Expression of High-Activity and High-SpecificityMicrobial L-Asparaginases from Naturally Occurring Microorganisms in the Alabama Black Belt - L-Asparaginase is an enzyme that converts L-asparagine to ammonia and L-aspartate and has health-related pharmaceutical and industrial applications. L-asparagine is one of the amino acids that most human cells can synthesize. However, acute lymphoblastic leukemia (ALL) cells are unable to synthesize L-asparagine and depend on circulating L-asparagine to survive. L-asparaginase can deplete asparagine in blood and is useful in the treatment of ALL. L-asparaginase has other uses in food processing and biosensor for L-asparagine detection. Acrylamide is a potential carcinogen and neurotoxin and can arise from the reaction of asparagine with sugars due to elevated temperatures in food processing. Thus, asparaginases are also useful in food processing to reduce the heat-initiated accumulation of acrylamide. The proposed study focuses on the expression and biochemical characterization of high enzyme activity and high substrate specificity L- asparaginases for inhibition of ALL proliferation and acrylamide formation in heat processed foods. Alabama Black Belt (ABB), so-named for its dark, fertile soil, may harbor unique microorganisms. We selected three naturally occurring microorganisms from the ABB for this work and will screen more ABB soil samples if needed. L-asparaginases will be partially purified from the three selected microorganisms and assessed for anti-cell proliferation and acrylamide reduction in heat-processing of carbohydrate-rich foods. The genome of selected isolates will be sequenced to identify L-asparaginase genes. We have identified three putative L- asparaginase genes from the genome of a biomass-hydrolyzing fungus, Trichoderma SG2, isolated from the ABB. Asparaginase genes from promising microbial isolates will be expressed using protein expression systems, purified, and characterized. Native and recombinant L-asparaginase that meet the overall objective of high enzyme activity and high substrate specificity will be tested for anti-cell proliferation and decreasing acrylamide formation in heat processing of food. High enzyme activity and high substrate specificity L- asparaginase may allow use of lower concentrations of the enzyme to potentially reduce side effects caused by current asparaginases and benefit the reduction of acrylamide formation in heat-processed foods as well as biosensor development for L-asparagine detection. Multipurpose L-asparaginase could reduce the cost of asparaginases for pharmaceutical and industrial applications. The project is a continuation of work started by undergraduate students. The students will gain research skills and professional development through conference presentations and networking with scientists. The project will encourage undergraduate participation in research, build research capacity, and contribute to the training of young scientists needed for the scientific future of the Nation.
