: Molecular weight of proteins; 1: Induced expression of the target protein.

[0020] FIG. 3. Gas chromatography of nitrilase Aa-Nit catalyzed kinetic resolution of IBSN.

[0021] FIG. 4. Optimization of the pH of the reaction system.

[0022] FIG. 5. Optimization of the temperature of the reaction system.

EXAMPLES

[0023] The following examples are for the illustration of the current invention and in no way represents the scope of the current invention.

[0024] The main experimental materials were purchased from the following sources:

TABLE-US-00001 E. coli host strain: E. coli BL21 (DE3) Invitrogen Expression vector pEt-28b(+) Novagen Restriction endonucleases Xho I and Xba I Fermentas T4 DNA ligase TaKaRa Kanamycin TaKaRa IPTG Promega DNA marker and stain GoldView TaKaRa DNA gel extraction kit Axygen PCR Clean-up kit Axygen Plasmid extraction kit Axygen

Example 1

Preparation of Nitrilase Aa-Nit

[0025] (1) Nitrilase Aa-Nit amino acid sequence and nucleic acid sequence. A nitrilase amino acid sequence (Genbank No. KFK44999.1) was obtained via screening nitrilase gene sequence from protein database PDB and NCBI. The nitrilase comes from Arabis alpina, a plant belong to genus Arabis, family Brassicaceae. Based on the amino acid sequence of nitrilase, optimized codons from E. coli preferred codons, and the characteristics of vector pET28b(+), restriction enzyme cutting sites Xho I and Xba I were selected. The nitrilase-coding nucleic acid (shown in SEQ ID No. 2) and the coded amino acid sequence (shown in SEQ ID No. 1) were synthesized.

[0026] (2) Construction of recombinant strain. The nucleic acid segment was treated with restriction endonucleases Xho I and Xba I and recovered. The recovered gene and commercial vector pET28b(+) (pre-treated with restriction endonucleases Xho I and Xba I) were treated with T4 DNA ligase for 16 hr at 16 C. to give Intracellular recombinant expression vector pET28b(+)-Aa-Nit, which was introduced into E. coli BL21 (DE3) (Invitrogen), which was then spread onto a LB agar-plate containing 50 g/ml of kanamycin and grown overnight at 37 C. The strains grown on the plate was randomly selected and the plasmid was extracted for agarose gel electrophoresis.

[0027] (3) Induced expression of nitrilase Aa-Nit. The recombinant genetically engineered E. coli BL21 (DE3)/pET28b(+)-Aa-Nit was inoculated into a liquid LB broth containing 50 g/mL of kanamycin and grown for 12 hr at 37 C. The LB broth was inoculated into a fresh liquid LB broth containing 50 g/mL of kanamycin at 2% (v/v). The strain was grown at 37 C. until the cell concentration (OD.sub.600) reached 0.6 and IPTG was then added to a final concentration of 0.2 mM to induce the protein expression 28 C. for 12 hr and then centrifuged at 4 C. at 12000 rpm for 5 min. The wet cells are collected (resting cells, used for hydrolysis). The wet cells were washed with physiological saline twice, mixed well, and the cell solution was analyzed with SDS-PAGE electrophoresis. The results are shown in FIG. 2.

Example 2

Catalysis with Nitrilase Aa-Nit-Containing Resting Cells

[0028] The optimal pH, temperature, pH stability and substrate tolerance were investigated.

[0029] Reaction mixture (10 mL) was composed of buffer solution (10 mL, buffer), racemic IBSN (substrate), and wet resting cells (catalyst). The substrate's concentration was 0.4 mol/L. The catalyst quantity was 20 g of wet resting cells/L. The resting cells contained 70-90% of water. The reaction was initiated in a water bath shaker at 150 rpm for 0.5 hr and terminated with 2M HCl. The conversion rate was obtained with gas chromatography to ascertian the catalytic activity of the resting cells under various conditions.

