Beta-galactosidase and application thereof

A technology of galactosidase and nucleotide sequence, applied in the fields of application, glycosylase, enzyme, etc., can solve problems such as protein sequence differences, and achieve the effect of high pH stability and thermal stability

Inactive Publication Date: 2014-11-05
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report that the β-galactosidase gene from Bacillus coagulans has been cloned and expressed, and the protein sequence of this β-galactosidase is very different from that of other reported β-galactosidases

Method used

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  • Beta-galactosidase and application thereof
  • Beta-galactosidase and application thereof
  • Beta-galactosidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: PCR amplification of Bacillus coagulans NL01β-galactosidase gene.

[0027] Genomic DNA of Bacillus coagulans NL01 was extracted using a genome extraction kit from Promega.

[0028] Design primers to introduce the EcoR I and Sal I restriction sites that can be inserted into the pETDuet-1 plasmid (Novagen), the sequences are as follows:

[0029] gal.f: 5'-GAATTCGATGTTAAAAAAACAAGAAAAATTTTATTATG-3'

[0030] gal.r: 5'-GTCGACCTATTTTTCAATTACCTGCAAAATTTTCA-3'

[0031] Using the extracted Bacillus coagulans NL01 genomic DNA as a template, the above primers were used for PCR amplification. The total PCR amplification system was 100 μL, containing 64 μL of ultrapure water, 20 μL of 5×FastPfu PCR Buffer, 0.25 mmol / L of dNTPs, 0.2-0.4 μmol / L of each primer, 50-200 ng of template, and FastPfu DNA Polymerase 5U from TransGen Company. PCR amplification conditions were: pre-denaturation at 95°C for 2 minutes, 30 cycles according to the following parameters (denaturation at...

Embodiment 2

[0032] Example 2: Cloning and sequence determination of PCR products.

[0033] The PCR product of Example 1 was recovered and connected to the pEasy-Blunt cloning vector of TransGen Company, transformed into Escherichia coli Mach1-T1 competent cells, coated with ampicillin plate, screened for positive recombinant strains and sequenced. The sequencing was completed by Beijing Liuhe Huada Gene Company, and the sequence analysis of the sequencing results showed that the PCR product was 1998bp, encoding 665 amino acids, as shown in SEQ ID No:1.

Embodiment 3

[0034] Example 3: Expression and purification of Bacillus coagulans NL01β-galactosidase gene in Escherichia coli

[0035] The correct recombinant strain screened in Example 2 was inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, cultured on a shaker at 37° C. for 8 to 12 hours, and the recombinant plasmid was extracted, digested with EcoR I and Sal I for 1 to 3 Hour. The pETDuet-1 plasmid expression vector was treated with the same digestion method, and the digested product was separated and purified by agarose gel electrophoresis. Use T4 DNA ligase from NEB Company for 1 hour at 22°C and then use CaCl 2 coli BL21 (DE3) competent cells were transformed by the method, ampicillin plates were spread, positive recombinant strains were screened and plasmids were extracted, EcoR I and Sal I double enzyme digestion identified the screened recombinant plasmids. The strain containing the correct recombinant plasmid was named E.coli BL21(pETDuet-gal). At the s...

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Abstract

The invention discloses beta-galactosidase and application thereof. The application method comprises the following steps: cloning beta-galactosidase genes bgl from bacillus coagulans into an expression vector pETDuet-1, transforming the beta-galactosidase genes into Escherichia coli BL21 (DE3), thus obtaining the beta-galactosidase with the expression quantity of 2031U/mg. The expressed beta-galactosidase has the optimal pH value of 6.0 and the optimal temperature of 60 DEG C. The recombinant beta-galactosidase can be used for lactose hydrolysis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a beta-galactosidase and its application. Background technique [0002] β-galactosidase (EC3.2.1.238) scientific name is β-D-galactoside galactohydrolase, commonly known as lactase, this enzyme can catalyze the hydrolysis of galactosidic bonds, hydrolyze lactose to generate glucose and half lactose. Lactose is a unique sugar in mammalian milk. The vast majority of sugar in milk is lactose, but lactose can only be absorbed by the small intestinal mucosa cells of the body after being hydrolyzed by β-galactosidase into glucose and galactose. In most mammals, the activity of β-galactosidase is high after birth, but then the activity gradually decreases with age, and eventually the lactase deficiency leads to lactose intolerance. Asians and Africans are the most prone to lactose intolerance in the world. After ingesting lactose, lactose cannot be absorbed by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12N15/70C12N1/21C12P19/02C12R1/19
CPCC12N9/2471C12P19/02C12P19/14C12Y302/01023
Inventor 郑兆娟石磊欧阳嘉徐颖勇强李鑫
Owner NANJING FORESTRY UNIV
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