Document Details

Document Type : Thesis 
Document Title :
MOLECULAR CLONING, PROPERTIES AND POTENTIAL APPLICATIONS OF ENZYMES FROM GEOBACILLUS THERMODENITRIFICANS STRAIN DSM-465
الاستنساخ الجزيئي والخصائص والتطبيقات المحتملة لإنزيمات من سلالة جيوباسليس ثيرمودنتريفيكانس DSM-465
 
Subject : Faculty of Sciences 
Document Language : Arabic 
Abstract : Enzymes are macromolecules, exclusively three dimensionally folded proteins which have specific catalytic activities. These macromolecules have been reportedly responsible for catalysing more than 5000 biochemical processes in the living systems. Use of enzymes in the human life started in parallel with the human civilization. Older Egyptians were unknowingly using enzymes in the preparation of cheese, first commercial application of yeast enzymes to produce alcoholic beverages by the Babylonians and Sumerians as early as 6000 BC. Commercial enzymes represent a growing industry worldwide. Present study describes the molecular cloning, E. coli expression, purification, biochemical, in silico analysis and potential applications of enzymes in pharmaceutical industry. The DNA of target bacterial strain Geobacillus thermodenirificans DSM 465 was obtained from DSMZ (Germany). The species with optimal growth at 65°C was isolated in Austria from the juice of sugar beet and it was unexplored for its enzymes and proteins. In the present study two enzymes, arginase and glutaminase were produced in E. coli. The genes coding for arginase (900 bp) and glutaminase (938 bp) were PCR amplified and cloned in E. coli strain BL21 (DE3) with pET21a (+) plasmid. The confirmed recombinant clones were used to produce enzymes under optimized conditions. The enzymes were purified by DEAE-Cellulose based anion exchange chromatography and characterized for physiochemical properties. In silico 3D models of each enzymes were built and their affinities with the protein partners and inhibitory molecules were studied. A structural comparison of recombinant enzymes was made with corresponding human enzymes. The enzymes were expressed in high quantities, representing more than 50% of all E. coli proteins. The molecular weight of purified recombinant arginase and glutaminase were 33 kDa and 34 kDa respectively on SDS-PAGE. The optimum pH for the activity of arginase and glutaminase was 9 and they exhibited maximum activity at 70°C. The KM value of arginase was 151.6 mM and its Vmax was 150 µmole per min of L-arginine. The KM value of glutaminase was 104 µM and Vmax was 238.1 µmole per mg per min. The 3D structural models of both enzymes were built in silico. The comparative structural analysis with corresponding human enzymes have shown significant identity and conserved domains in the enzymes from two systematically different species. The molecular docking of enzymes with protein partners and inhibitory molecules have shown their affinity with inhibitors. The inhibitors of arginases and glutaminases from human and Geobacillus have been ranked according to their binding free energies that can be further exploited for the in vivo anticancer studies. The present study has revealed the characteristics of recombinant enzymes from an unexplored moderately thermophilic microorganism. Kinetic studies have provided the insights in to properties and abilities of these enzymes to perform at relatively higher temperatures. The in silico structural analysis have revealed a series of conserved protein domains among the enzymes from taxonomically different organisms suggesting the studies for the selection of more versatile inhibitors. 
Supervisor : Prof. Jalaluddin A. Khan 
Thesis Type : Doctorate Thesis 
Publishing Year : 1440 AH
2018 AD 
Added Date : Monday, October 8, 2018 

Researchers

Researcher Name (Arabic)Researcher Name (English)Researcher TypeDr GradeEmail
لقمان شاهShah, Luqman ResearcherDoctorate 

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