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Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase

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Abstract

C-Terminal truncation mutagenesis was used to explore the functional and structural significance of the C-terminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, fluorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic efficiency, k cat/K M, of AcD1ChiAK606 with the 4MU-(GlcNAc)2 and the 4MU-(GlcNAc)3 chitin substrates was 15–26% decreased. When compared with AcD1ChiA, the truncated mutant AcD1ChiAK606 maintained 80% relative substrate-binding ability and about 76% of the hydrolyzing efficiency against the insoluble α-chitin substrate. Both fluorescence and CD spectroscopy indicated that AcD1ChiAK606 retained the same conformation as AcD1ChiA. These results indicated that removal of the C-terminal 259 amino acid residues, including the putative chitin-binding motif and the A region (a motif of unknown function) of AcD1ChiA, did not seriously affect the enzyme structure integrity as well as activity. The present study provided evidences illustrating that the binding and hydrolyzing of insoluble chitin substrates by AcD1ChiA were not absolutely dependent on the putative C-terminal chitin-binding domain and the function-unknown A region.

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Acknowledgments

We thank Dr. Chun-Hung Lin of the Institute of Biological Chemistry, Academia Sinica, for his considerable help in the CD spectroscopy analyses. Prof. Jui-Sen Yang of the Institute of Marine Biology, National Taiwan Ocean University was much gratitude for his thoughtful discussions to improve this manuscript. This work was supported by the National Science Council, Taiwan (grant NSC 94-2313-B019-029) and the Center for Marine Bioscience and Biotechnology (CMBB), National Taiwan Ocean University (grants 99529001A8 and 97529002E3).

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Authors and Affiliations

  1. Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan

    Fu-Pang Lin, Hsu-Han Chuang, Yi-Hsuan Liu, Chia-Yu Hsieh, Pei-Wen Lin & Hsu-Yang Lin

  2. Department of Life Science, National Taiwan Ocean University, Keelung, Taiwan

    Fu-Pang Lin

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  1. Fu-Pang Lin
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  2. Hsu-Han Chuang
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Correspondence to Fu-Pang Lin.

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Communicated by Jorge Membrillo-Hernandez.

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Lin, FP., Chuang, HH., Liu, YH. et al. Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase. Arch Microbiol 191, 265–273 (2009). https://doi.org/10.1007/s00203-008-0451-x

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  • DOI: https://doi.org/10.1007/s00203-008-0451-x

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