Abstract
L-Asparaginases (E.C. 3.5.1.1) are group of enzymes which catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Many microorganisms are known to produce two types of L-asparaginase. Considerable attention has been specifically drawn to type II L-asparaginases due to their potent tumor-inhibitory activity. Various limitations, such as high toxicity, poor stability and short half-life, are associated with the commercially available L-asparaginases used in cancer therapy. Additionally, L-asparaginases have a significant role in food industry owing to their ability to reduce the levels of acrylamide, which is a known neurotoxin and a potential carcinogen, in various food products. Knowing that less than 1% of microorganisms can be detected using standard culturing techniques, searching for new L-asparaginases II with improved properties using metagenomic approaches seems promising. The metagenomes of extreme environments serve as a potential source for novel biomolecules that can, in most cases, function optimally under the extreme conditions of their environments. Having a temperature of 68°C, salinity of 250 parts per thousand, high concentrations of heavy metals and very low oxygen levels, the lower convective layer (LCL) of the Atlantis II brine pool (ATII) in the Red Sea is considered one of the harshest marine environments, and hence an attractive site for mining for a novel L-asparaginase II. Several studies successfully isolated different L-asparaginases II from cultured isolates, however, to the best of our knowledge, this is the first study to isolate and characterize L-asparaginase II using metagenomic approach. In this study, a putative L-asparaginase II (AspATII) was identified through computational analysis of the ATII-LCL metagenomic database. The environmental DNA of the LCL was screened for the identified gene using sequence-based approach. Amplified genes were cloned into pET 20b(+) expression vectors, while pET-22b(+) vectors were used to express codon optimized genes. Protein expression was carried out in Escherichia coli (E.coli) BL21 (DE3) cells and the His-tagged protein was purified from the culture medium of cells transformed with recombinant pET 22b(+) vectors. Characterization of the purified protein revealed that it is a thermostable (optimum temperature, 50°C), halotolerant (maintains its activity up to 4 M NaCl) L- asparaginase II, which makes it highly beneficial for various biotechnological and industrial applications.
School
School of Sciences and Engineering
Degree Name
MS in Biotechnology
First Advisor
Moustafa, Ahmed
Second Advisor
Ferreira, Ari Jose
Third Advisor
Abou-Aisha, Khaled
Committee Member 1
Fouad, Walid
Committee Member 2
Abdel Latif, Ahmed
Committee Member 3
Moustafa, Ahmed
Document Type
Thesis
Rights
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Recommended Citation
APA Citation
Saleh, A. M.
().Isolation and characterization of a novel Thermostable, Halotolerant L-Asparaginase II from the Metagenome of the Red Sea; Atlantis II Brine Pool [Thesis, the American University in Cairo]. AUC Knowledge Fountain.
https://fount.aucegypt.edu/retro_etds/2566
MLA Citation
Saleh, Aya Medhat. Isolation and characterization of a novel Thermostable, Halotolerant L-Asparaginase II from the Metagenome of the Red Sea; Atlantis II Brine Pool. . American University in Cairo, Thesis. AUC Knowledge Fountain.
https://fount.aucegypt.edu/retro_etds/2566