Immobilized xylanase, preparation method and application of immobilized xylanase

A technology for xylanase and xylo-oligosaccharide, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of difficult acquisition of immobilization materials, complicated immobilization process, high price, etc., and achieves excellent immobilization effect, easy acquisition, and high cost. low cost effect

Inactive Publication Date: 2019-03-01
NANJING UNIV OF TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The acquisition of the immobilization materials used for these immobilization is relatively difficult, the price is relatively expensive, and the immobilization process is complicated and takes a long time.

Method used

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  • Immobilized xylanase, preparation method and application of immobilized xylanase
  • Immobilized xylanase, preparation method and application of immobilized xylanase
  • Immobilized xylanase, preparation method and application of immobilized xylanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] This example illustrates the method for preparing the immobilized support and extracting xylan.

[0024] Pretreatment of corncobs: crush corncobs (with a hemicellulose content of 30.5%), pass through a 20-40 mesh sieve, and take the under-sieve as crushed corncobs. Add distilled water ten times its mass to the crushed corncobs, place in a boiling water bath for 30 minutes, stir continuously during this period, centrifuge after cooling, discard the supernatant, rinse the precipitate with distilled water three times repeatedly, and dry.

[0025] Weigh 10 g of the pretreated corn cob into a 500 mL Erlenmeyer flask, add 200 mL, 40 g / L NaOH aqueous solution, stir at room temperature for 12 h, and obtain the filtrate and solid residue after suction filtration. The solid residue was washed with distilled water until neutral, and dried to obtain the corncob residue after alkali treatment, that is, the immobilized carrier. The pH of the filtrate was adjusted to 5.0-6.0 with 6M ...

Embodiment 2

[0027] This example illustrates the preparation method of expansin-xylanase fusion protein.

[0028] The amino acid sequence of the expansin-xylanase fusion protein is shown in SEQ ID NO: 1, and the fusion protein is named EXCL-EK2-XYN. Expansin-xylanase fusion protein (abbreviated as recombinant fusion protein) was prepared by engineering bacteria BL21(DE3) carrying the EXCL-EK2-XYN coding gene (see the invention patent application with application number 201710134735.7 and publication number CN108570107A for the construction method) ). The specific method is as follows: Pick a single colony of engineered bacteria from a plate cultivated overnight at 37°C, inoculate it into a 50mL Erlenmeyer flask containing 10mL of LB liquid medium, and cultivate it at 37°C until the logarithmic growth phase. Take 0.5 mL of the recombinant bacterial liquid and inoculate it into a 250 mL Erlenmeyer flask filled with 50 mL of LB liquid medium (containing 100 μg / mL of ampicillin kanamycin), an...

Embodiment 3

[0031] This example illustrates a method for highly efficient immobilization of recombinant fusion proteins.

[0032] The immobilized carrier (prepared in Example 1) was added to the recombinant fusion protein crude enzyme solution (232 U / mL) prepared in Example 2, and 50 g of the immobilized carrier was added to each liter of the crude enzyme solution. The mixture of the immobilized carrier and the crude enzyme solution was placed in a shaker at 20° C. and 150 rpm for adsorption reaction. After the adsorption reaction, the precipitate was collected by centrifugation, washed three times with pH 7.0 phosphate buffer, and the immobilized recombinant fusion protein was obtained. After the adsorption reaction time was 10min, 20min, 30min, 40min, 50min, and 60min, the immobilized yield of recombinant fusion protein, the recovery rate of immobilized recombinant fusion protein and the specific activity of xylanase of immobilized recombinant fusion protein were investigated.

[0033]...

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Abstract

The invention provides immobilized xylanase, a preparation method and application of the immobilized xylanase, and relates to the field of genetic engineering and enzyme engineering. The immobilized xylanase is obtained by immobilizing expansin-xylanase fusion protein on corncob residues. The preparation method of the immobilized xylanase comprises the following steps: immobilizing the expansin-xylanase fusion protein by using the corncob residues subjected to alkali treatment; and when preparing xylooligosaccharide from the immobilized xylanase, adding xylan and the immobilized xylanase in phosphate buffer, carrying out oscillatory reaction for 18-36 h at the temperature of 30-60 DEG C, centrifuging and then taking supernatant which is the xylooligosaccharide. According to the immobilizedxylanase, the fusion protein is immobilized on the corncob residues by the specific adsorption ability of expansin, a carrier is obtained easily, the cost is low, an immobilizing process is simple, consumed time is short, and when the xylooligosaccharide is prepared, operation is simple, and reaction conditions are mild.

Description

technical field [0001] The invention relates to the fields of genetic engineering and enzyme engineering, in particular to an immobilized xylanase, a preparation method and an application thereof. Background technique [0002] Xylooligosaccharide (Xylooligosaccharide, XOS) is a functional polymeric sugar composed of 2-7 xylose molecules combined with β-1,4 glycosidic bonds. It is the most stable and functional sugar among all functional sugars. The probiotic factor with the least intake” has pure sweetness, and the effect of value-added bifidobacteria is more than 20 times that of other functional sugars. After bifidobacteria ferment xylooligosaccharides during the growth process in the human intestinal tract It can produce organic acids such as lactic acid and acetic acid, make the intestinal tract acidic, control abnormal fermentation caused by harmful bacteria, stimulate intestinal peristalsis, improve the utilization rate of calcium, phosphorus, and iron while relieving ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/24C12P19/14C12P19/12C12P19/04C12P19/00
CPCC07K14/00C07K2319/00C12N9/2482C12P19/00C12P19/04C12P19/12C12P19/14C12Y302/01008
Inventor 吴斌韦利军李冰峰常思源何冰芳
Owner NANJING UNIV OF TECH
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