Cloning, expression, and characterization of an antifungal chitinase from Leucaena leucocephala de Wit.
Chitinase cDNAs from Leucaena leucocephala seedlings were cloned by PCR amplification with degenerate primers based on conserved class I chitinase sequences and cDNA library screening. Two closely related chitinase cDNAs were sequenced and inferred to encode precursor proteins of 323 (KB1) and 326 (KB2) amino acids. Expression of the KB2 chitinase from a pET32a plasmid in Origami (DE3) Escherichia coli produced high chitinase activity in the cell lysate. The recombinant thioredoxin fusion protein was purified and cleaved to yield a 32-kDa chitinase. The recombinant chitinase hydrolyzed colloidal chitin with endochitinase-type activity. It also inhibited growth of 13 of the 14 fungal strains tested.
Kaomek
Mana
M
Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Mizuno
Kouichi
K
Fujimura
Tatsuhito
T
Sriyotha
Poonsook
P
Cairns
James R Ketudat
JR
D000935:Antifungal Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D003593:Cytoplasm; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology*; D005658:Fungi / Q000187:drug effects; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity
D000935:Antifungal Agents; D004220:Disulfides; D011994:Recombinant Proteins; D002686:Chitin; D002688:Chitinases