Purification, characterization, and gene cloning of 46 kDa chitinase (Chi46) from Trichoderma reesei PC-3-7 and its expression in Escherichia coli.
We purified a chitinase (named Chi46), with a molecular mass of 46 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from the culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetylglucosamine (GlcNAc). The relative activity of this enzyme reduced when the degrees of acetylation (DA) of chitosan decreased. Furthermore, the enzyme was able to hydrolyze colloidal chitin and ethylene glycol chitin. The gene chi46 was cloned and sequenced. chi46 encodes a protein of 424 amino acid residues containing a 35-amino acid prepro-type secretion signal peptide. The molecular mass of mature Chi46 calculated from deduced amino acid sequence was 42,265 Da. The chi46 transcript was biphasic when the mycelia were grown on GlcNAc, suggesting that the multiple regulatory proteins are involved in the chi46 expression. The chi46 cDNA was expressed in Escherichia coli (ca. 0.23 mg/ml culture). To determine substrate cleavage fashion of Chi46 in more detail, we carried out high-performance liquid chromatography analysis and viscosimetric assay using recombinant Chi46 (rChi46). Chi46 was shown to release mainly (GlcNAc)(2) from colloidal chitin (insoluble chitin) as an exo-type manner and to act on chitosan 7B (DA ca. 30%) and N-acetylchitooligosaccharides (soluble chitins) in an endo-type one.
Ike
Masakazu
M
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
Nagamatsu
Kazuhisa
K
Shioya
Akiko
A
Nogawa
Masahiro
M
Ogasawara
Wataru
W
Okada
Hirofumi
H
Morikawa
Yasushi
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D011994:Recombinant Proteins; D048271:Chitosan; D002688:Chitinases