A kind of endoxylanase resistant to low pH value and its coding gene and application
A technology of xylan and gene encoding, applied in the field of genetic engineering, can solve problems such as denaturation and loss of function
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Embodiment 1
[0022] Embodiment 1 Preparation of endo-xylan hydrolase resistant to low pH value
[0023] 1. Cloning of low pH endo-xylan hydrolase gene
[0024] After the strain NJZ5 was cultured in the inorganic salt medium with 1% (w / v) glucose as the sole carbon source for 48 hours, the mycelium was transferred to the inorganic salt medium containing 1% (w / v) xylan The culture was continued for 12 hours, and then the total RNA of NJZ5 induced by xylan was extracted by an RNA extraction kit, and the cDNA sequence of the endoxylan hydrolase gene was obtained by PCR amplification after reverse transcription. The specific operation is to take out the pre-stored xylan-induced NJZ5 mycelium from the -80°C refrigerator, and grind it into powder with liquid nitrogen. The total RNA of NJZ5 was successfully extracted by using the Plant Mini Kit of QIAGEN in combination with the company's RNase-Free DNase set. The integrity and purity of the extracted total RNA were detected by electrophoresis. ...
Embodiment 2
[0050] Example 2 Detection of optimum reaction temperature when endo-xylan hydrolase hydrolyzes oat xylan
[0051] Add 2 μl of purified endo-xylan hydrolase to 1 ml of acetate buffer pH 5.5 containing 1% oat xylan or acetic acid buffer, and place at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C °C, 80 °C, 90 °C and 100 °C water bath for 10 min, and each temperature was set for 3 repetitions. Mix the reaction system thoroughly every 2 minutes during the reaction. Immediately after the reaction, add 1ml of DNS reagent and mix well. After 10 minutes in boiling water bath, dilute 5 times and absorb 200μl. Use a spectrophotometer at a wavelength of 540nm to detect the intensity of absorbance to determine the endo-xylan hydrolase. Optimum reaction temperature when sugar is the substrate. The results showed that when the endo-xylan hydrolase hydrolyzed oat xylan, as the reaction temperature gradually increased from 20℃, the enzyme activity also increased, and reached the highest activity at t...
Embodiment 3
[0052] Example 3 Detection of pH stability when endoxylan hydrolase hydrolyzes oat xylan
[0053] The purified endo-xylan hydrolase was placed in buffer solution with pH value (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0), stored at 4°C for 1 h, and then The enzyme activity was determined by DNS method under the optimal temperature and pH value conditions. For the low pH region, the enzyme was redesigned and stored at 4°C in pH 1.0 and pH 2.0 buffers (10min, 30min, 1h, 2h, 4h, 6h), and the enzyme was determined by DNS method at pH 1.0 and pH 2.0 respectively. The dynamic change curve of enzyme activity over time under the condition of pH 2.0. Finally, it was concluded that the low-pH-resistant endoxylan hydrolase X10C could exist stably at pH 2.0, and it existed more stably at pH 1.0.
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