Xylanase, gene coding same and application of xylanase to waste paper deinking

Abstract

The invention discloses xylanase, a gene coding the same and an application of xylanase to waste paper deinking. An amino acid sequence of xylanase is shown in SEQ ID NO.1. A nucleotide sequence of the gene coding xylanase is shown in SEQ ID NO.2. A recombinant plasmid comprises the xylanase gene. A recombinant bacterium contains the recombinant plasmid. When xylanase is applied to waste paper deinking, waste paper to be deinked is put in a buffer solution containing 0.6-1.2U / mg of xylanase and deinking reaction is carried out under the conditions that the temperature is 50-70 DEG C and the pH value is 6.0-10.0. Xylanase and the gene have the beneficial effects that the xylanase gene provided by the invention achieves high expression in escherichia coli by utilizing the codon optimization technology; expressed xylanase does not have cellulase activity and has high temperature and high alkalinity resistance; and xylanase can be applied to waste paper deinking, has higher efficiency than commercial enzymes and shows good industrial application values.

Description

technical field

[0001] The invention relates to the field of genetic engineering and microorganism technology, in particular to a xylanase, a gene encoding the enzyme 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 hydrolytic enzymes . The m...

Examples

Embodiment 1

[0038] Embodiment 1: the acquisition of xylanase gene, the construction of recombinant plasmid and recombinant engineered bacteria:

[0039] Obtaining the xylanase gene:

[0040] Analyze, screen, discover using the National Center for Biotechnology Information (NCBI) database Planomicrobium glaciei There is a sequence in CHR43 with BacillusThe sequence of xylanase (Genbank number: AAB70918) in sp.StrainNG-27 has high homology, and it is likely to be the gene sequence encoding xylanase. The sequence is screened and optimized (optimization includes mutation and insertion of some sites), and the nucleotide sequence with SEQ ID NO.2 is obtained. The xylanase gene was artificially synthesized by GenScript according to the nucleotide sequence of SEQ ID NO.2, and BamHI and XhoI restriction sites were added at both ends of the sequence, and then connected to the BamHI and XhoI restriction sites on the pETDuet-1 plasmid vector Between the sites, the recombinant plasmid pETDuet-1-xy...

Embodiment 2

[0044] Expression and purification of embodiment 2 xylanase xyn2

[0045] Express:

[0046] Recombinant bacteria were first inoculated in 10 ml LB liquid medium containing 100 μg / ml ampicillin, and cultured overnight at 37° C. and 220 rpm with shaking. Then transfer to 500ml LB liquid medium containing 100μg / ml ampicillin at 1% amount, shake culture at 37°C and 220rpm until OD 600 When it reaches about 0.6, add IPTG to a final concentration of 1 mM, shake and culture at 37° C. and 220 rpm for 6 hours. Bacteria were collected by centrifugation.

[0047] purification:

[0048] Add an appropriate amount of pH7.0PBSbuffer (137mmol / LNaCl, 2.7mmol / LKCl, 4.3mmol / LNa 2 HPO 4 , 1.4mmol / LKH 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 prote...

Embodiment 3

[0049] The enzymatic property determination of embodiment 3 xylanase

[0050] Use the DNS method to measure xylanase activity: add 200 μL of enzyme to the 2 mL reaction system, the substrate concentration is 0.9%, react at a temperature of 50 ° C and pH 4.8 for 10 min, add 2 mL of DNS to terminate the reaction, and bathe in boiling water for 10 min. After cooling to room temperature, add water to 15mL, and then detect its absorbance at a wavelength of 540nm, compare with 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 xylanase in the pH range of 3.0 to 12.0 was determined. The result is as figure 2 As shown, it can be seen from the figure that the optimal pH of xylanase is 7.0, and maintains a relatively 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 dete...

technical field

[0001] The invention relates to the field of genetic engineering and microorganism technology, in particular to a xylanase, a gene encoding the enzyme 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 hydrolytic enzymes . The m...

Examples

Embodiment 1

[0038] Embodiment 1: the acquisition of xylanase gene, the construction of recombinant plasmid and recombinant engineered bacteria:

[0039] Obtaining the xylanase gene:

[0040] Analyze, screen, discover using the National Center for Biotechnology Information (NCBI) database Planomicrobium glaciei There is a sequence in CHR43 with BacillusThe sequence of xylanase (Genbank number: AAB70918) in sp.StrainNG-27 has high homology, and it is likely to be the gene sequence encoding xylanase. The sequence is screened and optimized (optimization includes mutation and insertion of some sites), and the nucleotide sequence with SEQ ID NO.2 is obtained. The xylanase gene was artificially synthesized by GenScript according to the nucleotide sequence of SEQ ID NO.2, and BamHI and XhoI restriction sites were added at both ends of the sequence, and then connected to the BamHI and XhoI restriction sites on the pETDuet-1 plasmid vector Between the sites, the recombinant plasmid pETDuet-1-xy...

Embodiment 2

[0044] Expression and purification of embodiment 2 xylanase xyn2

[0045] Express:

[0046] Recombinant bacteria were first inoculated in 10 ml LB liquid medium containing 100 μg / ml ampicillin, and cultured overnight at 37° C. and 220 rpm with shaking. Then transfer to 500ml LB liquid medium containing 100μg / ml ampicillin at 1% amount, shake culture at 37°C and 220rpm until OD 600 When it reaches about 0.6, add IPTG to a final concentration of 1 mM, shake and culture at 37° C. and 220 rpm for 6 hours. Bacteria were collected by centrifugation.

[0047] purification:

[0048] Add an appropriate amount of pH7.0PBSbuffer (137mmol / LNaCl, 2.7mmol / LKCl, 4.3mmol / LNa 2 HPO 4 , 1.4mmol / LKH 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 prote...

Embodiment 3

[0049] The enzymatic property determination of embodiment 3 xylanase

[0050] Use the DNS method to measure xylanase activity: add 200 μL of enzyme to the 2 mL reaction system, the substrate concentration is 0.9%, react at a temperature of 50 ° C and pH 4.8 for 10 min, add 2 mL of DNS to terminate the reaction, and bathe in boiling water for 10 min. After cooling to room temperature, add water to 15mL, and then detect its absorbance at a wavelength of 540nm, compare with 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 xylanase in the pH range of 3.0 to 12.0 was determined. The result is as figure 2 As shown, it can be seen from the figure that the optimal pH of xylanase is 7.0, and maintains a relatively 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 dete...

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