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Method for synthesizing PAPS based on construction of bifunctional enzyme

A bifunctional enzyme and kinase technology, applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of expensive PAPS, limited application, limited purity of PAPS, etc., to reduce costs, simplify acquisition, shorten The effect of the transfer mass transfer process

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

AI Technical Summary

Problems solved by technology

[0005] Since the purification of PAPS is currently in its infancy, the purity of PAPS obtained is limited, which directly leads to the high price of PAPS with higher purity and limits its application.

Method used

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  • Method for synthesizing PAPS based on construction of bifunctional enzyme
  • Method for synthesizing PAPS based on construction of bifunctional enzyme
  • Method for synthesizing PAPS based on construction of bifunctional enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Expression and purification of ATP sulfurylase and APS kinase

[0073] ATP sulfurylase derived from Saccharomyces cerevisiae (Gene ID 853466), ATP sulfurylase derived from Penicillium chrysogenum (GenBank number CAP86100.1), and ATP sulfurylase derived from Kluyveromyces lactis ( Gene ID is 2894185), codon optimization was carried out according to the codon preference rule of Escherichia coli, and the optimized nucleotide sequence was connected between the Nde I and Hind III restriction enzyme sites of plasmid pET28a (+) to obtain recombinant plasmid.

[0074] APS kinase derived from Penicillium chrysogenum (Gen Bank No. U39393.1), APS kinase derived from Escherichia coli (Gen Bank No. M74586.1), and APS kinase derived from Saccharomyces cerevisiae (Gene ID 853869) were selected. , APS kinase of Mycobacterium tuberculosis (Gen Bank No. QGK78545.1), codon optimization was carried out according to the codon preference rule of Escherichia coli, and the optimize...

Embodiment 2

[0079] Example 2: Comparison of ATP sulfurylase and APS kinase from different sources

[0080] After constructing the ATP sulfurylase gene derived from Saccharomyces cerevisiae, Kluyveromyces lactis, and Penicillium chrysogenum on the carrier to obtain the target protein, add equimolar protein 0.1mM to the catalytic system, and the catalytic system is 50-100mM Tris -HCl buffer pH 7.0~8.5, then add 5mM ATP, 4mM MgSO 4 , catalyzed at 35-40°C for 60 hours, using high-performance liquid chromatography to detect the generation of APS, after comparison, the specific enzyme activity of ATP sulfurylase derived from Saccharomyces cerevisiae is higher, and it is more advantageous.

[0081] After obtaining APS kinase proteins from Escherichia coli, Saccharomyces cerevisiae, Penicillium chrysogenum, and Mycobacterium tuberculosis, ATP sulfurylases from Saccharomyces cerevisiae were catalyzed and compared together. Add APS kinase from different sources and ATP sulfurylase from Saccharomyc...

Embodiment 3

[0084] Example 3: Enzyme activity assay comparison of bifunctional enzymes with different linker sequences

[0085] Construction of a bifunctional enzyme: ATP sulfurylase derived from Saccharomyces cerevisiae and APS kinase derived from Escherichia coli were fused into one fragment according to genetic manipulation (the sequence of the two enzymes does not affect its expression), and different fusions were added to the linker part The linker sequence (linker) is fused into a fragment, so that both enzymes maintain a certain spatial position, and the catalysis is more orderly. Specifically, the stop codon of the previous gene is removed, directly connected to the linker, and then connected to another gene The sequence of the linker can be SEQ ID NO.1-5 (respectively GGGGGS, EAAAAK, GMALP, GA2PA3PAKQEA3PAPA2KAEAPA3PA2KA, KESGSVSSEQLAQFRSLD), and the number of repetitions n=1 is compared here.

[0086] Expression conditions: Pick a single colony and culture overnight in LB medium...

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Abstract

The invention discloses a method for synthesizing PAPS based on construction of a bifunctional enzyme, and belongs to the technical field of biology. The method comprises the following steps: firstly, optimizing ATP sulfating enzyme and APS kinase from different sources, and fusing a section of different protein linkers between the optimized ATP sulfating enzyme and APS kinase by utilizing a protein fusion technology to construct the bifunctional PAPS synthase. The linkers are further rationally designed and modified, so that the conversion efficiency is greatly improved, the acquisition of the enzyme catalyst is simplified, and the loss of the enzyme catalyst in the preparation process is reduced. The ATP conversion rate is improved by 25%, and the catalysis time is shortened by 59%. In addition, the ATP sulfating enzyme P ring HRAH sequence is mutated into HNGH and HAGH, the enzyme activity of the ATP sulfating enzyme P ring HRAH sequence is further improved, and the conversion rate of PAPS can reach 48%.

Description

technical field [0001] The invention relates to a method for synthesizing PAPS based on the construction of a bifunctional enzyme, which belongs to the field of biotechnology. Background technique [0002] 3'-Phosphoadenosine-5'-phosphosulfate (3'-phosphoadenosine-5'-phosphosulfate, PAPS) is the only known active sulfate group donor, which is the biologically "activated" sulfate form, is Substance essential for cellular uptake and metabolism of inorganic sulfates. In addition, it has been confirmed that the emergence and development of various diseases, such as cancer and HIV, will cause changes in the PAPS content in tissues. [0003] PAPS is the most direct sulfate donor such as chondroitin sulfate, heparin and dermatan sulfate. At present, the main sources of PAPS are extraction and enzymatic synthesis. Due to the low direct content of PAPS in most organisms, the extraction process of PAPS is cumbersome, and the by-products are more difficult to purify, which directly ...

Claims

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Application Information

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IPC IPC(8): C12P19/32C12N9/12
CPCC12P19/32C12N9/1205C12N9/1241C12Y207/07004C12Y207/01025C07K2319/00
Inventor 康振王阳胥睿睿堵国成陈坚
Owner JIANGNAN UNIV
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