A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae.
We have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consisting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%-44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN') composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.
Tatsumi
H
H
Research and Development Division, Kikkoman Corporation, Chiba, Japan.
Ogawa
Y
Y
Murakami
S
S
Ishida
Y
Y
Murakami
K
K
Masaki
A
A
Kawabe
H
H
Arimura
H
H
Nakano
E
E
Motai
H
H
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics*; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D013381:Subtilisins / Q000235:genetics
D004271:DNA, Fungal; D004792:Enzyme Precursors; D012697:Serine Endopeptidases; D013381:Subtilisins; C026109:oryzin