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Genes, plasmid, bacterial strain and application of xylanase

A xylanase and gene technology, applied in the application field of xylanase in pulp bleaching, can solve problems such as short half-life

Active Publication Date: 2011-01-26
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the xylanase can withstand high temperature and alkali to a certain extent, its half-life is short under the condition of high temperature and high alkali, which cannot meet the requirement of 1-1.5 hours of processing time in the pulp bleaching process

Method used

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  • Genes, plasmid, bacterial strain and application of xylanase
  • Genes, plasmid, bacterial strain and application of xylanase
  • Genes, plasmid, bacterial strain and application of xylanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 synthetic xylanase gene (S7-xyn)

[0024] The gene sequence and protein structure information of the endoxylanase gene S7-xyn of B.halodurans S7 comes from the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih .gov / ) and the Protein Database (PDB, http: / / www.rcsb.org / pdb / search / advSearch.do).

[0025] According to the S7-xyn gene sequence published on NCBI, the sequence was optimized and replaced with codons preferred by Pichia pastoris, and then the optimized gene sequence was used with DNAWORKS software ( http: / / mcll.ncifcrf.gov / lukowski.html ), designed primers for whole gene synthesis, and designed 34 primers (that is, corresponding to SEQ ID NO 2~SEQ ID NO 35), among which the first primer S7-1 and the 34th primer introduced EcoRI and NotI enzymes respectively See Table 1 for the cleavage sites and enzyme cleavage protection bases.

[0026] Table 1 Primers for the whole gene synthesis of S7-xyn gene

[0027]

[0028] T...

Embodiment 2

[0056] Embodiment 2 prepares the plasmid containing the S7-xyn gene of 3 copies

[0057] The PCR product obtained in Example 1 was electrophoresed, the gel was cut to recover the target band at about 1200bp, the recovered product was digested with EcoRI and NotI, and the expression vector pPICZαA was also digested with EcoRI and NotI and recovered from the gel with T4DNA Ligase ligation. The 10 μL volume reaction system is as follows: 1 μL (50ng) of pPICZαA, 6 μL of fully synthetic gene product, 1 μL of 10×Buffer containing ATP, 1 μL of T4 DNA ligase, and ddH 2 O to make up to 10 μL. Slightly centrifuge, connect in a water bath at 16°C overnight, and transform into E.coli Top10, and screen positive transformants on LB plates containing Zeocin (bleomycin, 25 μg / mL). Randomly pick a certain amount of transformants, prepare a small amount of plasmids, and perform electrophoresis analysis after double digestion with restriction endonucleases EcoRI and NotI. It is estimated that...

Embodiment 3

[0060] Example 3 High expression of xylanase S7-xyn in Pichia pastoris

[0061] The pPICZαA-(XYN) of Example 2 that was completely linearized by the SacI enzyme by the LiCl method 3 Transformation of Pichia pastoris X33. The transformants were spread on MD plates and cultured at 30°C for 2 days. Transformants on the MD plate were inoculated on YPD plates containing Zeocin 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, and cultured at 30°C for 3 days. Single clones were picked from the transformants appearing on the Zeocin-YPD plate with a higher concentration. Extract yeast genomic DNA as a template according to the Invitrogen operation guide, use the PCR primers of the target gene sequence for yeast genome PCR identification, the total reaction volume is 20 μL, and the amount of Taq enzyme is 2U. Take 2 μL of PCR products for 0.8% agarose gel electrophoresis identification.

[0062] Identification of the correct recombinant transformant X33 / pPICZαA-(XYN) 3 Fir...

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Abstract

The invention provides genes, an expression vector, a production bacterial strain and application of xylanase with high temperature resistance, strong base resistance, high efficiency and stability in pulp bleaching. The genes of the xylanase provided in the invention realize high expression in Pichia pastoris by utilizing a codon optimizing and multi-copy technique; the expressed xylanase has no cellulase activity, high temperature resistance and high base resistance; and the xylanase can be applied to wheat straw pulp bleaching; when applied to an elemental chlorine free (ECF) bleaching procedure, the xylanase saves chlorine dioxide by 10%; when applied to a hydrogen peroxide (OP) bleaching procedure, the xylanase saves hydrogen peroxide by 10%.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a gene of a high-temperature-resistant and strong-alkali-resistant xylanase, a production strain and the application of the xylanase encoded by the gene in pulp bleaching. Background technique [0002] The paper industry is one of the important industries supporting our national economy, but it is also one of the important sources of industrial pollution in our country. This is because the pulping and papermaking industry still mainly adopts kraft pulping and chlorine bleaching technology at present, and chlorine bleaching agent is a bleaching agent commonly used in the papermaking industry. Chlorine has long been considered the most cost-effective bleaching agent in pulp bleaching. However, in the traditional chlorine bleaching process, the bleaching wastewater contains a large number of harmful substances to the human body, among which organic chlorides represented by dioxins are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/81C12N1/19D21C9/10C12R1/84
Inventor 林影韩双艳林小琼郑穗平廖超登
Owner SOUTH CHINA UNIV OF TECH
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