Xylanase and the gene encoding the enzyme and its application in waste paper deinking
A technology of xylanase and waste paper, applied in the fields of genetic engineering and microorganisms
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Embodiment 1
[0038] Example 1: Obtaining of xylanase gene, construction of recombinant plasmid and recombinant engineering bacteria:
[0039] Acquisition of xylanase gene:
[0040] Use the database of the National Center for Biotechnology Information (NCBI) to analyze, screen, and discover Planomicrobium glaciei There is a sequence in CHR43 with Bacillus The xylanase (Genbank number: AAB70918) sequence in sp. Strain NG-27 has high homology, and it is likely to be a gene sequence encoding xylanase. The sequence is screened and optimized (optimization includes partial mutations, insertions, etc.) to obtain a nucleotide sequence with SEQ ID NO.2. The xylanase gene was artificially synthesized by GenScript according to the nucleotide sequence of SEQ ID NO.2, and BamH I and Xho I restriction sites were added at both ends of the sequence, and they were connected to the BamH on the pETDuet-1 plasmid vector. Between I and Xho I restriction sites, the recombinant plasmid pETDuet-1-xyn2 was obtained.
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Embodiment 2
[0044] Example 2 Expression and purification of xylanase xyn2
[0045] expression:
[0046] Recombinant bacteria were first inoculated into 10ml LB liquid medium containing 100μg / ml ampicillin, and cultured overnight at 37°C and 220rpm with shaking. Then transfer 1% to 500ml LB liquid medium containing 100μg / ml ampicillin, shake culture at 37℃, 220rpm to OD 600 When it reaches about 0.6, add IPTG to a final concentration of 1mM, and incubate with shaking at 37°C and 220rpm for 6 hours. Collect the bacteria by centrifugation.
[0047] purification:
[0048] Add an appropriate amount of pH7.0 PBS buffer (137mmol / L NaCl, 2.7mmol / L KCl, 4.3mmol / L Na 2 HPO 4 ,1.4mmol / L KH 2 PO 4 , Adjust the pH value of the solution to 7.0 with HCl) resuspend, ultrasonically disrupt the recombinant bacteria, centrifuge at high speed, and collect the supernatant. Incubate the supernatant at 65°C for half an hour to precipitate a large amount of non-heat-resistant host bacteria protein, and then collect...
Embodiment 3
[0049] Example 3 Determination of Enzymatic Properties of Xylanase
[0050] Use DNS method to determine xylanase activity: add 200μL of enzyme to a 2mL reaction system, with a substrate concentration of 0.9%, react for 10 minutes at a temperature of 50°C and pH 4.8, add 2mL of DNS to terminate the reaction and place in a boiling water bath for 10 minutes. After cooling to room temperature, add water to 15mLl and then measure its absorption value at 540nm wavelength. Compare the standard curve to get the reducing sugar content. Enzyme activity unit (U) is defined as the amount of enzyme that releases 1mM reducing sugar per minute. According to this method, the activity of xylanase in the pH range of 3.0 to 12.0 was determined. The result is figure 2 As shown, it can be seen from the figure that the optimum pH of xylanase is 7.0 and maintains high activity in the range of pH 6.0-10.0. The activity of xylanase in the range of 30°C to 90°C was also determined. The suitable reaction...
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