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Construction of a recombinant expression strain of nitrilase and its high-density fermentation method

A technology of nitrilase and concentration, applied in the direction of hydrolase, microorganism-based methods, fermentation, etc., can solve the problems of bacterial poisoning, increase process steps and production costs, etc., to ensure rapid growth, avoid inhibitory effects, and widely used foreground effect

Active Publication Date: 2020-05-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the nitrilase produced by traditional recombinant bacteria needs to be induced in vitro, which increases the process steps and production costs, and IPTG has certain toxicity to the bacteria

Method used

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  • Construction of a recombinant expression strain of nitrilase and its high-density fermentation method
  • Construction of a recombinant expression strain of nitrilase and its high-density fermentation method
  • Construction of a recombinant expression strain of nitrilase and its high-density fermentation method

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Construction of Escherichia coli CGMCC No.14254 producing constitutive recombinant nitrilase

[0027] In the following examples, the experimental methods of specific molecular construction conditions are not indicated, usually according to the conditions described in Sambrook et al.'s "Molecular Cloning Experiment Handbook" (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer.

[0028] 1) Amplification of the NIT sequence of the nitrilase gene: Genomic DNA was extracted from Pseudomonas putida CGMCC3830 and used as a template to design upstream and downstream primers 2PU, 3bD and restriction sites for PCR amplification. Primers:

[0029]2PU: 5'-GGAATTCCATATGATGGTTACGTACACGAATAAGTT-3'

[0030] 3bD: 5'-ATTGCTCAGCTCAGCCTCTCTTCATGGACCTTAAC-3'

[0031] PCR amplification system:

[0032]

[0033] PCR reaction process: cycle after pre-denaturation at 95°C for 3 minutes, denaturation...

Embodiment 2

[0041] Embodiment 2: Application of high-density fermentation technology in recombinant nitrilase fermentation

[0042] The slant strain CGMCC No.14254 was activated, transferred to a fresh seed medium with a 2% inoculation amount, and cultured at 37° C. for 8 hours. According to the 10% inoculation amount, transfer to the fermenter, use the pH electrode and the dissolved oxygen electrode to monitor the pH and dissolved oxygen value of the fermentation broth in real time, and maintain the dissolved oxygen at 10% by adjusting the rotation speed and ventilation. From the batch fermentation stage, adjust the pH by feeding ammonia water to make it not lower than 6.8; in the feeding stage, set the pH to be stable at 6.8, (1) In the early stage of feeding, start feeding the feeding medium when the pH changes by 0.01 (2) In the mid-feeding period, when the pH changes by 0.03, the feed medium or ammonia water is fed; (3) In the late feeding period, when the pH changes by 0.05, the fee...

Embodiment 3

[0043] Embodiment 3: Preparation and continuous conversion of free cell catalyst

[0044] CGMCC No.14254 was cultured in the fermentation medium for 8-10 hours, centrifuged at 12000×g for 1 min, discarded the supernatant, then resuspended the cells with sodium phosphate buffer (pH 7.2, 100 mM) and washed for 2- 3 times to obtain the cells after removal of the medium residue, and finally resuspend the cells with the same sodium phosphate buffer to obtain a resting cell suspension, measure the cell concentration and store in a 4°C refrigerator for later use.

[0045] Mix the prepared cell suspension (100mL, the dry weight of the bacteria is 2.26g / L) with 1.04g 3-cyanopyridine (the initial concentration of 3-cyanopyridine is 100mM), and transform at 30°C and 220rpm For the reaction, detect the remaining substrate in the conversion solution by HPLC, and when the substrate is completely converted, continue to add the next batch of substrate.

[0046] When the final concentration o...

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Abstract

The invention relates to construction of a nitrilase constitutive expression system and an application of nitrilase to synthesize nicotinic acid, and belongs to the technical field of industrial biology. A nitrilase constitutive expression vector is constructed by introducing a pseudomonas putida nitrilase gene coding sequence into pET-3b plasmids to obtain recombinant plasmids pET-3b-NIT, and the recombinant plasmids pET-3b-NIT are converted to E.coli BL21(DE3) to obtain constitutive recombination nitrilase expression bacterial strains free of in-vitro induction. The constitutive recombination nitrilase expression bacterial strains are named as Escherichia coli NIT-1, deposited with the China General Microbiological Culture Collection Center and assigned the accession number CGMCC No. 14254. A pH-stat feeding strategy is further adopted for accurate regulation and control to improve the bacterial density and the nitrilase yield. The Escherichia coli free cells are taken as a catalyst, 3-cyanopyridine is taken as a substrate, and a batch feeding manner is adopted for continuous conversion to produce nicotinic acid. The new provided constitutive recombination nitrilase expression bacterial strains can efficiently express nitrilase, and are low in production cost and short in period. An inducer is not required in a fermentation process, and the finally obtained bacteria are large in bacterial density, can efficiently catalyze 3-cyanopyridine to produce nicotinic acid, and have bright application prospects.

Description

technical field [0001] The invention belongs to the field of industrial biotechnology, and specifically relates to the use of Pseudomonas putida to amplify the nitrilase gene by PCR, to construct a constitutive recombinant expression system without in vitro induction, and to study its high-density fermentation process and its role in the synthesis of niacin in the application. Background technique [0002] Nicotinic acid, whose chemical name is 3-pyridinecarboxylic acid, can also be called nicotinic acid and vitamin B3. It plays an important role in the normal growth and development of the human body. It is often used in pharmaceutical intermediates, dyes, luminescent materials, food additives and Production of animal feed, etc. Niacin plays an important role in the oxidation-reduction process in body tissues, has the functions of promoting cell metabolism and dilating blood vessels, and can promote the growth and development of humans and animals. As a drug, niacin can pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P17/12C12R1/19
CPCC12N9/78C12P17/12C12Y305/05001
Inventor 许正宏史劲松龚劲松张强李恒蒋敏顾炳琛
Owner JIANGNAN UNIV
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