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A high-temperature-resistant and strong-alkali-resistant xylanase and its application in waste paper deinking

A xylanase and waste paper deinking technology, applied in the fields of genetic engineering and microorganisms, can solve the problems of temperature and pH that cannot meet industrial requirements, and achieve the effects of good high temperature resistance, high alkalinity, and good deinking efficiency

Active Publication Date: 2018-10-12
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to make up for the deficiency that the temperature and pH of the existing xylanase cannot meet the needs of the industry, the present invention provides a high-temperature-resistant and strong-alkali-resistant xylanase and its application in waste paper deinking

Method used

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  • A high-temperature-resistant and strong-alkali-resistant xylanase and its application in waste paper deinking
  • A high-temperature-resistant and strong-alkali-resistant xylanase and its application in waste paper deinking
  • A high-temperature-resistant and strong-alkali-resistant xylanase and its application in waste paper deinking

Examples

Experimental program
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Effect test

Embodiment 1

[0036]Example 1 Acquisition of high temperature and strong alkali resistant xylanase gene xyn1, construction of recombinant plasmid and recombinant engineered bacteria

[0037] Acquisition of High Temperature and Alkali Resistance Xylanase Gene xyn1

[0038] Analyze, screen, discover using the National Center for Biotechnology Information (NCBI) database Planomicrobium glaciei There is a sequence in CHR43 with Bacillus The sequence of xylanase (Genbank number: AAB70918) in sp. Strain NG-27 has high homology, and it is likely to be the gene sequence encoding xylanase. Screen and optimize the sequence (optimization includes mutation and insertion of some sites) to obtain the nucleotide sequence with SEQ ID NO.2. The 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 respectively, and connected to BamH I and Xho I on the pETDuet-1 plasmid vector Betw...

Embodiment 2

[0042] Example 2 Expression and purification of xylanase xyn1 Expression:

[0043] Recombinant bacteria were first inoculated in 10ml LB liquid medium containing 100μg / ml ampicillin, and cultured overnight at 37°C and 220rpm with shaking. Then transfer it to 500ml LB liquid medium containing 100μg / ml ampicillin at 1% (v / v), shake culture at 37°C and 220rpm until OD 600 When it reaches about 0.6, add IPTG to a final concentration of 1mM, shake and culture at 37°C and 220rpm for 6 hours. Bacteria were collected by centrifugation.

[0044] purification:

[0045] Add appropriate amount of pH7.0 PBS buffer (137mmol / L NaCl, 2.7mmol / L KCl, 4.3mmol / L NaCl) to the cells 2 HPO 4 , 1.4mmol / L KH 2 PO 4 , adjust the pH value of the solution to 7.0 with HCl), resuspend the recombinant bacteria by ultrasonication, centrifuge at high speed, and collect the supernatant. Incubate the supernatant at 65°C for half an hour to precipitate a large number of non-thermostable host bacterial ...

Embodiment 3

[0046] Example 3 Determination of enzymatic properties of high temperature and strong alkali resistant xylanase:

[0047] Use DNS method to measure xylanase activity: add 200μL enzyme to 2mL reaction system, substrate concentration is 0.9%, react at temperature 50°C, pH4.8 for 10min, add 2mL DNS to terminate the reaction, and bathe in boiling water for 10min. After cooling to room temperature, add water to 15mL, then measure the absorbance at 540nm wavelength, compare the standard curve to obtain the reducing sugar content, and the 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 thermostable and strong alkali tolerant xylanase in the range of pH 3.0 to 12.0 was determined. The result is as figure 2 As shown, it can be seen from the figure that the optimum pH of high temperature and strong alkali resistant xylanase is 9.0, and maintains a relatively high activity in the range of...

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Abstract

The invention provides high-temperature-resistant and strong-base-resistant xylanase and application of the xylanase to waste paper deinking. The amino acid sequence of the high-temperature-resistant and strong-base-resistant xylanase is shown as SEQ ID NO.1. A high-temperature-resistant and strong-base-resistant xylanase gene is encoded, and the nucleotide sequence of the gene is shown as SEQ ID NO.2. A recombinant plasmid comprises the high-temperature-resistant and strong-base-resistant xylanase gene. A recombinant bacterium contains the recombinant plasmid. The invention further discloses application of the xylanase to waste paper deinking. Waste paper to be deinked is placed into a buffering solution containing 0.6-1.2U / mg of the high-temperature-resistant and strong-base-resistant xylanase, and a deinking reaction is conducted under the condition that the temperature is 50-90 DEG C and the pH is 5.0-12.0. The xylanase gene achieves high expression in escherichia coli through the codon optimization technology. The expressed xylanase has no cellulose activity and has high temperature resistance and high alkali resistance. The xylanase can be applied to waste paper deinking, has higher efficiency compared with commercial enzyme and has good industrial application value.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and microorganisms, in particular to a high-temperature-resistant and strong-alkali-resistant xylanase and its application in waste paper deinking. Background technique [0002] Hemicellulose is the second most abundant renewable resource after cellulose, and its efficient application is of great significance for human beings to solve the current resource crisis, energy crisis, environmental pollution and other problems. The structure and composition of hemicellulose are very complex, including various components such as xylan, mannan, arabinan, arabinogalactan and dextran. Among them, xylan is the most representative hemicellulose in cells, and it can be utilized by people as a renewable resource. Xylan is a complex polypenta-carbon sugar, which is mainly connected by β-1,4-D-xylosidic bonds and has a variety of substituents. Its complete degradation requires the synergy of various ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21D21C5/02C12R1/19
CPCC12N9/248C12Y302/01D21C5/025Y02W30/64
Inventor 贾红华周俊邵婷婷李蘅香钟超韦萍
Owner NANJING TECH UNIV
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