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Efficient catalytic synthesis method of PAPS based on construction of ATP regeneration system

A catalyst and sulfurylase technology, applied in the field of bioengineering, can solve the problem that by-products cannot be fully utilized, and achieve the effects of simple preparation steps, good stability and high catalytic efficiency

Active Publication Date: 2021-06-29
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reutilization of by-products is an important research content for the efficient production of PAPS. In the current research, we mostly start from the perspective of polyphosphokinases in order to convert and reuse by-products. However, most polyphosphokinases can only convert ADP, and do not Pyrophosphoric acid cannot be converted, and there is still the problem that by-products cannot be fully utilized

Method used

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  • Efficient catalytic synthesis method of PAPS based on construction of ATP regeneration system
  • Efficient catalytic synthesis method of PAPS based on construction of ATP regeneration system
  • Efficient catalytic synthesis method of PAPS based on construction of ATP regeneration system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: Obtaining of polyphosphate kinase

[0067] (1) Obtain the Gene ID: 1020661 of the polyphosphokinase from Corynebacterium glutamicum on the NCBI website, obtain the nucleotide sequence through codon optimization, use polymerase chain reaction to amplify the target gene, and connect it to pRSFDuet-1 On the vector, a recombinant plasmid was obtained, and the verified correct recombinant plasmid was transferred into E.coli BL21(DE3) to obtain a transformant. The transformant was cultured and sequenced for verification, and the verified correct transformant was recorded as strain E.coli GluPPK.

[0068] (2) Find the Gene ID of the polyphosphokinase derived from Mycobacterium tuberculosis: 888760, obtain the nucleotide sequence through codon optimization, and use a method similar to step (1) to amplify and connect the target gene to the vector pET32a (+), and the verified correct recombinant plasmid was transferred into E.coli BL21(DE3) to obtain a transformant,...

Embodiment 2

[0072] Embodiment 2: Purification and enzymatic activity comparison of two kinds of polyphosphokinases

[0073] After the recombinant strain obtained in Example 1 was cultured in LB medium for 6-20 hours, the collected bacteria were ultrasonically disrupted and then centrifuged at high speed to remove cell debris. The supernatant was filtered through a 0.22 μm aqueous membrane, and the Ni-NTA affinity layer was used to analysis to purify the protein of interest. After equilibrating the column with solution A, load the crude enzyme solution, then use solution A to balance the chromatography column, and then wash the column with different concentrations of solution B and collect the washing solution, and use SDS-PAGE to verify the purified components ( figure 2 ), and the purest fraction was desalted with a PD-10 desalting column, and a low-salt buffer (10mM Tris-HCl, 0.1M NaCl; pH 6.0) was used for desalting, and the purified desalted protein was collected.

[0074] Solution ...

Embodiment 3

[0077] Example 3: Expression, purification and enzyme activity assay of a synthetic bifunctional enzyme

[0078] Construction of a bifunctional enzyme: ATP sulfurylase (Gene ID 853466) derived from Saccharomyces cerevisiae and APS kinase derived from Escherichia coli (Gen Bank No. M74586.1) were fused into a fragment according to genetic manipulation means (the sequence does not affect its expression), add different fusion linker sequences (linker) to the joint part respectively, and fuse them into one fragment, so that both enzymes maintain a certain spatial position, and the catalysis is more orderly. Specifically, the previous gene's Remove the stop codon, connect directly with the linker, and then connect with the start codon of another gene. The linker sequence is: (GGGGS) 6 , the fusion fragment was connected to the plasmid pET28a(+), and transformed into E.coli BL21(DE3) to obtain a transformant, the transformant was cultured and sequenced for verification, and the veri...

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Abstract

The invention discloses an efficient catalytic synthesis method of PAPS based on construction of an ATP regeneration system, and belongs to the technical field of bioengineering. PAPS is artificially constructed through microbial recombinant expression to synthesize a bifunctional enzyme, and high-efficiency production of PAPS is realized. On the basis, an ATP regeneration system of polyphosphokinase from corynebacterium glutamicum and mycobacterium tuberculosis is coupled, two by-products including pyrophosphoric acid and ADP can be recovered at the same time, equivalent conversion of a substrate and a product is achieved, PAPS generated in a catalytic system has high purity, and supply of sulfonic acid groups in most sulfonic acid transfer reactions can be achieved.

Description

technical field [0001] The invention relates to a method for efficiently catalytically synthesizing PAPS based on the construction of an ATP regeneration system, which belongs to the technical field of bioengineering. Background technique [0002] 3'-phosphoadenosine-5'-phosphosulfate (3'-phosphoadenosine-5'-phosphosulfate, PAPS) is the most important active sulfonic acid donor for in vitro sulfonic acid reactions, and can be used for chondroitin sulfate, heparin and sulfated skin Sulfonated group supply of prime. [0003] At present, the PAPS purchase channels are narrow, expensive and the purity is not high. The main reason is that the conversion rate of PAPS synthesized by in vitro enzymatic method is low, the by-products are difficult to remove, and the purification of PAPS is difficult. [0004] In the enzymatic synthesis of PAPS, two by-products, pyrophosphate and ADP, are mainly produced, so that the theoretical conversion rate of the substrate is only 50%. The reus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40
CPCC12P19/40Y02P20/584C12Y207/01C12Y207/07004C12N9/1241C12P19/32C12N9/1229C12N9/88
Inventor 康振堵国成王阳胥睿睿陈坚
Owner JIANGNAN UNIV
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