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Gene encoding beta-glucosidase

A technology of glucosidase and gene, applied in the field of enzyme gene, can solve the problems of cloning β-glucosidase gene and less microbial research

Inactive Publication Date: 2009-02-11
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few studies on microorganisms in polluted alkaline soil environments, and there has been no report on the cloning of related β-glucosidase genes in such extreme environments.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. A large amount of metagenomic DNA was directly extracted from soil samples.

[0065] 1) Weigh 4-4.5g of alkaline soil, grind it finely, add 10mL of 2 parts of TENS (each part is: 100mM Tris-HCL, 40mM EDTA, 200mMNaCI, 2% SDS, pH 8.0), in a shaker mix thoroughly;

[0066] 2) Add 100 μL of 25 mg / mL Protease-K (Protease-K), dissolve in a 70°C water bath for 30 minutes, and stir and mix once every 10 minutes;

[0067] 3) Then place it in liquid nitrogen for quick freezing for 2.5 minutes, quickly place it in a low-temperature refrigerator at -20°C for 5 minutes, and immediately dissolve it completely in a boiling water bath after taking it out. Step 3) Repeat 3 times in total;

[0068] 4) Put it still at room temperature for 3 minutes, then centrifuge, collect the supernatant and put it on ice, add 10mL of Buffer A (composition: 50mM Tris-HCL, 25mM EDTA, 3% SDS, 1.2% After PVPP, pH 8.0), centrifuge to collect the supernatant; mix all the supernatants together a...

Embodiment 2

[0073] Example 2. Purification of crude metagenomic DNA by electroelution.

[0074] This method can recover DNA fragments of any size, especially DNA fragments larger than 5kb. Moreover, the success rate is extremely high, and it is easy to purify.

[0075] (1) Treatment of dialysis bags

[0076] A. Cut out a dialysis bag with a length of 10 to 20 cm;

[0077] B, the dialysis bag was boiled in 2% (w / v) (mass / volume) sodium bicarbonate, 1mM EDTA (pH8.0) solution for 10 minutes;

[0078] C. Take out the dialysis bag and rinse thoroughly with distilled water;

[0079] D. Boil in 1mM EDTA solution (pH 8.0) for 10min (do not pour out the solution);

[0080] E. Allow the dialysis bag to cool naturally and store at 4°C (note that during storage, the dialysis bag should always be immersed in the solution to prevent drying;

[0081] F. Before use, fully rinse the inner and outer walls of the bag with distilled water;

[0082] Note: Wear medical latex gloves to operate.

[0083] ...

Embodiment 3

[0093] Embodiment 3, the construction of the genomic library of uncultivated microorganisms in alkaline polluted soil

[0094] Firstly, the modified alkaline denaturation method was used to extract a large amount of plasmid DNA from the cloning vector pGEM-3Zf(+) to obtain relatively high-purity plasmid DNA. Its main form is supercoiled (CCC), which was digested by EcoR I enzyme and turned into a linear DNA molecule. , the linear pGEM-3Zf(+) plasmid DNA was treated with CIP dephosphorylase, and then directly used to connect partially digested soil metagenomic DNA.

[0095] The purified metagenomic DNA was partially digested with EcoR I, the digested products were separated by agarose electrophoresis, these fragments were ligated with pGEM-3Zf(+) DNA treated with CIP dephosphorylase, and the ligated products were transformed by electrophoresis Methods Introduce Escherichia coli DH5 α in the presence of X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside) (40 μg / mL), IPTG (isopropy...

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Abstract

The invention provides a gene of coded beta-glucosaccharase, which is called Unbgl1B and is obtained by constructing Metagenome DNA library of uncultured microorganisms of alkality contaminated soil and by the detecting and screening method of the activity of the beta-glucosaccharase of clone library, so as to be one of the following nucleotide sequences: 1) DNA sequences and partial sequence thereof in sequence 1 of a sequence table; 2) DNA sequences having more than 80% of homoeology compared with the DNA sequences defined by the sequence 1 of the sequence table. The DNA in the sequence 1 of a sequence table is DNA sequences of a pGEM-3Zf(+) part of a cloning vector and DNA of exogenetic uncultured microorganisms cloned on the vector, and the exogenetic DNA segment consists of 838 basic groups; the GC content of the gene is 54.3%. The gene is used for producing the beta-glucosaccharase, so as to dissociate cellobiose into single glucose molecule.

Description

technical field [0001] The invention relates to an enzyme gene cloned from uncultivated microorganisms, in particular to an enzyme gene obtained by screening a metagenomic DNA library of uncultivated microorganisms in alkaline polluted soil. Background technique [0002] Cellulose is the most abundant renewable biomass resource on earth. According to reports, the global annual production of cellulose through photosynthesis is as high as 1.55×10 9 tons, 89% of which have not yet been exploited by humans. Cellulose is a polymer composed of multiple glucose residues connected by β-1,4-glucosidic bonds, and its basic repeating unit is cellobiose. The basic structure of natural cellulose is composed of microfibril bundles composed of fibrils. In natural cellulose, lignin and hemicellulose form a firmly bonded layer that tightly surrounds the cellulose. The utilization and transformation of cellulose is of great significance to solve the world's energy crisis, food shortage, e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12P19/02
Inventor 武波胡婷婷蒋承建
Owner GUANGXI UNIV
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