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Lactobacillus plantarum display linoleic acid isomerase, and preparation method and application thereof

The technology of linoleic acid isomerase and Lactobacillus plantarum is applied in the field of bioengineering, which can solve the problems of low expression efficiency, retention and low catalytic efficiency, and achieves the effect of reducing production cost and improving yield.

Inactive Publication Date: 2011-10-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During intracellular expression, the enzyme cannot contact the extracellular substrate, which greatly affects the catalytic efficiency of the enzyme, and the accumulation of enzyme molecules in the cell forms feedback inhibition, which also affects the expression efficiency
The main disadvantage of secretory expression is that the expression efficiency is not high, and complete secretory expression cannot be achieved, and a considerable part of the expression product is always retained in the cell, so that the complete contact between the enzyme and the extracellular substrate cannot be achieved
Both modes of expression lead to inefficient catalytic activity of the enzyme

Method used

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  • Lactobacillus plantarum display linoleic acid isomerase, and preparation method and application thereof
  • Lactobacillus plantarum display linoleic acid isomerase, and preparation method and application thereof
  • Lactobacillus plantarum display linoleic acid isomerase, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Fusion of linoleic acid isomerase gene, signal peptide gene and anchor gene

[0023] Use the primers in Table 1 to amplify the lai gene (Genbank No.: HQ831447) from Lactobacillus plantarum lp15-2-1, amplify the signal peptide gene usp45 (Genbank No.: LACUSP45) from Lactococcus lactis (Lactococcus lactis) and The M6 ​​anchor protein gene m6 (Genbank No: STRM6) was amplified from Streptococcus pyogenes; the PCR product of the gene sequence was connected to the pMD18-T simple (pMDS) vector, and the cloning vector pMDS- lai, pMDS-usp45 and pMDS-m6, and carry out PCR and enzyme digestion identification on the cloning vector, the reaction system and reaction procedure of PCR are as follows:

[0024] PCR reaction system (50μL):

[0025]

[0026] The reaction program was: 94°C for 5min; 94°C for 30s, 48°C for 30s (primers Usp45-5' and Usp45-3') / 50°C for 30s (primers M6-5' and M6-3') / 50°C for 40s (primers LAI -5' and LAI-3'), 72°C for 30s, 30 cycles; 72°C for 5min...

Embodiment 2

[0031] Example 2 Construction of Lactic Acid Bacteria Display Expression Vector

[0032] Lactococcus lactis universal expression plasmid pMG36e is a classic artificially constructed constitutive expression vector, which is constructed based on the transcription and translation signals of a Lactococcus lactis subsp. cremoris protease gene. It contains a strong promoter capable of expressing foreign proteins in a variety of bacteria. In this example, this expression vector was used. First, the plasmid pMDS-usp45 and the empty vector pMG36e were digested with Cla I and Sal I respectively. After electrophoresis detection, the DNA fragment usp45 and the vector pMG36e were recovered and ligated, and the ligated product was transformed into Escherichia coli DH5α Competent cells were screened on LB medium containing erythromycin to obtain the plasmid pMG36e-usp45 (pMU); then the plasmids pMDS-lai and pMU were subjected to Sal I and Pst I double enzyme digestion, respectively, after el...

Embodiment 3

[0038] Example 3 Display and expression of linoleic acid isomerase on the cell surface of Lactobacillus plantarum

[0039]The lactic acid bacteria display expression vector pMG36e-usp45-lai-m6 was transformed into Lactobacillus plantarum CGMCC NO.3782 cells by electroporation method, and the transformants were screened on the MRS plate containing erythromycin, and further obtained by using primers M6-5' and M6 -3' for PCR verification, the PCR reaction system and reaction procedures are as follows:

[0040] PCR reaction system (50μL):

[0041]

[0042] The reaction program was: 94°C for 5min; 94°C for 30s, 48°C for 30s (primers Usp45-5' and Usp45-3') / 50°C for 30s (primers M6-5' and M6-3') / 50°C for 40s (primers LAI -5' and LAI-3'), 72°C for 30s, 30 cycles; 72°C for 5min.

[0043] The result is as Figure 4 As shown, compared with the control negative bacterial strain, the Lactobacillus plantarum engineered strains can all amplify a band of about 500bp, indicating that the...

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Abstract

The invention discloses a Lactobacillus plantarum display linoleic acid isomerase, and a preparation method and application thereof. The method comprises the following steps: transforming a recombinant plasmid composed of Usp45 signal peptide gene, linoleic acid isomerase gene, M6 ankyrin gene and vector pMG36e into Lactobacillus plantarum CGMCC No.3782, inoculating in a culture medium containing erythromycin MRS (Methicillin-resistant Staphylococcus), culturing for 5-12 hours, centrifugalizing to collect Lactobacillus plantarum, flushing to obtain Lactobacillus plantarum display linoleic acid isomerase, adding the collected Lactobacillus plantarum display linoleic acid isomerase into a buffer solution containing linoleic acid, carrying out shake culture, and catalyzing to synthesize conjugated linoleic acid. In the invention, the Lactobacillus plantarum cell is subjected to gene engineering modification, so that the linoleic acid isomerase is expressed and secreted to the outside of the cell; and meanwhile, the M6 ankyrin cell is utilized to fix the linoleic acid isomerase to the cell surface, and the linoleic acid isomerase is utilized to carry out catalytic conversion to obtain the conjugated linoleic acid, thereby increasing the yield of the conjugated linoleic acid.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a plant lactobacillus displaying linoleic acid isomerase and a preparation method and application thereof. Background technique [0002] Conjugated Linoleic Acid (CLA) has important physiological functions, such as anti-cancer (colon cancer, gastric cancer, breast cancer, prostate cancer), improving cellular immunity, reducing body fat content, preventing the occurrence of diabetes and inhibiting Atherosclerosis, etc., so CLA has attracted widespread attention. Many microorganisms can use their own linoleic acid isomerase to convert linoleic acid (LA) into conjugated linoleic acid. However, the currently used linoleic acid isomerase mainly has the following problems: the recombinant expression of the enzyme is generally intracellular expression and secretory expression. During intracellular expression, the enzyme cannot contact the extracellular substrate, which greatly ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/61C12N15/11C12N9/90C12P7/64C12R1/25
Inventor 何国庆刘佩阮晖周倩
Owner ZHEJIANG UNIV
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