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Application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials

A technology for degrading cellulose and cellulase, applied in the field of bioengineering, can solve problems such as cumbersome steps of Expansin protein, difficult expression and purification of plant expansin, and cooperation with cellulase to improve cellulose degradation efficiency

Inactive Publication Date: 2011-05-25
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purification of a single Expansin protein from the total protein of plants is not only cumbersome and difficult, but also the amount of purification is relatively limited, and it is difficult to express and purify plant expansin in large quantities in bacteria. [11]
This limits the application of Expansin
[0004] The Expansins family of tomato includes LeEXP1, LeEXP2, LeEXP3 and other proteins. LeEXP2 is reported to be expressed in swollen and mature tissues, and participates in the physiological roles of cell elongation and other aspects [12-13] , but it has not been used to synergize with cellulase to improve the degradation efficiency of cellulose

Method used

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  • Application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials
  • Application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials
  • Application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of embodiment 1 tomato LeEXP2 gene

[0034] Use the Trizol reagent to extract the total RNA of tomato according to the kit instructions: get 2ug of total RNA, use the reverse transcription kit to reverse-transcribe it into cDNA (Promega Company), use the cDNA as a template, and use the SEQ ID NO. 1 and the nucleotide sequence shown in SEQID NO.2 carry out PCR for upstream primer and downstream primer, amplify the LeEXP2 gene of 744bp shown in SEQ ID NO.3 in the sequence listing (see figure 1 ). The PCR product was recovered and connected to the TA carrier pGEM-T (Promega Company) to obtain a new plasmid, which was confirmed by sequencing to contain the tomato LeEXP2 gene, and the plasmid was named pGEM-LeEXP2, that is, the plasmid pGEM-LeEXP2 containing the tomato LeEXP2 gene was obtained. build strategy see figure 2 .

Embodiment 2

[0035] Example 2 Construction of LeEXP2 gene to Pichia pastoris expression vector

[0036] Using the plasmid pGEM-LeEXP2 as a template, using the nucleotide sequence shown in SEQ ID NO.4 in the sequence listing as an upstream primer, and using the nucleotide sequence shown in SEQ ID NO.5 in the sequence listing as a downstream primer for PCR amplification, Obtain the 703bp fragment shown in SEQID NO.6 in the sequence listing, and connect the 703bp fragment with the TA cloning vector pGEM-T to transform the transformant obtained from Escherichia coli TOP10F'. It has been identified that the plasmid in the transformant is composed of LeEXP2 gene and pGEM -T connection, the new plasmid obtained from the transformant was named pTLeEXP2, and the plasmid pTLeEXP2 was extracted from the above-mentioned transformant, and the pPICZalpha A plasmid was extracted from Escherichia coli containing the yeast expression vector pPICZalpha A plasmid, and simultaneously enzyme Plasmids pTLeEXP2 ...

Embodiment 3

[0037] Example 3 Integration of LeEXP2 gene into Pichia pastoris chromosome:

[0038] Take 10-15 μg of pPICZalpha A-LeEXP2 plasmid, use PmeI enzyme to cut into the 4219bp fragment shown in the linear SEQ ID NO.9, electrophoresis detection is as follows Figure 7 A 4219bp fragment is generated as shown. After extraction with phenol / chloroform, ethanol precipitates the linear DNA fragment shown in the linear SEQ ID NO.9. After drying, it is dissolved in sterile water to make a linear DNA solution with a DNA concentration of 0.5-1μg / μL ;

[0039] Prepare the competent cells of Pichia pastoris host strain X-33 by referring to the electroporation transformation preparation yeast competent cell method in the refined Molecular Biology Experiment Guide (Fourth Edition) (P512-513), and 80 μL Bath The competent cells of Pichia host strain X33 were mixed with 10 μL of 0.5-1 μg / μL linear DNA solution, then transferred to a pre-cooled electrode cup at 4 °C, ice-bathed for 5 min, and the v...

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Abstract

The invention discloses application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials. Experiments prove that LeEXP2 can cooperate with cellulase to improve the efficiency of cellulose degradation to generate reducing sugar. A lot of waste lignocelluloses exist in the nature, and by adopting LeEXP2 protein to cooperate with cellulase to degrade cellulose, the environmental pollution caused by the methods for pre-treating cellulose through high temperature and high pressure as well as acid and alkali hydrolysis in the prior art is avoided, having fundamental significance in cleanly and efficiently utilizing the waste celluloses to produce energy to solve current energy crisis.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to an application of LeEXP2 to improve the efficiency of cellulase in degrading cellulose materials. Background technique [0002] Cellulose, the main component of plant cell walls, is the largest renewable carbon source on Earth. The use of biotransformation to produce fuel ethanol and other chemicals is of great significance for the current human beings to solve problems such as energy crisis, food shortage, and environmental pollution. The special crystal structure of cellulose makes it difficult for cellulase molecules to approach the glycosidic bonds inside the cellulose molecule, and enzymatic hydrolysis consumes a large amount of cellulase and time, which has become the bottleneck of biomass conversion [1] . At present, the use of chemical reagents or high temperature and high pressure to change the fiber structure is not only costly and time-consuming, but also ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14
Inventor 马媛媛洪解放皱少兰张鲲井欣张敏华
Owner TIANJIN UNIV
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