Heat-labile proteases in molecular biology applications

Abstract

Thermolabile proteases were identified in three Gram-negative psychrotrophic bacteria. The protease from the psychrotrophic strain A9 was purified and its application to common molecular biology techniques was demonstrated. Heat-stable molecular biology enzymes (Taq polymerase and PvuII) were digested by a heat-labile protease, which was then inactivated by a mild heat treatment. The clear benefit of using heat-labile proteases arises in situations where further reactions may be accomplished without an intermediate purification step, thereby saving time and avoiding the possibility of product loss.

Alkaline Proteases from Patagonian Bacteria

“…For example, high enzyme activity at low/ moderate temperatures is an important feature for energy saving in processes that would be performed at room or tapwater temperature. The use of thermosensible enzymes also allows their selective inactivation in complex mixtures (Gerday et al 2000 ;Moran et al 2001 ).…”

Advances in cold-adapted enzymes derived from microorganisms

“…SIB1 structure (Budiman et al, 2011(Budiman et al, ). 10.3389/fmicb.2023 Frontiers in Microbiology 07 frontiersin.org under mild thermal conditions and ensure the subsequent reactions are not interfered with (Moran et al, 2001). Alkaline phosphatase dephosphorylates DNA and is often used to clean up PCR products and prevent multiple cycles before cloning, but persistence of alkaline phosphatase activity can interfere with subsequent reactions.…”

“…Gentle thermal treatment of cold-adapted enzymes causes irreversible enzymatic inactivation without interfering with subsequent reactions during in vitro molecular biology. Moran et al (2001) isolated a strain of psychrophilic bacteria from seawater that secreted a cold-adapted protease (A9) that could digest restriction endonuclease PvuII to prevent it from cleaving DNA without interfering with DNA quality. However, the enzyme can also interfere with Taq polymerase activity, so A9 protease can be added to a reaction, then the enzyme can be inactivated to terminate its reaction under mild thermal conditions and ensure the subsequent reactions are not interfered with ( Moran et al, 2001 ).…”