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Method for producing xylanase at high yield from trichoderma reesei and application of xylanase

A technology of Trichoderma reesei and xylanase, applied in the field of bioengineering, can solve problems such as hindering the production of recombinant proteins, achieve the effects of improving saccharification efficiency, enhancing expression ability, and reducing production costs

Active Publication Date: 2021-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Heterologous expression of xylanase will be subject to many limitations, such as protein degradation and unfolded protein response caused by insufficient folding, etc., which will hinder the further improvement of recombinant protein production

Method used

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  • Method for producing xylanase at high yield from trichoderma reesei and application of xylanase
  • Method for producing xylanase at high yield from trichoderma reesei and application of xylanase
  • Method for producing xylanase at high yield from trichoderma reesei and application of xylanase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The construction of embodiment 1 recombinant plasmid and Trichoderma reesei recombinant strain

[0068] Using primers P1 and P2, the coding region of XYR1 was amplified using the Trichoderma reesei genome as a template, and connected to the pCAMBIA1301G vector sequence obtained by reverse amplification of primers P3 and P4 through InFusion to construct vector 1 containing the XYR1 expression framework , and use primers P5 and P6 to amplify the region containing the PgpdA promoter and XYR1 coding frame + terminator using vector 1 as a template to obtain the expression frame of XYR1. Next, primers P7 and P8 were used to amplify the URA3 screening marker using vector 2 as a template. Then, using the Trichoderma reesei genome as a template, primers P9 and P10 were used to amplify the upstream of the homology arm of ace1, and primers P11 and P12 were used to amplify the downstream of the homology arm of ace1. The upstream of the homology arm of ace1, the expression frame of...

Embodiment 2

[0072] Efficient secretory expression of embodiment 2 xylanase

[0073] Inoculate 1 μL of Trichoderma reesei spores to the center of the PDA plate and culture at 30°C for 7 days to form sporulation. Spores were collected with saline and diluted to a concentration of 10 7 / mL. Inoculate 0.5mL 10 7 / mL spores into 20mL SDB medium, cultivated at 28°C 200rpm for 40h, then transferred 5mL of mycelia to the cellulose-induced fermentation medium, cultured at 28°C 200rpm for 4-5 days, collected the fermentation broth by centrifugation, and detected the content of the supernatant of the fermentation broth Xylanase activity and xylosidase activity. The xylanase activity in the fermentation broth of the recombinant strain C30OExyr1 / 7IRxyn2Δcbh1 reached 6410U / mL ( Figure 4 A), the SDS-PAGE of the supernatant of the fermentation broth is as follows image 3 shown. In addition to the improvement of xylanase activity, the improvement of xylosidase activity of C30OExyr1 / 7IRxyn2Δcbh1 ha...

Embodiment 3

[0075] Example 3 Straw Saccharification Experiment Using Xylanase Fermentation Broth Cooperating with Commercial Cellulase

[0076] Inoculate 1 μL of recombinant Trichoderma reesei spores to the center of the PDA plate and culture at 30°C for 7 days to form sporulation. Spores were collected with saline and diluted to a concentration of 10 7 / mL. Inoculate 0.5mL 10 7 / mL spores into 20mL SDB medium, cultured at 28°C 200rpm for 40h, then transferred 5mL mycelia to cellulose-induced fermentation medium, cultured at 28°C 200rpm for 4-5 days, centrifuged to collect the fermentation broth, and obtained crude xylanase Enzyme solution.

[0077] The straw saccharification experiment was carried out with the xylanase crude enzyme solution and the commercial cellulase (Ningxia Xiasheng). The amount of straw added in the saccharification experiment was kept at a solid-to-liquid ratio of 5%, that is, 20 mL of the reaction solution contained 1 g of straw, and the total amount of added ...

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Abstract

The invention discloses a method for producing xylanase at high yield from trichoderma reesei and application of the xylanase, and belongs to the technical field of bioengineering. According to the invention, a transcription activator XYR1 is expressed to enhance the xylan degrading enzyme expression ability, and a transcription factor binding site on a promoter Pcbh1 is designed, so that XYR1 of constitutive expression can further activate the artificial promoter Pcbh1, and the artificial promoter Pcbh1 is used for expressing homologous xylanase XYNII to obtain recombinant trichoderma reesei. Under the cellulose induction condition, the expression quantity of the recombinant strain xylanase reaches 6410U / mL, which is 9.28 times higher than that of a parent; meanwhile, the activity of xylosidase is improved by 1.93 times and reaches 9.08 U / mL; and the recombinant strain can relieve carbon metabolism repression, soluble cheap carbon sources glucose and lactose are used as fermentation carbon sources, the activity of the extracellular xylanase of 2514 U / mL and the activity of the extracellular xylanase of 3446 U / mL can also be obtained, and meanwhile, the yield of the xylosidase is increased by 9.3 times and 2.3 times compared with that of the parent. Meanwhile, the xylanase produced with the method cooperates with commercial cellulose to perform straw saccharification, so that the saccharification efficiency can be improved by 35%.

Description

technical field [0001] The invention relates to a method for high-production xylanase in Trichoderma reesei and its application, belonging to the technical field of bioengineering. Background technique [0002] Xylan is a polysaccharide mainly composed of xylose through β-1,4-glucosidic bonds. It is commonly found in plants and is the second largest carbohydrate after cellulose. Different from cellulose, the β-1,4-main chain of xylan is mostly modified by side chain substituents such as acetyl group, arabinofuryl group and glucuronic acid to form heterogeneous xylan. Therefore, the xylan Sufficient degradation requires the synergistic action of multiple enzymes. Xylanase is a general term for a class of hydrolytic enzymes capable of degrading xylan, including endoxylanase, β-xylosidase, arabinofuranosidase, and acetylxylanase. Among them, endoxylanase mainly cuts the main chain of xylan, cooperates with β-xylosidase and other side chain hydrolases, and degrades xylan with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/31C12N9/24C12R1/885
CPCC12N9/248C12N15/80C07K14/37C12N2830/001Y02E50/10
Inventor 喻晓蔚闫肃徐岩
Owner JIANGNAN UNIV
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