[0030] (1) Determination of optimal pH. With the catalysis system defined above, the conversion rate of racemic IBSN was determined under various pH values (pH=5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10) at 30 C. with substrate concentration at 0.4 mol/L and wet resting cells at 20 g/L. The buffers for various pH were acetic acid buffer (pH=5.0-6.0), sodium phosphate buffer (pH=6.0-7.2), Tris-HCl buffer (pH=7.0-9.0), and Gly-NaOH buffer (pH=9.0-10.0).

[0031] (2) Determination of optimal reaction temperature. With the catalysis system defined above, the reaction was carried out at various temperatures (25, 30, 35, 40, and 45 C.) in Tris-HCl buffer (100 mM, pH=8.0) with the substrate concentration at 0.4 mol/L and wet resting cells of 20 g/L. The conversion rate of racemic IBSN was determined.

[0032] (3) Determination of tolerance for maximal substrate concentration. With the catalysis system defined above, the reaction was carried out at various substrate concentrations (150 mM, 300 mM, 450 mM, 600 mM, 700 mM, 1 M, 1.2 M, and 1.5 M) in 10 mL of Tris-HCl buffer (100 mM, pH=8.0) with wet resting cells of 20 g/L. The conversion rate of racemic IBSN was determined.

[0033] The results show that nitrilase Aa-Nit-containing resting cells exhibit highest catalytic activity at pH 8.0, and the optimal reaction temperature is 30 C. The maximal substrate concentration tolerated is 1.2 M.

Example 3

Application of Nitrilase Aa-Nit-Containing Resting Cells

[0034] Kinetic resolution of racemic IBSN. The reaction is shown in FIG. 1. To a Tris-HCl buffer (10 mL, 100 mM, pH 8.0) was added racemic IBSN to reach 1.2 M, and 0.5 g of wet resting cells prepared in Example 1. The mixture was shaken at 30 C. for 15 hr in a water bath shaker. A 500 L sample was taken every 3 hr and the reaction was quenched with 200 L of 2 M HCl. The mixture was extracted with 800 L of ethyl acetate, shaken, and centrifuged (12000g, 2 min). The supernatant was dried with anhydrous sodium sulfate and analyzed with gas chromatography.

[0035] (1) Determination of conversion rate and ee value with chiral gas chromatography. The amount of substrate and product present in the extract was determined with chiral gas chromatography (GC-14 C, Shimadzu, Japan). The capillary tube was BGB-174 (BGB Analytik, Switzerland). Gas chromatography conditions are below:

TABLE-US-00002 Sample amount 1 L Inlet and detector temperature 220 C. Column temperature 160 C. Carrier gas Helium Flow rate 1.6 mL/min Split ratio 30:1
The conversion rate and ee value were calculated according to literature method reported by Rakels et al. (Enzyme Microb Technol, 1993, 15: 1051).

[0036] (2) Isolation and purification of II. After the reaction, the reaction mixture is centrifuged to remove E. coli. The supernatant is evaporated to of the original volume. The temperature was maintained at 80 C. for 40 min to denature the proteins before the removal of the denatured proteins through centrifugation. The supernatant was vacuum filtered to remove more proteins. The filtrate was extracted with ethyl acetate (2 volume). The aqueous layer is acidified with 2 M HCl to pH 4.0. Extraction with ethyl acetate (2 volume), followed by evaporation of ethyl acetate on rotavap, affords II as an oil (ee>99.5%).

[0037] The results show that the resting cells containing nitrilase Aa-Nit can kinetically resolve IBSN at 1.2 M with the conversion rate at 42% in 15 hr and ee.sub.p>99%. Thus, nitrilase Aa-Nit catalysis disclosed in the current invention provides a mild method to manufacture II with high conversion rate and optical purity.

[0038] It is understood that the working examples are only for illustration so that those skilled in the art would understand the current invention and be able to reduce the current invention to practice. These examples are in no way to limit the scope and extent of the current invention. Any equivalent modifications or changes based on the current invention should be covered by the current invention.

REFERENCES CITED

[0039] Enzyme Microb Technol, 1993, 15: 1051 [0040] Org. Process Res. Dev. 2008, 12: 392-398 [0041] Angew. Chem. Int. Ed. 2008, 47: 3500-3504 [0042] J. Mol. Catal. B: Enzym. 2006, 41: 75-80 [0043] WO2005100580