Proteolytic enzymes from hyperthermophilic bacteria and processes for their production

US5391489A · kind A · utility

Assignee

• THE JOHNS HOPKINS UNIVERSITY · US

Inventors

• Robert M. Kelly · Ellicott City, US
• Anne K. S. Robinson · Philadelphia, US
• Ilse I. Blumentals · Four Corners, US
• Stephen Brown · Davis, US
• Christian B. Anfinsen · Baltimore, US

Key dates

• Filing date: Jun 30, 1993
• Grant date: Feb 21, 1995
• Expiry date: Jun 30, 2013

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N9/52
• WIPO field: Biotechnology
• WIPO sector: Chemistry

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.