To get Enterobacter aerogenes strains having high L-asparaginase activities via random mutation

ABSTRACT Master Thesis TO GET ENTEROBACTER AEROGENES STRAINS HAVING HIGH L-ASPARAGINASE ACTIVITIES VIA RANDOM MUTATION Miraç UÇKUN Department of Biology Institute of Natural Sciences Inonu University 55+ ix pp 2006 Supervisor: Hikmet GEÇKİL, Associate Professor While DNA mutations are the causes of malfunctions as cancer and various metabolic diseases in an organism or cell, mutations also can play important role in adaptation of organisms or cells to changing conditions for the continuing existence. Mutations are the inheritable changes occurring in nucleotide sequence of genome of an organism and they can be arisen spontaneously or induced by various physical and chemical agents. The DNA synthesis (replication) is not an error-free process. In Eschrichia coli there is about 1 error per million nucleotide introduced. Such a change (mutation) is completely random and can happen in any region on the genome. However, such a low mutation rate can be substantially increased by various chemical and physical agents. In this study, high L- asparaginase producing mutants of gram-negative bacterium Enterobacter aerogenes were selected through use of the mutagenic agent ethyl methane sulfonate (EMS). This enzyme which is not found in humans is widely used in cancer chemotherapy and is produced from several gram-negative bacteria including E. aerogenes. In this study, optimal EMS concentration for producing mutants was determined and the enzyme levels of these mutants were compared with the wild-type strain. An indicator medium for the selection of high L-asparaginase producing mutants was utilized and among about 100,000 colonies screened 3 showed a distinctly higher enzyme level than the wild- type strain. These 3 mutants were further isolated and used in the rest of study. In general, all three mutants showed considerably higher L-asparaginase activities, ranging from 30 to 65 %, than the wild-type strain. Among the 3 mutants E. aerogenes ansMU-2 was determined to have the highest enzyme level. However, not all mutants produced by EMS mutagenesis had high L-asparaginase levels. Most of the mutants showed lower L-asparaginase levels than the original wild-type strain and thus were not further included in the study. KEY WORDS: Random Mutagenesis; Ethyl methane sulfonate (EMS); L-asparaginase; Chemotherapeutic enzymes m