Elucidation of the thermal stability of the neutral proteinase II from Aspergillus oryzae.
The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.
Tatsumi
H
H
Research and Development Division, Kikkoman Corporation, Chiba, Japan.
Ikegaya
K
K
Murakami
S
S
Kawabe
H
H
Nakano
E
E
Motai
H
H
D001236:Aspergillus oryzae / Q000201:enzymology*; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D004220:Disulfides; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C070788:neutral protease II, Aspergillus oryzae