Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Propionibacterium acnes linoleate isomerase and application thereof

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 problems such as unfavorable conversion production, high price, etc., achieve good economic benefits, save costs, and improve enzyme live effect

Active Publication Date: 2015-04-22
NANJING FORESTRY UNIV
View PDF0 Cites 6 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Propionibacterium acnes linoleate isomerase and application thereof

Examples

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 Premix ExTaq, 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 ...

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 5 mL LLB liquid medium and culture to OD at 37°C and 180 rpm 600 At 0.6~0.8, add IPTG with a final concentration of 0.5 mmol / L, induce culture overnight at 25°C and 120 rpm. Centrifuge the induced bacterial solution at 4°C, 8000 rpm for 5 min, 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 place in an ice bath The bacterial cells were disrupted by medium ultrasonic wave, 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+ ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Propionibacterium acnes linoleate isomerase and its application. An amino acid sequence is as shown in SEQ ID NO.1. Through concerted catalysis of compound enzyme, conjugated linoleic acid can be directly prepared from plant oil containing high content of linoleic acid, thus overcoming the defect that conjugated linoleic acid must be produced by using expensive free linoleic acid as a substrate and saving costs greatly. Compound enzyme is prepared by the utilization of recombinant bacteria, thus raising enzyme activity and increasing enzyme output. There is no need to purchase commercial enzyme, and cost is saved effectively. The isomerization product conjugated linoleic acid contains single component and has a plurality of physiological activities. The scheme has good practicality. Good economic benefit can be produced. The propionibacterium acnes linoleate isomerase has a good industrial prospect.

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 series of isomers of octadecadienoic acid (octadecadienoic acid) containing conjugated double bonds derived from the essential fatty acid Linoleic Acid (LA). general term. 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 ruminants. Compared with...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com