A strain of Trichoderma hacinata that utilizes cellobiose to produce β-glucosidase

A technology of glucosidase and Trichoderma hamata, which is applied in the field of β-glucosidase preparation, can solve the problems of low activity, increasing the difficulty of suitable cellulase, and high demand for β-glucosidase, so as to reduce costs and solve The effect of feedback inhibition

Active Publication Date: 2016-07-06
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are already commercialized cellulase reagents, the activity of the key β-glucosidase is not high, and β-glucosidase is far from meeting the requirements when it is compounded with other enzymes, which is mainly due to the β-glucosidase - Problems caused by high demand but low activity of glucosidase, which greatly increases the cost of lignocellulose degradation
In addition, the degradation process of lignocellulose is generally acidic environment, which also increases the difficulty of finding suitable cellulase

Method used

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  • A strain of Trichoderma hacinata that utilizes cellobiose to produce β-glucosidase
  • A strain of Trichoderma hacinata that utilizes cellobiose to produce β-glucosidase
  • A strain of Trichoderma hacinata that utilizes cellobiose to produce β-glucosidase

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Experimental program
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Effect test

Embodiment 1

[0017] The preparation of embodiment 1 bacterial strain of the present invention

[0018] Take the soil collected in the paddy planting area of ​​Jiangyong County, Yongzhou City, Hunan Province as the test material, take 20g of the dried test material soil sample, shake and cultivate at 160rpm / min for 30min, and make 10 -1 double dilution. Then, divide the 10 -1 Dilute the bacterial suspension to 10 times -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 . Spread the dilutions of each treatment evenly on the PDA medium and culture at 28°C for 3 days, pick a single colony for isolation and purification of single spores, and repeat this until the strains are purified, and the strain of the present invention can be obtained.

[0019] The total DNA of the strain of the present invention was extracted by SDS method, and PCR amplification was carried out by using primers ITS1 / ITS4. The total volume of the PCR reaction system is 25 μL: (ITS1 primer volume 1 μL, ITS4 primer volum...

Embodiment 2

[0021] The mensuration of embodiment 2β-glucosidase

[0022] Insert the bacterial strain YYH13 of the present invention into an Erlenmeyer flask of 100mL cellobiose basal medium with an inoculation amount of 1%-2%, and measure the β-glucoside in the fermentation broth after fermentation at 28°C and 180r / min for 48h Enzyme content.

[0023] Result: The glucose standard curve made is as follows Figure 4 As shown, the measured β-glucosidase activity reaches 457.9U / mL, which has a higher β-glucosidase activity.

[0024] The mensuration of β-glucosidase: with salicin as substrate, adopt the method of Chahal (1985), get the crude enzyme liquid of 0.5ml suitably diluted, add 1mL1% salicin solution (0.2M sodium acetate buffer, pH4 .8), react at a certain temperature for 30 min, and use the DNS method (Miller, 1959) to measure the amount of reducing sugar generated. The amount of enzyme needed to produce 1 μmol of reducing sugar per minute was defined as 1 activity unit (IU).

[0...

Embodiment 3

[0026] Example 3 Optimization of Cellobiose Fermentation Process Conditions

[0027] Taking cellobiose as the sole carbon source, and taking the optimum cellobiose concentration, fermentation temperature, fermentation time and rotation speed as the influencing factors, a four-factor and three-level L 9 (4 3 ) Orthogonal optimization test (Table 1), with the fermented liquid β-glucosidase content as an investigation index, determine the best conditions for fermentation. Orthogonal test results are shown in Table 2. According to the extreme difference analysis, the relationship affecting the fermentation performance of the bacterial strain of the present invention is respectively A>D>C>B, and the factors affecting the generation of β-glucosidase are fiber Disaccharide concentration, rotational speed, fermentation time, fermentation temperature, the best combination of each factor is A 2 B 1 C 2 D. 3 . Therefore, the optimum fermentation conditions are: cellobiose concentra...

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Abstract

The invention discloses trichoderma hamatum producing beta-glucosidase through cellobiose. The trichoderma hamatum is classified and named as (Trichoderma hamatum) YYH13 and is conserved in the China Center for Type Culture Collection on June 30th, 2015. The conservation number is CCTCC NO:M2015416. The trichoderma hamatum is a natural bacterial strain producing the beta-glucosidase through cellobiose by means of single fermentation. When the trichoderma hamatum is used for fermenting the 15g / l cellobiose, the activity of the beta-glucosidase is 457.9 U / mL, the feedback inhibition function of the cellobiose on endoglucanase and exoglucanase can be effectively eliminated, and the trichoderma hamatum is crucial in the process that a cellulase system degrades cellulose to generate glucose.

Description

technical field [0001] The present invention relates to the field of preparation of β-glucosidase, in particular to a bacterial strain capable of fermenting cellobiose to produce β-glucosidase, in particular to a Trichoderma badenum that ferments cellobiose to produce β-glucosidase (Trichodermahamatum) YYH13 strain. Background technique [0002] At present, the widely accepted view of cellulase in cellulose degradation is that endoglucanase (EC3.2.1.4) and exoglucanase (EC3.2.1.91) synergistically degrade cellulose to produce a large amount of Cellobiose, the presence of excess cellobiose will cause feedback inhibition of exoglucanases and endoglucanases, reducing the efficiency of the entire cellulase system to degrade cellulose substrates. However, β-glucosidase (EC 3.2.1.21) is capable of degrading cellobiose, thereby releasing feedback inhibition of endo- and exo-glucanases by both cellobiose and cellooligosaccharides. [0003] β-glucosidase is considered to be the key...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/24C12R1/885
Inventor 程鹏
Owner HUNAN AGRICULTURAL UNIV
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