Ayesha Sadiqa2018-02-282018-02-282017https://escholar.umt.edu.pk/handle/123456789/2794Supervised by:Dr. Nouman RasoolThe present study describes the cloning, expression and characterization of alpha amylase from Pyrobaculum calidifontis. α-amylase is an extracellular enzyme and belongs to GH13 family. It is a starch degrading enzyme which hydrolyzes the α-1, 4-glycosidic linkages. In recent biotechnology applications the enzyme is being used in fermentation, food, paper and textile industry. Cloning of alpha amylase (Pcal_1039) gene was done in expression vector pET-21a(+). Pcal_1039 gene was expressed in E. coli BL21 and analysis of protein was done on SDS-PAGE. Recombinant protein was refolded by using in vivoand in vitro methods. The size of the gene was 1.37 kb and molecular mass of protein was 53.5 kDa. After expression the activity of α-amylase was measured by DNS method at OD540. Before refolding the inclusion bodies showed a minor activity while highest activity of 3.3 U/ml and specific activity of 23.6 U/mg/ml was measured by taking expression at 17°C (in vivo).enPyrobaculum calidifontis. α-amylaseSDS-PAGE. Recombinant proteinMS thesisThesis