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Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei

A technology of Trichoderma reesei and cellulase, which is applied in the field of microbial screening, can solve the problems of unsuitability for commercial production and low enzyme production level of Trichoderma strains, and achieve the effects of reducing production costs, reducing stirring speed, and increasing dissolved oxygen

Active Publication Date: 2015-02-04
QINGDAO VLAND BIOTECH GRP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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 capable of producing cellulase in high yield and application of trichoderma reesei
  • Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei

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Effect test

Embodiment 1

[0013] Screening of embodiment 1 bacterial colony " miniaturization " mutant strain

[0014] 1. Strain treatment:

[0015] Trichoderma reesei (this bacterial strain was screened by the inventor of this patent, Huang Yijun, in June 2013 from the dead bark of the Laoshan District forest farm in Qingdao) was inoculated on a PDA agar plate (200-300 grams of potatoes, glucose 20 grams, 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.

[0016] 2. Mutation screening:

[0017] Add the above-mentioned spore suspension into an empty plate, and carry out magnetic stirring (20-550rpm), and then use ultraviolet light to align the plate for mutagenesi...

Embodiment 2

[0019] Example 2 Screening of high-yield cellulase mutant strains

[0020] 1. Use 96 deep well plate to screen:

[0021] Add 2-3ml of induction medium to the wells of a 96-deep well plate, the composition of the induction medium (GLC medium) is CaCl 2 1.125g / L, glucose 10g / L, lactose 20g / L, corn steep liquor 15mlg / L, trace elements 10ml / L, KH 2 PO 4 20g / L, pH5.0; the composition of trace elements (ME) is FeSO 4 .7H 2 O 0.75g / L, ZnSO 4 .7H 2 O 0.06g / L, CuSO 4 .5H 2 O 0.012g / L, MnSO 4 .H 2 O0.053g / L,H 3 BO 3 0.003g / L. Then the mutant strains screened in Example 1 were respectively inoculated into each well, placed on a shaker, 30° C., 200 rpm, and cultured for 3 days. After the culture, the supernatant was collected by centrifugation for enzyme activity detection and SDS-PAGE electrophoresis detection. According to the test results, 4 mutant strains with significantly higher enzyme activity and extracellular protein content than the original strain were screened ou...

Embodiment 3

[0036] Purification of embodiment 3 mutant Trichoderma reesei VLKDN-1

[0037]Trichoderma reesei VLKDN-1 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 VLKDN-1 was 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.

[0038] After cultured on the PDA plate for 1 week, the colony morphology and hyphal morphology of the starting strain and the mutant Trichoderma reesei VLKDN-1 were significantly differen...

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Abstract

The invention provides a trichoderma reesei mutant strain VLKDN-1. The preservation number of the stain VLKDN-1 is CCTCC NO:M 2014506. A trichoderma reesei mutant strain capable of producing cellulase in high yield is obtained by adopting an ultraviolet-induced mutation technology combined with shake flask selection. Compared with a starting strain, the enzyme activity of the fermentation supernatant of mutant strain trichoderma reesei VLKDN-1 is increased by 235%, the content of protein is increased by 157%, the colonial morphology of the mutant strain is obviously smaller than that of the starting strain; the hyphae are shorter than that of the starting strain; the branches are more than that of the starting strain. The viscosity of a fermentation strain solution is remarkably reduced by virtue of the characteristics of short hyphae and more branches of the small colonial morphology of the mutant strain in the production process; the purposes of reducing the stirring rate and improving the dissolved oxygen are achieved; the production cost is reduced; the trichoderma reesei mutant strain VLKDN-1 is wide in application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbial screening, and in particular relates to a high-yield cellulase-producing Trichoderma reesei and its application in cellulase production. 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. Trichoderma reesei is widely distributed in nature, in decayed wood, seeds, plant residues, organic fertilizers, soil and even in the 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 deco...

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 黄亦钧吴佳鹏许丽红李瑞王玉明
Owner QINGDAO VLAND BIOTECH GRP
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