Novel system based on a new nitrile hydratase for highly efficient catalytic hydration reaction of aliphatic dinitriles

Abstract

The invention belongs to the technical field of green chemistry, and provides a novel system based on a new nitrile hydratase for highly efficient catalytic conversion of aliphatic dinitriles. The invention discloses a new application of nitrile hydratase using Rhodococcus erythropolis CCM 2595 in catalyzing aliphatic dinitrile. In particular, the enzyme can regioselectivity catalyze the formation of 5-cyanopyramides from adiponitrile with high reaction rate under mild reaction conditions, which provides a new method for the industrial production of 5-cyanopyramides.

Claims

1. A novel system based on a new nitrile hydratase for highly efficient catalytic hydration reaction of aliphatic dinitriles, wherein the concentration of the recombinant bacteria with nitrile hydratase derived from the Rhodococcus erythropohs CCM2595 is 1-3 g/L, the concentration of aliphatic dinitriles is 20-50 mM/L, and conversion system comprises phosphate buffer saline solution with pH 7-8, temperature of 25 C. and oscillation with 200 rpm for reaction; the reaction is then quenched by adding equal volume of methanol after 5 min cultivation; then supernatant is collected after centrifugation; the supernatant is filtered for high performance liquid chromatography detection; the regioselectivity of the recombinant bacteria with nitrile hydratase towards aliphatic dinitriles is more than 90%.

2. The system according to claim 1, wherein the preparation steps of the recombinant bacteria with nitrile hydratase are as follows: (1) plasmid construction: the gene sequence of nitrile hydratase from the strain Rhodococcus erythropohs CCM2595 contains 2596 nucleotides; plasmid pET-24a (+) is used as the expression vector, according to the characteristics of restriction sites of the plasmid, NdeI and Hind III restriction sites are selected to insert the nitrile hydratase gene which is obtained by PCR; after digestion, the corresponding DNA fragment is recovered and purified, and inserted the kanamycin KanR resistance gene fragment; T7 terminator is transformed into E. coli Top 10, recombinant plasmid is obtained, conformed with digestion and named as G0130349-1; (2) protein expression verification: the recombinant plasmid obtained in step (1) is transformed into two competent E. coli BL21(DE3) and Arctic Expression (DE3) respectively before adding to LB liquid medium for culture and expansion, then the obtained bacterial solution is coated on LB solid plate containing 50 g/ml kanamycin (kan) before inverted culture at 37 C. for 24 h; monoclone on the plate is selected and planted in LB liquid medium, cultured when OD value reaches 0.6-0.8, then 0.1-1 mM/L Isopropylthiogalactoside (IPTG) is added as inducer for 3-24 h; after the induction, the bacteria are collected by centrifugation; the bacteria are broken by ultrasonic after washing with PBS; SDS-PAGE analysis is carried out to verify the protein expression of nitrile hydratase; (3) preparation of bacterial solution: the monoclone picked from Arctic Expression (DE3) plate in step (2) is inoculate to LB liquid medium which contains 50 g/ml Kan; seed solution is obtained and collected after 37 C. and 220 rpm for 12-18 h of shake cultivation, the seed solution is then inoculated into LB liquid medium containing 50 g/ml Kan with volume ratio of 1%; when OD value reaches 0.6-0.8 after shake cultivation at 37 C., IPTG is added to reach a final concentration of 0.1 mM/L; after continuous shake cultivation at 16 C. and 220 rpm for 24 h, fermentation broth is collected; the bacteria is collected by centrifugation and washed with pH7-8 PBS buffer for 2-3 times and resuspended for use.

3. The system according to claim 1, wherein, the aliphatic dinitriles comprises adiponitrile, malononitrile, butanedinitrile and sebaconitrile.

4. The system according to claim 1, wherein the nitrile hydratase derived from Rhodococcus erythropolis CCM2595 is used in industrial production.

5. The system according to claim 1, wherein the nitrile hydratase derived from Rhodococcus erythropolis CCM2595 is used in the catalytic reaction of aliphatic dinitriles.

6. The system according to claim 1, wherein the nitrile hydratase derived from Rhodococcus erythropolis CCM2595 is used in the production of 5-cyanovaleramide.

Description

DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1: Map of ReNHase (nitrile hydratase from R. erythropolis) gene in Plasmid vector pET-24a (),

[0017] FIG. 2: Expression of the ReNHase from recombinant E. coli by the SDS-PAGE. [0018] Lane A: Negative control; [0019] Lane B: 37 C. temperature induced fragmentation (BL21(DE3)); [0020] Lane C: 37 C. temperature induced disrupted supernatant (BL21(DE3)); [0021] Lane D: 1.6 C. temperature induced fragmentation (Arctic Express (DE3)); [0022] Lane E: 16 C. temperature induced disrupted supernatant (Arctic Express (DE3).

