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L-asparaginase SaLA and coding gene and application thereof

An asparaginase and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of limiting the wide application of L-asparaginase, narrow temperature/pH range of activity, poor stability, etc. The effect of wide reaction temperature, wide reaction pH and high enzyme activity

Active Publication Date: 2021-10-26
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, there are few types of commercial L-asparaginases that people can choose, because most of the reported L-asparaginases have some disadvantages, such as low activity, poor stability, and the temperature at which they can maintain activity. / narrow pH range etc.
These disadvantages limit the wide application of L-asparaginase in medicine and food

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1, Isolation, Identification and Preservation of Halospiral JH

[0021] 1. Separation

[0022] The sample was taken from an alkali lake in the Ordos area of ​​Inner Mongolia. Take 50 μL of the alkali lake sample, add 450 μL of the alkali lake filtrate, and mix well by pipetting to obtain 10 -1 Concentration samples were diluted sequentially to obtain 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 concentrated samples. Take 20 μL, 40 μL of the original solution of the alkali lake water sample and the diluted sample, spread it on the LBH solid medium, culture it in a constant temperature incubator at 35°C for 3-4 days, and observe the growth.

[0023] 2. Identification

[0024] The purified strains were inoculated into LBH solid medium and cultured at a constant temperature of 30°C for 3 days, and the colony color, protrusion, edge regularity, size and transparency of the strains were recorded. The morphology, size and extracellular appendages of the strains were obser...

Embodiment 2

[0034] The preparation of embodiment 2, L-asparaginase (SaLA protein)

[0035] After a lot of sequence analysis, alignment and functional verification, a new protein was found from Halospira JH, which was named SaLA protein, as shown in sequence 1 of the sequence table. The gene encoding SaDL protein in Halospira JH is named as SaLA gene, and its coding frame is shown in sequence 2 of the sequence listing.

[0036] 1. Construction of recombinant plasmids

[0037] 1. Using the genomic DNA of Halospiral JH as a template, PCR amplification is carried out with a primer pair composed of LA-F and LA-R, and the PCR amplification product is recovered.

[0038] LA-F: 5'- CCGG AATTCATGGACACC-3';

[0039] LA-R: 5'-GCCAAGCTTTTACTGGTGCA-3'.

[0040] 2. Take the PCR amplification product obtained in step 1 and connect it to the pET-28a vector to obtain the recombinant plasmid pET-28a-SaLA.

[0041] pET-28a Vector (pET-28a Vector): Anolan (Beijing) Biotechnology Co., Ltd., catalog number...

Embodiment 3

[0054] The enzymatic properties of embodiment 3, L-asparaginase (SaLA protein)

[0055] PBS buffer (50mM, pH 8.0): Weigh 1.44g sodium dihydrogen phosphate, 0.24g potassium dihydrogen phosphate, 0.20g potassium chloride, 8.00g sodium chloride, dissolve in 800mL ultrapure water, adjust pH with HCl to 8.0, set the volume to 1L.

[0056] Substrate solution (25 mM asparagine): Weigh 3.303 g of L-asparagine, dissolve it in PBS buffer, and dilute to 1 L.

[0057] 1. The effect of pH on the activity of L-asparaginase

[0058] 1. Optimal pH

[0059] Take the SaLA protein solution prepared in Example 2, dilute it to 2 times the volume with PBS buffer (50 mM, pH 8.0), and use the diluted solution as the test solution.

[0060] 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 terminate the reaction, centrifuge at 10,000×g for 10 minutes, and th...

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Abstract

The invention discloses L-asparaginase SaLA and a coding gene and application thereof A protein provided by the invention is derived from Salinispirillum sp.JH, is L-asparaginase, is named as the SaLA protein, and is a protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table. The invention also protects the application of the SaLA protein as the L-asparaginase. The L-asparaginase SaLA has great application prospects in the related medical field, the food processing field and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to L-asparaginase SaLA and its coding gene and application. Background technique [0002] L-asparaginase is capable of catalyzing the hydrolysis of L-asparagine to L-aspartic acid and ammonia. In the medical field, L-asparaginases of microbial origin are useful as antineoplastic and antileukemic agents, such as in the treatment of lymphoid malignancies, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma, but their therapeutic utilization is subject to hypersensitivity reactions and other side effects. In addition, in the food field, people have detected acrylamide in fried and baked starchy foods such as French fries, and acrylamide is a neurotoxin, and it was listed as a neurotoxin by the International Agency for Research on Cancer of the World Health Organization in 2017. carcinogen. Studies have shown that L-asparaginase can effectively remove the important precur...

Claims

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Application Information

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IPC IPC(8): C12N9/82C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/82C12N15/70C12Y305/01001Y02A50/30
Inventor 刘建国李静王淼李子一辛文谭雯斐徐莹莹姜雪姣
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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