A high-yielding high-temperature-resistant alkaline xylanase strain and its production method
A technology of xylanase and alkali resistance, which is applied in the field of bioengineering, can solve problems such as the application of restriction enzyme preparations, and achieve the effects of low manufacturing cost, high extraction yield and stable performance
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Embodiment 1
[0027] The mutagenesis selection of embodiment 1 bacterial strain
[0028] Inoculate the Bacillus pumilus MJ3 strain preserved in the laboratory into the seed medium, cultivate it to the logarithmic phase at 37°C, then collect the bacteria, centrifuge at 8000rpm for 5min, and resuspend the bacteria with physiological saline to make the concentration of the bacteria 10 6 -10 7 / ml, and NTG mother liquor was added to make the final concentration 0.2g / L. Then react at 37°C for 30 minutes, after appropriate dilution, take 100 μL and spread it on a screening plate and incubate at 37°C for about 24 hours, and calculate the lethal rate. Pick a single colony with a large transparent circle on the screening plate and insert it into the seed bottle for cultivation. After the seeds grow well, insert them into the fermentation shaker flask. Finally, a high-yielding strain NGJ763 with a 2.3-fold increase in enzyme activity was screened out. Through the study of enzymatic characteristics,...
Embodiment 2
[0035] Embodiment 2 xylanase activity assay method
[0036] (1) Definition of xylanase activity unit
[0037] At 50°C and pH 8.0, the amount of enzyme required to release 1 μmol of reducing sugar per minute is defined as 1 enzyme activity unit.
[0038] (2) Enzyme activity assay method
[0039] Take 2ml of xylan substrate with a concentration of 1% (prepared with pH8.0 disodium hydrogen phosphate-citric acid buffer solution) into the colorimetric tube, preheat at 50°C for 10min, then add 2ml of pH8.0 buffer solution The diluted and preheated xylanase enzyme solution was mixed and reacted at 50°C for 30 minutes. After the reaction, add 5ml of DNS reagent, mix well to terminate the reaction, then boil in a boiling water bath for 10min, cool to room temperature with water, add water to make up to 25ml, use the standard blank as the blank control, and measure the absorbance A at 540nm.
[0040] Enzyme activity calculation formula:
[0041] In the formula: X is the activity o...
Embodiment 3
[0042] Embodiment 3 bacterial strain liquid fermentation produces xylanase and its extraction
[0043] 1. Seed cultivation
[0044] The composition of medium mass volume percentage is as follows:
[0045] (1) Plate separation medium
[0046] Beef extract 0.3%, peptone 1%, sodium chloride 0.5%, xylan 1.5%, Congo red 0.02%, agar 2%, the rest is water, pH 7.0.
[0047] (2) Seed medium
[0048] Corn cob 2%, beef extract 0.3%, peptone 1%, sodium chloride 0.5%, the rest is water, pH7.0.
[0049] (3) Fermentation Shake Flask Yeast Medium
[0050] 2% bean cake powder; 2% washed bran, 1.5% corn cob, 1.6% corn steep liquor, 0.2% dipotassium hydrogen phosphate, 0.25% magnesium sulfate, 0.3% ammonium sulfate, 0.8% sodium nitrate, the rest is water, pH7.0 .
[0051] (4) Culture conditions
[0052] Separation plate: culture at 37°C for 24 hours;
[0053] Liquid seeds: culture at 37°C for 10 hours, shaker speed 240r / min;
[0054] Fermentation shake flask: culture at 37°C for 72 hour...
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