title: Characterization of a Bacillus thuringiensis chitinase that binds to cellulose and chitin.
abstract: Bacillus thuringiensis is a Gram-positive soil bacterium that is known to be a bacterial biopesticide that produces insecticidal proteins called crystal proteins (Cry). In the insecticidal process, chitinases are suggested to perforate the peritrophic membrane barrier to facilitate the invasion of the Cry proteins into epithelial membranes. A chitinase gene from B. thuringiensis was successfully expressed in a soluble form in Escherichia coli, and the gene product was purified and characterized. The purified recombinant enzyme, BthChi74, hydrolyzed an artificial substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside [4NP-(GlcNAc)2], and the natural substrates, colloidal chitin and crystalline α-chitin, but it did not hydrolyze cellulose. BthChi74 exhibited catalytic activity under a weakly acidic to neutral pH range at 50 °C, and it was stable over a wide pH range for 24 h. Differential scanning fluorimetry (DSF) indicated a protein melting temperature (Tm) of 63.6 °C. Kinetic analysis revealed kcatand KMvalues of 1.5 s-1and 159 μM, respectively, with 4NP-(GlcNAc)2as a substrate. BthChi74 produced (GlcNAc)2and GlcNAc from colloidal chitin and α-chitin as substrates, but the activity toward the latter was lower than that toward the former. BthChi74 could bind similarly to chitin beads, crystalline α-chitin, and cellulose through a unique family 2 carbohydrate-binding module (CBM2). The structure-function relationships of BthChi74 are discussed in relation to other chitinases, such as Listeria chitinase, which possesses a family 5 carbohydrate-binding module (CBM5).
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Honda
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kunii
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Nohara
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Wakita
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.affiliation: Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Kawakita
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Oyama
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2, 665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan. bt11532@ns.kogakuin.ac.jp.
authors.forename: Masayoshi
authors.identifier: http://orcid.org/0000-0001-6724-9669
authors.initials: M
authors.lastname: Sakaguchi
keywords: Bacillus thuringiensis; Cellulose-binding module; Characterization; Chitin; Chitinase; Expression