Method for preparing triphosadenine by enzymic method

An enzymatic preparation technology of adenosine triphosphate, which is applied in the field of enzymatic preparation of adenosine triphosphate, can solve problems such as unclear classification, and achieve the effects of easy control, simple production process, and cost reduction

Active Publication Date: 2016-12-07
BEIJING TIANKAI YIDA BIOLOGICAL SCI & TECH
2 Cites 21 Cited by

AI-Extracted Technical Summary

Problems solved by technology

"ATP-producing enzymes" used include polyphosphate kinase (EC 2.7.4.1, Ppk), adenylate kinase (EC 2.7.4.3, Adk) and polyphosphate-adenylate phosphotransferase (EC 2.7.4.- ...
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Abstract

The invention discloses a method for preparing triphosadenine by an enzymic method. The method comprises the following steps of (1) preparing ATP production enzymes: obtaining the ATP production enzymes in gene engineering transformation, fermentation, purification or direct extraction modes; (2) preparing ATP in reaction liquid; adding adenosine, the ATP production enzymes and polyphosphoric acid or salts of the polyphosphoric acid for ATP preparation; (3) separating products and ATP production enzymes; directly separating immobilized ATP production enzymes in a reaction tank, and separating free ATP production enzymes through an ultrafiltration membrane in a filtering device; (4) preparing a finished product: preparing the reaction liquid subjected to the enzyme separation into finished product ATP through the steps of ion-exchange column chromatography, concentration, crystallization; drying and the like. The method has the advantages that the novel ATP production enzymes are used; the production process is simple; the control is easy; the adenosine is used as a substrate; the cost is reduced; an APT production enzyme recovery system is built; the method is applicable to industrial production; the content of impurities such as impurity protein and pigment in the enzymic method preparation reaction liquid is low; the subsequent purification is easy.

Application Domain

Fermentation

Technology Topic

Ion exchangeUltrafiltration +10

Image

  • Method for preparing triphosadenine by enzymic method
  • Method for preparing triphosadenine by enzymic method
  • Method for preparing triphosadenine by enzymic method

Examples

  • Experimental program(9)

Example Embodiment

[0046] Example 1 Preparation of AK, Ppk, Adk and Pap enzymes
[0047] The AK, Ppk (Ppk1 and/or Ppk2), Adk and Pap enzymes in the method of the present invention can be obtained commercially, or they can be artificially modified enzymes with the same catalytic function.
[0048] The preparation process of AK, Ppk1, Ppk2, Adk and Pap enzymes is as follows:
[0049] According to the sequences of the five enzyme genes, five pairs of amplification primers are designed. Extract the genomic DNA of Saccharomyces cerevisiae strain, use it as a template, PCR amplify the ado1 (AK enzyme gene) fragment, and ligate it to the pET22b vector (purchased from Novagene); extract Escherichia coli K12 strain ( (Purchased from Tiangen Biochemical Technology Co., Ltd.) genomic DNA, using it as a template, PCR amplified ppk1 and adk gene fragments, and ligated them to pET 22b vector (purchased from Novagene); Pseudomonas aeruginosa (Pseudomonas aeruginosa) strain (CICC 10419) genomic DNA, using it as a template, PCR amplified ppk2 gene fragments, and ligated to pET22b vector (purchased from Novagene); extract Acinetobacter johnsonii (Acinetobacter johnsonii) strain (CGMCC1. 8030) Genomic DNA, using it as a template, PCR amplifies the pap gene fragment, and ligates it to the pET22b vector (purchased from Novagene). After the five connecting sequences were sequenced correctly, they were transferred to E.coli BL21(DE3) strain (purchased from Tiangen Biochemical Technology Co., Ltd.).
[0050] The transformed E.coli BL21(DE3) single clone was connected to LB medium, after culturing to log phase, 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction for 5 hours, Bacteria were collected and sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to screen high expression strains.
[0051] The selected high-expression strains are connected to the seed culture medium under aseptic conditions, cultured to the logarithmic growth phase and then connected to the fermentor containing 5L fermentation medium, and then cultured to the logarithmic growth phase and then connected to the fermentation containing 50L In the fermentation tank of the culture medium, after 5 hours of culture, 1 mM IPTG was added to induce 5 hours, and about 1 kg of bacteria were collected by centrifugation.
[0052] The LB medium components are: 1% peptone, 0.5% yeast powder and 1% sodium chloride; the seed medium components are: 1% peptone, 0.5% yeast powder, and 1% sodium chloride; the fermentation medium components are: 1 % Peptone, 0.5% yeast powder, 1% sodium chloride, 5% disodium hydrogen phosphate, 1% sodium dihydrogen phosphate, 0.01% magnesium sulfate and 1% glycerin.
[0053] After the harvested cells were homogenized by ultrasound or high pressure, the supernatant was collected by centrifugation. Add 40-60% saturated ammonium sulfate to the supernatant and centrifuge. After the collected precipitate was dissolved in Tris buffer (pH 8.0), it was desalted using a G25 column (purchased from General Electric Medical Bioscience Co., Ltd.), and then subjected to CM- or DEAE-Sepharose FF (purchased from General Electric Medical Bioscience Co., Ltd.) Chromatography can get preliminary purified free enzyme.
[0054] figure 1 The SDS-PAGE chart of the prepared AK enzyme is shown in the figure: Lane 1 is the protein marker 14.4-116kDa (purchased from Beijing Zhongkeruitai Biotechnology Co., Ltd.); Lane 2 is the AK enzyme, about 40kDa.
[0055] figure 2 The SDS-PAGE diagrams of other enzymes prepared are shown in the figure: Lane 1 is the protein marker 14.4-116kDa (purchased from Beijing Zhongkeruitai Biotechnology Co., Ltd.); Lane 2 is the Ppk1 enzyme, about 60kDa; Lane 3 It is Ppk2 enzyme, about 40kDa; Lane 4 is Adk enzyme, about 25kDa; Lane 5 is Pap enzyme, about 55kDa.
[0056] Using the well-known method of measuring enzyme activity described in the prior art, the 1mg/ml AK, Ppk1, Ppk2, Adk and Pap enzyme solution activities were detected to be approximately 800U, 100U, 500U, 1000U and 800U, respectively, and the 1μM substrate was Complete conversion within 1 minute is defined as 1 activity unit (U).

