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Optimized alkaline pectinase gene and application thereof

A pectinase and alkaline technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low expression of alkaline pectinase gene, excessive residual toxic substances, consumption of large chemical substances and heat energy, etc. To achieve the effect of simplifying the downstream purification work, simple production process and high product yield

Inactive Publication Date: 2013-02-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional textile pretreatment can be roughly divided into four steps: sizing, desizing, refining and bleaching. Each step consumes a lot of chemical substances and heat energy, and the waste water produced is also rich in high COD, causing serious pollution to the environment. , has not adapted to the development of the whole society
At present, the problems that need to be solved are: in the traditional textile treatment process, the excessive use of various chemicals forms a large amount of industrial wastewater, the consumption of water resources and energy is huge, and the residual toxic substances in the treated fabrics exceed the standard. Many governments have issued relevant provisions and laws, expressly Point out toxic and harmful substances in textiles and ban them
However, according to reports, so far, the expression level of alkaline pectinase gene in Bacillus subtilis is very low, and the output is only about 60U / mL

Method used

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  • Optimized alkaline pectinase gene and application thereof
  • Optimized alkaline pectinase gene and application thereof
  • Optimized alkaline pectinase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: the acquisition of alkaline pectinase gene sequence

[0016] The alkaline pectinase gene (pgl) sequence was derived from Bacillus sp.WSHB04-02, and the sequence of the alkaline pectinase coding region was synthesized by chemical total synthesis.

Embodiment 2

[0017] Example 2: Construction of recombinant plasmid vector pP43NMK1217

[0018] The alkaline pectinase gene obtained in Example 1 and the cloning vector pET-20 (+) were subjected to double digestion with SalI and NcoI respectively. The enzyme digestion system was as follows: 40 μL of plasmid, 5 μL of 10×buffer, 2.5 μL of SalI, 2.5 μL of NcoI . Gently mix the above components, and digest at 37°C for 20min. Then 0.8% agarose gel electrophoresis was used to verify and recover the PCR amplification products. Afterwards, the cloning vector obtained by digestion was ligated with the target gene, and the ligation reaction system (10 μL): 4 μL of the target gene pgl, 1 μL of the cloning vector pET-20b (+), 5 μL of ligase SolutionI, 16°C, ligated overnight. Then the ligation product was transformed into competent Escherichia coli JM109, and the transformation method was as follows:

[0019] (1) Take 100 μL of frozen-preserved competent cells and melt them in an ice bath;

[0020]...

Embodiment 3

[0028] Embodiment 3: the cultivation of recombinant bacterial strain

[0029] Glycerol tube: the concentration of glycerol is 20%; the composition of the seed medium is (g / L): sucrose 20, peptone 10, corn steep liquor 30, dipotassium hydrogen phosphate 18.4, potassium dihydrogen phosphate 6, pH 7.0, 50 μg / mL card Namycin; fermentation medium composition (g / L): corn starch 15, peptone 8, yeast extract 10, dipotassium hydrogen phosphate 9.2, potassium dihydrogen phosphate 3, pH 7.0, 50 μg / mL kanamycin;

[0030] Seed culture: Inoculate 0.4% of the inoculum from the glycerol tube into a 25mL / 250mL Erlenmeyer flask, rotate the rotary shaker at 200rpm, cultivate at a temperature of 37°C, and cultivate for 14 hours; The inoculum amount of (v / v) was inoculated into a 25mL / 250mL Erlenmeyer flask for cultivation, the cultivation temperature was 37°C, the shaker speed was 200rpm, and after cultivation for 2-3 days, the production of alkaline protease could reach 460U / ml, as shown in SDS-...

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Abstract

The invention discloses an optimized alkaline pectinase gene and application thereof. The optimized alkaline pectinase gene has a nucleotide sequence as shown in SEQ ID NO.1. Through an alkaline pectinase gene (pg1) sequence in synthetic Bacillus sp.WSHB04-02 and bacillus subtilis alkaline protease signal peptide (bpr), a recombinant plasmid pP43NMK1217 is constructed, an RB S sequence of the recombinant plasmid pP43NMK1217 is optimized, the recombinant plasmid pP43NMK1217 can be used in efficient expression of the alkaline pectinase; and a gene engineering bacterium WB600-pg1 constructed by using the recombinant plasmid pP43NMK1217 and used for efficiently secreting and expressing the alkaline pectinase has an alkaline pectinase yield reaching 460U / Ml which is 8 times higher than that of the existing technology, and has a wide application prospect.

Description

technical field [0001] The invention relates to an alkaline pectinase (alkaline polygalacturonate lyase, E.C.4.2.2.2, PGL for short) and its application, in particular to an optimized high-yield alkaline pectinase and its genetically engineered bacteria. Background technique [0002] In recent years, with the large-scale expansion of the chemical industry, the global ecological balance has been seriously threatened, and the environmental quality has deteriorated rapidly. Almost every country is vigorously developing green environmental protection technology and clean production industry. The textile industry has a huge scale and is closely related to the lives of all human beings. Traditional textile pretreatment can be roughly divided into four steps: sizing, desizing, refining and bleaching. Each step consumes a lot of chemical substances and heat energy, and the waste water produced is also rich in high COD, causing serious pollution to the environment. , has not adapted...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N15/63C12N9/88C12N1/21C12R1/125
Inventor 陈坚张俊娇康振堵国成
Owner JIANGNAN UNIV
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