L-asparaginase as well as encoding gene and application thereof

A coding gene and coding technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of lack of characteristic L-asparaginase products, etc., and achieve the effect of reducing the production amount and good thermal stability

Inactive Publication Date: 2014-05-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are fewer research reports on the enzymatic properties and applications of L-asparaginase derived from fungi, and only one patent discloses the gene sequence and preparation method of L-asparaginase deriv

Method used

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  • L-asparaginase as well as encoding gene and application thereof
  • L-asparaginase as well as encoding gene and application thereof
  • L-asparaginase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the acquisition of L-asparaginase gene

[0048] 1. PCR amplification of the conserved region of the L-asparaginase gene

[0049] (1) According to the amino acid sequence of L-asparaginase reported in GenBank, use the online software BlockMaker (http: / / blocks.fhcrc.org / blocks / blockmkr / make_blocks.html) to search for the conserved region, and then use the online primer design Software CODEHOP (http: / / blocks.fhcrc.org / codehop.html) designed degenerate primers AsnDF (forward primer): 5'-TCGTCCTGCACGGACNGAYACNATG-3', AsnDR (reverse primer): 5'-CGTTGCCGGCGCCAAANGTYTC-3' .

[0050] (2) Genomic DNA of Rhizomucor miehei CAU432 was used as template, AsnDF and AsnDR were used as primers, and Ex Taq DNA polymerase was used to amplify to obtain PCR amplification products.

[0051] PCR reaction conditions: 94°C for 5min; 94°C for 30s, 60°C-55°C (0.5°C for each cycle) for 30s, 72°C for 1min, a total of 10 cycles; 94°C for 30s, 55°C for 30s, 72°C for 1min, a total of 20...

Embodiment 2

[0067] Embodiment 2, expression of recombinant L-asparaginase

[0068] 1. Construction of recombinant strains

[0069] (1) Primer design

[0070] Upstream primer RmAsnAF: 5'-GGGTTT CATATG GATTCGAGAACGACTGCT-3';

[0071] The underlined sequence is the recognition site for Nde I digestion;

[0072] Downstream primer RmAsnAR: 5'-ATTCCG CTCGAG TTCTTTTCCTAGAAGTTGTGC-3';

[0073] The underlined sequence is the Xho I restriction recognition site.

[0074] (2) Extract the mRNA of Rhizomucor miehei CAU432 and reverse transcribe it into cDNA, use the cDNA as a template, and use RmAsnAF and RmAsnAR as primers to carry out PCR amplification to obtain the PCR amplification product, and the agarose gel electrophoresis image of the product Such as figure 1 shown. figure 1 Among them, M is a DNA marker, and 1 is a PCR amplification product.

[0075] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 7...

Embodiment 3

[0092] Embodiment 3, the property of recombinant L-asparaginase (RmAsnase)

[0093] 1. The optimal reaction pH and pH stability of RmAsnase

[0094] Determination of the optimum reaction pH: the RmAsnase prepared in Example 2 was used as the enzyme solution to be tested, and the enzyme activity of L-asparaginase was measured.

[0095] The only difference lies in the use of the following different buffers (both at a concentration of 50mmol / L): citric acid buffer (pH3.0-6.0), MES (2-(N-morpholine)ethanesulfonic acid) buffer ( pH5.5-6.5), phosphate buffer (pH6.0-8.0), Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid) buffer (pH7.5-9.0), CHES (2-cyclohexylamino ethanesulfonic acid) buffer (pH8.0-10.0). The same amount of RmAsnase was dissolved in the same volume of five buffer systems with different pH values. Then the enzyme activity of L-asparaginase was measured at 45°C, and the highest enzyme activity was taken as 100%, and the relative enzyme activity in each syst...

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Abstract

The invention discloses L-asparaginase as well as an encoding gene and application thereof. A protein disclosed by the invention is shown in the following formula (1) or (2): (1) the protein is a protein shown in SEQ ID No.2; and (2), the protein is a protein which is obtained through substitution and/or deficiency and/or addition of one or more amino acid residues on an amino acid sequence shown in SEQ ID No.2 and has the same function. The L-asparaginase disclosed by the invention is suitable for being used as a food additive applied to bakery products so that the production amount of acrylamide as a cancerogen in the product is reduced, and is also suitable for being used as a medicine applied to adjuvant therapy of leucocythemia.

Description

technical field [0001] The invention relates to an L-asparaginase, its coding gene and application. Background technique [0002] L-asparaginase (EC3.5.1.1, L-asparaginase), also known as L-asparagine amidohydrolase, can specifically hydrolyze asparagine to generate L-aspartic acid and ammonia, which can be widely used In food, medicine and other fields. In the food industry, L-asparaginase can be used as a high-efficiency food additive. L-asparagine and reducing sugar contained in baked food raw materials can generate carcinogenic acrylamide through Maillard reaction during high-temperature baking. Adding L-asparaginase to baked food raw materials can hydrolyze L-asparagine therein, thereby reducing the amount of acrylamide in baked foods and improving the safety of such foods. In terms of medicine, L-asparaginase can be used as a drug for treating cancer. L-asparagine is a kind of nutrient required for cell proliferation. Normal cells have the ability to synthesize L-asp...

Claims

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Application Information

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IPC IPC(8): C12N9/82C12N15/55C12N15/70C12N1/21A21D8/04A61K38/50A61P35/00A61P35/02C12R1/645
CPCA21D2/26A61K38/00C12N9/82C12Y305/01001
Inventor 闫巧娟黄林华江正强杨绍青刘昱
Owner CHINA AGRI UNIV
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