Method for synthesizing D-psicose by combining phosphorylation and ATP (adenosine triphosphate) regeneration system

A technique of psicose and dephosphorylation, applied in the field of synthetic biology, can solve problems such as low material conversion rate, achieve the effects of low cost, increase ATP content, and reduce preparation cost

Active Publication Date: 2022-04-15
湖南成大生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to previous studies, there is a spatial distance between enzymes, and the product of the previous enzyme is the substrate of the next enzyme, resulting in a lower material conversion rate

Method used

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  • Method for synthesizing D-psicose by combining phosphorylation and ATP (adenosine triphosphate) regeneration system
  • Method for synthesizing D-psicose by combining phosphorylation and ATP (adenosine triphosphate) regeneration system
  • Method for synthesizing D-psicose by combining phosphorylation and ATP (adenosine triphosphate) regeneration system

Examples

Experimental program
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Effect test

Embodiment 1

[0032] This example is an implementation example of a method for synthesizing D-psicose by combining phosphorylation and ATP regeneration system, and the specific steps are as follows:

[0033] (1) Construction of recombinant Escherichia coli:

[0034] Construction of the recombinant plasmid pRSFDuet-gi: The gi gene fragment derived from Bacillus.sp was used as a template, gi-F: ccacagccaggatccgaattcaatgagcctgaccaccgc (BamHI) was used as an upstream primer, and gi-R: gcattatgcggccgcaagcttttaaccacgcgcgccc (HindIII) was used as a downstream primer for PCR amplification , the plasmid pRSFDuet-1 was double-digested at 37°C with restriction endonucleases BamHI and HindIII, and then the gene fragment was recombined with the plasmid pRSFDuet-1 using a seamless cloning kit to obtain the recombinant plasmid pRSFDuet-gi.

[0035] Construction of the recombinant plasmid pRSFDuet-gi-dpe: the dpe gene fragment derived from Clostridium bolteae ATCCBAA-613 was used as a template, dpe-F: agat...

Embodiment 2

[0044] The temperature of the whole reaction system was 30° C. during the whole-cell catalytic reaction, and the rest of the operations were the same as in Example 1.

Embodiment 3

[0046] The temperature of the whole reaction system was 37° C. and the pH was 8.0 during the whole-cell catalyzed reaction, and the rest of the operations were the same as in Example 1.

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Abstract

The invention discloses a method for synthesizing D-psicose by combining phosphorylation and an ATP (adenosine triphosphate) regeneration system, which comprises the following steps: by taking D-glucose as a raw material, sequentially carrying out recombinant escherichia coli construction, wet cell preparation, whole-cell catalytic reaction and dephosphorylation to prepare the D-psicose. Wherein the construction process of the recombinant escherichia coli comprises the step of introducing an ATP regeneration system gene. According to the method, the equilibrium conversion rate of synthesizing the D-psicose from the D-glucose is increased, and the addition of exogenous ATP (adenosine triphosphate) is reduced. The method has the characteristics of simple preparation process, high product conversion rate, greenness and economy, and can be used for industrial production.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology, and in particular relates to a method for efficiently synthesizing D-psicose from D-glucose through a multi-enzyme cascade system constructed by combining phosphorylation and ATP regeneration systems. Background technique [0002] D-psicose is the epimer of D-fructose at the C-3 position. It is rare in nature, with a sweetness of 70% of sucrose and only 0.3% of energy. At the same time, D-psicose is rated as "GRAS" by the US Food and Drug Administration and is recommended as an ideal substitute for sucrose in food. In recent years, studies have shown that D-psicose has the ability to lower blood sugar levels, prevent atherosclerosis and scavenge reactive oxygen species (ROS), so it has received more and more attention. In addition, some derivatives of D-psicose have anticancer and antiviral activities. Therefore, developing a method for the efficient production of D-psicose is a must ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/24C12P19/02C12N1/21C12N15/54C12N15/61C12N15/70C12R1/19
Inventor 刘继栋郭燕王志琦冯婷婷李宏伟
Owner 湖南成大生物科技有限公司
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