Polyphosphokinase mutant, engineering bacterium and application thereof

A polyphosphate kinase, mutant technology, applied in application, genetic engineering, enzyme and other directions, to achieve the effect of broad industrial application prospects

Pending Publication Date: 2022-06-10
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a polyphosphate kinase mutant and use the polyphosphate kinase mutant to solve the problem that the existing polyphosphate kinase activity is not high. Gene recombinant bacteria or its crude enzyme solution are used as biocatalysts for the construction of ATP regeneration systems to solve the problem of low efficiency of existing ATP regeneration systems

Method used

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  • Polyphosphokinase mutant, engineering bacterium and application thereof
  • Polyphosphokinase mutant, engineering bacterium and application thereof
  • Polyphosphokinase mutant, engineering bacterium and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of wild-type Ecoli.BL21(DE3)-ChPPK

[0035] According to the PPK protein sequence (ChPPK, GenBank number: ABG57400.1) from Cytophaga hutchinsonii in GenBank, it was optimized for the codon preference of Escherichia coli, and a 6His tag was fused to the C-terminus of the sequence, provided by Beijing Qing Kebio Company (Beijing, China) synthesized a recombinant gene ChPPK sequence with a length of 930bp, its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

[0036] The recombinant gene ChPPK was inserted under the T7 promoter of pET-28a(+) to obtain the expression plasmid pET28-ChPPK. Transform the expression plasmid into Escherichia coli E.coli BL21(DE3), smear it on an LB plate containing 50 μg / mL kanamycin resistance, cultivate it at 37°C for 8-12 hours, and pick positive clones, which are wild Type Ecoli.BL21(DE3)-ChPPK for expression of recombinant ChPPK.

[0037] SEQ...

Embodiment 2

[0041] Example 2: Induced expression of wild-type Ecoli.BL21(DE3)-ChPPK and extraction of wild-type polyphosphate kinase

[0042] (1) Crude enzyme solution: Inoculate the wild-type Ecoli.BL21(DE3)-ChPPK obtained in Example 1 into LB liquid medium containing 50 μg / mL kanamycin resistance, culture at 37°C for 12 hours at 200 rpm, and then Inoculate 1% (v / v) inoculum into fresh LB liquid medium containing 50 μg / mL kanamycin resistance, cultivate at 37°C and 150 rpm until the OD600 of the bacteria = 0.6, and add a final concentration of 0.1 mM IPTG, induced at 28°C for 12h, centrifuged at 4°C, 8000rpm for 10min, discarded the supernatant, collected the precipitate, and obtained the wet cells expressing the recombinant ChPPK. The collected wet bacteria were resuspended in 50mM potassium phosphate buffer (PBS) with a pH of 7.2 in an amount of 40g / L to form a bacterial suspension, and crushed using an ultrasonic cell pulverizer with a crushing power of 50W, each working for 1s, and i...

Embodiment 3

[0044] Embodiment 3: the mensuration of enzyme activity

[0045] The crude enzyme liquid prepared by 0.4mg embodiment 2 method or the polyphosphoric acid (PPA) of 0.05mg pure enzyme, final concentration 1.6g / L, the adenosine phosphate (AMP) of final concentration 2.25mM, the MgCl of final concentration 10mM 2 Added to 10mL of 50mM potassium phosphate buffer (pH7.5). The reaction solution was incubated at 37° C. for 5 min, and then 10 mL of 0.2 M phosphoric acid aqueous solution was added to terminate the reaction. The ATP content in the final solution was determined by HPLC method.

[0046] The instrument used for HPLC is Agilent 1260 Infinity II (Agilent Technologies Co., Ltd., USA), equipped with Agilent 2414 UV detector, Agilent 1525 pump, and Agilent 717 injector. The chromatographic column is XBridge C18column (C18, 5μm, 4.6×250mm, Waters, California, USA). The mobile phase rate was 1 mL / min, the UV detection wavelength was 254 nm, the mobile phase was potassium phosph...

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Abstract

The invention discloses a polyphosphate kinase mutant, an engineering bacterium and application of the polyphosphate kinase mutant and the engineering bacterium. The polyphosphate kinase mutant is obtained by performing single mutation or multiple mutation on the 79th site, the 106th site, the 108th site, the 111th site or the 285th site of an amino acid sequence shown in SEQ ID NO: 2. The invention provides a plurality of polyphosphokinase mutants derived from Cytophaga hutchinsonii, the specific enzyme activity of the mutants is increased by 2.7-17.9 times compared with that of female parent polyphosphokinase, an ATP regeneration system formed by the mutants can reduce the consumption of ATP in ATP-dependent biocatalytic synthesis reaction by 70% or more, and the mutant has a wide industrial application prospect.

Description

(1) Technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a polyphosphokinase (PPK) mutant, engineering bacteria and applications thereof, and to develop an efficient and cheap ATP regeneration system. (2) Background technology [0002] Adenosine triphosphate (ATP) is a key molecule in the regulation of various biological processes such as energy metabolism, RNA and DNA synthesis, and signal transduction in organisms. The activity of a large number of potential biocatalysts, including ligases, kinases and synthetases, is also dependent on ATP. Introducing an ATP regeneration system during these biotransformations can significantly reduce ATP consumption. Most ATP regeneration systems include a phosphate donor and a phosphotransferase that catalyzes the connection between ADP and the phosphate donor. However, the biosynthesis of many valuable products requires a regenerative system that directly generates ATP from AMP, such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N1/21C12N15/70C12P19/30C12P19/02C12R1/19
CPCC12N9/1229C12N15/70C12P19/30C12P19/02C12N9/1205C12Y207/04001C12Y207/01001C12Y207/01022C12P19/32C12N9/12
Inventor 薛亚平薛语真张诗嘉沈其郑裕国
Owner ZHEJIANG UNIV OF TECH
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