Recombinant strain capable of expressing thermostable Beta-galactosidase and construction method and application

A technology of galactosidase and recombinant bacteria, which is applied in the field of applied microorganisms, can solve the problems of poor thermal stability and low reaction temperature, and achieve the effects of wide reaction range, strong tolerance and good enzyme activity

Inactive Publication Date: 2013-04-17
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Generally speaking, most of the enzymes produced in industry have low reaction temperature and poor thermal stability, which have certain limitations in the application of dairy products

Method used

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  • Recombinant strain capable of expressing thermostable Beta-galactosidase and construction method and application
  • Recombinant strain capable of expressing thermostable Beta-galactosidase and construction method and application
  • Recombinant strain capable of expressing thermostable Beta-galactosidase and construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Extraction of total DNA of flora and cloning of thermostable β-galactosidase gene:

[0024] Use the improved CTAB / NaCl method to extract the bacterial flora YTY-70 enriched from Yongtai Hot Spring (use a deep water sampler to take a deep water sample at 2-3 meters from the hot spring at 70°C, and store the sample in an anaerobic bottle immediately. Refrigerated treatment. Use the Hugate anaerobic culture method to add 1% inoculum to 100 mL enrichment medium, change to the screening medium after one week, and transfer for 15 generations to enrich the relevant cellulose-using flora , to obtain the total DNA of the flora YTY-70), using the following degenerate primers: GlaF: 5'-ATG AGT TTT(A,C) CCA AAA GGA TT-3', GlaR: 5'-TTA C(T)GA G(A)TT TTC CTT TAT AT-3' amplifies the gene for the thermostable β-galactosidase enzyme. The PCR reaction system was as follows: 5 min at 95°C; 30 more cycles: 1 min at 95°C; 1 min at 57°C, 2 min at 72°C; and a final extension at 72°C for 10 m...

Embodiment 2

[0026] Construction of heat-resistant β-galactosidase recombinant engineering bacteria:

[0027] The expression vector pGEX-4T-2 was used Bam HI and xho I was subjected to double enzyme digestion, and digested at 37°C for 3h. Large fragments were recovered from agarose gels. The target fragment amplified with embodiment 1 is also used Bam HI and xho I carry out double enzyme digestion, and agarose gel recovers the target gene fragment. Link the recovered gene fragment of β-galactosidase to the expression vector PGEX-4T-2, transform it into Escherichia coli competent cells, spread it on the LB plate containing Amp100ug / ml, and pick 10 positive recombinants Clones were put into fresh LB medium containing Amp100ug / ml and plasmids were extracted. Then double enzyme digestion and sequencing were used to identify whether the target gene was successfully inserted. The cloned plasmids successfully inserted into the target fragments were sent out for sequencing again.

[...

Embodiment 3

[0030] Expression and purification of recombinant thermostable β-galactosidase:

[0031] (1) Pick a single colony containing the recombinant plasmid (containing the heat-resistant β-galactosidase gene), inoculate it in 2×YT medium, and culture it overnight at 37°C and 250 rpm. Transfer the overnight bacteria to 100 mL of fresh 2×YT liquid medium containing 100 mg / mL Amp according to 1% inoculum, cultivate to OD at 37°C, 250 rpm 600is 0.8. Add IPTG to a final concentration of 1 mmol / L, shake and culture at 37°C for 4-6 h to induce expression of the recombinant protein.

[0032] (2) Centrifuge the culture solution at 10,000 rpm for 5 minutes at 4°C, discard the supernatant, collect the cells, and place them on ice. Collect the cells that induce expression.

[0033] (3) Add ice-cold 10 ml PBS buffer per mL of culture. Bacteria were lysed by ultrasonic in an ice bath, ultrasonic condition: 150W, working for 10 s, interval of 10 s, 30 times.

[0034] ⑷ Add 20% Triton X-100 to ...

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Abstract

The invention provides a recombinant strain capable of expressing thermostable Beta-galactosidase, a construction method and application, and belongs to the technical field of applied microbiology. The recombinant strain can express and produce thermal-stability Beta-galactosidase, and is classified and named as Escherichia coli. BL21-LA1, wherein the Escherichia coli. is registered and preservedin China General Microbiological Culture Collection Center on July 27th, 2011, and the preservation number is CGMCC No. 5097. The thermal-stability Beta-glycosidase expressed and secreted by the recombinant bacteria disclosed by the invention has the advantages that: 1, the optimum reaction temperature reaches up to 75 DEG C, the reaction range is very wide, and good enzyme activity is expressed at the temperature of 50-90 DEG C; 2, and the pH range to which the Beta-galactosidase is adaptive is wider (pH 5.0-9.0); and 3, the thermal-stability Beta-glycosidase has high tolerance on detergent,inhibitor and metal ions.

Description

technical field [0001] The invention relates to the technical field of applied microorganisms, in particular to a recombinant bacterium capable of expressing heat-resistant β-galactosidase, its construction method and application. Background technique [0002] β-galactosidase (EC3.2.1.23) scientific name is β-D-galactoside galactohydrolase, and the trade name is lactase (lactase). It is hydrolyzed into galactose and glucose, and has the function of transferring galactoside to generate galactooligosaccharides, which are mainly used in the processing of dairy products. [0003] β-galactosidase mainly comes from animals, plants, bacteria, yeasts, and molds. The β-galactosidases that have been commercialized abroad are mainly derived from Escherichia coli, Aspergillus niger, Aspergillus oryzae, yeast, and Lactobacillus. At present, only β-galactosidase derived from microorganisms has industrial application value. The β-galactosidase is produced by microbial fermentation, whic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/56C12N15/63C12N9/38C12R1/19
Inventor 刘斌刘宗保黄一帆邓云金赵超
Owner FUJIAN AGRI & FORESTRY UNIV
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