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Trichoderma strain capable of generating cellulase and application of trichoderma strain

A technology of cellulase and cellulose, applied in the field of microbial engineering, can solve the problem that the production of cellulase cannot meet the needs of large-scale industrialization

Inactive Publication Date: 2015-12-23
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in reality, its ability to produce cellulase still cannot meet the needs of large-scale industrialization, especially the application needs of the cellulosic ethanol industry.

Method used

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  • Trichoderma strain capable of generating cellulase and application of trichoderma strain
  • Trichoderma strain capable of generating cellulase and application of trichoderma strain
  • Trichoderma strain capable of generating cellulase and application of trichoderma strain

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1. Determine the optimal irradiation time of Trichoderma reesei (T.reesei) RUT-C30 ultraviolet mutagenesis

[0029] First, fresh RUT-C30 spores were washed with spore eluent and counted with a hemocytometer. Then diluted with spore eluent to 1~2×10 6 individual / mL. Take 2mL of the diluted spore suspension and pour it into the plate, shake gently to make the spore suspension cover the entire plate, and then place it 30cm away from the 20W UV lamp.

[0030] Seven time gradients of 0min (control), 0.5min, 1min, 2.5min, 5min, 15min and 30min were irradiated.

[0031] Dilute the mutated spores 1000 times to 1~2×10 3 1 / mL, respectively take 100 μL of diluted spore liquid in each time period and spread it on the glucose basic medium (+0.5% Triton) plate and culture at constant temperature at 30°C, three groups in parallel. (operated in a dark room)

[0032] After culturing for 4 days, the number of colonies on each plate was counted, and the lethality of RUT-C3...

Embodiment 2

[0034] Embodiment 2. Exploration of Trichoderma reesei (T.reesei) RUT-C30 mutant strain screening conditions

[0035] The screening conditions of Trichoderma reesei (T.reesei) RUT-C30 mutant strain were determined by single factor method, and the concentration of cellulose and Triton in the cellulose double-layer plate were mainly determined. Since T. reesei RUT-C30 is a glucose derepressor strain, 1% glucose was added to the cellulose bilayer plates.

[0036] First consider the concentration of Triton as a fixed value of 1.5%, and determine the concentration of cellulose. Dilute the RUT-C30 spore suspension to 1×10 2 each / mL, and then spread on the Triton double-layer plate containing 1.5%, the cellulose concentration is respectively (0, 0.1%, 0.5%, 1%, 1.5%, 2%) each three parallel, put in 30 ℃ Incubator cultivation. After cultivating for 6 days, the observation results determined that 1.5% cellulose concentration was the best (see figure 2 ).

[0037] Then determine t...

Embodiment 3

[0040] Example 3. Primary screening and secondary screening of Trichoderma reesei (T.reesei) RUT-C30 mutant strain

[0041] Preliminary screening: Pick mutant strains with a larger transparent circle than the starting strain from the cellulose double-layer plate, inoculate them on the PDA slope, number them, and put them in a 30°C incubator to cultivate sporulation.

[0042] Re-screening: wash out the cultivated mutant strains with spore eluent. Take 1 μL and spot it on a cellulose double-layer plate, cultivate it in a 30°C incubator, and use the original strain as a control. After culturing for 7 days, the mutant strain was compared with the original strain, and the strain with a significantly enlarged transparent circle was screened again, and the T. reesei mutant strain numbered CU7-4 was finally determined.

[0043] Purification of single spores: Dilute the spore suspension of the mutant strains screened out to 100 / mL, spread 100 μL of the bacterial solution onto a cellul...

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Abstract

The invention discloses a Trichoderma strain capable of generating cellulase. The Trichoderma strain is obtained by ultraviolet-induced mutation of Trichoderma reesei RUT-C30, named as Trichoderma reesei CU7-4, collected at the China General Microbiological Culture Collection Center on 10th July, 2015 and numbered as CGMCC N0.11065. The invention further discloses application of the Trichoderma strain to fermenting production of cellulase liquid. Experiments confirm that highest filter paper activity of the strain is 8.28IU / mL which is 3.43 times that of an original strain, highest total protein is 4.14mg / mL which is 1.92 times that of the original strain, and it is indicated that the strain has good application prospect in cellulase production.

Description

technical field [0001] The invention belongs to the field of microbial engineering; specifically, the invention relates to a cellulase-producing Trichoderma strain and its application in fermenting and producing cellulase enzyme liquid. Background technique [0002] It is known that Trichoderma reesei (Trichoderma reesei) RUT-C30 (preservation number is ATCC56765) is bred from the wild type strain Trichoderma reesei (T. reesei) QM6a through multiple mutagenesis procedures, and it has a strong cellulose Enzyme production capacity, and release of glucose repression. However, in reality, its ability to produce cellulase still cannot meet the needs of large-scale industrialization, especially the application needs of the cellulosic ethanol industry. Currently reported Trichoderma reesei (Trichodermareesei) RUT-C30 filter paper enzyme activity in shake flasks is only 2.41IU / mL, total protein is only 2.16mg / mL, further improvement is needed. [0003] Ultraviolet light is one of ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12R1/885
Inventor 钟耀华吕珊珊侯运华杨少龙任美斌钱远超闫孟节钟立霞张静
Owner SHANDONG UNIV
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