Production of recombinant stenotrophomonas maltophilia L-asparaginase for potential medical application / by Nada Anwar Abdelrazek, Master of Pharmaceutical Sciences (Microbiology and Immunology), Faculty of Pharmacy, Ain Shams University, 2018 ; Under Supervision of Prof. Dr. Mohammad Mabrouk Aboulwafa, Professor of Microbiology and Immunology and Dean of Faculty of Pharmacy-King Salman International University; Assoc. Prof. Marwa Mahmoud Raafat Associate Professor and Acting Head of Microbiology and Immunology Department, Faculty of Pharmacy-Future University in Egypt; Prof. Dr. Amal Emad Eldin Ali, Professor of Microbiology and Immunology and Vice Dean of Faculty of Pharmacy, Future University in Egypt, Assoc. Prof. Sarra Ebrahim Saleh, Associate Professor and Acting Head of Microbiology and Immunology Dep, Faculty of Pharmacy- Ain Shams University

Under Supervision of Prof. Dr. Mohammad Mabrouk Aboulwafa, Professor of Microbiology and Immunology and Dean of Faculty of Pharmacy-King Salman International University; Assoc. Prof. Marwa Mahmoud Raafat Associate Professor and Acting Head of Microbiology and Immunology Department, Faculty of Pharmacy-Future University in Egypt; Prof. Dr. Amal Emad Eldin Ali, Professor of Microbiology and Immunology and Vice Dean of Faculty of Pharmacy, Future University in Egypt, Assoc. Prof. Sarra Ebrahim Saleh, Associate Professor and Acting Head of Microbiology and Immunology Dep, Faculty of Pharmacy- Ain Shams University

Thesis (Ph. D.)--Ain Shams University, Faculty of Pharmacy, Department of Microbiology and Immunology, 2024

Includes bibliographical references.

L-asparaginase is an important enzyme used in the combination chemotherapy regimens for the treatment of acute lymphoblastic leukemia and since its incorporation in the pediatric treatment protocols, it helped in achieving a high cure rate. Owing to its therapeutic efficacy, L-asparaginase is considered as a milestone in cancer treatment, however, its use has been associated with a high rate of hypersensitivity and toxicity reactions. There is a current need for the identification of novel L-asparaginase enzyme with improved properties and lower adverse effects compared to those available in the market. The present study aimed at the production of two forms (recombinant & native) of Stenotrophomonas maltophilia L-asparaginase that exhibit comparable efficiency with the commercially available E. coli L-asparaginase which might be useful in pharmaceutical, and food industries. Stenotrophomonas maltophilia was previously isolated and reported with promising L-asparaginase activity. L-asparaginase gene from Stenotrophomonas maltophilia was successfully cloned in E. coli DH5α and expressed in E. coli BL21 (DE3). Investigations of different conditions on the induction of expression of the recombinant L-asparaginase in E. coli BL21 (DE3) using response surface methodology (Box–Behnken central composite design) predicted maximum enzyme expression at temperature 37°C, 250 rpm, isopropylthio-β-D-galactoside (IPTG) concentration of 0.83 mM and incubation period of 17 h. the optimized induction conditions were validated using L-asparaginase activity assay. The obtained recombinant protein was purified using Ni-NTA spin column. Gene amplification was performed for Stenotrophomonas maltophilia isolate using different concentrations of chloramphenicol to enhance the productivity of the isolate, The microbial production of L-asparaginase was increased at 200 µg/ml concentration. The native Stenotrophomonas maltophilia L-asparaginase could be purified to homogeneity by ammonium sulphate precipitation followed by gel exclusion chromatography. This resulted in L-asparaginase homogenate with purity up to 5.48 folds, a total activity of 96.4375 IU/ml and specific activity of 36.251 IU/mg protein. SDS-PAGE analysis of the native and recombinant purified enzymes revealed that the purified form of the enzyme is separated at an apparent molecular weight of 17 KDa. The kinetic parameters for the recombinant and native protein were determined and it showed a low Km value of 2.94 ×10-2 M and 4.16×10-2 M and Vmax of 14.73 IU/ml and 10.67 IU/ml, respectively. The antitumor activity of the purified recombinant and native L-asparaginase was evaluated in comparison to the commercial E. coli L-asparaginase (L-ASAP Neova, Biogene) on different cell lines. The results revealed low IC50 of 1.92 IU/ml and 2.9 IU/ml against Hepatocellular cancer cell line (HepG-2) for the recombinant and commercial enzyme, respectively. At the same time the native and commercial enzymes, the IC50 was of 2.2 IU/ml and 2.4 IU/ml, respectively. By testing the antitumor activity of the recombinant and commercial L-asparaginase on the human leukemia cancer cell line (K-562), the IC50 was of 2.03 IU/ml and 2.22 IU/ml, respectively. While in case of the native and commercial L-asparaginase, the IC50 was 2.83 IU/ml and 1.86 IU/ml, respectively. Whereas no cytotoxic effect for both forms of L-asparaginase (recombinant and native) could be detected on normal human lung fibroblast cells (MRC-5). Furthermore, mice treated with recombinant and native L-asparaginase showed lower immune response compared to commercial E. coli L-asparaginase. These results highlight the potential of the recombinant and native L-asparaginases from Stenotrophomonas maltophilia for the development of chemotherapeutic drugs.

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