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L-asparaginase XiDL and coding gene and application thereof

An asparaginase, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of high tolerance, wide reaction pH, high enzyme activity

Active Publication Date: 2019-09-20
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] But currently, only several L-asparaginases are available in the food industry, such as and Both are from Aspergillus sp., with the deepening of the research and application of L-asparaginase, seek novel L-asparaginases with different activities and properties, which is beneficial to expand its Applications in the medical field and food industry

Method used

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  • L-asparaginase XiDL and coding gene and application thereof
  • L-asparaginase XiDL and coding gene and application thereof
  • L-asparaginase XiDL and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, Isolation, Identification and Preservation of Xingfangfangbacterium DLY26

[0037] 1. Separation

[0038] Take about 1ml of activated sludge sample (taken from the petrochemical refinery sewage treatment system), and dilute to 10 times volume, 20 times volume, 50 times volume, 100 times volume and 1000 times volume in turn with sterile distilled water. Spread 100 μl of the diluted sample on the surface of the solid medium by the coating plate method, incubate at 30°C, and observe the growth of the colonies on the surface every day. Pick colonies with different colors and shapes, and purify and culture them by the three-section line method until a single colony with the same shape and size can be observed on the surface of the culture medium.

[0039] 2. Identification

[0040] The purified bacterial strains were inoculated on TSA plates, cultivated at 30°C for 24h, and then observed the morphology, size and other characteristics of the cells using a transmi...

Embodiment 2

[0057] The preparation of embodiment 2, L-asparaginase (XiDL protein)

[0058] After a large number of sequence analysis, alignment and functional verification, a new protein was found from DLY26 of Xingfangxiang bacteria, and it was named as XiDL protein, as shown in sequence 1 of the sequence table. The gene encoding the XiDL protein in Xylorrhizae DLY26 is named XiDL gene, and its coding frame is shown in sequence 2 of the sequence list.

[0059] 1. Construction of recombinant plasmids

[0060] 1. Using the genomic DNA of A. syringae DLY26 as a template, a primer pair composed of DL-F and DL-R is used for PCR amplification, and the PCR amplification product is recovered.

[0061] DL-F: 5'- CACC GCAGAAAAATCGCACATCT-3';

[0062] DL-R: 5'-CGCGTGAATGGGAACAGGCG-3'.

[0063] 2. Take the PCR amplification product obtained in step 1 and connect it to the pDE1 vector to obtain the recombinant plasmid pDE1-XiDL.

[0064] The pDE1 vector (pDE1Vector) is a component of the pDE1 D...

Embodiment 3

[0077] Embodiment 3, the enzymatic property of L-asparaginase (XiDL protein)

[0078] Tris-HCl buffer solution (50mM, pH 7.4): Weigh 6.06g Tris, dissolve it in ultrapure water, and adjust the pH to 7.4 with HCl.

[0079] Substrate solution (25 mM asparagine): Weigh 3.303 g of L-asparagine, dissolve it in Tris-HCl buffer, and dilute to 1 L.

[0080] 1. The effect of pH on the activity of L-asparaginase

[0081] 1. Optimal pH

[0082] Take the XiDL protein solution prepared in Example 2, dilute it to 2 times the volume with buffer, and use the diluted solution as the test solution.

[0083] Detection method: Add 200 μL of test solution and 700 μL of substrate solution (prepared with pH buffer solution), react at 37°C for 15 minutes, add 100 μL of 15% TCA (trichloroacetic acid) to stop the reaction, centrifuge at 10000g for 10 minutes, and then take the supernatant 200μL, add 4.8mL ammonia-free water and 200μL Nessler's reagent in turn, let stand at room temperature for 10min,...

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Abstract

The invention discloses L-asparaginase XiDL and a coding gene and application thereof. The protein, L-asparaginase XiDL, provided herein is originated from Xinfangfangia sp., and is named as XiDL protein, which is a protein composed of an amino acid sequence shown as a sequence 1 in a sequence table, or a protein composed of an amino acid sequence shown as a sequence 3 in the sequence table. Invention also claims application of the XiDL protein as L-asparaginase, as well as application of the XiDL protein in treatment of acute lymphocytic leukemia, lymphoid malignancies, Hodgkin lymphoma and the like. The invention also claims a preparation to prevent formation of acrylamide in oil-fried or baked foods, with the preparation having the XiDL protein as an active ingredient. The L-asparaginase XiDL has a major application prospect in the related medical field, food industry field and other fields.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to L-asparaginase XiDL and its coding gene and application. Background technique [0002] L-asparaginase (L-asparagine amidohydrolase, E.C 3.5.1.1) catalyzes the hydrolysis of L-asparagine to produce L-aspartic acid and ammonia. In the medical field, L-asparagine of microbial origin Amidases are used as antineoplastic and antileukemic agents, for example, in the treatment of lymphoid malignancies, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma, and are also one of the main drugs in the treatment of acute lymphoblastic leukemia, but their therapeutic utilization is subject to Adverse complications of hypersensitivity reactions and some other toxic side effects are hampered. In the field of food, in April 2002, researchers from the Swedish National Food Administration and Stockholm University took the lead in detecting acrylamide in fried and grilled starchy foods s...

Claims

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Application Information

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IPC IPC(8): C07K9/00C12N9/82C12N15/62C12N15/55A61K38/50A61P35/00A61P35/02A23L29/00
CPCA61K38/50A23L29/06A61P35/00A61P35/02C07K2319/00C12N9/82C12Y305/01001
Inventor 刘建国郗丽君刘德健窦珂谭雯斐贾庆珠苏石晶
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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