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Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof

A galactosidase, alkali-resistant technology, applied in the field of genetic engineering, to achieve the effect of wide action temperature range, wide application, good thermal stability and protease resistance

Inactive Publication Date: 2011-10-19
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the α-galactosidases reported so far are mesophilic or high-temperature enzymes and acid-resistant or neutral pH-resistant enzymes. never reported

Method used

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  • Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof
  • Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof
  • Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: α-galactosidase gene agaAJB13 clone

[0037] Extract the genomic DNA of Sphingomonas: centrifuge the 2-day cultured bacteria liquid to get the bacteria, add 1mL lysozyme, treat at 37°C for 60min, then add the lysate, lyse in a water bath at 70°C for 60min, and mix every 10min. Centrifuge at 10000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0038]According to the conserved sequence of the 36th family of glycoside hydrolases ([F / L / V]-[L / V]-[L / M / V]-D-D-G-W-F and E-P-E-M-[V / I]-[N / S]-[ P / E]) designed and synthesized degenerate primers GH36F and GH36R (Table ...

Embodiment 2

[0043] Example 2: Preparation of recombinant α-galactosidase

[0044] The expression vector pET-28a (+) was double digested ( Eco RI and Hind III), while the gene encoding α-galactosidase agaAJB13 double enzyme digestion ( Eco RI and Hind III), the α-galactosidase digested by the above enzyme agaAJB13 Connect with the expression vector pET-28a (+) to obtain the gene containing α-galactosidase agaAJB13 The recombinant plasmid pET- agaAJB13 And transform Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli strain BL21(DE3) / agaAJB13 .

[0045] Take the recombinant plasmid pET- agaAJB13 of E. coli BL21(DE3) strains and those containing only pET-28a(+) empty plasmid E. coli BL21 (DE3) strain was inoculated in LB (50 μg / mL Kan) culture medium with 0.1% inoculum, and shaken rapidly at 37°C for 16 hours. Then inoculate the activated bacterial solution into fresh LB (50 μg / mL Kan) culture solution with 1% inoculum, and culture with rapid shaki...

Embodiment 3

[0046] Example 3: Activity analysis of recombinant α-galactosidase

[0047] The enzyme activity assay method used p NPG method. Will p NPG was dissolved in 0.1 M buffer to make a final concentration of 2 mM. The reaction system contains 50 μL of appropriate enzyme solution and 450 μL of 2 mM substrate. After the substrate was preheated at the reaction temperature for 5 minutes, add the enzyme solution and react for 10 minutes, then add 1.5mL 1M Na 2 CO 3 The reaction was terminated, and after cooling to room temperature, the released p NP. 1 enzyme activity unit (U) is defined as decomposing per minute p NPG produced 1 μmol p Amount of enzyme required for NP. The method for determining the activity of the substrate raffinose, soybean meal and cotton meal is 3,5-dinitrosalicylic acid (DNS) method: the substrate is dissolved in 0.1M buffer to make the final concentration 0.5% (w / v); the reaction system contains 100 μL of appropriate enzyme solution and 900 μL of subst...

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Abstract

The invention relates to an Alkali-resistant low-temperature alpha-galactosidase AgaAJB13 and genes thereof. The invention provides an alpha-galactosidase AgaAJB13 derived from Sphingobium sp., a coding gene agaAJB13 for encoding the alpha-galactosidase AgaAJB13, a recombinant vector containing the gene agaAJB13 and a recombination strain containing the gene agaAJB13, wherein, the amino acid sequence of the alpha-galactosidase AgaAJB13 is shown in SEQ ID No.1. The alpha-galactosidase AgaAJB13 has the following properties that: the optimal PH is 5.0; the optimal temperature is 60 DEG C, the enzyme activity of more than 10% and the enzyme activity of more than 20% can be achieved respectively at the temperature of 10 DEG C and 20 DEG C; the activity of about 40% can be maintained after processing for 1h at the temperature of 37 DEG C by using a buffer with 0.1M and pH 11.0; and the alpha-galactosidase AgaAJB13has good thermal stability, good proteinase resistance and good hydrolysis to various natural supports, thus being capable of being used as a feed or food additive in fields of feed and food.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an alkaline-resistant low-temperature α-galactosidase AgaAJB13 and its gene. Background technique [0002] α-galactosidase, melibiase (1,6-α-d-galactoside galactohydrolase; α-galactosidase; melibiase; EC 3.2.1.22), removes α-linked terminal non-reducing properties in different substrates D-galactose, including oligosaccharide substrates such as melibiose, raffinose, stachyose and verbascose, and polysaccharide substrates such as galactomannan. These polysaccharides are widely found in food and feed ingredients, especially in the seeds of legume plants, such as soybean meal, cotton meal and rapeseed meal (Karr-Lilienthal et al. Livest Prod Sci, 2005, 97: 1–12.). [0003] α-galactosidase can be used in feed, food, papermaking and medical industries. In the feed industry, α-galactosidase preparations can promote the digestion of nutrients and eliminate or reduce the si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/40C12N15/56C12N15/63C12N1/21C12N1/19
Inventor 黄遵锡董岩岩周峻沛许波唐湘华李俊俊高雅洁潘璐
Owner YUNNAN NORMAL UNIV
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