Effects of C-terminal amino acids truncation on enzyme properties of <Emphasis Type="Italic">Aeromonas caviae</Emphasis> D1 chitinase |
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| Authors: | Fu-Pang Lin Hsu-Han Chuang Yi-Hsuan Liu Chia-Yu Hsieh Pei-Wen Lin Hsu-Yang Lin |
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| Institution: | (1) Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan;(2) Department of Life Science, National Taiwan Ocean University, Keelung, Taiwan |
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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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| Keywords: | Aeromonas caviae D1 Chitinase C-terminal truncation |
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