Effects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase
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ORIGINAL PAPER
EVects of C-terminal amino acids truncation on enzyme properties of Aeromonas caviae D1 chitinase Fu-Pang Lin · Hsu-Han Chuang · Yi-Hsuan Liu · Chia-Yu Hsieh · Pei-Wen Lin · Hsu-Yang Lin
Received: 17 October 2008 / Revised: 24 November 2008 / Accepted: 28 November 2008 / Published online: 17 December 2008 © Springer-Verlag 2008
Abstract C-Terminal truncation mutagenesis was used to explore the functional and structural signiWcance of the Cterminal region of Aeromonas caviae D1 chitinase (AcD1ChiA). Comparative studies between the engineered full-length AcD1ChiA and the truncated mutant (AcD1ChiAK606) included initial rate kinetics, Xuorescence and circular dichroism (CD) spectrometric properties, and substrate binding and hydrolysis abilities. The overall catalytic eYciency, kcat/KM, 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 eYciency against the insoluble -chitin substrate. Both Xuorescence 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 aVect 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
Communicated by Jorge Membrillo-Hernandez. F.-P. Lin · H.-H. Chuang · Y.-H. Liu · C.-Y. Hsieh · P.-W. Lin · H.-Y. Lin Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan F.-P. Lin (&) Department of Life Science, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]
absolutely dependent on the putative C-terminal chitinbinding domain and the function-unknown A region. Keywords Aeromonas caviae D1 · Chitinase · C-terminal truncation
Introduction The family of chitinases can be classiWed on the basis of their hydrolytic mechanisms and the similarity of amino acid sequences in their catalytic domains (Henrissat 1999). Family 18 chitinases are characterized as having the (/)8 barrel catalytic domains, whereas family 19 chitinases have a high -helical content and a structure similar to that of chitosanases and lysozymes. Bacterial family 18 chitinases have subfamilies A, B, and C (Suzuki et al. 1999). Serratia marcescens QMB1466 chitinase A and chitinase B (SmChiA and SmChiB) and Bacillus circulans WL-12 chitinase A1 (BcChiA1), which belong to bacterial family 18 chitinases, have known three-dimensional (3D) structures (Perrakis et al. 1994; Matsumoto et al. 1999; van Aalten et al. 2000). SmChiA comprises an N-terminal domain, a catalytic (/)8 barrel domain and a small ( + ) domain inserted within the (/)8 barrel. In SmChiB, an additional C-terminal chitin-bin
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