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Trichoderma reesei mutant strain and application thereof

A technology of Trichoderma reesei and cellulase, which is applied in the field of microbial screening, can solve problems such as being unsuitable for commercial production and low level of enzyme production by Trichoderma strains, and achieve the effects of lowering viscosity, lowering production costs and having broad application prospects.

Active Publication Date: 2015-02-25
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] However, the enzyme production levels of Trichoderma strains directly screened from nature are often very low, which is not suitable for commercial production

Method used

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  • Trichoderma reesei mutant strain and application thereof
  • Trichoderma reesei mutant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The mutagenesis screening of embodiment 1 Trichoderma reesei strain

[0016] 1. Strain treatment:

[0017] Starting bacterial strain Trichoderma reesei L-10 (this bacterial strain is that the inventor of the application, Huang Yijun, screened from the soil of the forest farm in Laoshan District, Qingdao City in 2010) was inoculated on a PDA agar plate (200-300 grams of potatoes, 20 grams of glucose , 15-20 grams of agar, 1000 milliliters of tap water, natural pH), 28-30 ° C constant temperature culture for 1 week to a stable period. After the spores are mature, add 5-10ml of sterile water to the plate to wash the spores, draw the spore suspension and count it, and adjust the concentration to 10 according to the spore content. 5-6 cells / ml, followed by subsequent mutagenesis treatment.

[0018] 2. Nuclear radiation mutagenesis screening:

[0019] Take 50ul of the above-mentioned spore suspension and put it into sterile 1.5ml centrifuge tubes, a total of 100 tubes, and...

Embodiment 2

[0020] Example 2 Screening of high-yield cellulase mutant strains

[0021] 1. Use 24 deep well plate to screen:

[0022] Add 3-4ml of induction medium to each well of the 24 deep-well plate (the composition of the induction medium (TIM) is glucose 15.0g / L, lactose 16.0g / L, corn steep liquor 25.0ml / L, (NH 4 ) 2 SO 4 5.0g / L, MgSO 4 1.0g / L, KH 2 PO 4 20g / L, CaCl 2 0.4g / L, Tween-80 0.2ml / L, trace elements 0.2ml / L, polypropylene glycol-2000 0.2ml / L, NaOH 1.0g / L, pH 5.0. Wherein the composition of trace element solution (g / L) is: CuSO 4 ·5H 2 O 0.048mol / L, FeSO 4 ·7H 2 O 0.18mol / L, CoCl 2 ·6H 2 O 0.034mol / L, MnCl 2 4H 2 O 0.08mol / L). Then the mutant strains screened in Example 1 were inoculated in each well respectively, placed on a shaker, and the culture method was that the fermentation temperature was 28°C, and the shaker speed was 200r / min, and cultivated for 2 days; then cultivated at 25°C for 2 days . After the culture, the supernatant was collected by cent...

Embodiment 3

[0038] Purification of embodiment 3 mutant Trichoderma reesei LA2-7

[0039] Trichoderma reesei LA2-7 was inoculated on a PDA agar plate, and cultured at a constant temperature of 28-30° C. for 1 week to a stable period. After the spores mature, add 5-10ml of sterile water to the plate to wash the spores; then absorb an appropriate amount of spore suspension and spread it on the PDA plate, and finally culture the plate at a constant temperature of 30°C until a single colony grows; pick a single colony , that is, the purified mutant Trichoderma reesei LA2-7 was streaked and inoculated on a PDA plate. At the same time, with the starting bacteria as the control group, the same operation as above was used for cultivation and purification, and a single colony was picked and inoculated on the PDA plate.

[0040] After culturing on the PDA plate for 1 week, it was observed that the colony morphology and mycelium morphology of the starting bacterium L-10 and the mutant bacterium Tric...

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Abstract

The invention provides a trichoderma reesei mutant strain LA2-7 which is capable of highly producing cellulose by use of a radiation induced mutation technology. The collection number of the trichoderma reesei mutant strain LA2-7 is CCTCC NO: 2014564; the enzyme activity of the fermentation supernatant of the mutant strain LA2-7 is 3282U / mL which is increased by 276% in contrast with that of the beginning bacteria, and the protein content of the mutant strain LA2-7 is 12.2g / L which is increased by 237% in contrast with that of the beginning bacteria. Besides, the bacterial colony of the mutant strain LA2-7 is obviously smaller than that of the beginning bacteria, and the hyphae of the mutant strain LA2-7 are thicker and shorter than those of the beginning bacteria; the mutant strain LA2-7 is highly branched so as to reduce the viscosity of a fermentation bacterial liquid in the production process, so that the stirring speed can be reduced, the dissolved oxygen can be increased and the production cost can be effectively reduced; in short, the mutant strain LA2-7 has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbial screening, and in particular relates to a mutant strain of Trichoderma reesei and an application thereof. Background technique [0002] Trichoderma reesei (Trichoderma reesei) is a filamentous fungus, belonging to multicellular eukaryotic microorganisms, is the anamorph of Hypocrea jecorina (Hypocrea jecorina) Hyphospora, Polycystis, Trichoderma. T. reesei is widely distributed in nature, in decayed wood, seeds, plant residues, organic manure, soil and even air (Montenecourt & Eveleigh, 1979, Adv Chem Ser.). [0003] Trichoderma reesei can express a variety of extracellular cellulase and hemicellulase, including exocellulase (CBH1 and CBH2), endocellulase (EG1, EG2, EG3, EG4 and EG5, etc.), beta - Glucosidases, xylanases and mannanases, etc. Through the synergistic action of these cellulase and hemicellulase, the cellulose is completely decomposed into monosaccharides (Biely & Tenkanen.1998, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/42C12R1/885
CPCC12N9/2437C12N1/145C12R2001/885
Inventor 黄亦钧许韦吴佳鹏许丽红李瑞王华明
Owner QINGDAO VLAND BIOTECH GRP
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