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Gene engineering bacterium for generating PVA dehydrogenase (Polyvinyl Alcohol Dehydrogenase) as well as construction method and application thereof

A construction method and dehydrogenase technology are applied in the field of genetic engineering bacteria and construction of high-yield PVA dehydrogenase, and can solve the problems of few PVA dehydrogenase genetically engineered bacteria, inability to act on secondary and tertiary alcohols, low similarity, and the like, To achieve the effect of easy industrial application, simple extraction process and high yield

Active Publication Date: 2011-06-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein encoding the PVA dehydrogenase gene has 639 amino acid residues, a heme binding site and a PQQ binding site were found, and the amino acid sequence showed low similarity with other quinone protein dehydrogenases (Shimao M et al. , Cloning and characterization of the gene encoding pyrroloquinoline quinonone- dependent poly (vinyl alcohol) dehydrogenase of Pseudomonas sp . st rain VM15C); Mamoto cloned the The full gene of PVA dehydrogenase is 1965bp in length and 655 amino acids in total. The recombinase has 51% homology with the PVA dehydrogenase produced by Pseu domonas sp1 VM15C. The enzyme can degrade In addition to PVA, it can also degrade diols, such as polypropylene alcohol, 1.3-butanol, 2.4-pentanediol, etc., but the enzyme cannot act on secondary and tertiary alcohols
[0006] At present, foreign studies on PVA dehydrogenase mainly focus on its gene report and degradation mechanism, and there are few research reports on PVA dehydrogenase genetically engineered bacteria. There are fewer reports about high-yield PVA dehydrogenase genetically engineered bacteria

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the synthesis of PVA dehydrogenase gene

[0039] PVA dehydrogenase ( PVADH ) genes derived from Sphingopyxis.sp .113P contains PVA dehydrogenase ( PVADH ) gene operon (GenBank No. AB190288), of which the 3177th to 5141st are coding PVADH gene. After cutting off the signal peptide of the gene itself, modifying it with codon bias, etc., adding EcoR I and not I restriction site, newly designed PVADH The nucleotide sequence of the gene is shown in SEQ ID NO. 1, which was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 2

[0040] Example 2: Recombinant plasmid pPIC9K- PVADH build

[0041] with PVADH Gene vector and expression vector pPIC9K were carried out separately not I and EcoR I double enzyme digestion, after the kit is recovered, use T4 ligase for ligation. The ligation reaction system is (10 μL): target gene fragment 2 μL, carrier DNA 2 μL, 10×T4 ligase Buffer 1 μL, T4 DNA ligase 1 μL, ddH 2 O 4 μL.

[0042] The ligation product was transformed into competent Escherichia coli JM109 for transformation. The conversion method is as follows:

[0043] (1) Under sterile conditions, take 200 μL of competent cells and place them in a sterile microcentrifuge tube;

[0044] (2) Add 1-2 μL of recombinant plasmid to each tube, swirl gently to mix the contents, and place on ice for 30 min;

[0045] (3) Heat shock at 42°C for 90 s (accurate), do not shake the centrifuge tube;

[0046] (4) Quickly transfer the centrifuge tube to an ice bath to cool the cells for 1-2 min;

[0047] (5) Add...

Embodiment 3

[0050] Embodiment 3: the construction of PVA dehydrogenase genetically engineered bacteria

[0051] The expression vector pPIC9K- PVADH use Sal I Restriction linearization. Enzyme digestion system (50 μL system): recombinant plasmid 10 μL, Buffer 5 μL, Sal I 3 μL, ddH 2 O 32 μL. Water bath at 37 °C for 3 h, purify and recover the linearized product with a PCR product purification kit, and transform Pichia pastoris GS115 by electric shock method, the specific method is as follows:

[0052] (1) Inoculate a loop of activated Pichia pastoris GS115 in 25 mL YPD liquid medium, and culture overnight at 28°C-30°C with shaking;

[0053] (2) Transfer the above culture solution into 100 mL YPD (500 mL Erlenmeyer flask) liquid medium, culture with shaking at 28 ℃-30 ℃, measure every 1 h, and cultivate until the cell concentration OD 600 1.3-1.5;

[0054] (3) Cool in ice water for more than 10 minutes;

[0055] (4) Collect the cells by centrifugation at 4°C at 8000 r / min, suspe...

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Abstract

The invention discloses a gene engineering bacterium for generating PVA dehydrogenase (Polyvinyl Alcohol Dehydrogenase, EC1.1.99.23, PVADH for short) as well as a construction method and application thereof, belonging to the field of genetic engineering. In the invention, optimized PVA dehydrogenase gene is connected to pichia pastoris GS115 (Pichiapastoris) chromosome, so that the gene engineering bacterium for expressing the PVA dehydrogenase by high-efficiency secretion is constructed, therefore, the problem of low yield of the PVA dehydrogenase is solved. Due to the application of the PVA dehydrogenase generated by the strain, the yield is up to 110U / mL, the process is simple, and convenient industrial application is realized.

Description

technical field [0001] The present invention relates to a genetically engineered bacterium producing PVA dehydrogenase (polyvinyl alcohol dehydrogenase, EC1.1.99.23, referred to as PVADH) and its construction method, in particular to a genetically engineered bacterium producing high PVA dehydrogenase and its construction method and The invention belongs to the field of genetic engineering. Background technique [0002] PVA (full name polyvinyl alcohol), that is, polyvinyl alcohol, is a synthetic water-soluble polymer compound with the following excellent physical and chemical properties: Good solubility: PVA is soluble in water, the higher the water temperature, the greater the solubility, but almost Insoluble in organic solvents; strong film-forming properties: PVA is easy to form films, the mechanical properties of the film are excellent, and the tensile strength of the film increases with the degree of polymerization and alcoholysis; good adhesion: PVA and hydrophilic Ce...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N9/04C02F3/00
Inventor 陈坚堵国成贾东旭张东旭
Owner JIANGNAN UNIV
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