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Recombinant expression vector, genetically engineered bacterium, and culture method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and expression vectors, which is applied in the field of genetically engineered bacteria and bacterial cultivation, and recombinant expression vectors, can solve the problems of low substrate tolerance, poor stability, and poor practicability, and achieve low production costs and excellent preparation conditions. Mild, pollution-reducing effect

Pending Publication Date: 2022-01-18
SHANGHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The engineered bacteria obtained in this patent have low substrate tolerance, can only adapt to a small number of environments, and have poor practicability
[0008] In summary, although there are currently studies on the production of nicotinamide by microbial transformation, there are usually problems of low activity, poor stability, and high cost. Therefore, a strain with high expression of nitrile hydratase and high product concentration tolerance is constructed to increase the production of nitrile hydratase. The stability and activity of hydratase have important industrial application significance and value for improving production efficiency and reducing production cost

Method used

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  • Recombinant expression vector, genetically engineered bacterium, and culture method and application of genetically engineered bacterium
  • Recombinant expression vector, genetically engineered bacterium, and culture method and application of genetically engineered bacterium

Examples

Experimental program
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Embodiment 1

[0040] The recombinant plasmid pET24A (+) - NHTABC was synthesized from all gene, then converted to E. coli DH5α, selecting a positive clone, and obtain a large number of recombinant plasmid PETs 44A (+) - NHTABC, which contains a nitrile hydrated enzyme. Gene, the nitrile hydr enzyme gene sequence is SEQ ID NO.1.

[0041] 100 μl of E.Coli BL21 (DE3) perfosure cells were placed in a pre-cooled sterilized EP tube, and the ice is opened, and the cells are evenly suspended. Add 10 μL recombinant plasmid PET24A (+) - NHTABC, gently mix, ice bath for 30 min, and open the thermostat preheating. The EP tube was transferred to a dry thermostat of 42 ° C and then transferred to an ice bath and allowed cells to cool for 15 min. 900 μl of LB liquid medium containing non-antibiotics was added, and oscillated at 37 ° C, 250 rpm was oscillated for 1 h. Then, the 400ul bacteria was applied to the antibiotic LB agar plate. When there was no liquid flow in the surface of the gel, it was inverted i...

Embodiment 2

[0043] Seed Liquid Culture: Seeds from the transformed single bacteria in the plate inoculated to a shake flask containing 30 ml of seed liquid medium (containing kanamycin 50 μg / mL), TB medium, oscillated at 37 ° C, 200 rpm, 12h . The seed liquid medium formulation is: trypsin 10g / L, yeast extract 10 g / L and NaCl 5 g / L, the initial pH is 7.0.

[0044] Shake flask fermentation: The cultured seed fluid was inoculated into fermentation medium containing kanamycin (50 μg / ml) in a fermentation medium containing 1%, and added a final concentration of 0.6 mm. IPTG was added after 37 ° C, 200 rpm The final concentration was 0.1 g / L Cobalt chloride, and then induced at 22 ° C for 12 h, and the final OD was determined. 600 . The culture solution was centrifted and the cell precipitate was washed twice with physiological saline to give a wet bacterium. The formulation of fermentation medium is: trypsin 16 g / L, yeast extract 10 g / l, NaCl 5 g / L, lactose 10 g / L, the initial...

Embodiment 3

[0049] Seed Liquid Culture: Seeds from the transformed single bacteria in the plate inoculated to a shake flask containing 30 ml of seed liquid medium (containing kanamycin 50 μg / mL), TB medium, oscillated at 37 ° C, 200 rpm, 12h . The seed liquid medium formulation is: trypsin 10g / L, yeast extract 10 g / L, NaCl 5g / L, initial pH of 7.0.

[0050] Shake flask fermentation: The cultured seed fluid was inoculated into fermentation medium containing kanamycin (50 μg / ml) in a fermentation medium containing 1%, and added a final concentration of 0.6 mm. IPTG was added after 37 ° C, 200 rpm The final concentration was 0.1 g / L Cobalt chloride, and then induced at 28 ° C for 12 h, and the final OD was measured. 600 . The culture solution was centrifted and the cell precipitate was washed twice with physiological saline to give a wet bacterium. The formulation of fermentation medium is: trypsin 16 g / L, yeast extract 10 g / l, NaCl 5 g / L, lactose 10 g / L, the initial pH of the...

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Abstract

The invention relates to a genetically engineered bacterium expressed by a nitrile hydratase gene, and particularly relates to a recombinant expression vector, a genetically engineered bacterium, and a culture method and application of the genetically engineered bacterium. The recombinant expression vector contains a section of nitrile hydratase gene, and the sequence of the nitrile hydratase gene is shown as SEQ ID NO. 1; the genetically engineered bacterium is a host microorganism carrying the recombinant expression vector; the culture method comprises the following steps of: culturing the host microorganism in an LB culture medium to obtain a seed solution, and then inoculating the seed solution into a fermentation culture medium for culturing to obtain the genetically engineered bacterium. Compared with the prior art, the nitrile hydratase gene can realize efficient expression in the engineered bacterium and has excellent enzymatic activity, and the specific enzyme activity can reach 50U / mg or above through determination and can reach 100U / mg under a preferred condition, so that the catalytic synthesis efficiency of nicotinamide is high, the conversion rate can reach 99% or above, and the production efficiency reaches 720g / (L.h).

Description

Technical field [0001] The present invention relates to a nitrile hydratase gene expression in genetically engineered bacteria, particularly relates to a recombinant expression vector, and culture methods using gene engineering bacteria and bacteria. Background technique [0002] Nicotinamide, also known as nicotinamide, vitamin B3, English Nicotinamide, nicotinic acid amide compound, as a white crystalline powder, odorless or almost odorless, bitter, slightly hygroscopic. Soluble in water, ethanol and glycerol. Mainly used in clinical prevention and treatment of pellagra, stomatitis, glossitis, sick sinus syndrome, atrioventricular block and so on. Industrial mainly as a nutritional feed additives, but also for food, medicine, dye intermediate, and as additives and biochemical reagents plating solution. In addition, nicotinamide in dermatology related industries have more applications, and is a popular ingredient in recent years, skin whitening products. [0003] Nicotinamide in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/60C12N9/88C12N1/21C12P17/12C12R1/19
CPCC12N15/70C12N9/88C12P17/12C12Y402/01084C12N2800/101
Inventor 徐毅王自稳吴小梅马宝娣高峰
Owner SHANGHAI INST OF TECH
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