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Method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase

A technology of linoleic acid isomerase and conjugated linoleic acid, which is applied in the field of bioengineering, can solve the problems of expression product retention, low expression efficiency, and inability to achieve secreted expression, so as to reduce production costs and improve transformation efficiency Effect

Inactive Publication Date: 2013-11-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the currently used linoleic acid isomerase mainly has the following problems: the recombinant expression of the enzyme is generally intracellular expression and secreted expression, and the enzyme cannot be in contact with the extracellular substrate during intracellular expression, which greatly affects the catalysis of the enzyme efficiency, and the accumulation of enzyme molecules in the cell forms feedback inhibition, which also affects the expression efficiency; the main disadvantage of secreted expression is that the expression efficiency is not high, and it cannot achieve complete secreted expression. Inability to achieve complete contact of the enzyme with the extracellular substrate
Both modes of expression lead to inefficient catalytic activity of the enzyme

Method used

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  • Method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase
  • Method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase
  • Method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of Yeast Display Recombinant Plasmid

[0021] Lactobacillus plantarum (Lactobacillus plantarum) lp15-2-1 and Saccharomyces cerevisiae K601 genomic DNA were respectively used as templates, and the primers in Table 1 were used for PCR amplification. The PCR reaction system was: 10×PCR buffer (containing Mg 2+ ) 5 μL, dNTPs (25mM) 4 μL, primer 1 and primer 2 (10 μM) 1 μL each, genomic DNA 1 μg, ExTaq DNA polymerase 1U, add sterile ultrapure water to a final volume of 50 μL; PCR reaction conditions: 94 ° C pre-denaturation for 5 min Denaturation at 94°C for 30s, annealing at 48°C for 30s (gene mfα1) / annealing at 50°C for 40s (linoleic acid isomerase gene lai and yeast α-lectin C-terminal anchor region gene sag containing 321aa), extension at 72°C for 1min , reacted for 30 cycles; extended at 72°C for 5 min. The signal peptide gene mfα1 (SEQ ID NO: 3), the linoleic acid isomerase gene lai (SEQ ID NO: 1) and the anchor region gene sag (SEQ ID NO: 2). ...

Embodiment 2

[0025] Example 2 Obtaining and Screening of Recombinant Yeast

[0026] SD (uracil-deficient) solid screening medium (g / L): SD+2% agar powder

[0027] SD medium composition (g / L): 16.8g yeast nitrogen source; 0.77g uracil-deleted amino acid; 2g galactose.

[0028] There is URA gene on the vector pYES2, and the recombinant yeast can grow on the medium lacking uracil, so the SD solid screening medium is selected to screen the transformants.

[0029] The above-mentioned recombinant plasmid was transformed into Saccharomyces cerevisiae K601 by electric shock method, and the electric shock cup was transferred to the electric shock seat with Bio-Rad's gene electric shock transformation instrument (BTX), and the electric shock transformation was carried out. The conditions were: the voltage was 1.5KV; 25μF; resistance 200Ω; electric shock time 5s; then cultured upside down on SD solid screening medium, 30°C, cultured for 48 hours, and screened to obtain transformants; finally, the ob...

Embodiment 3

[0030] Example 3 Determination of Recombinant Yeast Enzyme Activity and Transformation Product Analysis

[0031] YPG induction medium composition (g / L): 10g peptone; 5g yeast extract; 10g galactose.

[0032] The recombinant yeast obtained above was inoculated into YPG induction medium, and after cultivating at 30°C for 48h, centrifuged (3000r / min, 10min,) to collect the bacterial cells, the collected bacterial cells were washed with physiological saline (0.9% v / v ) after washing twice, add to the phosphate buffer solution (0.1mol / l, pH7.0) containing 3mg / ml linoleic acid; 30 ℃, under the condition of 180rpm, carry out conversion reaction, measure linoleic acid by sampling every 6 hours The enzymatic activity of isomerase was compared with the transformation product of yeast cells carrying the empty vector pYES2, and the results were as follows: image 3 As shown, it can be seen from the figure that with the increase of culture time, the enzyme activity of the recombinant yeas...

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Abstract

The invention discloses a method for catalytically synthesizing conjugated linoleic acid by saccharomyces cerevisiae display linoleic acid isomerase, which comprises the following steps of: inoculating saccharomyces cerevisiae K601 containing recombinant plasmid into a yeast peptone glucose (YPG) culture medium, inducing and culturing, collecting thalli, and washing to obtain the saccharomyces cerevisiae display linoleic acid isomerase; adding the obtained saccharomyces cerevisiae display linoleic acid isomerase into a buffer solution containing linoleic acid, and performing oscillating reaction. The linoleic acid isomerase is displayed and expressed on the outer surface of the cell of the saccharomyces cerevisiae, and is directly contacted with the linoleic acid substrate, so that the conversion efficiency of the conjugated linoleic acid is improved and the production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing conjugated linoleic acid catalyzed by Saccharomyces cerevisiae displaying linoleic acid isomerase. Background technique [0002] Conjugated linoleic acid (hereinafter referred to as CLA) is octadecadienoic acid containing cis or trans conjugated double bonds, and is derived from the essential fatty acid linoleic acid (hereinafter referred to as LA). A general term for a group of positional and conformational isomers. Many microorganisms can use their own linoleic acid isomerase to convert linoleic acid 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 secreted expression, and the enzyme cannot be in contact with the extracellular substrate during intracellular expression, which greatly affects the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/64C12N9/90C12N15/63C12R1/865
Inventor 何国庆刘佩阮晖周倩
Owner ZHEJIANG UNIV
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