Method for producing nicotinamide ribose phosphate transferase by fermentation

A technology of phosphoribose and nicotinamide, applied in the field of bioengineering, to achieve the effects of optimizing fermentation and culture conditions, inducing culture conditions, and increasing expression

Active Publication Date: 2021-11-26
XINTAI JIAHE BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the large-scale production of NAMPT by fermentation.

Method used

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  • Method for producing nicotinamide ribose phosphate transferase by fermentation
  • Method for producing nicotinamide ribose phosphate transferase by fermentation
  • Method for producing nicotinamide ribose phosphate transferase by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Construction of NAMPT production strain

[0033] The plasmid pLLP-ompA was double-digested with BamHI and NhelI, and then the nucleotide fragment encoding the NAMPT enzyme shown in SEQ ID NO. A recombinant expression vector (pLLP-ompA-NAMPT).

[0034] Carry out double enzyme digestion verification on the constructed first recombinant expression vector (pLLP-ompA-NAMPT). Carry out double enzyme digestion of pLLP-ompA-NAMPT with BamHI and NhelI. If two bands of 1.5kb and 7.5kb are produced, it proves the recombinant The expression vector was constructed successfully. For the results of the double enzyme digestion verification of the constructed first recombinant expression vector, see figure 1 . The results showed that the nucleotide fragment shown in SEQ ID NO.5 had been successfully inserted into the pLLP-ompA vector.

[0035] Plasmid pET-42a(+) was double digested with SacII and SphI, and then the nucleotide fragment encoding PARP1 protein shown in SE...

Embodiment 2

[0041] Embodiment 2: the fermentative production of NAMPT enzyme

[0042] (1) Strain activation: Streak the production strain of NAMPT enzyme prepared in Example 1 preserved at low temperature on an LB plate containing 100 μg / ml KAN and 100 μg / ml AMP, culture at 33°C for 24 hours; pick a single colony of the production strain and repeat Transfer to LB plates containing 100 μg / ml KAN and 100 μg / ml AMP by streaking, culture at 33°C for 24 hours, and set aside.

[0043] The medium formula of LB plate: peptone 10.0g, yeast powder 5.0g, NaCl 10.0g, agar 15.0g, water 1.0L.

[0044] (2) Preparation of primary seed liquid: use the inoculation loop to scrape the lawn of the production strain activated in the second step (1), inoculate it in LB liquid medium, 33°C, 200r / min, shake the flask for 12h to obtain the primary seed liquid.

[0045] LB liquid medium formula: peptone 10.0g, yeast powder 5.0g, NaCl 10.0g, water 1.0L.

[0046] (3) Preparation of secondary seed liquid: the prima...

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Abstract

The invention discloses a method for producing nicotinamide ribose phosphate transferase by fermentation, and belongs to the technical field of bioengineering. The method comprises the following steps: (1) inoculating an NAMPT production strain into a culture medium for fermentation culture under the fermentation culture conditions that the fermentation temperature is 32-34 DEG C, the dissolved oxygen is 20-40%, and the fermentation culture time is 5-8 hours; and (2) reducing the temperature of the fermentation system to 21-23 DEG C, adding IPTG (isopropyl-beta-d-thiogalactoside), and carrying out induced culture for 20-30 hours to obtain the NAMPT. By adopting the method disclosed by the invention, large-scale and industrial production of the NAMPT can be realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for fermenting and producing nicotinamide phosphoribosyltransferase. Background technique [0002] Nicotinamide phosphoribosyl transferase (nicotinamide phosphoribosyl transferase, NAMPT) is the rate-limiting enzyme of the NAD salvage synthesis pathway, which participates in important processes such as cellular material and energy metabolism, protein modification, and DNA repair through the synthesis of NAD. Previous research on NAMPT mainly focused on aging, immune response, inflammatory response, diabetes, oxidative stress, and metabolism. Recent studies have found that NAMPT is highly expressed in malignant tumors, speculating that it may be one of the potential therapeutic targets. NAMPT has become one of the research hotspots of anti-tumor treatment strategies, and its biological functions have been continuously explored and utilized. [0003] Therefore, NA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N1/21C12N15/54C12N15/70C12R1/19
CPCC12N9/1077C12N15/70C12Y204/02012
Inventor 岳明瑞曹华杰谢沛郭永胜滕义卫
Owner XINTAI JIAHE BIOTECH CO LTD
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