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Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof

A technology of alcohol dehydrogenase and crystal structure, which is applied in the fields of molecular biology and applied microbiology, and can solve the problem of not finding the crystal structure of thermophilic long-chain alkanol dehydrogenase in public literature reports and the like

Inactive Publication Date: 2013-02-20
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] After literature search, no public literature report identical to the crystal structure of the thermophilic long-chain alkanol dehydrogenase of the present invention was found

Method used

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  • Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof
  • Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof
  • Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, protein expression and purification of thermophilic long-chain alkanol dehydrogenase

[0037] 1. Induced expression of thermophilic long-chain alkanol dehydrogenase

[0038] 1) The bacterial fluid containing the thermophilic long-chain alkanol dehydrogenase gene was provided by our cooperative research group. Take 15 μL of the bacterial solution and inoculate it into 5 ml of high-temperature sterilized LB liquid medium supplemented with 100 mg / l kanamycin antibiotic, and culture overnight at 37°C and 200 rpm with shaking;

[0039] 2) Transfer 1% of the inoculum to 1L liquid LB medium supplemented with 100mg / l kanamycin antibiotic, culture at 37°C and 200rpm for 5-6h, add 100μL of IPTG with a final concentration of 0.1mM and induce at 45°C for 2.5h.

[0040] 2. Purification of thermophilic long-chain alkanol dehydrogenase

[0041] 1) Cell collection

[0042] a. Centrifuge the liquid culture solution in 1 at 5000rpm for 15min to collect the bacteria, and rin...

Embodiment 2

[0067] Example 2. Crystallization, data collection and analysis of thermophilic long-chain alkanol dehydrogenase protein

[0068] 1. Crystallization and Structure Analysis of Purified Alcohol Dehydrogenase

[0069] 1) Alcohol dehydrogenase crystallization

[0070] a. Dilute the concentration of the purified alcohol dehydrogenase to 10mg / ml and 20mg / ml;

[0071] b. The crystallization kit from Emerald BioSystems was used for primary screening of crystals, and the crystal plate was placed at 16°C; after a period of observation, it was found that there were crystals growing in WizardI No. 31, and the growth was good; based on this condition, alcohol removal Optimization experiment of hydrogenase crystallization conditions;

[0072] 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;

[0073] d. The pH buffer conditions in the crystallization conditions ...

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Abstract

The invention relates to a crystal structure of thermophilic alphanol alcohol dehydrogenase under tail end degradation of thermophilic denitrified bacillus n-alkane. According to the crystal structure, the alcohol dehydrogenase is subjected to heterogenous expression by using escherichia coli E.coli; the pure alcohol dehydrogenase is obtained by a protein purification method; the crystal, proper for diffraction, of the alcohol dehydrogenase is obtained by a hanging-drop crystallizing method; and the crystal structure of the alcohol dehydrogenase is analyzed by an X-ray diffraction method. As the results shown, one subunit includes two structural domains, and one structural domain is separated by a crack; the N-end structural domain includes an Alpha / Beta fold similar to Rossman fold, and the structural domain also includes a binding site of NAD (Nicotinamide Adenine Dinucleotide) + coenzyme; the C-end structural domain includes an Alpha structural domain distribution similar to that of dehydro-quinate synthetase; and one iron ion is bonded to the C-end structural domain and exposed in the intermediate crack. According to the results, the spatial conformation of the alcohol dehydrogenase can be universally shown, and the theoretical guidance is provided for further searching the relationship between the structure and the function of the alcohol dehydrogenase and improving the degrading activity of the alcohol dehydrogenase.

Description

Technical field: [0001] The invention relates to a thermophilic long-chain alkanol dehydrogenase crystal structure determined by X-ray crystal diffraction method, and belongs to the fields of molecular biology and applied microbiology. Background technique: [0002] Alcohol dehydrogenase (alcohol dehydrogenase, ADHs, EC 1.1.1.1) is one of the important redox agents in organisms, and many alcohol metabolisms are catalyzed by alcohol dehydrogenase. Alcohol dehydrogenases have been found in animals, plants, microorganisms, eukaryotes and prokaryotic bacteria, and enzymes with at least 25 EC numbers are called alcohol dehydrogenases. [0003] Alcohol dehydrogenases catalyze the reversible oxidation of alcohols to aldehydes. ADHs have a wide range of substrates, and their physiological roles are diverse. In the terminal degradation pathway of microbial alkanes, ADHs catalyzes the second step in the reaction, and the terminal degradation pathway is as follows image 3 shown. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04
Inventor 马克·巴特兰姆王莹莹纪玉蕊
Owner NANKAI UNIV
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