High-yield strain of high temperature resistant 1,4-beta-D-xylanase, method for producing high temperature resistant 1,4-beta-D-xylanase through fermentation of high-yield strain, and high temperature resistant 1,4-beta-D-xylanase
A technology for xylanase and high-yielding strains, applied in the fields of biotechnology and engineering, can solve problems such as application limitations, and achieve the effects of good stability and broad application prospects
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Embodiment 1
[0057] Screening Example 1: Screening and identification of xylanase-producing strains
[0058] (1) Enrichment culture
[0059] Take 0.1g soil sample, add it to 10mL sterile water, shake and mix, then pipette 1mL bacterial suspension into 5mL enrichment medium, and shake culture in 37℃ water bath for 48h.
[0060] (2) Primary screening by transparent circle method
[0061] Dilute the enriched culture 10 times step by step, and take the dilution factor as 10 -3 ,10 -5 And 10 -7 Spread 0.5mL of the bacterial suspension on a selection medium plate, culture at 37°C for 24h, pick a single colony with a relatively large transparent circle for separation and purification ( figure 1 ), insert the inclined plane culture medium to preserve the original strain.
[0062] (3) Shake flask fermentation re-screening
[0063] Inoculate the first screened bacteria in 50mL seed culture medium, cultivate overnight at 37℃, 180rpm, take 1mL of the bacterial solution after overnight culture and connect it to 5...
Embodiment 2
[0066] Enzyme Activity Example 2: Study on fermentation conditions and enzyme production activity of P. campinasensis G1-1
[0067] (1) The determination method of xylanase activity is (DNS method):
[0068] Pipette 0.1 mL of the appropriately diluted enzyme solution into a 5 mL graduated test tube with stopper, then add 0.1 mL of 10 g / L xylan substrate solution, close the test tube stopper, and react in a water bath at 50°C for 10 minutes. Add 0.6 to the test tube immediately mLDNS reagent and mix well to terminate the reaction, then boil it in boiling water for 10 minutes, add water to make the volume up to 5mL after cooling, shake well, and calculate the sugar content of the reaction system according to the regression equation of the xylose standard curve.
[0069] The definition of the activity unit of xylanase in the experiment is: 1mL enzyme solution produces 1μmoL of reducing sugar (calculated as xylose) per minute as an activity unit, expressed in IU:
[0070] IU=N×R / 10min×0.1...
Embodiment 3
[0075] Enzyme Separation Example 3: Separation and Purification of Thermostable Xylanase
[0076] ⑴ Centrifuge the fermentation broth at 8000 rpm for 10 min at 4°C, and take the supernatant;
[0077] (2) Slowly add ammonium sulfate to the fermentation supernatant to make the ammonium sulfate saturation reach 70%, and salt out overnight at 4°C;
[0078] (3) Centrifuge the salted out overnight fermentation supernatant at 4°C, 8000 rpm, for 10 min, take the precipitate, and dissolve the precipitate with an appropriate volume of pH 7.0 PBS buffer to obtain a high-temperature resistant xylanase crude enzyme solution for use;
[0079] ⑷OctylSepharoseFastFlow hydrophobic interaction chromatography: adjust the ammonium sulfate saturation of the crude enzyme solution to 40%, perform hydrophobic interaction chromatography, column type: Equilibrium solution: 0.02mol / L PBS buffer (pH7.0) containing 40% saturated ammonium sulfate; flow rate: 2mL / min, after loading, linearly eluted with 40% to 0% s...
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