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Beta-galactosidase galRBM20_1 and preparation method and application thereof

A technology of galactosidase and BL-21, which is applied in the field of β-galactosidase galRBM20_1 and its preparation, can solve the problems of low practical application value, achieve high pH stability, thermal stability, and good salt tolerance Effect

Active Publication Date: 2019-06-07
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The study of salt-tolerant enzymes is a key part of the enzymatic properties section. Although β-galactosidase has a wide range of sources, few of them have practical application value.

Method used

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  • Beta-galactosidase galRBM20_1 and preparation method and application thereof
  • Beta-galactosidase galRBM20_1 and preparation method and application thereof
  • Beta-galactosidase galRBM20_1 and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of embodiment 1β-galactosidase gene galRBM20_1

[0039] Primers galRBM20_1F1 (5′-ATGGCCTGCCGGCGTAGGGGCGTT-3′) and galRBM20_1R1 (5′-CCACTGGTTGTCCAGGTTTTTCGTA-3′) were designed to amplify β-galactoside by Touch-down PCR using the Yunnan golden monkey faecal microbial metagenomic Fosmid library mixed plasmid as a template Enzyme gene galRBM20_1.

[0040] PCR reaction system: 2×GC buffer Ⅰ 25.0μL, 2.5mmol / L dNTP Mix 4.0μL, LA-Taq DNAPolymerase 1U, 10μmol / L galRBM20_1DNA F11.0μL, 10μmol / L galRBM20_1DNA R11.0μL, Fosmi d mix of metagenomic library Plasmid 0.5 μL, ddH 2 O to make up to 50.0 μL.

[0041] The PCR reaction program was: 94°C for 5 min; 94°C for 30 s; 63°C for 30 s (0.5°C per cycle); 72°C for 1 min, 20 cycles; 94°C for 30 s, 53°C for 30 s, 72°C for 1 min, 10 cycles; ℃ 10min.

[0042] PCR reaction products were analyzed by 1% agarose gel electrophoresis (such as figure 1 ). After a large number of target genes were obtained by PCR, the PCR products were...

Embodiment 2

[0044] Example 2 Construction of Escherichia coli recombinant expression system

[0045] According to the full-length sequence of the β-galactosidase gene galRBM20_1 and the instructions of the one-step rapid cloning kit, the expression primers were designed: galRBM20_1Nde F2 (5′-TAAGAAGGAGATATACATATGGAATTGGCTGCCGGCGTAGGGGCG-3′) and galRBM20_1Xho R2 (5′-GTGGTGGTGGTGGTGCTCGAGACAAATTGCAAAGGAATAT-3′) galRBM20_1NdeF and galRBM20_1XhoR contain recognition sites for restriction enzymes NdeI (CATATG) and XhoI (CTCGAG), respectively. The β-galactosidase gene galRBM20_1 was used as a template, and galRBM20_1 was used.

[0046] NdeF and galRBM20_1XhoR two primers were used for PCR, and the gene fragment galRBM20_1 / NdeⅠ / XhoI containing the recognition sites of endonuclease NdeⅠ and XhoⅠ was obtained. The expression vector pEASY-E2 was cut and linearized with restriction endonucleases NdeI and XhoI. The gene fragment galRBM20_1 / NdeI / XhoI was cloned into the linearized expression vector ...

Embodiment 3

[0048] The mensuration of embodiment 3 optimum temperature and optimum pH

[0049] Dissolve 2-nitrophenyl β-D-galactopyranoside (oNPG) in 0.1mol / L citric acid-disodium hydrogen phosphate buffer (pH 5.0) to prepare a substrate with a concentration of 2mmol / L solution. Take 450 μL of the substrate and preheat it in a 45°C water bath for 5 minutes, add 50 μL of purified and appropriately diluted enzyme solution, and react for 10 minutes. Then add 1mL 1mol / L NaCO 3 Terminate the reaction, and measure the absorbance at a wavelength of 405 nm.

[0050] Definition of enzyme activity unit: One enzyme activity unit (U) is the amount of enzyme required to hydrolyze the substrate oNPG to generate 1 μmol oNP per minute at pH 5.0 and 45°C.

[0051] In buffer solution with pH 7.0, at different temperatures (0°C, 10°C, 20°C, 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C) The enzyme activity of β-galactosidase galRBM20_1 was determined.

[0052] Prepare different buffers with pH 2...

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Abstract

The invention discloses beta-galactosidase galRBM20_1 and a preparation method and application thereof. The amino acid sequence of the beta-galactosidase galRBM20_1 is as shown in SEQ ID NO.1. Variousenzyme genes are obtained from excrement and urine microorganisms of Yunnan snub-nosed monkeys, and a metagenome Fosmid library of the excrement and urine microorganisms of the Yunnan snub-nosed monkeys is constructed. The beta-galactosidase gene galRBM20_1 is obtained through cloning from the metagenome of the excrement and urine microorganisms of the Yunnan snub-nosed monkeys, and after heterologous expression of escherichia coli, enzymatic property and salt tolerance are researched. The recombinase has higher pH stability and heat stability, and besides, also has favorable salt tolerance.A new source is provided for obtaining the beta-galactosidase gene galRBM20_1, and through efficient recombinant expression, a large number of beta-galactosidase genes galRBM20_1 are provided for industrial production, and can be widely applied to dairy product trade.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a beta-galactosidase galRBM20_1 and a preparation method and application thereof. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12P19/14C12P19/02
Inventor 许波张文洪黄遵锡杨云娟李俊俊唐湘华周峻沛
Owner YUNNAN NORMAL UNIV
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