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Trichoderma reesei strain for producing cellulase, and applications thereof

A technology of Trichoderma reesei and cellulase, applied in the directions of enzymes, enzymes, fungi, etc., can solve problems such as poor stability, and achieve the effect of improving stability and having broad application prospects.

Active Publication Date: 2018-01-05
QINGDAO VLAND BIOTECH GRP +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There have been nearly 40 years of research on cellulase at home and abroad, and the organisms that produce cellulase are also very extensive. Most of the cellulase is mainly produced by microbial fermentation. Although the activity of cellulase is high, the stability is poor. Both are a major technical problem in the application of cellulase in industrial production. Screening stable cellulase production strains is the fundamental measure to obtain stable cellulase. Therefore, it is urgent to improve the stability of cellulase by transforming strains.

Method used

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  • Trichoderma reesei strain for producing cellulase, and applications thereof
  • Trichoderma reesei strain for producing cellulase, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Construction of Metalloprotease Gene Knockout Cassette

[0017] First, Trichoderma reesei ( Trichoderma reesei ) of the total genomic DNA, the specific operation is as follows:

[0018] Trichoderma reesei was inoculated on PDA medium and cultured for 7 days. Take 1cm×1cm, 12000r bacteria block in a 1.5mL centrifuge tube, add 400μl lysis buffer (60mM Tris-HCl, pH7.8, 20mM Na-Ac, 1mM EDTA, 1.5% SDS), shake vigorously with a bead mill for 2.5min; bathe in water at 65°C for 20min, add 200μl 10M ammonium acetate solution and mix well, bathe in ice for 10min, centrifuge at 12000rpm for 10min, take the supernatant; add an equal volume of phenol to extract Once, centrifuge at 12000rpm for 2 minutes, take the supernatant; add an equal volume of isopropanol to precipitate for 5 minutes, centrifuge at 12000rpm for 5 minutes; wash twice with 70% ethanol; finally dissolve the dried DNA in ddH2O.

[0019] Using Trichoderma reesei genomic DNA as a template to amplify the ...

Embodiment 2

[0035] Example 2 Transformation and screening

[0036] 2.1 Protoplast preparation

[0037] Inoculate the mycelia of auxotrophic Trichoderma reesei FN1 expressing cellulase (the strain was a mutant strain obtained by Wu Jiapeng, an employee of Qingdao Weilan Biological Group through mutagenesis in December 2013) and grow on PDA plates for 4 days; Colonies with a diameter of about 3 cm were placed in about 100 ml of YEG+U (0.5% yeast powder, 1% glucose, 1% uridine) liquid medium, 30 ° C, 200 rpm shaking culture overnight; multi-layer gauze filter to collect mycelia; Place the mycelium in 20 ml of lysing enzyme solution (Sigma L1412) for 2 hours; take out the enzymatic solution, add 0.7 M NaCl solution, shake gently, pour it on three layers of sterilized lens paper to filter, and collect the filtrate. Centrifuge at 3000 rpm for 10 min; discard the supernatant, add 10-20 ml STC solution (20% sucrose, 50mM Tris-Cl, 50mM CaCl2 ) suspension, 3000 rpm, centrifuged for 10 min; add app...

Embodiment 3

[0048] Example 3 Fermentation Verification and Enzyme Activity Determination

[0049] Trichoderma reesei MPASE-FN1 and starting strain Trichoderma reesei FN1 were inoculated in MM fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH 4 ) 2 SO 4 , 0.09%MgSO 4 , 2%KH 2 PO 4 , 0.04%CaCl 2 , 0.018% Tween-80, 0.018% trace elements, 0.018% polypropylene glycol-2000), cultured at 30°C for 48 hours, then cultured at 25°C for 48 hours, and the supernatants were taken for SDS-PAGE electrophoresis analysis and enzyme activity detection . The results of electrophoresis analysis were as figure 1 As shown, the cellulase is indicated by the arrow, indicating that Trichoderma reesei MPASE-FN1 can effectively express cellulase as the starting strain, and the enzyme activity of the fermentation supernatant of the two strains is about 80 U / mL.

[0050] (1) Enzyme activity assay method

[0051] Under the conditions of 50°C and pH 4.8 (neutral is pH 6.0), the...

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Abstract

The invention relates to the technical field of enzyme preparations, particularly to a Trichoderma reesei strain for producing cellulase, and applications thereof. According to the present invention,the Trichoderma reesei MPASE-FN1 strain obtained by knocking down metalloprotease gene has the preservation number CCTCC NO: M2016338, and can significantly improve the stability of the cellulase production; after the 40 DEG C water bath treatment for 40 days, the cellulase product produced by the starting strain Trichoderma reesei FN1 is significantly degraded while only a small amount of the cellulase protein produced by the Trichoderma reesei MPASE-FN1 is degraded, such that the stability of the Trichoderma reesei MPASE-FN1 is significantly superior to the starting strain, and the unexpected technical effect is achieved; and the Trichoderma reesei MPASE-FN1 strain can be widely used for the production of cellulase, and has wide application prospect.

Description

technical field [0001] The invention relates to the technical field of enzyme preparations, in particular to a cellulase-producing Trichoderma reesei strain and an application thereof. Background technique [0002] Cellulose is the most widespread type of carbohydrate in nature, and it is also the largest renewable resource on earth. At present, only a small part of cellulose in nature has been utilized, and the vast majority of cellulose is not only wasted, but also causes environmental pollution. Using cellulase produced by microorganisms to convert cellulose into energy, food and chemical raw materials urgently needed by humans has great practical significance for human society to solve environmental pollution, food shortage and energy crisis. [0003] Cellulase refers to the general term for small molecules that can degrade cellulose to generate cellobiose and glucose, and is a compound induced hydrolytic enzyme. It is generally believed that the decomposition of cellu...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12R1/885
Inventor 许丽红吴佳鹏黄亦钧王华明
Owner QINGDAO VLAND BIOTECH GRP
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