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Cloning, Site-Directed Mutagenesis, and Functional Analysis of Active Residues in Lymantria dispar Chitinase

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Abstract

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc)3. Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc)3, while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k cat and k cat / K m. A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31101490) and Key Research and Development Project of Shanxi Province (Grant No. 201603D321094).

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  1. Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No. 79 West Yingze Street, Taiyuan, Shanxi, 030024, People’s Republic of China

    Xiao-Jun Fan, Chun Yang, Chang Zhang, Hui Ren & Jian-Dong Zhang

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Correspondence to Jian-Dong Zhang.

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Fan, XJ., Yang, C., Zhang, C. et al. Cloning, Site-Directed Mutagenesis, and Functional Analysis of Active Residues in Lymantria dispar Chitinase. Appl Biochem Biotechnol 184, 12–24 (2018). https://doi.org/10.1007/s12010-017-2524-2

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  • DOI: https://doi.org/10.1007/s12010-017-2524-2

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