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High-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and application thereof

A technology of glucanase and Trichoderma harzianum, which is applied in the biological field, can solve the problems of low yield of target protein, cumbersome protein purification process, limited source of inducers, etc., and achieves the effect of strong protein expression and secretion ability and improved positive rate.

Active Publication Date: 2018-11-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using natural strains to ferment and produce α-1,3-glucanase has problems such as low yield of target protein, limited sources of inducers, and cumbersome protein purification process.

Method used

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  • High-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and application thereof
  • High-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and application thereof
  • High-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. A recombinant bacterium that efficiently secretes and expresses Trichoderma harzianum α-1,3-glucanase and its application in the production of α-1,3-glucanase

[0065] 1. Construction of Trichoderma harzianum α-1,3-glucanase expression vector

[0066] 1. Optimization of Trichoderma harzianum α-1,3-glucanase gene

[0067] According to the codon preference of Trichoderma reesei, codon optimization was carried out on the α-1,3-glucanase gene mutAW of Trichoderma harzianum CCM F-340, and the α-1,3-glucanase gene mutAW of Trichoderma harzianum The 6His-tag is added to the C-terminus of the glycanase to facilitate subsequent protein purification. The optimized coding gene sequence is shown in sequence 1, wherein the 1-111th position of sequence 1 is the signal peptide coding gene sequence, and the 1903-1920th position is 6His -tag encoding gene sequence.

[0068] 2. Preparation of vector backbone fragments containing cbh1 promoter and cbh1 terminator

[0069] Th...

Embodiment 2

[0109] Example 2. Activity determination and enzymatic properties of Trichoderma harzianum α-1,3-glucanase

[0110] 1. Activity determination of Trichoderma harzianum α-1,3-glucanase

[0111] 1. Preparation of water-insoluble dextran mutan

[0112] (1) Inoculate S.mutans UA159 into BHI medium at an inoculation amount of 1 / 100, and culture statically under anaerobic conditions at 37°C;

[0113] (2) Transfer the overnight cultured S.mutans UA159 to fresh BHI medium at an inoculum size of 1 / 50, and culture it under anaerobic conditions at 37°C for 24 hours;

[0114] (3) Centrifuge at 12,000g for 10min, remove the bacteria, collect the supernatant, and filter the supernatant with a 0.22um sterile filter;

[0115] (4) Then add sucrose to the supernatant to a final concentration of 3%, mix well, and place at 37°C for 48 hours;

[0116] (5) Centrifuge at 12,000 g for 30 minutes, discard the supernatant, collect the precipitate, and wash the precipitate repeatedly with sterile wate...

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Abstract

The invention discloses a high-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and an application thereof. The high-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and the application thereof are characterized in that alpha-1, 3-glucanase genes from trichoderma harzianum CCM F-340 conduct codon optimization; with pyr4 genes used as screening marker genes, trichoderma reesei cbh1 strong promoter and signal peptide sequence thereof and cbh1 terminator are used to construct alpha-1, 3-glucanase expression vector pSK-mutAW; highly secreted expression of the trichoderma harzianum alpha-1, 3-glucanase in trichoderma reesei is achieved. Experiments show that the high-yield recombinant strain of trichoderma harzianum alpha-1, 3-glucanase and the application thereof have the advantages that recombinant expression of the trichoderma harzianum alpha-1, 3-glucanase can effectively degrade non-water-soluble glucan mutan with fermentation enzyme activityunit reaching 2.3 milliliters and alpha-1, 3-glucanase content in fermentation broth being about 68 micrograms per milliliter; fermentation process and target protein purification process of the sameare simple and very suitable for industrial production and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant bacterium with high production of Trichoderma harzianum α-1,3-glucanase and application thereof. Background technique [0002] Oral dental plaque is a highly diverse and complex biofilm, and dental plaque is an important cause of oral diseases such as gingivitis, periodontitis, and dental caries. At present, commonly used plaque control methods include mechanical methods and pharmaceutical methods. Mechanical methods mainly include brushing teeth and using dental floss and toothpicks. The drug rule is to utilize some chemical drugs to remove or inhibit the formation of dental plaque. The main chemical drug currently on the market is chlorhexidine. Chlorhexidine has good antibacterial activity, but long-term use can easily lead to the destruction of beneficial bacteria in the oral cavity and the accumulation of drug-resistant bacteria. [0003] Polysaccha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/56C12N9/24C12P19/14C12R1/885
CPCC12N9/2405C12P19/14C12Y302/01084
Inventor 董志扬张吓妹陈秀珍林洁
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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