초록
An exo-inulinase gene was cloned from Arthrobacter sp. MN8, a cold-adapted bacterium isolated from lead-zinc-rich soil. The gene was expressed in Escherichia coli BL21(DE3). The resultant 505-residue polypeptide (InuAMN8) showed the highest identity (81.1%) with the putative levanase from Arthrobacter phenanthrenivorans Sphe3 (ADX73279) and shared 57.8% identity with the exo-inulinase from Bacillus sp. snu-7 (AAK00768). The purified recombinant InuAMN8 (rInuAMN8) showed an apparently optimal activity at 35<SUP>o</SUP>C, and 75.3%, 39.4%, and 15.8% of its maximum activity at 20<SUP>o</SUP>C, 10<SUP>o</SUP>C, and 0<SUP>o</SUP>C, respectively. After pre-incubation for 60 min at 50<SUP>o</SUP>C and 55<SUP>o</SUP>C, the rInuAMN8 exhibited 69.8% and 17.7% of its initial activity, respectively. The apparent K<SUB>m</SUB> values of rInuAMN8 towards inulin were 2.8, 1.5, 1.2, 5.3, and 8.2 mM at 0<SUP>o</SUP>C, 10<SUP>o</SUP>C, 20<SUP>o</SUP>C, 30<SUP>o</SUP>C, and 35<SUP>o</SUP>C, respectively. Inulin and Jerusalem artichoke tubers were effectively hydrolyzed to release fructose by rInuAMN8 at 0<SUP>o</SUP>C, 10<SUP>o</SUP>C, and 35<SUP>o</SUP>C. Compared with its hyperthermophilic and thermophilic counterparts, the exo-inulinase had less aromatic amino acid F and more hydrophobic amino acid A. In addition, the purified rInuAMN8 retained 127.9%-88.4% inulinase activity at 3.5%-15.0% (w/v) NaCl concentrations. Zn<SUP>2+</SUP> and Pb<SUP>2+</SUP> at 10 mM exhibited little or no effect on the enzyme activity. This paper is the first to report a cold-active and/or NaCl-tolerant exo-inulinase from the genus Arthrobacter. The exo-inulinase rInuAMN8 shows a potential for use in the production of fructose at low temperatures.