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Recombinant escherichia coli engineering bacterium realizing high yield of cutinase and fermentation process thereof

A technology for recombining Escherichia coli and cutinase, applied in the fields of genetic engineering and fermentation engineering, can solve the problems such as the production of cutinase is not significantly improved, lack of specific optimization of the cutinase fermentation process, low cutinase activity, etc., and achieves low production cost. , the effect of high yield and wide source of raw materials

Inactive Publication Date: 2018-11-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, from the perspective of genetically engineered bacteria, the existing genetically engineered bacteria obtained by heterologously expressing the cutinase derived from Humicola insolens in bacteria has not significantly improved its cutinase output. The activity of the cutinase produced is also very low; from the perspective of the fermentation process, there is also a lack of strategies for specific optimization of the fermentation process for the functional characteristics of the cutinase itself

Method used

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  • Recombinant escherichia coli engineering bacterium realizing high yield of cutinase and fermentation process thereof
  • Recombinant escherichia coli engineering bacterium realizing high yield of cutinase and fermentation process thereof
  • Recombinant escherichia coli engineering bacterium realizing high yield of cutinase and fermentation process thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Construction of E.coli BL21(DE3) / pET-20b(+) / hic

[0071] The cutinase gene derived from Humicola insolens inserted Nco I and Hind III on both sides of the cutinase gene, and was connected to the commercial plasmid pET-20b(+), located downstream of the signal peptide sequence, to construct the expression vector pET-20b(+ ) / hic, and transform it into the host strain E.coli BL21(DE3), the recombinant strain E.coli BL21(DE3) / pET-20b(+) / hic is constructed.

[0072] ( figure 1 It is a recombinant plasmid pET-20b(+) / hic double enzyme digestion gel electrophoresis. The electrophoresis results show that there is a band consistent with the theoretical molecular weight of the cutinase gene at about 500p, and a band with the vector pET-20b(+ ) Bands with consistent theoretical molecular weight. )

Embodiment 2

[0073] Example 2: Detection of the enzyme activity of the recombinant engineering strain E.coli BL21(DE3) / pET-20b(+) / hic of the present invention

[0074] The E.coli BL21(DE3) / pET-20b(+) / hic strain obtained in Example 1 was transferred to LB medium and cultured at 36-38°C for 8-10 hours, and then the resulting culture solution was inoculated at 4-6% The amount is connected to the TB fermentation medium, 36-38℃, 200r / min cultured for 1.5-2.5h, then induced with IPTG with a final concentration of 0.01-0.03mM, and cultured for 24h. Measure the cell concentration, collect the fermentation broth, and determine the enzyme activity in the fermentation supernatant of the sample after centrifugation. ( figure 2 This is the SDS-PAGE protein electrophoresis diagram of the extracellular supernatant of fermentation in the shake flask for 24 hours. The protein electrophoresis result shows that there is a band consistent with the theoretical molecular weight at about 20kDa. At this time, the c...

Embodiment 3

[0075] Example 3: Influence of different induction time on enzyme production of recombinant engineering bacteria of the present invention

[0076] 1. Seed culture: The E.coli BL21(DE3) / pET-20b(+) / hic strain obtained in Example 1 is inserted into the seed culture medium, and cultivated using a constant temperature shaker at a temperature of 37°C and a rotation speed of 200 rpm. 8h.

[0077] 2. Fermentation enzyme production: connect the seed liquid with a 10% inoculum to a 3.6L fermentor for fermentation, control the rotation speed in the fermenter to 300rpm, and the ventilation to 1.5vvm, so that the dissolved oxygen of the fermentation liquid is maintained at 30%. The control temperature is 37℃, and 25% (v / v) ammonia water is added to control the pH at 7.0. After the initial glycerol is consumed, the dissolved oxygen rises to 80-100%, then the batch fermentation and cultivation ends; The method of supplementing the feeding medium for feeding fermentation; after the fermentation c...

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Abstract

The invention discloses a recombinant escherichia coli engineering bacterium realizing high yield of cutinase and a fermentation process thereof, and belongs to the technical field of gene engineeringand fermentation engineering. Humicola isnolens sourced cutinase gene is used as a target gene; pET-20b(+) is used as an expression vector; E.coli BL21(DE3) is used as an expression vector; a gene engineering bacterium E.coli BL21(DE3) / pET-20b(+) / hic capable of realizing efficient active expression of the cutinase is successfully built; meanwhile, the recombinant escherichia coli engineering bacterium E.coli BL21(DE3) / pET-20b(+) / hic is creatively used as a production strain; a production process of combining a two-stage temperature control process, a constant dissolved oxygen control process,a pH control process, a supplemented material batch fermentation process, and a high induction thallus concentration combined high induction intensity induction control process is used for producingthe cutinase; the advantages of high yield, wide raw material sources, low production cost and the like are realized.

Description

Technical field [0001] The invention relates to a recombinant Escherichia coli engineered bacteria with high cutinase production and a fermentation process thereof, belonging to the technical fields of genetic engineering and fermentation engineering. Background technique [0002] Cutinase is a multifunctional hydrolase that can be used to hydrolyze short-chain or long-chain fatty acid esters and triglycerides, and it can also be used to degrade plant polymer cutin. [0003] Compared with lipase, cutinase has no lid structure and is easier to degrade polyester. Therefore, cutinase has been widely used in the deep processing of agricultural products, care products industry, textile industry, etc., especially in the bio-refining and artificial cotton fabrics. In the surface modification of fibers, cutinase is expected to replace traditional chemical alkali treatment, which is of great significance to environmental protection. [0004] There are many sources of cutinase. Among them, th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/18C12R1/19
CPCC12N9/18C12Y301/01074
Inventor 吴敬宿玲恰孙益荣洪若宇
Owner JIANGNAN UNIV
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