Thesis (Ph.D.) - Cairo University - Faculty of Science - Department of Biochemistry

In this study we report the isolation of two new oligopeptidases B (opdB) producer bacteria. The 16s rRNA analysis shows that the two newly isolated strains are Xenophilus sp.SN213 and Pseudomonas Lubricans strain SF168. The two genes, named opdB xeno and opdB seudo were cloned and overexpressed in E. coli. We managed to express the opdB xeno in a soluble form at low temperature but the opdB seudo was expressed in an insoluble form. The opdB xeno gene encodes a 703-residue peptide with high homology to the oligopeptidase B family in prokaryotes. The isolated opdB xeno gave the highest similarity score to oligopeptidase B of stenotrophomonas maltophilia strain K279a (Gen Bank AM743169). In this work, the two oligopeptidase B genes were cloned, expressed in E. coli expression host BL21(DE3) RIL using the vector pET28a. A method for purification of the recombinant enzyme (His6-oligopeptidase B) was developed to produce sufficient material to help in physical investigations. Enzyme assay of OpdB xeno was done to establish the identity of the recombinant enzyme. We then investigated whether the enzyme isolated is a thermophilic enzyme. We measured the activity at different temperatures and the enzyme ranging 37-95 degree. Our data show that the enzyme has an oligopeptidase activity even at a temperature higher than 65 degree centigrade. This indicates that the new enzyme is a thermostable. Further characterization of the soluble opdB xeno was performed. The inhibition of oligopeptidase B plays an important role to treat trypanosomiasis (the drugs most commonly used in sleeping sickness treatment reduce the activity of serine oligopeptidases), but there is little drugs are currently available for treatment the human African sleeping sickness caused by Trypanosoma brucei

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