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Nitrilase capable of preparing paracyanobenzoic acid by hydrolyzing p-benzenedicarbonitrile

A technology of terephthalonitrile and cyanobenzoic acid, which is applied in hydrolytic enzymes, genetic engineering, plant genetic improvement, etc., can solve the problems of low substrate concentration, low enzyme catalytic efficiency, long reaction time, etc., and achieve high reaction speed Fast, mild reaction conditions, and non-polluting process routes

Active Publication Date: 2018-01-30
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of low substrate concentration, long reaction time, and low enzyme catalytic efficiency in the reported preparation methods, the present invention adopts the method of gene mining, and the obtained nitrilase can efficiently catalyze the selective hydrolysis of terephthalonitrile to prepare p-cyanogen benzoic acid

Method used

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  • Nitrilase capable of preparing paracyanobenzoic acid by hydrolyzing p-benzenedicarbonitrile
  • Nitrilase capable of preparing paracyanobenzoic acid by hydrolyzing p-benzenedicarbonitrile
  • Nitrilase capable of preparing paracyanobenzoic acid by hydrolyzing p-benzenedicarbonitrile

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Experimental program
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Embodiment 1

[0023] Embodiment 1: the acquisition of highly expressed genetically engineered bacteria

[0024] The whole gene synthesis was completed by Shanghai Xuguan Company.

[0025] According to the gene NIT1 (WP_021186296.1) of Pantoea sp.AS-PWVM4, the gene NIT2 (NP_190016) of Arabidopsis thaliana, and the gene NIT3 (ABD98457.1) of Acidovorax facilis72W ), the gene NIT4(U9VXK5) of Leptolyngbya sp., the gene NIT5(XP_013627177.1) of Brassica oleracea var. oleracea and the gene NIT6( XP_010514769.1) were codon-optimized in order to enable the gene to be expressed in the E. coli expression host, and the sequence is shown in the attached table. And add corresponding enzyme cutting sites at both ends of the gene, and construct them into corresponding vectors to obtain genetically engineered bacteria N1, N2, N3, N4, N5, N6.

[0026] Transform the prepared recombinant vector into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct a genetically engineered bacteri...

Embodiment 2

[0027] The cultivation of embodiment 2 genetically engineered bacteria and the preparation of resting cells

[0028] Pick a single colony on the plate and inoculate it into 5ml of fermentation medium containing corresponding antibiotics, cultivate it for about 15 hours as a seed solution, inoculate it into 600ml of fermentation medium according to the inoculation amount of 1%, and cultivate it on a shaker at 37°C and 200rpm to OD 600 = about 0.6-0.8, add IPTG with a final concentration of 0.1 mM to induce for more than 10 h, and collect the bacteria by centrifuging the culture solution at 8000 rpm.

Embodiment 3

[0029] Embodiment 3 Utilizes resting cell of N1 to catalyze the single hydrolysis of terephthalonitrile

[0030] Take 2.0g N1 resting cells and resuspend in 90mL sodium phosphate buffer (100mM, pH 7.2), add 10mL dimethyl sulfoxide in terephthalonitrile (10.0g) suspension, and then at 30℃, 200rpm The shaking table reacted for 6 hours, and HPLC detection showed that the reaction was complete (liquid chromatography column: Agilent Eclipse XDB-C18 5 μm, 4.6 × 150mm, mobile phase: water (0.5% trifluoroacetic acid) / methanol=75:25, detection wavelength: 230nm, flow rate: 1.0mL / min), centrifuge to recover resting cells, collect the supernatant, acidify to pH 2 with 6M hydrochloric acid, filter with suction, and recrystallize from ethanol to obtain 10.28g of p-cyanobenzoic acid with a yield of 90%.1 H NMR (400MHz, DMSO-d 6 ): δ8.10 (d, J=8.3Hz, 2H), 7.99 (d, J=8.3Hz, 2H). 13 C NMR (100MHz, DMSO-d 6 ): δ166.52, 135.33, 133.13, 130.38, 118.65, 115.53.

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Abstract

The invention discloses a nitrilase N1 derived from pantoea sp.AS-PWVM4 and a gene thereof, a nitrilase N2 derived from arabidopsis thaliana and a gene thereof, a nitrilase N3 derived from acidovoraxfacilis 72W and a gene thereof, a nitrilase N4 derived from leptolyngbya sp. and a gene thereof, a nitrilase N5 derived from brassica oleracea var.oleracea and a gene thereof and a nitrilase N6 derived from camelina sativa and a gene thereof, and a method for preparing paracyanobenzoic acid as p-aminomethylbenzoic acid intermediate by using the nitrilase as a biological catalyst; resting cells ofthe corresponding nitrilases can be used for catalyzing 100g / L of substrate; the conversion rate is greater than 99%; the method has the obvious characteristics of mild reaction conditions, no pollution and simple process route, and has broad industrial application prospects.

Description

technical field [0001] The present invention relates to genes and protein products thereof, in particular to nitrilase N1 and its gene from Pantoea sp.AS-PWVM4, nitrilase N2 and its gene from Arabidopsis thaliand, agile Nitrilase N3 and its gene from Acidovorax facilis 72W, Nitrilase N4 and its gene from Leptolyngbya sp., Nitrile from Brassica oleracea var. oleracea The hydrolase N5 and its gene, the nitrilase N6 of Camelina sativa and its gene, and its biocatalytic preparation of p-cyanobenzoic acid belong to the field of applied microorganism and enzyme engineering. Background technique [0002] p-Aminomethylbenzoic acid is a hemostatic agent, suitable for abnormal bleeding during operations on the lung, liver, pancreas, prostate, thyroid, adrenal gland, etc. Hypertrophic bleeding, upper gastrointestinal bleeding, etc. In industry, the catalytic hydrogenation of p-cyanobenzoic acid is mainly used to prepare p-aminomethylbenzoic acid. [0003] The production method of p-...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/55C12P13/00
Inventor 姚培圆于珊珊张慕姣冯进辉吴洽庆朱敦明马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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