Abstract
Microbial l-asparaginases which catalyze the conversion of l-asparagine to l-asparate and ammonia, have been proved to be useful in medical and food industries. In the present work, a thermostable l-asparaginase was characterized from a hyperthermophilic archaeon strain, Thermococcus gammatolerans EJ3. Cloning and recombinant expression of Tco. gammatoleransl-asparaginase was performed in Escherichia coli. The recombinant enzyme was purified to homogeneity by nickel-affinity chromatography, and was characterized as a homodimer composed of two identical subunits of approximately 36.5kDa. The optimum pH and temperature were 8.5 and 85°C, respectively. The purified enzyme had specific activities of 7622 and 2926Umg−1 for l-asparagine and l-glutamine, respectively, and exhibited promising thermostability at all tested temperatures from 70 to 95°C. In addition, it displayed very high catalytic efficiency toward substrate l-asparagine. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) values for substrate l-asparagine were estimated to be 10.0mM, 5721s−1, and 572.1mM−1s−1, respectively.
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