D-lactic dehydrogenase SaDLD as well as encoding gene and application thereof

A technology of lactate dehydrogenase and gene, applied in application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2021-11-23
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, D-lactate dehydrogenase derived from Salinispirillum sp. microorganisms has not been reported

Method used

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  • D-lactic dehydrogenase SaDLD as well as encoding gene and application thereof
  • D-lactic dehydrogenase SaDLD as well as encoding gene and application thereof
  • D-lactic dehydrogenase SaDLD as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1, Isolation, Identification and Preservation of Halospiral JH

[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, prese...

Embodiment 2

[0034]Embodiment 2, the preparation of D-lactate dehydrogenase (SaDLD protein)

[0035] After a lot of sequence analysis, alignment and functional verification, a new protein was found from Halospira JH, which was named SaDLD protein, as shown in sequence 1 of the sequence listing. The gene encoding SaDLD protein in Halospira JH is named as SaDLD 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 performed using a primer pair composed of DL-F and DL-R, and the PCR amplification product is recovered.

[0038] DL-F: 5'-GGAATTCATGAAAATCGCCGTCT-3';

[0039] DL-R: 5'-CCCAAGCTTTTATATCTTAACGACGTGA-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-SaDLD.

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

Embodiment 3

[0054] Embodiment 3, the enzymatic property of D-lactate dehydrogenase (SaDLD protein)

[0055] PBS buffer (100mM, pH 6.0): Weigh 2.88g sodium dihydrogen phosphate, 0.48g potassium dihydrogen phosphate, 0.40g potassium chloride, 16.00g sodium chloride, dissolve in 800mL ultrapure water, adjust pH with HCl to 6.0, set the volume to 1L.

[0056] Sodium alanine reaction solution (20mM): Weigh 2.2g of sodium alanine, dissolve in PBS buffer, and dilute to 1L.

[0057] NADH solution (10 mM): Weigh 6.6343 g of NADH, dissolve in PBS buffer, and dilute to 1 L.

[0058] 1. The effect of pH on the activity of D-lactate dehydrogenase

[0059] 1. Optimal pH

[0060] Get the SaDLD protein solution prepared in Example 2, dilute to 2 times the volume with PBS buffer (100mM, pH 6.0), and use the diluted solution as the test solution.

[0061] Detection method: Add 10 μL of test solution, 10 μL of sodium pyruvate solution (20 mM), 10 μL of NADH solution (10 mM), 170 μL of PBS buffer solutio...

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Abstract

The invention discloses D-lactic dehydrogenase SaDLD as well as an encoding gene and application thereof. The protein provided by the invention is derived fromSalinispirillum sp.JHis D-lactic dehydrogenase, is named as SaDLD 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 SaDLD protein as the D-lactic dehydrogenase. The protein has great application prospects in the fields of related medicines, foods, cosmetics and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to D-lactate dehydrogenase SaDLD and its coding gene and application. Background technique [0002] D-Lactate Dehydrogenase (DLD, EC 1.1.1.28) can catalyze the conversion of pyruvate into lactic acid, which is a valuable organic acid widely used in food, medicine, cosmetics, chemical and other industries . In recent years, there has been increasing interest in the use of lactic acid as a monomer to synthesize the polymer polylactic acid (PLA), as PLA is a biodegradable and environmentally friendly alternative to petrochemically-derived plastics. In addition, D-lactate dehydrogenase can also be used as a diagnostic biosensor to determine the type of disease associated with elevated D-lactate concentration in urine or serum, because lactate measurement is crucial in clinical diagnosis, and elevated blood lactate concentration can lead to Patients with multiple organ failure a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N15/70C12Y101/01028
Inventor 刘建国李静姜雪姣谭雯斐王淼李子一
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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