Document Details

Document Type : Thesis 
Document Title :
MOLECULAR CLONING AND CHARACTERIZATION OF α-AMYLASE FROM THERMOPHILIC GEOBACILLUS STRAIN DSM-465
الاستنساخ الجزيئي وخصائص أنزيم ألفا أميليز المستخلص من سلالة جيوباسيلس DSM-465 المحبة للحرارة
 
Subject : Faculty of Science 
Document Language : Arabic 
Abstract : Alpha-amylase is the enzyme of primary metabolism that catalyzes the hydrolysis of α-1, 4-glycosidic linkage in the starch molecules. The enzyme is ubiquitous in nature, found in the human saliva, pancreatic secretions, plants, fungi and bacteria. There is a big market for this enzyme in the baking, beverage, paper, textile and pharmaceutical industries. The present study describes cloning, E. coli expression, partial purification, kinetic properties, in silico structural and molecular docking analysis of alpha amylase from Geobacillus thermodenitrificans DSM-465. A 1698 bp gene coding for 565 amino acid protein was PCR amplified, T/A cloned in pTZ57R/T plasmid and subcloned in pET21a (+) expression plasmid. The enzyme was expressed in BL21 (DE3) RIL codon plus strain of E. coli under 0.5mM IPTG in LB broth containing 100µg of ampicillin per milliliter of medium adjusted at 200 rpm at 37°C. The enzyme was purified by DEAE-Sephadex based anion exchange chromatography. It gave a protein band at about 63 kDa on SDS-PAGE with optimum pH and temperature at 8 and 70°C respectively. The KM value of recombinant alpha amylase was 157.7µM and Vmax was 333 U/mg. The enzyme retained considerable activity at 90 to 100°C when incubated for 5 minutes. A 3D protein model comprising of 31% α-helix, 15% β-Sheet, and 52% loop was built by Raptor-X, validated by Ramachandran plot and selected for molecular docking studies. Molecular Operating Environment (MOE) software was used for docking of protein model with selected ligands. The best bonding affinity of enzyme was found with amylopectin as indicated by the lowest docking energy (∆G -10.59). The main interacting residues Asp232, Arg448, Glu385, Asp99 and, Arg175 with a distance less than 2.86Å make the potential active site of enzyme. In conclusion, the study provides an insight into the biochemical and structural properties of the enzyme. Stability and activity at a broad range of pH and temperature makes it a strong candidate for industrial applications. 
Supervisor : Dr. Muhammad Shahid Nadeem 
Thesis Type : Master Thesis 
Publishing Year : 1441 AH
2020 AD 
Co-Supervisor : Prof. Jalaluddin A. Khan 
Added Date : Wednesday, June 24, 2020 

Researchers

Researcher Name (Arabic)Researcher Name (English)Researcher TypeDr GradeEmail
عبدالعزيز ضيف الله العمريAlamri, Abdulaziz DhaifallaResearcherMaster 

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