Molecular cloning, functional expression, and mutagenesis of cDNA encoding class I chitinase from rye (Secale cereale) seeds.
A cDNA encoding rye seed chitinase-a (RSC-a) was cloned by rapid amplification of cDNA ends and PCR procedures. It consists of 1,191 nucleotides and encodes an open reading frame of 321 amino acid residues. Recombinant RSC-a (rRSC-a) was produced in the oxidative cytoplasm of Escherichia coli Origami(DE3) in a soluble form by inducing bacteria at a low temperature (20 degrees C). Purified rRSC-a showed properties similar to the original enzyme from rye seeds in terms of chitinase activity toward a soluble substrate, glycolchitin, and an insoluble substrate, chitin beads, in chitin-binding ability to chitin, and in antifungal activity against Trichoderma sp. in vitro. rRSC-a mutants were subsequently produced and purified by the same procedures as those for rRSC-a. Mutation of Trp23 to Ala decreased the chitinase activity toward both substrates and impaired the chitin-binding ability. Furthermore, the antifungal activity of this mutant was weakened with increasing of the NaCl concentration in the culture medium. Complete abolishment of both activities was observed upon the mutation of Glu126 to Gln. The roles of these residues in both activities are discussed.
Laboratory of Protein Chemistry and Engineering, Graduate School of Bioresource and Environmental Science, Kyushu University, Fukuoka, Japan.
Takayuki
T
Ohnuma
Toki
T
Taira
Takeshi
T
Yamagami
Yoichi
Y
Aso
Masatsune
M
Ishiguro
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics / Q000502:physiology; D011061:Poly A / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012434:Secale / Q000235:genetics*; D014242:Trichoderma / Q000187:drug effects
D017931:DNA Primers; D018076:DNA, Complementary; D002686:Chitin; D011061:Poly A; D002688:Chitinases