Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1989-09-12
1993-09-07
Cashion, Jr., Merrell C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435212, 435 712, 435219, 4352521, C12N 952, C12N 950
Patent
active
052428176
ABSTRACT:
Cell-free extracts from Pyrococcus furiosus were found to possess unusually high levels of proteolytic activity as measured by hydrolysis of azocasein; loss in activity was only 30% after incubation for 24 hours at 98.degree. C. and the half-life of proteolytic activity at that temperature was about 60 hours. Furthermore, cell-free extracts incubated at 98.degree. C. in 1% sodium dodecyl sulfate (SDS) for 24 hours yielded an SDS-resistant protease having a temperature optimum of at least 100.degree. C. The enzyme retained at least 40% of its activity when tested at 98.degree. C. by azocasein hydrolysis in the presence of 4M urea, 2M guanidinium chloride, 10 mM dithiothreitol or 150 mM .beta.-mercaptoethanol. The protease was found to have a pH optimum of 6.8 at 98.degree. C. and retained more than 45% of its activity at pH 9.3 and 82% of its activity at pH 4.5 in assays performed at those values. The protease was classified as a metalloprotease through inhibitor studies, and peptide hydrolysis showed trypsin-like cleavage with additional activities.
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Anfinsen Christian B.
Blumentals Ilse I.
Brown Stephen H.
Kelly Robert M.
Robinson Anne K. S.
Cashion Jr. Merrell C.
Celsa B.
Johns Hopkins University
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