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Alkaline pectinase PelN, as well as encoded gene and application thereof

A technology encoding gene and pectinase, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as increasing difficulty and increasing the hardness of plant cell walls.

Active Publication Date: 2013-07-24
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The presence of pectin substances can increase the firmness of plant cell walls, which is conducive to maintaining the specific shape and structure of plants, but increases the difficulty of industrial operations for deep processing of plants

Method used

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  • Alkaline pectinase PelN, as well as encoded gene and application thereof
  • Alkaline pectinase PelN, as well as encoded gene and application thereof
  • Alkaline pectinase PelN, as well as encoded gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the acquisition of protein PelN and its coding gene

[0051]Using the genomic DNA of Paenibacillus sp. SJN-PL0602 with the preservation number CGMCC NO.5696 in the General Microbiology Center of the China Microbiological Culture Collection Management Committee as a template, PCR amplification was performed with primers F and R, and the amplified The product was subjected to agarose gel electrophoresis, and a 1.4kb fragment was recovered and purified. Sequencing showed that the fragment was 1440bp in size, and its sequence was shown in Sequence Listing Sequence 2, wherein the 7th to 1434th positions of Sequence 2 coded the Sequence Listing sequence The protein shown in 1 is named as PelN, and its coding gene is named as PelN.

[0052] The sequences of the above primers F and R are as follows:

[0053] 5'- CCATGG ATGAAAAAGAACAGTACGAAGCT-3' (the underlined base is the NcoI enzyme recognition sequence);

[0054] 5'- CTCGAG TTAGTAGGAAGTCTTCAGCCAC-3' (the ...

Embodiment 2

[0055] Embodiment 2, the construction of recombinant expression vector and recombinant bacteria

[0056] 1. Digest the DNA fragment of sequence 2 in the sequence listing with NcoI and XhoI double enzymes, and connect it to the backbone fragment of the vector pET22b (purchased from Novagen, catalog number 69744-3) after NcoI and XhoI double enzyme digestion to obtain the recombinant The vector pET22b-PelN was confirmed by sequencing. The recombinant vector pET22b-PelN was inserted between the NcoI and XhoI sites of the vector pET22b by inserting the DNA fragment shown in the 7th to 1434th positions of Sequence 2 of the Sequence Listing.

[0057] 2. Transform the recombinant vector pET22b-PelN obtained in step 1 into Escherichia coli BL21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd., the product catalog number is CD601-01), and the obtained recombinant vector pET22b-PelN containing The recombinant bacterium was named BL21(DE3)PelN. The recombinant bacterium w...

Embodiment 3

[0059] Embodiment 3, the preparation of protein PelN

[0060] 1. Preparation of seed solution

[0061] Inoculate the recombinant strain BL21(DE3)PelN obtained in Example 2 into a 250mL Erlenmeyer flask filled with 25mL of seed medium, culture at 37°C, 200r / min, and shake for 6-12h to OD 600nm =6.0, to obtain seed solution;

[0062] The solvent of described seed culture medium is water, and solute and its final concentration in described seed culture medium are respectively: tryptone 12g / L, yeast extract 7g / L, sodium chloride 5g / L; The pH of the base is 7.2.

[0063] 2. Fermentation culture

[0064] Take the seed solution obtained in step 1 and inoculate it into a 250mL Erlenmeyer flask containing 20-30mL of fermentation medium at an inoculum size of 5%; first shake culture at 37°C until OD 600nm =0.6, then add IPTG (inducer, to induce the expression of the target protein PelN) to a final concentration of 200 μM, and continue to shake at 30°C for 40 hours to obtain a fermen...

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Abstract

The invention discloses alkaline pectinase PelN, as well as encoded gene and an application of the alkaline pectinase PelN. The PelN is protein formed by an amino acid sequence shown as sequence 1 in a sequence table, and has alkaline pectinase activity, and the preservation number of a recombinant bacterium (escherichia coli) BL21 (DE3) PelN containing the encoded gene of the protein in China General Microbiological Culture Collection Center (CGMCC) is CGMCC No. 7166. Experiments prove that the enzyme activity of the protein PelN for degrading polygalacturonic acid is 4590-4950U / mg; the heat stability is better; the relative enzyme activity is above 90% through heat preservation for 120 minutes at 45 DEG C; the optimum pH value of the alkaline pectinase is 9.8; the optimum temperature is 65 DEG C; and production methods of the protein PelN, the recombinant bacterium and the alkaline pectinase provide efficient paths for industrial large-scale production of the alkaline pectinase.

Description

technical field [0001] The invention relates to an alkaline pectinase PelN and its encoding gene and application. Background technique [0002] Pectin is an important component of plant cell walls, including protopectin, pectic acid and pectin ester acid. The presence of pectin substances can increase the firmness of plant cell walls, which is conducive to maintaining the specific shape and structure of plants, but increases the difficulty of industrial operations for deep processing of plants. [0003] Pectinase refers to a class of enzymes that can decompose pectin. It is commonly found in higher plants and microorganisms in nature and is an important industrial enzyme preparation. [0004] Alkaline pectinase (EC: 4.2.2.2) is a kind of enzyme that breaks the main chain of pectin by trans-elimination under alkaline conditions to produce unsaturated oligogalacturonic acid. It is used in the textile industry It has a wide range of applications in the fields of hemp degummin...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N5/10C12N1/21C12R1/19
Inventor 李小曼王辉林宋江宁马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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