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Recombinant escherichia coli for producing cyclic adenosine monophosphate with high yield and application thereof in synthetization of cyclic adenosine monophosphate

A technology for recombining Escherichia coli and cyclic adenosine monophosphate, which is applied to the application field of synthesizing cyclic adenosine monophosphate, can solve problems such as inability to meet large-scale industrial production, complex enzyme preparation, etc., and achieve the effects of cost saving and process simplification

Pending Publication Date: 2019-08-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Invention patent CN 104342468 discloses that the adenylyl cyclase gene is cloned into Bacillus subtilis to obtain a strain of genetically engineered bacteria, and the highest fermentation level of 12.1g / L CAMP can be obtained through cultivation, which cannot meet the requirements of large-scale industrial production
At present, there are few reports on enzymatic production of CAMP, because the preparation of enzymes is complicated.

Method used

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  • Recombinant escherichia coli for producing cyclic adenosine monophosphate with high yield and application thereof in synthetization of cyclic adenosine monophosphate
  • Recombinant escherichia coli for producing cyclic adenosine monophosphate with high yield and application thereof in synthetization of cyclic adenosine monophosphate
  • Recombinant escherichia coli for producing cyclic adenosine monophosphate with high yield and application thereof in synthetization of cyclic adenosine monophosphate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of BL21(DE3)-PET22b-CYA446 Expression Strain

[0026] Using the whole genome of Escherichia coli MG1655 as a template, the 1-446 nucleotide coding sequence of adenylyl cyclase protein was amplified by conventional PCR.

[0027] The upstream primer used has an Nde I restriction site, and the sequence is: GGAATTCCATATGatgTACCTCTATATTGAGACTCTGAAAC.

[0028] The downstream primer has an Xho I restriction site, and the sequence is CCGCTCGAGTTCCGAGAGATCGGGTGAA.

[0029] The reaction conditions are: 95°C for 2min, 95°C for 20s, 50°C for 20s, 72°C for 50s, a total of 30 cycles; 72°C for 5min. The obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragments were recovered. The sequence and the expression vector pET22b were digested with Nde I and Xho I of Takara Company. The enzyme digestion reaction system was: 10×buffer H 2 μl, Nde I 0.5 μl, Xho I 0.5 μl, gene fragment or pET22b vector 3 μl, H 2 O14 μl. The enzyme...

Embodiment 2

[0031] Example 2 Induced expression and cell disruption of BL21(DE3)-PET22b-CYA446

[0032] 1. Induced expression of BL21(DE3)-PET22b-CYA446

[0033]The positive strain BL21(DE3)-PET22b-CYA446 was inoculated into 100ml LB / Amp liquid medium, and cultured with shaking at 37°C and 200rpm until OD600≈1. Inoculate in 500mL fresh LB / Amp liquid medium at a ratio of 10:100, shake and culture at 37°C and 200rpm until OD600≈0.5~0.7 (such as image 3 As shown, the enzyme activity is better between 0.5 and 0.7, but it can be seen from the figure that when the OD is 0.5, the enzyme activity of the enzyme is also the strongest). Add IPTG to a final concentration of 0.5‰~1‰ (eg Figure 4 As shown, the enzyme activity is better when the concentration is between 0.5‰~1‰, but the IPTG with a concentration of 1‰ has the best effect); at 20~25°C (such as Figure 5 As shown, the enzyme activity is better between 20~25°C, and 20°C is the best), shake culture at 200rpm for 18~20h. Centrifuge at ...

Embodiment 3

[0036] Example 3 Catalytic synthesis of CAMP

[0037] Mix the following reaction system in the test tube: add adenosine triphosphate with a concentration of 10-50mM, 10-20mM ZnCl 2 (like Image 6 As shown, the enzyme activity between 10-20mM is better, 10 mM is the best), Tris HCl (pH8.0), after stirring evenly, it will be at pH7.0-9.0 (such as Figure 7 As shown, the enzyme activity is better between pH7.0~9.0, and pH8.0 is the best), 30~35℃ (such as Figure 8 As shown, the enzyme activity is better between 30~35°C, and 30°C is the best) to react for 4~6h to complete the enzymatic reaction to synthesize cyclic adenosine monophosphate. Agilent HC-C18 chromatographic column was used to detect the generation of cAMP in the supernatant, the mobile phase was phosphoric acid + triethylamine (6mL+18.4mL to 1L):methanol=75:25, the flow rate was 0.4ml / min, and the detection wavelength was 254nm.

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Abstract

The invention discloses recombinant escherichia coli for producing cyclic adenosine monophosphate with high yield and application of the recombinant escherichia coli in synthetization of cyclic adenosine monophosphate. An adenylate cyclase gene is cloned from a cAMP production strain, a crude enzyme solution obtained after recombinant bacteria are broken has good catalytic activity and stability,adenosine triphosphate (ATP) and Zn2 + are added, a reaction system is simple, the conditions are mild, a period is short, few byproducts are produced, the method is clean and pollution-free and is asimple, rapid and efficient production way, and the conversion rate of substrate ATP reaches 90% or above.

Description

technical field [0001] The invention belongs to the field of cyclic adenosine monophosphate preparation, and in particular relates to a high-yielding cyclic adenosine monophosphate recombinant Escherichia coli and its application in synthesizing cyclic adenosine monophosphate. Background technique [0002] Cyclic adenosine monophosphate (CAMP) is an important nucleic acid derivative with various physiological functions and widely used in medicine and animal husbandry. In medical applications, CAMP is the second messenger in human cells, involved in the regulation of glucose metabolism, lipid metabolism, nucleic acid synthesis, and protein synthesis. Studies have shown that the occurrence of more than 40 diseases including coronary heart disease, myocardial infarction, and cancer are all related to the abnormal metabolism of CAMP. Exogenous CAMP has physiological functions such as relaxing smooth muscle, dilating blood vessels, improving myocardial hypoxia, and improving liv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/32C12R1/19
CPCC12N9/88C12Y406/01001C12P19/32
Inventor 王昕王静陈可泉马琛王雪麟欧阳平凯
Owner NANJING UNIV OF TECH
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