Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli.
4-Methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine oligosaccharides were used as substrates to detect expression of a Streptomyces chitinase in Escherichia coli. Low levels of enzyme were detected when S. plicatus DNA was cloned into a bacteriophage lambda vector (EMBL-4). Subcloning into E. coli plasmids also gave low but detectable levels of enzyme expression. High level expression was achieved by resection of the cloned S. plicatus DNA with Bal31 followed by in-frame fusion to the amino-terminal peptide sequence of beta-galactosidase found in the pUC vectors. The Streptomyces chitinase was secreted into the periplasmic space of E. coli, and its signal sequence was removed. We characterized the activity of the cloned enzyme and compared it to three other purified Streptomyces plicatus chitinases with respect to hydrolysis of the 4-MU oligosaccharides. We found that two of the enzymes form 4-methylumbelliferone much more rapidly from the 4-MU disaccharide than from the trisaccharide. These same enzymes convert the 4-MU trisaccharide primarily to diacetylchitobiose and the 4-MU monosaccharide, a nonfluorescent product. The latter compound is not hydrolyzed appreciably by any of the enzymes. On the basis of these results, we suggest a new definition of "exo" and "endo" chitinase that differs from that found in the literature. We propose that exochitinase activity be defined as processive action starting at the nonreducing ends of chitin chains with release of successive diacetylchitobiose units, and that endochitinase activity be defined as random cleavage at internal points in chitin chains.
Robbins
P W
PW
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Albright
C
C
Benfield
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D002688:Chitinases