Preparation method of fusion enzyme and fusion enzyme

A technology of fusion enzyme and crude enzyme liquid, applied in the field of fusion enzymes, can solve the problems of lack of fusion enzyme preparation and purification scheme, low reaction catalytic efficiency, low catalytic activity, etc., and achieves good stability, improved enzyme catalytic activity, and efficient catalysis. effect of reaction

Pending Publication Date: 2022-05-13
HEBEI UNIV OF TECH
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Problems solved by technology

However, in the existing technical practice, there is a lack of feasible and effective preparation and purification schemes for fusion enzymes; moreover, the catalytic activity of the enzymes obtained by the existing preparation methods is not high, and the reaction catalytic efficiency in the preparation process is relatively low. Low

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  • Preparation method of fusion enzyme and fusion enzyme
  • Preparation method of fusion enzyme and fusion enzyme
  • Preparation method of fusion enzyme and fusion enzyme

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preparation example Construction

[0051] This embodiment relates to a preparation method of a fusion enzyme, and an exemplary preparation schematic diagram thereof is as follows figure 1 Shown; This preparation method mainly comprises following three steps:

[0052] 1. Construct fusion enzyme plasmid;

[0053] 2. Extract the fusion enzyme plasmid and transfer it into Pichia pastoris for fermentation and expression;

[0054] 3. Utilize Pichia pastoris to prepare crude enzyme solution, and carry out separation and purification of fusion enzyme.

[0055] The main body of the fusion enzyme is UPO mutants JaWa, SoLo, WamPa ​​and the C-terminal 6×His-SUMO tag. The specific affinity between histidine and metal ions makes the separation and purification process of the enzyme simple and efficient.

[0056] Based on the overall setting principle of the preparation and purification method of the fusion enzyme mentioned above in this embodiment, the following specific steps can be referred to for the preparation.

[00...

Embodiment 1

[0120] This example relates to the preparation of 6×His-SUMO-JaWa, 6×His-SUMO-SoLo, 6×His-SUMO-WamPa ​​fusion enzyme plasmids. The principle of plasmid extraction from Escherichia coli, and subsequent linearization and purification is as follows: figure 1 shown.

[0121] (1) Construct mutants JaWa, SoLo, and WamPa ​​of non-specific peroxidase (AaeUPO) from Agrocybe aegerita, and fuse 6×His-SUMO gene at their C-terminus. Construct the fusion enzyme gene sequence, and then use restriction endonucleases BamHI and NotⅠ to connect the fusion enzyme gene sequence to plasmid pPIC9K to construct recombinant plasmids pPIC9k-6×His-SUMO-JaWa, pPIC9k-6×His-SUMO-SoLo , pPIC9k-6 x His-SUMO-WamPa.

[0122] The schematic diagram of the constructed plasmid is shown in figure 2 shown. The following table 1 lists the protein sequence of the fusion enzyme

[0123] Table 1. Protein sequences of fusion enzymes

[0124]

[0125] (2) Transfer the above constructed plasmid into the competent...

Embodiment 2

[0130] This example involves the electroporation of fusion plasmids into competent Pichia cells and obtaining positive bacteria. The colony picture of positive bacteria on the MD plate is as follows Figure 4 shown. The plasmid extraction map and PCR verification electrophoresis map of positive bacteria are as follows: Figure 5 In a, b shown.

[0131] (1) Firstly, Pichia pastoris was activated with YPD medium, and the culture medium was inoculated from Glycerol bacteria at an inoculum size of 1%.

[0132] (2) Continue to inoculate into two new YPDs with 1% inoculum size, expand and cultivate Pichia pastoris, and wait until OD 600 To 1.2, the bacteria solution was centrifuged. The centrifugation conditions were 5000 rpm, 5 min, 4°C. After centrifugation, the supernatant was discarded, leaving the pellet. Use 10-15 mL of pre-cooled sterile water to mix the precipitate by pipetting, continue to centrifuge, and repeat the operation 2-3 times. Finally, it was resuspended wi...

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Abstract

The invention provides a preparation method of a fusion enzyme and the fusion enzyme, and the preparation method of the fusion enzyme comprises the following steps: firstly constructing a fusion enzyme plasmid, then extracting the fusion enzyme plasmid, and transferring the fusion enzyme plasmid into pichia pastoris for fermentation expression; and preparing a crude enzyme solution by using pichia pastoris, and separating and purifying the fusion enzyme. A 6 * His-SUMO tag is fused at the C ends of mutants JaWa, SoLo and WamPa of non-specific peroxidase (UPO), the fusion enzyme is subjected to induced expression in pichia pastoris and later separation and purification of the fusion enzyme, the JaWa can remove peroxidation activity of the enzyme, the WamPa has good stability in an organic solvent, and the SUMO tag can effectively improve the expression quantity of the enzyme, so that the reaction is efficiently catalyzed. In addition, after JaWa, SoLo and WamPa are fused with a 6 * His-SUMO label, separation and purification can be carried out by using the chelation of histidine and metal ions Ni < 2 + >, and the catalytic activity of the separated and purified enzyme is greatly improved.

Description

technical field [0001] The invention relates to the technical field of fusion enzyme construction and fermentation, in particular to a preparation method of fusion enzyme. Meanwhile, the present invention also relates to a fusion enzyme. Background technique [0002] The selective monooxygenation reaction of inert carbon-hydrogen bonds is one of the main challenges in organic synthesis. At present, chemical and enzymatic methods are mainly used for catalysis. Chemical catalysis often has harsh reaction conditions, while enzymatic catalysis is relatively mild. Heme-dependent oxygenase is one of the commonly used selective oxygen functionalization catalysts, among which unspecific peroxygenase (UPO) has become an efficient catalyst for single oxygenation reaction due to its simple electron transfer process and high economic benefit. catalyst. UPO has a wide range of sources, among which non-specific peroxidase (AaeUPO) from Agrocybe aegerita is a glycosylated (20%-40%) extra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N9/08C07K19/00C12R1/84
CPCC12N15/815C12N9/0065C12Y111/02001C07K2319/95C07K2319/21
Inventor 高静李彦洁姜艳军周丽亚
Owner HEBEI UNIV OF TECH
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