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Low-temperature-resistant phospholipase D derived from Antarctic bacteria and preparation method and application thereof

A technology of Antarctic bacteria and phospholipase, which is applied in the field of enzyme genetic engineering, can solve the problems of limiting the large-scale preparation of phospholipase D, low enzyme activity, and high price, and achieve good enzyme activity, storage stability, and high expression. Effect

Active Publication Date: 2021-06-04
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Internationally commercialized phospholipase D is mainly monopolized by Amano Corporation of Japan, and its source is mostly Streptomyces, which is rare and expensive
Domestic research on phospholipase D has just started, and commercial PLD has not yet been developed.
Second, the expression level and enzyme activity of the reported phospholipase D are generally low
Natural phospholipase D is difficult to extract and purify, and the yield obtained is low, the production cost is high, and it is difficult to industrialize production
However, phospholipase D heterologously expressed by genetically engineered bacteria mainly exists in the form of inclusion bodies and has low enzyme activity, which limits the large-scale preparation of phospholipase D.
The third is that most of the reported phospholipase D enzyme activity will be inhibited at low temperature, and the storage period is short, which seriously affects the application and preservation of the enzyme

Method used

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  • Low-temperature-resistant phospholipase D derived from Antarctic bacteria and preparation method and application thereof
  • Low-temperature-resistant phospholipase D derived from Antarctic bacteria and preparation method and application thereof
  • Low-temperature-resistant phospholipase D derived from Antarctic bacteria and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Recombinant expression and purification of MsPLD

[0042] Transform the constructed MsPLD recombinant expression plasmid into Escherichia coli SHuffle T7 competent cells by heat shock, smear the plate to obtain the recombinant, pick a single colony to culture and extract the plasmid, and send it to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing verification If the sequencing is correct, the MsPLD recombinant expression strain will be obtained. Inoculate the recombinant strain in LB (containing 100 μg / mL Amp) liquid medium, cultivate overnight at 37°C, expand to 100mL LB medium according to the inoculum size of 5%, when the culture solution OD 600nmWhen = 0.8, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2mM, induce at 16-20°C for 20h, centrifuge at 10000rpm for 5min, and collect the bacteria. Cells were obtained and resuspended in buffer A (50 mM Tris-HCl, 500 mM NaCl, pH 8.0) and sonicated. After crushing, the lysate was c...

Embodiment 2

[0044] Optimum Reaction Temperature and Temperature Stability of Phospholipase D

[0045] In order to explore the effect of different temperature conditions on the activity of MsPLD enzyme, at different temperatures (4°C, 10°C, 20°C, 30°C, 35°C, 40°C, 50°C, 60°C), the enzyme-linked colorimetric assay its enzyme activity. The reaction system (100 μL) contains 0.1MTris-HCl (pH8.0), 5mM soyPC, 15mM SDS, 15mM Tritonx-100 and 10μL purified enzyme solution, react at each temperature for 5min, heat to terminate the reaction, after the solution is cooled, Add a chromogenic solution containing 10U / mL choline oxidase, 1U / mL peroxidase, 5mM 4-aminoantiline and 7mM phenol, incubate at 30°C for 30min, and finally add 1% TrionX-100 to terminate the color reaction. Absorbance was measured at 490 nm. The experiment was repeated three times, and the experimental results were expressed by relative enzyme activity, and the maximum enzyme activity measured was set as 100%. The results showed t...

Embodiment 3

[0049] Optimum pH and pH Stability

[0050] In order to explore the effect of pH on the enzyme activity of MsPLD, the enzyme activity of MsPLD was determined by enzyme-linked colorimetry under different pH conditions (pH4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0). Refer to Example 2 for the enzyme activity assay reaction system and reaction conditions. Buffers of various pHs used were: 50 mM citrate (pH 4.0-5.0), 50 mM sodium phosphate (pH 6.0-7.0), 50 mM Tris-HCl (pH 8.0) and 50 mM glycine-NaOH (pH 9.0-10.0). The experiment was repeated three times, and the experimental results were expressed by relative enzyme activity, and the maximum enzyme activity measured was set as 100%. The results show that the optimum pH of MsPLD is 8.0 (see Figure 6 ).

[0051] In order to evaluate the pH stability of MsPLD, it was treated for 12 hours under the conditions of different pH values ​​(pH4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0), and the residual enzyme activity of MsPLD was determined. The enzy...

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Abstract

The invention discloses low-temperature-resistant phospholipase D derived from Antarctic bacteria and a preparation method and application thereof. The amino acid sequence of the low-temperature-resistant phospholipase D is shown as SEQ ID NO: 3; and the gene sequence of the low-temperature-resistant phospholipase D is shown as SEQ ID NO: 2. An escherichia coli recombinant expression strain of the low-temperature-resistant phospholipase D is obtained, by means of the strain, a large amount of soluble expression of recombinant protein can be achieved, and later purification and protein obtaining are easy. The expression product has good low-temperature storage stability and good enzyme activity, and meanwhile has certain tolerance to organic solvents and surfactants. The obtained phospholipase D can be suitable for phospholipid modification, phosphatidic acid and various natural rare phospholipid and non-natural phospholipid compounds are produced, and the phospholipase D is applied to the fields of biology, food, medicine, cosmetics, agriculture, industry and the like.

Description

technical field [0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to an optimized Antarctic bacteria-sourced low-temperature-resistant phospholipase D recombinant Escherichia coli strain and an enzyme protein preparation method; the invention also relates to the expressed low-temperature-resistant phospholipase D Applications. Background technique [0002] Phospholipase D (Phospholipase D, PLD, EC3.1.4.4) is a class of enzymes that hydrolyze the phosphodiester bonds in the phospholipid structure to generate phosphatidic acid (PA) and corresponding hydroxyl-containing compounds. PLD can function as a second messenger through its hydrolyzed product PA and participate in various physiological activities, such as cell proliferation, inflammation, virus infection, etc., and can also lead to neurodegenerative diseases, human cancer and plant stress response. In addition, phospholipase D can also catalyze the phosphatidyl t...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12P7/64C12R1/19
CPCC12N9/16C12N15/70C12Y301/04004C12P7/6481Y02A50/30
Inventor 王永华王方华刘思雨杨博蓝东明
Owner SOUTH CHINA UNIV OF TECH
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