Polyphosphokinase mutants

A polyphosphate kinase and mutant technology, applied in the field of enzyme engineering, can solve the problem that the enzyme activity cannot reach the industrialized large-scale production of glutathione

Pending Publication Date: 2021-08-17
洛阳华荣生物技术有限公司
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  • Abstract
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Problems solved by technology

[0004] The inventors have conducted a lot of research on the use of polyphosphate kinases derived from various microorganisms for the ATP cycle regeneration system. Polyphosphate kinase (PPK2) and its mutants can be used as ATP regeneration agents to promote glutathione synthetase catalyzed production of glutathione, but the enzyme activity of the mutants is still not up to industrialized large-scale production of glutathione Glutathione Requirements

Method used

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  • Polyphosphokinase mutants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of recombinant Escherichia coli expressing wild-type polyphosphate kinase

[0040] For the amino acid sequence SEQ ID NO:1 of the polyphosphate kinase (GenBank accession number: AB092983.1) derived from Acinetobacter johnsonii, codon optimization was performed, and the optimized gene sequence was SEQ ID NO:2. The nucleotide sequence of the whole gene is synthesized as SEQ ID NO: 2, and the restriction endonuclease sites XhoI and NdeI are designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET24a-ajPPK2 ,like figure 1 shown.

[0041] The recombinant plasmid pET24a-ajPPK2 was electrotransformed into host Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli PET24a-ajPPK2 / BL21(DE3) expressing wild-type polyphosphate kinase.

Embodiment 2

[0042] Example 2: Induced expression and purification of polyphosphate kinase

[0043] For the Escherichia coli ajPPK2 constructed in Example 1, pick a single colony on its LB culture plate, inoculate it into a test tube containing 4 ml of 50 μg / mL kanamycin sulfate LB medium, and cultivate it at 37°C for 15 hours to become the first grade For seed solution, inoculate the primary seed solution into a Erlenmeyer flask containing 100ml of TB medium according to the inoculation amount of 1v / v%. When the OD600 is 0.6-0.8, add 0.1mM IPTG and cultivate overnight at 28°C and 200rpm. Then at 4°C, 10000rpm, centrifuge for 10min, collect the bacterial cells respectively, and freeze overnight at -20°C.

[0044] The collected cells were resuspended with 10ml of 0.1M Tris-HCl buffer (pH8.0), and ultrasonically disrupted (200W, super 1.5s, stop 3.5s, 10min in total). Centrifuge at 10,000 rpm for 30 minutes at 4°C to obtain crude enzyme solution of PPK2.

[0045] The crude enzyme liquid wa...

Embodiment 3

[0047] Embodiment 3: the investigation of ATP regeneration catalyzed by crude enzyme

[0048] In order to investigate the practicability of using low-cost crude enzyme solution to catalyze ATP regeneration, the crude enzyme PKK2 in Example 2 was investigated.

[0049] Enzyme reaction system and reaction conditions: 0.1M Tris-HCl buffer (PH8.0), 20mM MgCl2, 2.5mM AMP, 2.5mM sodium hexametaphosphate, after the dissolution is complete, use sodium hydroxide to correct pH8.0, constant volume to 8ml. Then add 2ml of the PKK2 crude enzyme in Example 2, react at 30°C for 2h, and quantitatively analyze AMP, ADP and ATP by HPLC, wherein AMP1mM, ADP1M, ATP0.5mM, that is, the ATP generation rate is 20% .

[0050] The detection results prove that polyphosphate kinase can use sodium hexametaphosphate as a substrate to catalyze the transfer of high-energy phosphate bonds to AMP to generate ADP and further generate ATP.

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Abstract

The amino acid sequence of the polyphosphate kinase mutant is SEQ ID NO: 3, the enzyme activity of the mutant is improved by at least three times compared with that of wild enzyme, and the polyphosphate kinase mutant can be used for improving the ATP regeneration capacity of microorganisms.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a polyphosphate kinase mutant and its use in improving the ability of microorganisms to regenerate ATP. Background technique [0002] Adenosine triphosphate (ATP) is the most important high-energy phosphate compound in the body. It can be used clinically as a coenzyme drug for the treatment of cerebrovascular diseases, progressive muscular atrophy, brain injury, cardiac insufficiency, nephritis, myocarditis and hepatitis. In the process of biocatalysis, ATP, as the energy currency in biosynthesis, participates in the catalysis of multi-enzymes in the cell, and ATP acts as an energy donor to promote the smooth progress of the reaction, so it is commonly used in industry to participate in many biocatalytic reactions for high-value compounds Synthesis. However, the high price of ATP limits its application in industry. [0003] Developing a reaction system th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12P21/02C12R1/19
CPCC12N9/1229C12N15/70C12P21/02C12Y207/04001Y02P20/584
Inventor 范文超高书良丁鹏
Owner 洛阳华荣生物技术有限公司
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