Gene recombinant plasmid, gene recombinant pichia pastoris and straw fiber degumming application

A gene recombination, Pichia pastoris technology, applied in application, fiber processing, genetic engineering and other directions, can solve the problems of surface adhesion of impurities, uneven thickness, fiber damage, etc., to achieve high degumming efficiency, low cost, and fiber residue. low rate effect

Pending Publication Date: 2022-04-01
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention solves the technical problems in the prior art that the degumming of the straw fiber causes damage to the fiber, and the processed fiber is relatively rough, uneven in thickness, and has impurities adhered to the surface

Method used

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  • Gene recombinant plasmid, gene recombinant pichia pastoris and straw fiber degumming application
  • Gene recombinant plasmid, gene recombinant pichia pastoris and straw fiber degumming application
  • Gene recombinant plasmid, gene recombinant pichia pastoris and straw fiber degumming application

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Embodiment 1

[0035] The present invention uses the ppic9k plasmid as a carrier to design and synthesize the gene pathway for fiber degumming. The specific plasmid structure is as follows: figure 1shown. Select the ppic9k plasmid vector, select the pectin degrading enzyme gene pelA (pectate lyase) found in Aspergillus niger EIM-6, the multifunctional peroxidase gene VP (Versatile peroxidase) found in Pleurotus ostreatus, and the small laccase gene SLAC (Small laccase), Kozak ribosome binding sequence, self-designed basic gene Alkali. Each gene, promoter and terminator (TT) were sequentially connected to the ppic9k plasmid vector by using Gibson Assembly technology. like image 3 As shown, the sequence is pGAP constitutive promoter, Kozak ribosome binding site, FLO10-pro signal peptide gene modified pelA gene, terminator (TT); acid-sensitive promoter Pgpda, Kozak ribosome binding site, FLO10 Signal peptide gene modified SLAC gene, terminator (TT); acid-sensitive promoter Pgpda, Kozak ribo...

Embodiment 2

[0037] Select Pichia pastoris GS115 as the biological chassis, and use electroporation technology to introduce the recombinant plasmid pGAPZαA into GS115, including the following steps:

[0038] a. Take out 80-100 μL of Pichia pastoris medium for continuous culture into a centrifuge tube and put it in an ice water bath;

[0039] b. Add 7-15 μL of linearized pGAPZαA recombinant plasmid, and put it in an ice-water bath for 5 minutes;

[0040] c. Connect the pre-cooled electrodes, and stimulate with 1500v, 5ms, 200Ω and 25μF electricity;

[0041] d. Immediately after stimulation, add 1mL, 1mol / L sorbitol solution (to increase the permeability of the cell membrane, in the Pichia pastoris expression system, adding sorbitol also has the effect of carbon source), and mix thoroughly;

[0042] e. 30°C water bath for 1-2h, add 1mL medium, continue water bath for 1-2h;

[0043] f. Centrifuge at 12000rpm for 0.5min, remove the supernatant, then add 1mL of culture medium and antibiotics ...

Embodiment 3

[0045] Straw fiber degumming of the present invention such as figure 2 as shown, figure 2 The six steps shown are:

[0046] (1) Raw material straw;

[0047] (2) Carry out pretreatment to straw, use mechanical rolling to process;

[0048] (3) Carry out constant turbidity continuous culture together with the cultivated engineering yeast thallus;

[0049] (4) Obtain the processed fiber, and extract by-products such as hemicellulose and galacturonic acid from the fermented liquid taken out from constant turbidity culture;

[0050] (5) bleaching, drying, etc. are carried out as required by the fiber obtained;

[0051] (6) Obtain straw fiber crude product.

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Abstract

The invention relates to a gene recombinant plasmid, gene recombinant pichia pastoris and straw fiber degumming application, and belongs to the technical field of renewable resource utilization. The gene recombinant plasmid comprises a constitutive promoter, a ribosome binding site, a pectin degrading enzyme gene modified by a first signal peptide gene, a terminator, a first acid-sensitive promoter, a ribosome binding site, a small laccase gene modified by a second signal peptide gene, a terminator and a first acid-sensitive promoter which are sequentially connected from upstream to downstream, the kit comprises a first acid-sensitive promoter, a ribosome binding site, a peroxidase gene, a terminator, a second acid-sensitive promoter, a basic gene and a terminator. According to the method, pectin and cellulose in the straw are degraded by using the recombinant pichia pastoris, the comprehensive utilization rate of the straw is improved, high-quality and zero-pollution straw fibers for spinning and derivative chemical products are produced, and the yield of industrial products with high additional value can be increased while the waste straw treatment cost and labor force can be reduced.

Description

technical field [0001] The invention belongs to the technical field of renewable resource utilization, and in particular relates to a gene recombination plasmid, gene recombination Pichia pastoris and the application of straw fiber degumming. Background technique [0002] Banana stalks are a kind of bulk agricultural waste in the tropics of our country, and the output is relatively large. At present, they are mainly returned to the fields directly and piled up on the side of the fields and roadsides, which not only causes environmental pollution, but also causes a lot of waste of resources. At the same time, a small amount of banana straw is used for composting, feed production, energy conversion, and fiber extraction and processing for textiles. Banana fiber will also become an important source of natural plant fiber for high-quality textiles due to its strength and toughness exceeding ordinary plant fibers. However, banana stalks contain a large amount of colloids, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/67C12N15/62C12N15/53C12N1/19D01C1/00C12R1/84
Inventor 樊昌鑫裘豪王博涵霍修楠石家诚梁元浩张东方占艺谢尚县闫云君宁康
Owner HUAZHONG UNIV OF SCI & TECH
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