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Propionibacterium acnes linoleic acid isomerase and its application

A technology of linoleic acid isomerase and Propionibacterium acnes, which is applied in the field of biological enzymatic preparation of conjugated linoleic acid, can solve the problems of high price, unfavorable transformation and production, and achieves good economic benefits, cost savings, and good industrialization Foreground effect

Active Publication Date: 2017-11-14
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, free linoleic acid rarely exists in nature, and most of it is obtained through chemical synthesis, so the price is relatively expensive and it is not conducive to expanding the conversion production

Method used

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  • Propionibacterium acnes linoleic acid isomerase and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Design and synthesis of PAI gene:

[0026] According to the codon usage frequency database information Codon Usage Database, the gene PAI encoding linoleic acid conjugating enzyme was optimized according to the preferred codons of Escherichia coli. Under the premise of not changing the amino acid sequence, the JCat codon optimization software was used to select the optimal codon, and at the same time, fine-tuning was performed with reference to the secondary structure of the mRNA to make the activation energy as high as possible, so that the mRNA was easier to express. The codon-optimized pai gene was synthesized by Shanghai Jierui Bioengineering Co., Ltd.

Embodiment 2

[0027] Embodiment 2: Cloning of recombinant PAI:

[0028] Primer: Upstream primer P1: 5'-cgccatatgtctatctccaaagacagccg-3' (with Nde I restriction site) downstream primer P2: 5'-ccg ctcgag aacgaagaagcgggtaacc-3' (incl. Xhol I restriction site).

[0029] PCR reaction system: 1 μL synthetic sequence, 1 μL upstream primer P1, 1 μL downstream primer P2, 25 μL PremixExTaq, 22 μL ddH 2 O.

[0030] PCR reaction conditions: Denaturation at 94°C for 10 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min, 30 cycles; extension at 72°C for 10 min; incubation at 4°C.

[0031] The yield and specificity of the PCR products were detected by 1% agarose gel electrophoresis, and purified with a PCR product recovery kit (BIOMIGA, Shanghai).

[0032] Purified PCR amplification products were Nde I. Xhol After I double enzyme digestion, it was connected with the same double enzyme digestion expression plasmid pET-20b, and transformed into E...

Embodiment 3

[0033] Example 3: Induced expression and purification of recombinant PAI:

[0034] The identified positive recombinant plasmid pET-20b-pai was transformed into Escherichia coli BL21 (DE3) competent cells, and positive clones were screened on LLB plates containing Amp antibiotics (100 μg / mL). Transfer the positive clones to fresh 5mL LLB liquid medium and cultivate to OD at 37°C and 180 rpm 600At 0.6~0.8, add IPTG with a final concentration of 0.5mmol / L, and induce culture overnight at 25°C and 120 rpm. Centrifuge the induced bacterial solution at 4°C and 8000 rpm for 5 minutes, wash the cells twice with 20 mmol / L Tris-HCl buffer (pH 8.0), and finally resuspend the bacterial cells with 6 mL of this buffer and store in an ice bath The cells were disrupted by ultrasonic waves, and centrifuged at 12000 rpm for 20 min at 4°C to obtain the supernatant crude enzyme solution. The supernatant crude enzyme solution was passed through the affinity Ni by the AKTA protein purifier 2+ Th...

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Abstract

Propionibacterium acnes linoleic acid isomerase and application thereof, the amino acid sequence is as described in SEQ ID NO.1. The present invention adopts complex enzyme synergistic catalysis, can directly prepare conjugated linoleic acid from vegetable oil containing higher linoleic acid, and overcomes that linoleic acid isomerase must use expensive free linoleic acid as a substrate to produce conjugated linoleic acid. The downside of oleic acid is that it saves a lot of money. Using recombinant bacteria to prepare compound enzymes can improve enzyme activity and increase enzyme production, without the need to purchase commercial enzymes, effectively saving costs. The isomerization product, conjugated linoleic acid, has a single component and has various physiological activities. The scheme has good practicability, can produce good economic benefits, and has good industrialization prospects.

Description

technical field [0001] The invention belongs to the technical field of preparing conjugated linoleic acid by a biological enzymatic method, and relates to a method and a reaction system for preparing conjugated linoleic acid through joint catalysis of lipase and linoleic acid isomerase. Background technique [0002] Conjugated Linoleic Acid (CLA) is a general term for a series of isomers of octadecadienoic acid (octadecadienoic acid) containing conjugated double bonds derived from the essential fatty acid linoleic acid (Linoleic Acid, LA) . Among them, cis-9, trans-11 CLA and trans-10, cis-12 CLA are considered as the main biologically active monomers, which have various physiological activities, such as anti-cancer, anti-atherosclerosis, lowering blood fat, weight loss, It can promote growth, etc., and has broad application prospects in industries such as medicine, food, health care products and cosmetics. [0003] Natural CLA is mainly found in the meat and milk of rumin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P7/64C12R1/19
CPCC12N9/90C12P7/6427C12Y502/01005
Inventor 李迅顾华祥王飞
Owner NANJING FORESTRY UNIV
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