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Method for producing PAPS by whole-cell catalysis

A technology of adenosine triphosphate sulfurase and recombinant bacteria, which is applied in the field of bioengineering, can solve the problems of limited scale production of PAPS, unsuitability for industrial production, lack of high efficiency and simplicity, and achieve improved substrate utilization efficiency, Convenient separation and purification, eliminating the effect of reaction by-product inhibition

Active Publication Date: 2021-04-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, adenosine 3,5-diphosphate is expensive, and the synthesis of PAPS catalyzed by acyl sulfonate transferase is only tested on a small scale in the laboratory, which is not suitable for industrial production
ATP sulfurylase and APS kinase catalyze complex enzyme purification process, product inhibition and other problems, which lead to the limitation of large-scale production of PAPS
At present, the commercial reagent PAPS is synthesized by enzymatic method, and the price is very expensive (20559.40 yuan / 25mg, the purity is only 60%), much higher than chondroitin sulfate and heparin
Therefore, and also lack efficient, easy, cheap method for preparing PAPS

Method used

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  • Method for producing PAPS by whole-cell catalysis
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  • Method for producing PAPS by whole-cell catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Codon optimization and vector construction

[0038] The ATP sulfurase atps used in the present invention is derived from Saccharomyces cerevisiae, the 5'-phosphoadenosine sulfatase kinase apsk is derived from Penicillium Chrysogenum, and the inorganic pyrophosphate hydrolase enzyme ppa is derived from Escherichia coli (Escherichia coli) and polyphosphokinase gene ppk are derived from Corynebacterium glutamicum, codon optimization and artificial synthesis were carried out respectively, and the sequences are as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO. 3. Shown in SEQ ID NO.4.

[0039] Construction of catalytic module expression vectors: Link the genes encoding atps and apsk to vectors pACYCDuet-1, pETDuet-1, pRSFDuet-1, and pCDFDuet-1, respectively, so that atps and apsk are respectively activated by the T7 promoter, and four kinds of dual Enzyme expression vectors pACYCDuet-ATPS-APSK, pETDuet-ATPS-APSK, pRSFDuet-ATPS-APSK, pCDFDuet-ATPS-APSK.

[0040] Constructi...

Embodiment 2

[0041] Embodiment 2: Construction of recombinant escherichia coli

[0042]The 4 main catalytic modules and 4 coenzyme modules in Example 1 were combined, and the combined results are shown in Table 1. Transform the double-plasmid combination of 12 combinations into E.coli BL21(DE3), pick a single colony grown on the LB-resistant plate, and verify the colony by PCR. After the verification is correct, send it for sequencing to obtain a transformed expression host with the correct sequence There are 12 strains in total, numbered 1-12, and the specific strains are shown in Table 1.

[0043] Table 1 Combination optimization of dual-vector expression strains

[0044]

Embodiment 3

[0045] Example 3: Recombinant Escherichia coli induces expression of enzymes and thalline treatment

[0046] 12 kinds of recombinant Escherichia coli in Example 2 are picked single bacterium colony from plate, inoculate in the seed medium, cultivate overnight, transfer to 50mL fermentation medium with the inoculum size of 5% (v / v), 37 ℃ , 200rpm / min culture to OD 600 0.6-0.8, add IPTG with a final concentration of 0.6mM for induction, collect the bacteria after induction at 30°C for 12h, and treat the active cells with 0.3mM Triton for 30min for whole cell transformation.

[0047] Whole-cell transformation: Use Tris-HCl solution with pH=7.5 as the medium for whole-cell transformation, add 10mM (NaPO 3 ) 6 , 100mM LiCl, 50mM MgSO 4 , the concentration of the substrate ATP disodium salt was 40mM, 15g / L of bacteria was added, and transformed at 30°C for 10h, and the transformation efficiency was measured after the transformation was completed.

[0048] Such as figure 2 As s...

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Abstract

The invention discloses a method for producing PAPS through whole-cell catalysis, and belongs to the technical field of bioengineering. According to the method, coenzyme PPA and PPK are introduced based on ATP sulfatase and APS kinase, so that simultaneous synthesis of the four enzymes in microbial cells can be realized. The ATP sulfurase gene atps derived from saccharomyces cerevisiae, a 5'-adenosine phosphate phosphokinase gene apsk derived from penicillium chrysogenum, an inorganic pyrophosphate hydrolase enzyme gene ppa derived from escherichia coli and a polyphosphate kinase gene ppk derived from corynebacterium glutamicum are synthesized in microbial cells at the same time due to the fact that different genes are expressed by different vectors to obtain recombinant bacteria. The recombinant bacterium is utilized to perform whole-cell catalysis to produce PAPS by taking ATP disodium salt as a substrate, and the conversion rate can reach 96.12 percent through an optimized catalytic condition after catalytic reaction is carried out for 15 hours; and the material supplementation condition is optimized in the reacting process, and the conversion rate can reach 97.8 percent after 16 hours of catalysis.

Description

technical field [0001] The invention relates to a method for whole-cell catalyzed production of PAPS, which belongs to the technical field of bioengineering. Background technique [0002] 3'-Phosphoadenosine-5'-phosphoryl sulfate (PAPS) is the direct sulfate group donor in the synthesis of heparin and chondroitin sulfate catalyzed by GAG sulfotransferase. The production methods of PAPS include fermentation and biocatalysis. The fermentation method is based on the cell's own metabolic pathway, and realizes the synthesis from ATP to PAPS by introducing foreign genes. However, under normal conditions, the concentration of ATP in the cell is low, and most of the ATP is used for the energy metabolism of the cell itself, resulting in extremely low final production. Moreover, different types of by-products will be produced during the fermentation process, which is not conducive to the separation and purification of later products. [0003] At present, researchers generally use i...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/55C12P19/32C12R1/19
CPCC12P19/32C12N9/14C12N9/1205C12N9/1229C12Y306/01003C12Y207/01025C12Y306/01009C12Y207/04001Y02A50/30
Inventor 刘立明刘开放梁光杰高聪刘佳陈修来宋伟
Owner JIANGNAN UNIV