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Method for preparing cyclic adenosine monophosphate by adenylate cyclase

A technology of adenylate cyclase and cyclic adenosine monophosphate, applied in biochemical equipment and methods, botanical equipment and methods, enzymes, etc., can solve the problem of high-efficiency expression mechanism that has not been clarified, further research is needed, and the yield is low and other problems, to achieve the effect of short cycle, mild conditions and simple industry

Pending Publication Date: 2020-12-11
杭州美亚药业股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are also limitations in the production of cAMP by fermentation methods such as Brevibacterium liquefaction, Bacillus liquefaction, Corynebacterium, Arthrobacter, etc., such as low yield
[0004] The enzymatic method uses adenylate cyclase as the enzyme source or uses recombinant bacteria containing adenylate cyclase as the catalyst to catalyze the production of cAMP. Adenylate cyclase (E.C.4.6.1.1) 2+ It can catalyze ATP to generate cAMP and PPi, which is the key enzyme of cAMP biosynthesis, but the mechanism of high-efficiency expression of adenylate cyclase has not been clarified, and the relationship between ion environment and cell activity and the regulation mechanism need to be further studied

Method used

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  • Method for preparing cyclic adenosine monophosphate by adenylate cyclase
  • Method for preparing cyclic adenosine monophosphate by adenylate cyclase
  • Method for preparing cyclic adenosine monophosphate by adenylate cyclase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Screening for efficient adenylyl cyclase (AC)

[0057] Using the amino acid sequence of known adenylyl cyclase (asAC) as a template, six potential adenylyl cyclase sequences were obtained by NCBI-Blastp online alignment, namely ag30AC, gmc3AC, naAC, kiAC, biAC and tcAC , the homology between the screened six potential adenylyl cyclases and the template adenylyl cyclase was 89%, 77%, 65%, 56%, 46%, 36%, respectively. The amino acid sequences and nucleotide sequences of the six screened adenylyl cyclases are shown in Table 1.

[0058] Table 1

[0059]

[0060]

[0061] Subsequently, the corresponding nucleotide sequences were synthesized in vitro and connected into the expression vector pET28a to obtain six recombinant plasmids pET28a-ag30AC, pET28a-gmc3AC, pET28a-naAC, pET28a-kiAC, pET28a-biAC and pET28a-tcAC, which will be obtained 6 kinds of recombinant plasmids were transformed into E.coli BL21(DE3), spread on LB plates containing 50 μg / mL kanamycin, and scree...

Embodiment 2

[0067] IPTG induced expression of adenylate cyclase

[0068] The recombinant bacteria E.coli BL21(DE3) / pET28a-ag30AC, E.coli BL21(DE3) / pET28a-gmc3AC and E.coli BL21(DE3) / pET28a-tcAC screened in Example 1 were selected to inoculate to 50 μg / In the LB medium of mL kanamycin, culture at 37°C and 220rpm for 8 hours, inoculate into fresh LB medium at 2% inoculum size, and cultivate to OD at 37°C and 220rpm 600 When it reaches about 0.6, add IPTG with a final concentration of 0.5mM, induce expression at 28°C and 220rpm for 14h, then centrifuge at 4°C and 8000rpm / min for 10 minutes to obtain wet cells containing adenylyl cyclase. Subsequently, the wet bacteria were resuspended with pure water, and crushed according to the crushing method provided in Example 1 to obtain a crude enzyme solution to measure the enzyme activity.

[0069] The enzyme activity assay system was as described in Example 1. After testing, the enzyme activities of ag30AC, gmc3A, and tcAC induced by IPTG were 14...

Embodiment 3

[0071] Optimization of Catalytic Conditions for Adenylyl Cyclase

[0072] According to the method described in Example 2, the expression of recombinant bacteria E.coli BL21(DE3) / pET28a-tcAC was induced, and the crude enzyme solution was obtained after centrifugation and crushing, and the optimum temperature in the catalytic process was optimized.

[0073] The optimization system was carried out in a 1mL reaction tube. Take 900 μL of the ATP substrate solution prepared in Example 1 as the base solution, incubate it with the crude enzyme solution at 25-90°C for 5 minutes, and then add 100 μL of the crude enzyme solution (based on the volume before crushing). Wet bacterial concentration meter, the final concentration is 10g / L), react in a water bath at 25-90°C for 15min, take 100μL of the reaction solution and add 5μL of 6M hydrochloric acid solution to terminate the reaction. Add 900 μL of pure water to dilute 10 times, mix well, then take 100 μL of it and add 900 μL of pure wat...

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Abstract

The invention discloses a method for preparing cyclic adenosine monophosphate by adenylate cyclase. The method comprises the following steps that the adenylate cyclase serves as a catalyst, adenosinetriphosphate serves as a substrate, and in the presence of Mg <2+>, a reaction solution containing the cyclic adenosine monophosphate is obtained. According to the adenylate cyclase, an amino acid sequence as shown in SEQ ID NO.14 is provided, the stability is good, the half-life period is long at medium and low temperatures, ATP can be efficiently catalyzed to generate cAMP in one step at mediumtemperature, a substrate conversion rate reaches 90 % or above, the advantages of simple industry, mild conditions, short period, few by-products and the like are achieved, the clean and pollution-free effects are achieved, and the energy conservation, consumption reduction and emission reduction in the cAMP production process can be realized.

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to a method for preparing cyclic adenosine monophosphate by adenylate cyclase. Background technique [0002] Cyclic adenosine monophosphate (cAMP) is a substance widely present in the human body with important physiological activities. play an important regulatory role. Clinically, cAMP is used to treat chronic congestive heart failure, pulmonary heart disease, myocardial infarction, myocarditis and cardiogenic shock; it has a certain effect on improving symptoms such as palpitations, shortness of breath, and chest tightness in rheumatic heart disease; it can improve the efficacy of acute leukemia combined with chemotherapy. It can also be used to induce remission of acute leukemia; in addition, it also has certain curative effect on elderly chronic bronchitis, various hepatitis and psoriasis. cAMP can also be used as a drug intermediate to prepare dibutyryl cyclic adenosine monophosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12N9/88C12N15/60C12N15/70
CPCC12N9/88C12N15/70C12P19/32C12Y406/01001
Inventor 李晓燕李恒王宏傅得响张燕
Owner 杭州美亚药业股份有限公司
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