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KMID : 0545120020120020242
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Journal of Microbiology and Biotechnology
2002 Volume.12 No. 2 p.242 ~ p.248
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A Microbial D-Hydantoinase is Stabilized and Overexpressed as a Catalytically Active Dimer by Truncation and Insertion of the C-Terminal Region
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Kim Geun-Joong
Kim Hak-Sung
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Abstract
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Previously, it was reported that the nonhomologous C-terminal regions of the D-hydantoinases are nonessential for catalysis, but affect the oligomeric structure of the enzyme [3]. In an effort to further confirm the above observation, the C-terminal region-inserted enzyme was constructed by attaching a peptide (22 residues) at the C-terminal of the D-hydantoinase from Bacillus thermocatenulatus GH2, and its structural and biochemical properties were compared with both the wild-type and C-terminal region-truncated enzymes. As a result, native tetrameric D-hydantoinase was dimerized as the truncated enzyme, and the inserted mutant with a new sequence was expressed as a catalytically active form in E. coli. Expression level of the inserted and truncated enzymes were found to be significantly increased compared to the level of the wild-type enzyme, and this appears to be due to the reduced toxic effect of the mutant enzymes on host cells. Dimerized enzymes exhibited increased thermo- and pH stabilities considerably when compared with the corresponding wild-type enzyme. Comparison of the substrate specificity between the mutant and wildtype enzymes suggests that the substrate specificity of the D-hydantoinase is closely linked with the oligomeric structure.
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