Cloning and high-level production of a chitinase from <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. and the role of conserved or nonconserved residues on its catalytic activity |
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Authors: | Seur Kee Park Chi Wook Kim Hoon Kim Jae Sung Jung G E Harman |
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Institution: | (1) Department of Agricultural Biology, Sunchon National University, Sunchon, 540-742, South Korea;(2) Technical Research Institute, Dong Bang Agro Corp., Puyeo Gun 323-930, ChungNam, South Korea;(3) Department of Agricultural Chemistry, Sunchon National University, Sunchon, 540-742, South Korea;(4) Department of Biology, Sunchon National University, Sunchon, 540-742, South Korea;(5) Department of Horticultural Sciences, Cornell University, Geneva, NY 14456, USA |
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Abstract: | A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and
a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37°C and was about fourfold higher than that from C-61. Chi54 from the culture
supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding
affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging
to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in
loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process,
a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of K
cat values. |
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Keywords: | Chitinase Site-directed mutagenesis Enzyme purification Enzyme enhancement |
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