Novel glucose-resistant beta-glucosidase gene and application thereof

A technology of glucosidase and glucose, applied in the field of genetic engineering, can solve the problems of unfavorable hydrolysis of resveratrol glycosides in industrialized production, narrow stability ranges such as temperature and pH, high price of β-glucosidase and the like, and achieve large-scale industrialization. Production and application potential, good glucose tolerance, good organic solvent resistance

Inactive Publication Date: 2018-03-23
GUANGDONG PHARMA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the β-glucosidase currently on the market mainly comes from fungi such as Trichoderma, Aspergillus, etc., the enzyme activity is low and the stability range of temperature and pH is relatively narrow, resulting in high price of β-glucosidase, which is not conducive to Industrialized Production of Retrol Glycoside Hydrolysis

Method used

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  • Novel glucose-resistant beta-glucosidase gene and application thereof
  • Novel glucose-resistant beta-glucosidase gene and application thereof
  • Novel glucose-resistant beta-glucosidase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0064] 1. Extraction of total DNA:

[0065] (1) Take 4 autoclaved 50ml centrifuge tubes, weigh the cornfield soil samples in the Suihua area of ​​Heilongjiang, add 6g of DNA extraction buffer to each tube, vortex and mix well, put on a shaker at 220rpm, 37 ℃ activation for 30min.

[0066] (2) Add 1.5ml of 20% SDS to each tube to make the final concentration reach 2% (m / V).

[0067] (3) Bath in a water bath at 65°C for 2 hours, gently invert up and down several times every 30mm to mix well.

[0068] (4) 25° C., 6,000 rpm, centrifuge for 10 minutes.

[0069] (5) Transfer the supernatant to an autoclaved 50ml high-speed centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), and gently invert up and down to mix.

[0070] (6) 25° C., 11,000 rpm, centrifuge for 30 minutes.

[0071] (7) Collect the supernatant, add 0.6 times the volume of is...

Embodiment 2

[0126] Example 2 Optimization of Induced Expression Conditions for Recombinant β-glucosidase

[0127] (1) Optimization of induction time

[0128] Pick and verify the correct recombinant strain in 20ml LB liquid medium, culture overnight at 37°C and 200rpm, as the seed solution. Transfer the inoculum solution to 50ml LB liquid medium at a ratio of 1:100, culture at 37°C and 220rpm with shaking until the cell density OD 600 When = 0.8-1.2, add IPTG to make the final concentration 1.0mM, under the condition of 220rpm, culture at 25°C for 8h, 11h, 13h, 15h, 18h, 21h respectively, prepare crude enzyme solution, quantitatively measure β-glucosidase For enzyme activity, the highest enzyme activity was defined as 100, and the optimal induction temperature and induction time were determined. (if attached Figure 4 shown).

[0129] (2) Optimization of IPTG concentration

[0130] The verified recombinant strains were picked and cultured overnight in 20ml LB medium at 37°C and 200rpm a...

Embodiment 3

[0131] Example 3 Recombinant β-glucosidase enzyme activity assay

[0132] (1) Determination of enzyme activity

[0133] The invention uses pNPG as a substrate to detect the enzyme activity of β-glucosidase. Take 10 μl of crude enzyme solution with a certain dilution factor, 80 μl of B-R buffer at the optimum pH, and 10 μl of 50 mM pNPG, mix them, bathe in water for 20 minutes at the optimum temperature, and take 200 μl of 1M Na 2 CO 3 solution to terminate the enzymatic reaction, take 200 μl of the above mixture, and measure the OD 405 . Each enzymatic reaction was assayed in triplicate. The inactivated crude enzyme solution treated under the same conditions was used as blank control. One enzyme activity unit (U) is defined as the amount of enzyme needed to decompose pNPG to produce 1 μmol pNP per minute.

[0134] (2) Drawing of p-nitrophenol standard curve

[0135] Accurately weigh 0.6956g of pNP, dissolve it with 100ml of B-R tri-acid buffer solution with pH 6.10, dil...

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Abstract

The invention discloses a novel glucose-resistant beta-glucosidase gene and an application thereof. The novel glucose-resistant beta-glucosidase gene has a nucleotide sequence shown in SEQ ID NO.1, and has an amino acid sequence shown in SEQ ID NO.2. The invention also discloses a preparation method of the novel glucose-resistant beta-glucosidase gene and a recombinant plasmid pET32a-Bgl2238 containing the novel glucose-resistant beta-glucosidase gene; the invention further discloses a recombinant beta-glucosidase and a preparation method thereof; the recombinant beta-glucosidase gene is over-expressed in an escherichia coli prokaryotic expression system, the recombinant beta-glucosidase is subjected to high-efficiency solution expression in an escherichia coli expression system. The invention also discloses an application of the recombinant beta-glucosidase in hydrolysis of polydatin in polygonum cuspidatum; the recombinant beta-glucosidase obtained by the method is indicated to haverelatively high catalytic activity and glucose tolerance activity and have great industrialized production and application prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a novel glucose-resistant β-glucosidase gene and its encoded product, in particular to obtain a novel β-glucosidase gene and its encoded product from corn soil microorganisms in Heilongjiang by using the metagenomic library screening method. product. Background technique [0002] β-glucosidase (taxonomy number EC 3.2.1.21), also known as β-D-glucoside hydrolase, is one of the three cellulase enzymes, which can hydrolyze the glycosidic bond between hydrocarbon group or aromatic group and sugar group Generates glucose, belongs to the glycoside hydrolase family, and plays a decisive role in cellulose degradation. β-glucosidase has a wide range of applications in industry. It is used in the food industry to improve food flavor; in the pharmaceutical industry, it is used in the transformation of resveratrol glycosides and soybean isoflavone glycosides, the preparation of oligosacchar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12N15/66C12P7/22
CPCC12N9/2445C12N15/66C12N15/70C12P7/22C12Y302/01021
Inventor 李荷张雪玲夏玉林
Owner GUANGDONG PHARMA UNIV
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