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Recombinant beta-galactosidase and construction method and application thereof

A technology of galactosidase and construction method, applied in the field of recombinant β-galactosidase and its construction, can solve problems such as loss of enzyme activity, contamination by miscellaneous bacteria, difficulty in separation and purification, etc. The effect of broad market prospects

Active Publication Date: 2020-08-28
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, β-galactosidase has poor stability, is prone to bacterial contamination, and is difficult to separate and purify, which will increase the initial cost of production and cause loss of enzyme activity.

Method used

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  • Recombinant beta-galactosidase and construction method and application thereof
  • Recombinant beta-galactosidase and construction method and application thereof
  • Recombinant beta-galactosidase and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction of recombinant expression plasmid

[0025] The β-Gal gene comes from ( Lactobacillus sp.B164, accession number JF345715.1), the β-Gal nucleotide sequence synthesized by the whole gene of the biological company, and the 5' and 3' were respectively equipped with Spe I and Nhe I, and the whole gene was synthesized with Spe I The β-Gal nucleotide sequence of the Nhe I restriction site was constructed on the pet28a (+) plasmid, named pet28a (+)-β-Gal.

[0026] Biological company synthesized the Linker-ELP nucleotide sequence from the whole gene, with Nhe I restriction site at the 5' of linker, Hind I restriction site at the 3' of ELP, and Sac I restriction site between Linker and ELP site. Then the above sequence was constructed on the pUC18 plasmid, named as pUC18-Linker-ELP plasmid.

[0027] Use Nhe I and Hind I to double-digest pet28a(+)-β-Gal and PUC18-Linker-ELP, and the double-digestion system is:

[0028] NheI: 1 μl;

[0029] Hind I: 1 ...

Embodiment 2

[0040] Example 2: Expanded culture of Escherichia coli expressing β-Gal-LEH

[0041] (1) Transfer freshly transformed recombinant Escherichia coli to solid LB medium supplemented with 50 μg / mL kanamycin (agar powder 15g / L, tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, pH7.4), and cultured at 37 ℃, and then transfer the single colony after overnight culture to 5ml liquid LB medium containing 50 μg / mL kanamycin (tryptone 10g / L, Yeast extract 5g / L, sodium chloride 10g / L, pH 7.4), and cultured overnight at 37°C in an orbital shaker at 200 rpm, then inoculated 3 mL of overnight cultured bacteria into 300 mL containing 50 μg Grow the liquid LB medium of kanamycin / mL at 37 °C, 200 rpm for 3 hours or the OD600 of the bacteria reaches between 0.4-0.6.

[0042](2) Place the liquid medium finally obtained in step (1) on ice for 20 minutes, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to it to a final concentration of 0.4 mM, Then shake culture at 180 rpm at 16°C, 25°C ...

Embodiment 3

[0045] Example 3: Purification of β-Gal-LEH by Reversible Phase Change Cycle (ITC)

[0046] In this example, a reversible phase transition cycle was used to purify β-galactosidase, so as to verify the self-purification performance and purification efficiency of β-galactosidase prepared in Example 2. An appropriate concentration of 1.0-2.0 M (NH 4 ) 2 SO 4 Add to 500 μL of clarified sample lysate, then shake at 1000 rpm at 25 °C for 20 minutes, then spin the sample at 14,000 rpm at 25 °C for 30 minutes, discard the supernatant, and resuspend the pellet by pipetting extensively. Suspend in 1 mL of cold Tris-HCl buffer (50 mM, pH 8.0). The samples were then placed on ice for 60 minutes, followed by centrifugation at 13,000 rpm for 30 minutes at 4 °C to obtain the supernatant containing the purified protein of interest, which was transferred to a new EP tube. Save a portion of the purified sample for SDS-PAGE analysis.

[0047] figure 2 for different concentrations (NH 4 )...

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Abstract

The invention provides recombinant beta-galactosidase and a construction method and application thereof, and belongs to the technical field of bioengineering. The recombinant beta-galactosidase beta-Gal-LEH is constructed in the invention, and beta-Gal and ELP (VPGVG) 50 are connected to construct the recombinant beta-galactosidase beta-Gal-LEH which can be self-purified and has good stability andhigh lactose hydrolysis efficiency.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically includes a recombinant β-galactosidase and its construction method and application. Background technique [0002] Lactose is a disaccharide composed of D-glucose and β-D-galactose monomers, mainly found in dairy products such as milk, and β-galactosidase (β-galactosidase, [EC 3.2.1.23]) is the hydrolysis The main enzyme needed for lactose, which is expressed in high levels in human infancy but declines with age. [0003] The widespread prevalence of lactase deficiency is a concern, and lactose indigestion and malabsorption, which often results in lactose indigestion and malabsorption in people who are deficient in consuming milk or other dairy products, can cause abdominal pain, bloating, inflammation, flatulence, and Diarrhea and other symptoms. In order for patients with lactase deficiency to consume dairy products containing lactose, it is necessary to hydrolyze the l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/38C12N15/62C12N15/70C12N1/21C12R1/19
CPCC12N9/2471C12N15/70C12Y302/01023C07K2319/21C07K2319/00
Inventor 周阳施海锋高力顾杰王莹莹任真
Owner JIANGSU UNIV
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