Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof
What is Al technical title?
Al technical title is built by PatSnap Al team. It summarizes the technical point description of the patent document.
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
View PDF2 Cites 5 Cited by
Summary
Abstract
Description
Claims
Application Information
AI Technical Summary
This helps you quickly interpret patents by identifying the three key elements:
Problems solved by technology
Method used
Benefits of technology
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
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more
Image
Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
Click on the blue label to locate the original text in one second.
Reading with bidirectional positioning of images and text.
Smart Image
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 ...
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
PUM
Login to view more
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
the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more
Application Information
Patent Timeline
Application Date:The date an application was filed.
Publication Date:The date a patent or application was officially published.
First Publication Date:The earliest publication date of a patent with the same application number.
Issue Date:Publication date of the patent grant document.
PCT Entry Date:The Entry date of PCT National Phase.
Estimated Expiry Date:The statutory expiry date of a patent right according to the Patent Law, and it is the longest term of protection that the patent right can achieve without the termination of the patent right due to other reasons(Term extension factor has been taken into account ).
Invalid Date:Actual expiry date is based on effective date or publication date of legal transaction data of invalid patent.
Login to view more 
Login to view more