A kind of lcd-pct gene tandem recombination expression vector and its application

A technology for expressing vectors and genes, which is applied in the field of LCD-PCT gene tandem recombination expression vectors, can solve the problems that other substances cannot be used, achieve high production and application value, reduce production costs, and reduce environmental pollution.

Inactive Publication Date: 2015-10-28
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Therefore, the current problem is to construct a strain of genetically engineered bacteria, which contains an enzyme that can effectively catalyze the conversion of D-lactic acid into acrylic acid, and acrylic acid is the final metabolite and cannot be used to produce other substances

Method used

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  • A kind of lcd-pct gene tandem recombination expression vector and its application
  • A kind of lcd-pct gene tandem recombination expression vector and its application
  • A kind of lcd-pct gene tandem recombination expression vector and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Acquisition of LCD and PCT coding genes

[0047] Clostridium propionicum (purchased from American Standard Collection center, No. ATCC25522) Genomic DNA, and using Clostridium propionicum genomic DNA as a template for PCR amplification of LCD-PCT gene.

[0048] The amplification primers are:

[0049] Upstream primer: 5'-GAATGAGTTTAACACAAGGCATGAAAGCAAAA-3';

[0050] Downstream primer: 5'-TCAGGACTTCATTTTCCTTCAGACCCATTAAG-3'.

[0051] The PCR product was connected to the pESAY-E1 vector, and the connection condition was 25°C for 20 minutes.

[0052] The connection product was transformed into competent cells of host bacteria E.coli Top10 (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.), and coated with LB plates (Amp + ), select multiple clones, and culture them in shake flasks for 16 hours. Cells were harvested, and a small amount of plasmid was extracted for sequencing verification. The positive clones with the correct connection directi...

Embodiment 2

[0053] Implementation Example 2: Expression of LCD and PCT and Preparation of Catalytic Active Liquid

[0054] The tandem vector pESAY-E1-lcd-pct was transformed into E.coli BL21 competent cells, and positive clones (namely LCD-PCT recombinant genetically engineered bacteria) were picked and cultured.

[0055] The culture conditions are shaker culture in a liquid medium containing peptone 10g / L, yeast extract 5g / L, and sodium chloride 10g / L for 6-12 hours, the temperature is 37°C, the speed is 200rmp / min, and the culture container is sterile infusion bottle, the liquid volume is 50mL, and the mouth of the bottle is covered with sterile gauze.

[0056] After the cultivation, IPTG with a final concentration of 0.3mmol / L was added, the bottle mouth was replaced with a butyl rubber stopper to block oxygen, and the two enzymes were induced to express for 5 hours under anaerobic conditions.

[0057] At the end of the induction, add filtered and sterilized vitamin C (VC) aqueous sol...

experiment example 3

[0058] Experimental example 3: Determination of PCT and LCD enzyme activity

[0059] (1) Determination of PCT enzyme activity by spectrophotometry (refer to K K Tung, W A Wood. Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. Journal of Bacteriology. 1975, 124(3): 1462.) .

[0060] The reaction system is as follows: at pH 7.0, 2umol sodium acrylate, 5nmol acetyl-CoA, 10umol potassium phosphate, 1 unit of crotonase, 0.1ml of cell disruption solution was added, reacted at 25°C for 20min, and measured the absorbance at 245nm. A standard curve of the absorbance of acrylic acid is made, and 1 unit of PCT enzyme activity is defined as the amount of enzyme that converts 1 μmol of acrylic acid to lactic acid per minute under the above reaction conditions.

[0061] (2) Determination of LCD enzyme activity by spectrophotometry (refer to Antje E, Wolfgang B. (R)-Lactyl-CoA dehydratase from Clostridium propionicum. Eur. J. Biochem. 1992,...

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Abstract

The invention relates to an LCD (Lactoyl Coenzyme A Dehydratase)-PCT (Propionic Acid Coenzyme A Transferase) gene serial recombinant expression vector and an application thereof. The vector is established by connecting a PCT gene for encoding a PCT and an LCD gene for encoding an LCD in series, wherein the PCT gene and the LCD gene are respectively cloned from a clostridium propionicum genome; the vector is converted to an escherichia coli competent cell, and the expression of the PCT and the LCD is realized in the escherichia coli competent cell, so that an LCD-PCT recombinant genetically engineered bacterium is obtained; and an LCD-PCT catalytic active liquid obtained after the engineered bacterium is cultured and broken contains the PCT and the LCD which can be used for effectively catalyzing D-lactate to be converted into acrylic acid with the yield of about 41%, so that the efficient biological conversion of the acrylic acid can be realized, the production cost can be reduced, the environment pollution can be relieved, and the vector has very high production and application values.

Description

technical field [0001] The invention belongs to the technical field of gene recombination, and relates to an LCD-PCT gene tandem recombination expression vector and application thereof. Background technique [0002] Acrylic acid is an important chemical raw material, and its molecule has a special double bond structure and acidic functional groups. Because the polymers produced by acrylic acid and its derivatives are colorless, transparent, viscous, elastic, stable to light, and not easy to weather, they are widely used in coatings, textiles, adhesives, pulp treatment, glazing agents, leather, etc. , fibers, detergents and superabsorbent materials and other fields. [0003] The production method of acrylic acid has developed from the initial Reppe method and acrylonitrile hydrolysis method using acetylene and carbon monoxide as raw materials to the currently mainly used propylene oxidation method. There are two kinds of propylene oxidation methods. The one-step method is t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/64C12N1/21C12P7/40C12R1/19
Inventor 王凤寰孟青青杨慧敏王玉海
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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