Biochemical characterization of an extremely thermostable L-asparaginase from Thermococcus gammatolerans EJ3

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. gammatolerans L-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 36.5 kDa. The optimum pH and temperature were 8.5 and 85 degrees C, respectively. The purified enzyme had specific activities of 7622 and 2926 Umg(-1) for L-asparagine and L-glutamine, respectively, and exhibited promising thermostability at all tested temperatures from 70 to 95 degrees C. In addition, it displayed very high catalytic efficiency toward substrate L-asparagine. The Michaelis-Menten constant (K-m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K-m) values for substrate L-asparagine were estimated to be 10.0 mM, 5721 s(-1), and 572.1 mM(-1) s(-1), respectively. (C) 2014 Elsevier B.V. All rights reserved.