Enzymatic synthesis method and application of 3',5'-adenosine diphosphate (PAP)

A kind of technology of adenosine diphosphate and synthesis method, which is applied in the direction of biochemical equipment and methods, enzymes, transferases, etc., to achieve the effect of low price, excellent stability, and reduced enzyme cost

Pending Publication Date: 2020-04-10
孙军亭 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some APSKs have the ability to catalyze the synthesis of PAP using AMP as a substrate has been reported, so far, there is no published literature or published patents on the application of APSK to AMP as a substrate for the one-step enzymatic synthesis of PAP

Method used

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  • Enzymatic synthesis method and application of 3',5'-adenosine diphosphate (PAP)
  • Enzymatic synthesis method and application of 3',5'-adenosine diphosphate (PAP)
  • Enzymatic synthesis method and application of 3',5'-adenosine diphosphate (PAP)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] (1) Construction and fermentation expression of 5'-phosphosulfatase kinase (SEQ01) and acetate kinase (SEQ06) co-expression bacteria

[0029] The APSK with the amino acid sequence of SEQ01 and the AK with the amino acid sequence of SEQ06 were respectively optimized for Escherichia coli codons to construct the pETDuet-SEQ01-SEQ06 expression vector, and the constructed and verified correct expression vector was heat-shock transformed Escherichia coli BL21 (DE3) , after the transformation is completed, smear a plate containing AMP, pick a single clone to express, and measure the enzyme activity verification; amplify the strain with better expression and save the seed bank seeds; take a seed bank seed to amplify, inoculate the fermenter for fermentation, When the OD reaches 22-40, cool down to 28-30°C, add IPTG with a final concentration of 0.6mM to induce, continue to feed the culture for no less than 8 hours, put the tank, and collect the wet cells by centrifugation, which...

Embodiment 2

[0037] (1) Construction and fermentation expression of 5'-phosphosulfatase kinase (SEQ01) and acetate kinase (SEQ06) individual expression bacteria

[0038] The APSK with the amino acid sequence of SEQ01 and the AK with the amino acid sequence of SEQ06 were codon-optimized in Escherichia coli to construct pET22b-SEQ01 and pET22b-SEQ06 expression vectors respectively, and the constructed and verified correct expression vectors were transformed into Escherichia coli by heat shock BL21(DE3), after the transformation is completed, spread the plate containing Amp, pick a single clone to express, measure the enzyme activity verification; amplify the strain with better expression and save the seed bank seeds; take a seed bank seed to amplify and inoculate Ferment in a fermenter, cool down to 28-30°C when the OD reaches 22-40, add IPTG at a final concentration of 0.6mM to induce, continue to feed the culture for no less than 10 hours, put the tank, and collect the wet cells by centrifu...

Embodiment 3

[0046] (1) Construction and fermentation expression of 5'-phosphosulfatase kinase (SEQ02) and acetate kinase (SEQ07) individual expression bacteria

[0047] The APSK with the amino acid sequence of SEQ02 and the AK with the amino acid sequence of SEQ07 were codon-optimized in Escherichia coli respectively, and the pET28a-SEQ02 and pET28a-SEQ07 expression vectors were respectively constructed, and the constructed and verified correct expression vectors were heat-shocked and transformed into Escherichia coli For BL21(DE3), after the transformation is completed, apply a Kan-containing plate, pick a single clone for expression, and measure the enzyme activity for verification; perform the expression of the two enzymes and the storage of the bacteria according to the procedures in 1 in Example 1;

[0048] (2) crude enzyme extraction

[0049] Using the method of step (2) in Example 1 to carry out the extraction and preservation of 5'-phosphosulfatase kinase (SEQ02) and acetate kinas...

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Abstract

The invention provides an enzymatic synthesis method of PAP. The enzymatic synthesis method comprises the following steps: generating PAP with 5'-adenosine monophosphate (AMP) as a substrate, 5'-phosphosulfated kinase (APSK) as a catalyst and 5'-adenosine triphosphate (ATP) or 5'-adenosine diphosphate (ADP) as a coenzyme; and synthesizing ATP with acetylphosphoric acid or polyphosphoric acid as aphosphate group donor and acetokinase (AK) or polyphosphoric kinase (PK) as a catalyst, thereby realizing ATP circulation (as shown in Figure 1). The method has the advantages that the PAP is synthesized in one step by taking AMP as a substrate, and that the theoretical utilization rate of phosphorus reaches 100%.

Description

technical field [0001] The invention relates to the technical field of enzyme catalysis, in particular to an enzyme synthesis method of 3',5'-adenosine diphosphate (PAP) and its application. Background technique [0002] 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is a sulfate group donor widely used in organisms and plays a very important role as a coenzyme of sulfotransferases in organisms. 3',5'-di Adenosine phosphate (PAP) differs from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) by one sulfate group in structure. During the synthesis of sulfated compounds, 3',5'-diphosphate adenosine (PAP ) can replace 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to produce high value-added sulfated compounds. [0003] In 2000, Michael D. Burkart published a document entitled "Regeneration of PAPS for the Enzymatic Synthesis of Sulfated Oligosaccharides", reporting a method for PAPS regeneration, making low-cost enzyme-catalyzed synthesis of high value-added sulfated compounds a Poss...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/32C12N9/12
CPCC12P19/32C12N9/1205C12N9/1217C12N9/1229C12Y207/01025C12Y207/02001C12Y207/04001
Inventor 孙军亭陈江徐光彩
Owner 孙军亭
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