Construction and application of genetic engineering strain of yak rumen anaerobic fungus xylanase

A technology of rumen anaerobic fungi and genetically engineered bacteria, applied in the direction of genetic engineering, application, fungi, etc., can solve the problems of incomplete anaerobic fermentation technology of rumen fungi, few genetically engineered bacteria, and limited output

Active Publication Date: 2022-01-11
GANSU ACAD OF SCI INST OF BIOLOGY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

With the development of biotechnology, research on the cloning and expression of xylanase genes has become more and more in-depth. The expression in the medium will encounter problems such as proteolytic enzymes and glycosylation, so there are still relatively few genetically engineered bacteria that are actually transformed into production practice
[0005] Rumen anaerobic fungi can secrete highly active cellulase, which is even better than industrial strains, but its output is limited. In addition, the anaerobic fermentation technology of rumen fungi is not complete, which affects the large-scale production of rumen anaerobic fungal xylanase. Large-scale industrial application

Method used

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  • Construction and application of genetic engineering strain of yak rumen anaerobic fungus xylanase
  • Construction and application of genetic engineering strain of yak rumen anaerobic fungus xylanase
  • Construction and application of genetic engineering strain of yak rumen anaerobic fungus xylanase

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 The extraction of total RNA of anaerobic fungi and the cloning of target genes

[0046] The samples in this experiment were collected from the anaerobic fungus Piromyces Yak TZ isolated from the rumen juice of grazing yaks in Tianzhu County, Gansu Province. After the bacterial liquid was centrifuged at 39°C for 4 days, the bacterial pellet was ground with liquid nitrogen, and the total RNA extraction kit ( Tiangen, DP4 19) Extract the total RNA of PiromycesYak TZ, and use the reverse transcription kit after passing the quality inspection III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novazyme, R312-01) was used as a template to synthesize cDNA by reverse transcription. Further design primers to amplify the xylanase gene. All experimental operations were carried out in strict accordance with the product instructions of the above-mentioned kits.

[0047] In order to obtain the full-length CDS sequence of xylanase, primers were designed according to the sequ...

Embodiment 2

[0050] Embodiment 2 Construction of recombinant plasmid pPIC9K-xyn

[0051] The target gene synthesized in the previous step was digested with restriction endonucleases and connected to the expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-Beta-xylanase. The steps are as follows:

[0052] 1. Extraction of plasmid pPIC9K

[0053] Get the E.coli Top10 single bacterium colony of pPIC9K, be 50mg / L (kanamycin) in the LB liquid medium of 3mL final concentration Cultivate overnight. Centrifuge at 12000r / min for 1min, and collect the bacterial cells for extraction with the plasmid extraction kit. Specific steps:

[0054] (1) Add 500 μL of equilibrium solution BL to the adsorption column CP3, discard the waste liquid in the collection tube, and put CP3 back into the collection tube; (2) Centrifuge the bacterial solution at 12000r / min for 1 min, remove the supernatant; Add 250 μL of solution P1 to the precipitate, and use a pipette to suspend the bacteria; (4) Add 250...

Embodiment 3

[0070] Example 3 Electrotransformation of Pichia pastoris GS115 with recombinant plasmid

[0071] 1. Preparation of Pichia Competent Cells

[0072] Take the Pichia pastoris GS115 plate at -80°C and culture it at 30°C for 2 days.

[0073] (1) Pick a single colony and culture it in 5mLYPD medium (without antibiotic), 30°C, 220r / min for 2 days; (2) Add the bacterial solution to the YPD medium at a ratio of 1:100, 30°C, 220r / min Cultivation; (3) detection of bacterial solution OD 600nm When the temperature is 1.3, place the Erlenmeyer flask on ice for 30 minutes; (4) shake the Erlenmeyer flask gently after ice bathing, pour the bacteria into a 50mL sterilized centrifuge tube, and centrifuge at 5000r / min for 10min at 4°C; (5 ) to remove the supernatant, put the 50mL centrifuge tube on ice, take 10mL of pre-cooled sterile water to suspend the bacteria, add sterile water to 50mL; (6) remove the supernatant, wash the bacteria with pre-cooled sterile water 2 times; (7) Collect the p...

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Abstract

The invention relates to yak rumen anaerobic fungus xylanase and construction of a genetic engineering strain of the yak rumen anaerobic fungus xylanase. The invention provides the yak rumen anaerobic fungus xylanase and provides the genetic engineering strain P.p(xyn-N.f) capable of efficiently expressing the yak rumen anaerobic fungus xylanase, and the enzymatic activity of the xylanase produced by culture of the genetic engineering strain is as high as 500.70IU / mL. Compared with currently-reported anaerobic fungi or other xylanase genetic engineering strains, the genetic engineering strain P.p(xyn-N.f) disclosed by the invention has the advantages that not only is the expression of the anaerobic fungus xylanase under aerobic conditions realized, but also the enzyme activity of the xylanase is improved; and the obtained xylanase has a relatively wide pH application range, and has important research and application values in the industries of pulp bleaching, leather manufacturing, cleaning agents, textile fade-proof agents, dyes, detergents, feeds, biofuels, deinking agents and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to the construction of a yak rumen anaerobic fungus xylanase and its genetically engineered bacteria. Background technique [0002] Lignocellulose refers to biomass comprising three major components (cellulose, hemicellulose and lignin). Lignocellulose is widely found in crop wastes such as corncobs, bagasse, wheat bran, and straw. Xylan is the main component of plant hemicellulose, a highly branched heterosaccharide, and the second most abundant renewable resource after cellulose. The basic structural unit of xylan is β-1, 4 glycosidic bonds or β-1,3 glycosidic bonds of polyxylan chains, different plant sources, xylan branch composition is also different, the second oxygen of D-xylose is connected to D-glucuronic acid or 4 -O-Methylglucuronic acid, or L-arabinofuranose connected at the third position, some xylose is also acetylated at the second or third oxygen, and the ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2482C12N15/815C12Y302/01008C12N2800/22
Inventor 魏亚琴武小龙赵疆何国林贺敏婕张静荣毛婷
Owner GANSU ACAD OF SCI INST OF BIOLOGY
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