Example Embodiment

[0057] Example 2 Preparation of ATP using free enzyme
[0058] image 3 It is a process flow diagram of the method of the present invention for producing ATP using free enzymes. See image 3 , The operation steps of using free enzyme to prepare ATP are as follows:
[0059] (1) Synthesis of ATP in the reaction tank:
[0060] In the reaction tank, the reaction system of 100L sterile water contains 2.5kg adenosine as the substrate, as well as 0.1kg ATP, 0.5kg sodium dihydrogen phosphate, 0.3kg sodium chloride, 0.3kg ammonium sulfate, 1.0kg magnesium chloride hexahydrate and A solution of 2.0kg sodium hexametaphosphate, stir evenly during preparation to prevent precipitation. Adjust the pH to 7.5, add 500U/LAK enzyme, 800U/LPpk2 enzyme and 800U/L Pap enzyme to the reaction system to start the reaction. During the reaction, the pH value was 7.5 and the temperature was 45°C.
[0061] Figure 5 , Image 6 Respectively are the 3 hours and 6 hours reaction HPLC detection chart.
[0062] After 6 hours of reaction, the amount of ATP produced was about 44g/L, and the adenosine conversion rate was over 90%. HPLC detection conditions are: Kromasil C18 column (purchased from AKZO NOBEL) (150×4.6mm), detection wavelength 210nm, detection temperature 30℃, detection flow rate 1ml/min, mobile phase containing 5% methanol and 50mM phosphate Buffer, pH=3.0.
[0063] (2) Separate ATP production enzyme in the filter:
[0064] Through the ultrafiltration method, the reaction solution of the reaction system in step (1) is passed through a filter to separate the three ATP production enzymes of AK, Ppk2 and Pap, see image 3. The filter is equipped with a membrane package (purchased from Pall, with a molecular weight cut-off of 20kDa). The filtrate is the reaction solution after the enzyme is separated and contains ATP, a small amount of ADP, a small amount of AMP and salt ions.
[0065] It was detected that the activity of the recovered AK, Ppk2 and Pap enzymes was reduced by 5%-10% compared with the previous reaction, and the corresponding new enzymes were added and used again in the step (1) reaction.
[0066] (3) Separation product ATP:
[0067] After the filtrate of step (2) is passed through ion exchange chromatography, concentrated, crystallized, and dried, the finished product ATP is prepared with a purity of more than 98% and a total yield of 85%.

Example Embodiment

[0068] Example 3 Preparation of ATP using free enzyme
[0069] See image 3 , The operation steps of using free enzyme to prepare ATP are as follows:
[0070] (1) Synthesis of ATP in the reaction tank:
[0071] In the reaction tank, the reaction system of 100L sterile water contains the substrate 3.0kg adenosine, and 0.1kg AMP, 0.5kg disodium hydrogen phosphate, 0.3kg potassium chloride, 0.25kg ammonium chloride, 2.5kg heptahydrate sulfuric acid A solution of magnesium and 3.0kg of tetrapolyphosphoric acid should be evenly stirred during preparation to prevent precipitation. Adjust the pH to 7.0, add 500U/LAK enzyme, 500U/LPpk2 enzyme, 300U/LAdk enzyme and 500U/L Pap enzyme to the reaction system to start the reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 40°C.
[0072] After 8 hours of reaction, the amount of ATP produced was about 50g/L, and the adenosine conversion rate reached 85%. The HPLC detection conditions were the same as the step (1) of Example 2.
[0073] (2) Separate ATP production enzyme in the filter:
[0074] Through the ultrafiltration method, the reaction solution of the reaction system in step (1) is passed through a filter to separate the four ATP producing enzymes of AK, Ppk2, Adk and Pap, see image 3. The filter is equipped with a membrane package (purchased from Pall, with a molecular weight cut-off of 8kDa). The filtrate is the reaction solution after the enzyme is separated, and contains ATP, a small amount of ADP, a small amount of AMP and salt ions.
[0075] It was detected that the activities of the recovered AK, Ppk2, Adk and Pap enzymes were reduced by 5%-10% compared with that before the reaction, and the corresponding new enzymes were added and used again in the step (1) reaction.
[0076] (3) Separation product ATP:
[0077] After the filtrate of step (2) is passed through ion exchange chromatography, concentrated, crystallized, and dried, the finished product ATP is prepared with a purity of more than 98% and a total yield of 80%.

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