Alanine dehydrogenase SaAD as well as coding gene and application thereof

An alanine dehydrogenase, gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of wide reaction temperature, wide reaction pH, and great application prospects

Active Publication Date: 2021-11-12
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on alanine dehydrogenase derived from Salinispirillum sp.

Method used

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  • Alanine dehydrogenase SaAD as well as coding gene and application thereof
  • Alanine dehydrogenase SaAD as well as coding gene and application thereof
  • Alanine dehydrogenase SaAD as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1, Isolation, Identification and Preservation of Halospira NM

[0020] 1. Separation

[0021] Take 50 μL of the alkali lake sample, add 450 μL of the alkali lake filtrate, and mix 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 samples, and spread them on solid medium such as YMAH, TSAH, LBH, 2216EH, etc., culture them in a constant temperature incubator at 35°C for 3-4 days, and observe the growth conditions. Colonies with different colors and shapes were picked, purified and cultivated by the three-section line method, and after repeating the line purification process 3-4 times, pure colonies were obtained.

[0022] 2. Identification

[0023] The purified strains were lined in three zones in LBH solid medium, cultured at 35°C for 2 days, and the morphology, size, presen...

Embodiment 2

[0034] Embodiment 2, the preparation of alanine dehydrogenase (SaAD protein)

[0035]After a lot of sequence analysis, alignment and functional verification, a new protein was found from Halospira NM, which was named SaAD protein, as shown in sequence 1 of the sequence listing. The gene encoding the SaAD protein in Halospira NM is named SaAD 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 NM as a template, PCR amplification is carried out with a primer pair composed of AD-F and AD-R, and the PCR amplification product is recovered.

[0038] AD-F: 5'-CCCAAGCTTTCAGACCGGC-3';

[0039] AD-R: 5'-CGGGATCCATGGACTTCG-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-SaAD.

[0041] pET-28a Vector (pET-28a Vector): Novagen, catalog number 69864-3.

[0042] After ...

Embodiment 3

[0054] Embodiment 3, the enzymatic property of alanine dehydrogenase (SaAD 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] Alanine reaction solution (10mM): Weigh 1.1g of L-alanine, dissolve it in PBS buffer, and dilute to 1L.

[0057] NAD + Solution (80μM): weigh 0.0531g NAD + , dissolved in PBS buffer, and dilute to 1L.

[0058] Reaction principle: Alanine dehydrogenase can catalyze the oxidative deamination reaction of L-alanine, the product is pyruvate, and NAD + It is reduced to NADH, and NADH has a maximum absorption value at 340nm.

[0059] 1. The effect of pH on the activity of alanine dehydrogenase

[0060] Take the SaAD protein solution prepared in Example 2, dilute it to 2 times the volume with buffer, and use the diluted solution as the test sol...

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Abstract

The invention discloses alanine dehydrogenase SaAD as well as a coding gene and application thereof. The protein provided by the invention is derived from Salinispirillum sp.NM (Salinispirillum sp.NM), is alanine dehydrogenase, is named as an SaAD protein, and is a protein consisting of an amino acid sequence as shown in a sequence 1 in a sequence table. The invention also protects the application of the SaAD protein as alanine dehydrogenase. The invention has great application prospects in the fields of related medicines, food industry, health care and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to alanine dehydrogenase SaAD and its coding gene and application. Background technique [0002] Alanine dehydrogenase (Alanine Dehydrogenase, EC 1.4.1.1) belongs to the amino acid dehydrogenase class, which can reversibly catalyze the oxidative deamination of alanine to generate pyruvate and reduce NAD at the same time + Generate NADH. The reaction product, pyruvate, is often used in the pharmaceutical industry to synthesize anti-inflammatory and analgesic drugs, anti-tuberculosis drugs, anti-viral drugs and anthelmintic drugs, etc. It can also be used as a food additive in the food industry. In addition, pyruvic acid is also widely used in biotechnology diagnostic reagents, weight loss and health care and other fields. [0003] Alanine dehydrogenase is widely distributed and has been reported in plants, microorganisms and algae, but more alanine dehydrogenases from microo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N1/20C12N15/70C12N1/21C12P7/40C12R1/01
CPCC12N9/0016C12N15/70C12P7/40C12Y104/01001
Inventor 刘建国李静徐莹莹谭雯斐王淼姜雪姣
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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