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A kind of polyphosphate kinase mutant and its application

A polyphosphate kinase and mutant technology, applied in the fields of genetic engineering and enzyme catalysis, can solve the problems of poor substrate tolerance, low enzyme activity, and high substrate inhibition

Active Publication Date: 2021-06-08
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the low polyphosphate kinase enzyme activity at present, the poor substrate tolerance is the defect that the substrate inhibition is high, the present invention utilizes genetic engineering technology to the polyphosphate kinase (PPK2) of Corynebacterium glutamicum ATCC 13032 source ) was transformed and screened to construct a high-performance polyphosphate kinase mutant with sodium hexametaphosphate as a substrate, which is conducive to the industrial application of the ATP regeneration system with polymetaphosphate as a substrate

Method used

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  • A kind of polyphosphate kinase mutant and its application
  • A kind of polyphosphate kinase mutant and its application
  • A kind of polyphosphate kinase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of wild-type polyphosphate kinase gene recombinant Escherichia coli

[0055] For the wild-type polyphosphate kinase (GenBank sequence number is NP_601909) derived from Corynebacterium glutamicum ATCC 13032, namely SEQ ID NO: 3, codon optimization is carried out on this basis, and the whole gene synthesis gene sequence SEQ ID NO: 4. Design restriction endonuclease sites Nde I and XhoI at both ends of the gene, subclone into the corresponding sites of the vector pET24a (Novagen), and obtain the recombinant plasmid pET24a-cgPPK2, see the plasmid map figure 1. The recombinant plasmid pET24a-cgPPK2 was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli PET24a-cgPPK2 / BL21(DE3) expressing wild-type polyphosphate kinase.

Embodiment 2

[0056] Example 2 Construction of Escherichia coli high-throughput screening host

[0057] The gshB in BL21(DE3) was knocked out by insertion and knockout by Red homologous recombination, and the combined fragments of glutamic acid-cysteine ​​ligase gene gshA and chloramphenicol gene were inserted into glutathione Peptide synthase gene gshB site.

[0058] According to the gene sequence (NCBI accession number: AM946981.2) of the glutamate-cysteine ​​ligase gene gshA derived from Escherichia coli BL21 (DE3), the primers were designed as follows:

[0059] Forward GshA-NcoI-F: 5'-CATGCCATGGGAATCCCGGACGTATCACAGGC-3',

[0060] Reverse GshA-Xho I-R: 5'-CGCTCGAGTCAGGCGTGTTTTTCCAGCC-3'.

[0061] The gshA fragment was amplified using BL21(DE3) genomic DNA as a template.

[0062] The PCR reaction system includes: 50pmol each primer, 100ng BL21(DE3) DNA template, 1×KOD neo plusbuffer, 0.2mM dNTP, 1.5mM MgSO 4 , KOD neo plus 1U, add ddH 2 O to 50 μL of the total system. The PCR amplif...

Embodiment 3

[0083] Embodiment 3 error-prone PCR method constructs random mutation library and screening method

[0084] 3.1 The first round of error-prone PCR method to construct random mutation library

[0085] Using the gene SEQ ID NO:4 of the wild enzyme as a template, an error-prone PCR technique was used to construct a random mutant library. The forward primer cgPPK2-F is 5'-ATGGTTGGTAAACTGCCGATC-3', and the reverse primer cgPPK2-R is 5'-TTAGTCACCGATCTGGTCACG-3'.

[0086] 50μL error-prone PCR reaction system includes: 10ng plasmid template pET24a-cgPPK2, 50pmol pair of primers cgPPK2-F and cgPPK2-R, 1×Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mMMgCl 2 , (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (Fermentas). The PCR reaction conditions were: 95°C for 5 minutes; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 72°C for 10min. The 1kbp random mutant fragment was recovered from the gel as a large primer, and MegaPrimer PCR was perf...

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Abstract

The invention discloses a polyphosphate kinase mutant SEQ ID NO: 1, which has significantly improved ATP regeneration ability compared with the wild-type sequence SEQ ID NO: 3, and adopts the polyphosphate kinase mutant as ATP regeneration Agent, can promote glutathione synthase catalyzed production of glutathione.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme catalysis, and in particular relates to a polyphosphoric acid kinase mutant and its use for converting ADP into ATP by using polymetaphosphoric acid as a substrate. Background technique [0002] ATP (Adenosine triphosphate, referred to as adenosine triphosphate) is a high-energy phosphate compound that participates in many enzyme catalysis, protein metabolism, biosynthesis processes in cells, and provides energy. In industry, ATP also participates in the biosynthesis of many important biologically active substances and chemical drugs. However, due to the high cost and price of ATP raw materials, the industrial application of the enzymatic reaction in which ATP participates is limited. Therefore, the synthesis of ATP and the recycling and regeneration of ATP become the main problem to be solved in this direction. [0003] At present, there are mainly three kinds of literatur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12P21/02C07K5/037
CPCC07K5/0215C12N9/1229C12N15/70C12P21/02C12Y207/04001
Inventor 范文超王金刚梁岩高书良
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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