[0023] FIG. 3: The HPLC analysis results of substrate and products. [0024] (a) standard sample-adipamide; [0025] (b) standard sample-5-CVAM; [0026] (c) The products of catalytic reaction of adiponitrile by ReNHase after 5 min; [0027] (d) The products of catalytic reaction of adiponitrile by ReNHase in after 30 min.

[0028] FIG. 4: Curve of enzymatic activity of product 5-CVAM with time.

DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are further described below in conjunction with the drawings and technical solutions.

Embodiment 1: Expression Validation of the ReNHase

[0030] 1 l plasmid was added to 100 l BL21. (DE3) and Arctic expression (DE3) competent E. coli respectively. After placed in ice bath for 20 min, it was heated and shocked at 42 C. for 90 sec. It was then put into ice quickly for 3 min, then 600 l LB liquid culture medium was added, and it was vibrated at 37 C. and at 220 rpm for 1 h. 200 l bacterial solution was taken and coated on the LB plate containing 50 g/ml Kan, and finally carried out inverted culture at 37 C. for 24 h. On the next day, two colonies of BL21 (DE3) and one clone of Arctic expression (DE3) were inoculated into the 4 ml shaker tube with LB culture medium containing 50 g/ml Kan respectively. It was cultured under the condition of 37 C. and 220 rpm until the OD value was about 0.6. One tube of BL21 (DE3) without IPTG was used as negative control, one another tube with IPTG to the final concentration of 1 mMIL was induced at 37 C. for 3 h, IPTG was added to the single tube of Arctic expression (DE3) until the final concentration reached 0.1 mM/1, then it was induced at 16 C. for 24 h. On the next day, the supernatant was centrifuged at 12000 rpm for 1 min to collect the bacteria, and the buffer solution (20 mkt PB, 150 mM NaCl, pH7.4) was added, then the bacteria was crushed at 300 W power for 4 S, with an interval of 6 s. The bacteria were broken for a total of 30 cycles. SDS-PAGE analysis was carried out. 12% separation gel and 5% concentration gel were selected. The electrophoresis conditions were 80 V for 20 min and then 160 V for 100 min. As shown in FIG. 2, the target protein subunit (about 27 kDa) was expressed successfully in the supernatant.

Embodiment 2: The Reaction of Adiponitrile Catalyzed by ReNHase

[0031] (1) Seed culture: a monoclone of Arctic expression (DE3) was selected and inoculated in a shaker tube with 4 ml LB culture medium containing 50 g/ml Kan, and was shaken at 37 C. and at 220 rpm for 24 h.

[0032] (2) Induction culture: 2 ml bacterial solution in a flask containing 200 ml LB culture solution and 50 g/ml Kan was inoculated, shaken at 37 C., at 220 rpm for about 3 h until the OD value reached 0.6-0.8, then IPTG was added to reach its final concentration of 0.1 mM/L, and finally it was incubated at 16 C., at 220 rpm for 24 h.

[0033] (3) Bacteria collection: the solution was centrifuged at 3000 rpm for 10 min. The supernatant was discarded, washed twice with PBS buffer of pH=7.4, and resuspended the bacteria with 10 ml PBS buffer.

[0034] (4) High performance liquid chromatography detection: 150 l resuspended bacteria was added to 300 l PBS buffer, then 50 l 200 mM adiponitrile was added, and reacted at 25 C.; 200 rpm. The reaction time was 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 15 h, 24 h, respectively. After the reaction, 500 l methanol was added to quench the reaction. The supernatant was collected after centrifuged at 10 min, 13000 rpm; then filtered by 0.22 m strainer for HPLC detection. HPLC detection method: Ultimate 5 m 4.6250 min LP-C18 column was used, and the following solvent system was 25 mM H.sub.3PO4 buffer and methanol (89:11, vol:vol); detection wavelength was 200 ran, column temperature was 25 C., flow rate was 1 ml/min. As shown in FIG. 3, (a) the peak time of standard sample adipamide was 4.4 min; (b) the peak time of standard sample 5-CVAM was 7.1 min; (c) The conversion rate of adiponitrile was 100% and the regioselectivity of 5-CVAM was more than 90% when the reaction was carried out for 5 min. following step (4) of experiment 2. (d) With the increase of reaction time, the production of 5-CVAM decreased, the production of adipamide increased, indicating that the nitrile hydratase catalyze adiponitrile with step reaction.

[0035] As shown in FIG. 4, when the final concentration of adiponitrile was 50 mM, the product 5-CVAM with the primary total enzyme activity could reach 4269 U. With the increase of reaction time, the enzyme activity gradually decreased. It showed that the ReNHase can catalyze the formation of 5-CVAM from adiponitrile with a high efficiency.