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Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof

A technology for alkanal dehydrogenase and aldehyde dehydrogenase, which is applied in the fields of molecular biology and applied microbiology, and can solve the problems of not finding the crystal structure of thermophilic long-chain alkanal dehydrogenase, such as public literature reports and the like

Active Publication Date: 2014-03-05
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After literature search, no public literature report identical to the crystal structure of the thermophilic long-chain alkanal aldehyde dehydrogenase of the present invention was found

Method used

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  • Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof
  • Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof
  • Thermophilic long-chain alkyl aldehyde dehydrogenase and crystal structure thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Molecular construction and protein purification of thermophilic long-chain alkanal aldehyde dehydrogenase

[0052] 1. Extraction of Genomic DNA from Geobacillus thermodenitrificans NG80-2

[0053] 1) Inoculate Geobacillus thermodenitrificans NG80-2 in LB liquid medium and culture at 37°C and 200rpm for 12 hours;

[0054] 2) Take 3ml of the fresh culture cultured overnight, and collect the bacteria by centrifugation;

[0055] 3) The cells were suspended in 250 μL of 50 mM Tris (pH 8.0) buffer, 10 μL of 0.4M EDTA (pH 8.0) was added, mixed well and incubated at 37°C for 20 min;

[0056] 4) Then add 30μL 20mg / ml lysozyme, mix well and keep warm at 37℃ for 20min;

[0057] 5) Add 5 μL 20mg / ml proteinase K, mix gently, then add 20 μL 10% SDS, keep warm at 50°C until the solution is clear;

[0058] 6) Use equal volumes of phenol: chloroform: isoamyl alcohol to extract twice, chloroform: isoamyl alcohol to extract once, add 2.5 times the volume of pre-cooled absol...

Embodiment 2

[0130] Example 2. Crystallization and data collection and analysis of thermophilic long-chain alkanal aldehyde dehydrogenase protein

[0131] 1. Crystallization and Structure Analysis of Purified Aldehyde Dehydrogenase

[0132] 1) Aldehyde dehydrogenase crystallization

[0133] a. Dilute the purified aldehyde dehydrogenase concentration to 10mg / ml and 20mg / ml;

[0134] b. The crystallization kit from Hampton Research was used for preliminary crystal screening, and the crystal plate was placed at 16°C;

[0135] After a period of observation, it was found that there was crystal growth in Index 43, and the growth was good; based on this condition, the optimization experiment of the crystallization condition of aldehyde dehydrogenase was carried out;

[0136] c. Fill the syringe with high-vacuum silicone grease, and draw a 2mm-wide silicone grease circle on the side of each culture well on the 24-well hanging drop crystal plate;

[0137] d. The pH buffer conditions in the ...

Embodiment 3

[0148] Example 3. Molecular construction and protein purification of thermophilic long-chain alkanal aldehyde dehydrogenase mutant

[0149] 1. Construction and protein purification of recombinant plasmid of aldehyde dehydrogenase mutant (Cys301Ala)

[0150] The construction process of the recombinant plasmid of the aldehyde dehydrogenase mutant is the same as the construction of the recombinant plasmid of the wild type of aldehyde dehydrogenase, except that the PCR part of the target fragment is different, and the overlap extension method is used. The specific steps are as follows:

[0151] 1) Using the nucleotide sequence of ALDH as a template, use the two pairs of primers F / RM301 and R / FM301 to carry out PCR reaction to amplify the front and back fragments of the mutation site. The reaction system is as follows:

[0152]

[0153] The PCR procedure is the same as that of ALDH wild-type recombinant plasmid. After the PCR products were recovered by gel cutting, they were r...

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Abstract

The invention relates to thermophilic long-chain alkyl aldehyde dehydrogenase in an n-alkane end degradation process of thermophilic denitrified bacillus and a crystal structure thereof. A molecular clone method is utilized, so that a target gene is cloned into escherichia coli (E. coli) so as to be heterologously expressed. A purified product of aldehyde dehydrogenase is obtained through a protein purification method. A crystal of the aldehyde dehydrogenase, which is suitable for diffraction, is obtained through a hanging droplet crystallization method. The crystal structure of the aldehyde dehydrogenase is analyzed by utilizing an X-ray diffraction method. The results show that the aldehyde dehydrogenase contains a characteristic ALDH fold which is formed by three structural domains. Site-specific mutagenesis is carried out on conserved residues on an enzyme activity part, and the influence of the conserved residues on enzyme activity is proved through wild type of the enzyme and enzyme activity experiment of the mutant thereof. The results can comprehensively reflect space conformation of the enzyme, and provide theoretical guidance for further researching relation between structures and functions of the aldehyde dehydrogenase, and improving the degradation activity of the enzyme.

Description

Technical field: [0001] The invention relates to a thermophilic long-chain alkanal aldehyde dehydrogenase crystal structure determined by X-ray crystal diffraction method, and belongs to the fields of molecular biology and applied microbiology. Background technique: [0002] Aldehyde Dehydrogenase (ALDH) is ubiquitous in all organisms, and this type of enzyme utilizes NAD + or NADP + As a coenzyme factor, it oxidizes aldehydes to carboxylic acids. In organisms, aldehyde dehydrogenase oxidizes the toxic aldehydes produced in the process of cell metabolism to complete the detoxification process. In addition, aldehyde dehydrogenase is also involved in the biotransformation process of the degradation of alkane substances. In this process, alkyl aldehyde appears as an intermediate product of the reaction, which is converted into carboxylic acid under the action of aldehyde dehydrogenase and enters the bacteria. step in the metabolic process. [0003] ALDH of many bacterial or...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/70G01N23/20C12R1/01
Inventor 马克·巴特兰姆王莹莹纪玉蕊郝振宇毛冠男
Owner NANKAI UNIV
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