L-asparaginase sala and its encoding gene and application

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: 2022-07-12
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 its encoding gene and application
  • L-asparaginase sala and its encoding gene and application
  • L-asparaginase sala and its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Separation, identification and preservation of Halospira JH

[0021] 1. Separation

[0022] The sample was taken from an alkaline lake in the Ordos area of ​​Inner Mongolia, 50 μL of the alkaline lake sample was taken, 450 μL of the alkaline lake filtrate was added, and 10 was obtained by pipetting and mixing. -1 concentration samples, diluted sequentially to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 concentration of the sample. Take 20 μL, 40 μL of alkaline lake water sample stock solution and the samples obtained by dilution, spread them into LBH solid medium, and cultivate them in a constant temperature incubator at 35 °C for 3-4 d to observe the growth.

[0023] 2. Identification

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

Embodiment 2

[0034] Example 2. Preparation of L-asparaginase (SaLA protein)

[0035] After extensive sequence analysis, alignment and functional verification, a new protein was discovered from Halospira JH, which was named SaLA protein, as shown in sequence 1 of the sequence listing. The gene encoding SaDL protein in Halospira JH was named 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 Halospira JH as a template, PCR amplification was carried out using a primer pair composed of LA-F and LA-R, and the PCR amplification product was 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 with the pET-28a vector to obtain the recombinant plasmid pET-28a-SaLA.

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

Embodiment 3

[0054] Example 3. Enzymatic properties of 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, and make up to 1L.

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

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

[0058] 1. Optimum pH

[0059] The SaLA protein solution prepared in Example 2 was taken and diluted with PBS buffer (50 mM, pH 8.0) to 2 times the volume, and the diluted solution was used as the test solution.

[0060] Detection method: Add 200 μL of test solution and 700 μL of substrate solution (prepared with pH buffer), 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 then take the...

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Abstract

The invention discloses L-asparaginase SaLA and its encoding gene and application. The protein provided by the present invention is derived from Salinispirillum sp. JH, is a L-asparaginase, named SaLA protein, and is a protein composed of the amino acid sequence shown in Sequence 1 in the sequence table. The present invention also protects the use of SaLA protein as L-asparaginase. The present invention has great application prospects in the related medical fields, food processing fields and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to L-asparaginase SaLA and its encoding gene and application. Background technique [0002] L-asparaginase can catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. In the medical field, microbial-derived L-asparaginases can be used as antineoplastic and antileukemia agents, such as in the treatment of lymphoid malignancies, acute lymphoblastic leukemia and non-Hodgkin lymphoma, but their therapeutic utilization is limited by hypersensitivity reaction and other toxic 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 was listed by the World Health Organization International Agency for Research on Cancer in 2017. carcinogen. Studies have shown that L-asparaginase can effectively remove the important precursor of acrylamide (L-asparagine), th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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