Cloning and characterization of an endo-beta-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938.
We describe the identification and expression cloning of two novel enzymes, a beta-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-beta-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0-6.0, and a temperature activity optimum around 40 degrees C. Both enzymes show only low sequence identity to other known enzymes.
Bang
M L
ML
Novo Nordisk A/S, Bagsvaerd, Denmark. bang@embl-heidelberg.de
Villadsen
I
I
Sandal
T
T
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D002480:Cellulase; D016282:Aspartic Acid Endopeptidases