Purification, characterization, and molecular modeling of pyrolysin and other extracellular thermostable serine proteases from hyperthermophilic microorganisms.

Published on Jan 1, 2001in Methods in Enzymology1.862
· DOI :10.1016/S0076-6879(01)30390-7
W.M. de Vos136
Estimated H-index: 136
W.G.M. Voorhorst1
Estimated H-index: 1
+ 3 AuthorsRoland J. Siezen66
Estimated H-index: 66
Publisher Summary Currently several serine proteases have been purified from hyperthermophiles with a variety of approaches and characterized at the enzyme and, in most cases also the gene level. Those that are genetically characterized all belong to the subtilisin-like family of serine proteases, also known as “subtilases.” Moreover, a variety of homologs of subtilases can be detected in the present databases of sequenced genomes of hyperthermophiles. The first of these serine proteases to be characterized at the enzyme and gene level has been pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus , which is the most thermostable protease to date and retains half of its activity after boiling for several hours. A characteristic feature of pyrolysin and other proteases is the fact that these enzymes are substrates of their proteolytic activity and degrade themselves in a process termed autoproteolysis. This has for a long time hampered the purification of pyrolysin and prevented its further characterization at the molecular level. This chapter discusses the procedures for the biochemical and genetic characterization of pyrolysin and other related thermostable serine proteases and the prediction of their properties by homology comparisons, database searches, and molecular modelling.
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