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A gene encoding thermostable Lon protease fromBrevi-bacillus thermoruberWR-249 was cloned and characterized. TheBr. thermoruberLon gene (Bt-lon) encodes an 88 kDa protein characterized by an N-terminal domain, a central ATPase domain which includes an SSD (sensor- and sub-strate-discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putativeBacillus subtilisr A -dependent promoter, but in theabsenceofCIRCE(controlling inverted repeat of chaperone expression) | Eur. J. Biochem. 271 834-844 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.03988.x Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR-249 and biochemical characterization of its thermostable recombinant enzyme Alan Y.-L. Lee1 2 San-San Tsay3 Mao-Yen Chen1 and Shih-Hsiung Wu1 2 1 Institute of Biological Chemistry Academia Sinica Taipei Taiwan 2Institute of Biochemical Sciences National Taiwan University Taipei Taiwan 3Department of Life Science and Institute of Plant Biology National Taiwan University Taipei Taiwan A gene encoding thermostable Lon protease from Brevi-bacillus thermoruber WR-249 was cloned and characterized. The Br. thermoruber Lon gene Bt-lon encodes an 88 kDa protein characterized by an N-terminal domain a central ATPase domain which includes an SSD sensor- and substrate-discrimination domain and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis ơA-dependent promoter but in the absence of CIRCE controlling inverted repeat of chaperone expression . Bt-lon was expressed in Escherichia coli and its protein product was purified. The native recombinant Br. thermoruber Lon protease Bt-Lon displayed a hexameric structure. The optimal temperature of ATPase activity for Bt-Lon was 70 C and the optimal temperature of peptidase and DNA-binding activities was 50 C. This implies that the functions of Lon protease in thermophilic bacteria may be switched depending on temperature to regulate their physiological needs. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore the substrate specificity of Bt-Lon is different from that of E. coli Lon in using fluo-rogenic peptides as substrates. Notably the Bt-Lon protein shows chaperone-like activity by preventing aggregation of denatured insulin B-chain in a dose-dependent and ATP-independent manner. In thermal denaturation experiments Bt-Lon was found to display an indicator of .
