New role of C-terminal 30 amino acids on the insoluble chitin hydrolysis in actively engineered chitinase from Vibrio parahaemolyticus.
A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments, respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His-Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy of fluorescence and circular dichroism, chitin-binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had very similar physicochemical properties such as the optimum pH (6), temperature (40 degrees C), and kinetic parameters of Km and kcat against the 4MU-(GlcNAc)(2) or 4MU-(GlcNAc)(3) soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589 toward the insoluble alpha-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids.
Chuang
Hsu-Han
HH
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Lin
Fu-Pang
FP
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D014736:Vibrio parahaemolyticus / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases