Secretion of an enzymatically active Trichoderma harzianum endochitinase by Saccharomyces cerevisiae.
A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENCI) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENCI amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 degrees C.
Draborg
H
H
GeneSearch, Novo Nordisk A/S, Bagsvaerd, Denmark.
Christgau
S
S
Halkier
T
T
Rasmussen
G
G
Dalbøge
H
H
Kauppinen
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology*
D011994:Recombinant Proteins; D002688:Chitinases