Beta-glucosidase mutant and application thereof

A glucosidase and amino acid technology, applied in the field of β-glucosidase mutants, can solve the problems of low activity and low β-glucosidase content, and achieve the effect of a wide range of applications

Active Publication Date: 2015-03-25
QINGDAO VLAND BIOTECH GRP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the content of β-glucosidase in the cellulase component is low and the

Method used

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  • Beta-glucosidase mutant and application thereof
  • Beta-glucosidase mutant and application thereof
  • Beta-glucosidase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Cloning of β-glucosidase gene and construction of its mutant

[0019] 1.1 Extraction of Aspergillus fumigatus total genomic DNA

[0020] Incubate Aspergillus fumigatus overnight, place an appropriate amount of bacteria in a centrifuge tube, centrifuge at 13000 rpm for 5 min, discard the supernatant; add 400 μl extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl, 1% SDS) ; Then add 100mg quartz sand or glass beads, vigorously shake in the beading instrument for about 2min; after 20min at 65℃, add 200μl 10M NH4AC, ice bath for 10min; centrifuge at 13000rpm for 10min, take the supernatant; add 2 times the volume of absolute ethanol, Place at -20°C for 30 minutes; centrifuge at 13000 rpm for 10 minutes, discard the supernatant; wash twice with 70% ethanol; dry, add water to dissolve, and store at -20°C.

[0021] 1.2 Gene cloning

[0022] Using the total genomic DNA extracted in 1.1 as a template, PCR amplification was performed using primers BG-F and BG-R:

[002...

Embodiment 2

[0033] Example 2 Transformation and Screening

[0034] Pipette the spore suspension of Trichoderma reesei strain in the center of the PDA plate (9cm petri dish). When the colony grows over the entire petri dish, cut the medium of 1cm×1cm each, and place it in 120mL YEG+U liquid medium at 30℃ , 200rpm, culture for 14-16h.

[0035] Collect the mycelium with a sterile Miracloth filter cloth and wash it once with solution A. Transfer the washed mycelium to 40mL protoplastization solution under aseptic conditions, incubate at 30℃, 90rpm, for 1-2h, observe with a microscope Detect the progress of protoplast transformation.

[0036] Filter the above warm bath liquid with a sterile Miracloth filter cloth, and the obtained filtrate is the protoplast solution. Divide the protoplast solution into two 50 mL sterile disposable centrifuge tubes, and dilute the volume of each tube to 45 mL with solution B, centrifuge at 3000 rpm for 10 min, discard the supernatant to obtain a precipitate; use 5 m...

Embodiment 3

[0054] Example 3 Fermentation verification

[0055] The host bacteria of Trichoderma reesei, Trichoderma reesei TR-BG5 and Trichoderma reesei TR-BG that have not been introduced into foreign genes were respectively inoculated into 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) cultured at 30°C for 48 hours, and then at 25°C for 48 hours. The fermentation broth was centrifuged separately, and the supernatant was taken for SDS-PAGE electrophoresis detection. The result is figure 1 As shown, the theoretical molecular weight of the β-glucosidase derived from Aspergillus fumigatus in the present invention is about 95KDa, namely figure 1 The position indicated by the middle arrow, lane 1 is the host bacteria fermentation supernatant, which has almost no protein band at the position indicated by the arrow, while the T. reesei TR-BG in lane 2 and the T. r...

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Abstract

The invention aims at providing a beta-glucosidase mutant and application thereof. The amino acid sequence of the beta-glucosidase mutant is as shown in SEQ ID NO: 3, a nucleotide sequence of a coding gene is as shown in SEQ ID NO: 4, the optimum operative temperature is 50 DEG C and enzyme activity can be kept above 80% within 30-60 DEG C, and the mutant is stronger than a wild type in heat resistance; the optimum operative pH is 5.0 and the enzyme activity can be kept above 70% within 3.0-6.0, and the mutant is wider than the wild type in application scope under an acid condition; therefore, the beta-glucosidase mutant is conducive to the application in a cellulose raw material degrading process.

Description

Technical field [0001] The invention belongs to the technical field of gene screening and transformation, and specifically relates to a β-glucosidase mutant and its application. Background technique [0002] β-glucosidase, also known as β-D-glucoside glucohydrolase, is also called gentiobiase, cellobias (CB or β-G) and amygdalase. It belongs to the cellulase class and is an important component of the cellulolytic enzyme system. It can hydrolyze the non-reducing β-D-glucose bond bound to the terminal and release β-D-glucose and the corresponding ligand at the same time. [0003] Cellulose is a D-glucopyranose polymer connected by β-1,4-glycosidic bonds, which is the most abundant and renewable resource on earth. It is estimated that 1.5×101.1 billion tons of cellulose are produced by photosynthesis on the earth every year (Ryu, D.D, etc., 1980, Enzyme and Microbial Technology). In most cases, to make high-value utilization of cellulose, it must first be effectively degraded by cel...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/80C12N1/15C12R1/885
CPCC12N9/2445C12Y302/01021
Inventor 张青李宾曹体爽董计巧唐波黄亦钧王华明
Owner QINGDAO VLAND BIOTECH GRP
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