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KMID : 0545120000100020238
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 2 p.238 ~ p.243
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Cloning and Characterization of Pseudomonas mucidolens Exoinulinase
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Kwon, Young Man
Kim, Hwa Young/Choi, Yong Jin
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Abstract
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An exoinulinase (β-D-fructofuranosidase) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. The exoinulinase gene consisted of an ORF of 1,506bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli DH5α strain was also purified to homogeneity as determined by SDSPAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed sucrose, raff nose, levan, in addition to inulin, with an S/I activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90min at 55℃. Taken together, these results indicate that the purified β-D-fructofuranosidase was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and 55℃, respectively. The enzyme had no apparent requirement for a cofactor, and its activity was completely inactivated by Ag^+, Hg^2+ , and Zn^2+. Kinetic experiments gave K_m, V_max, and K_cat, values for inulin of 11.5mM, 18nM/s, and 72s^-1, respectively. The exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4h incubation at 55℃.
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KEYWORD
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