Cloning, expression and decoding of the cold adaptation of a new widely represented thermolabile subtilisin-like protease
Acevedo, J. P.
Asenjo de Leuze, Juan
Andrews Farrow, Bárbara
Cita de ítem
Journal of Applied Microbiology 114, 352--363
Artículo de publicación ISI
Aims: Cloning, expression and characterization of a new cold-adapted
protease with potential biotechnological application, isolated from Antarctic
Method and Results: A subtilisin-like gene was isolated from several Antarctic
bacterial genus using CODPEHOP-designed primers and a genome walking
method. This gene encodes a precursor protein, which undergoes an
autocatalytic cleavage resulting in a 34 6 kDa active cold-adapted protease with
a maximum activity at 25–35°C and optimum pH of 8 0–9 0. It showed a
higher catalytic efficiency at lower temperatures compared to its mesophilic
counterpart. Heat-induced inactivation resulted in a very low melting point.
Local packing analysis using the homology model indicated Ala284 as an
important cold-adaptation determinant, which was corroborated by the sitedirected
Conclusions: A new thermolabile subtilisin-like protease has been successfully
cloned and analysed, and an important hot spot in the evolution of the cold
adaptation and substrate specificity of this enzyme was identified and tested.
Significance and Impact of the Study: This work reports a new cold-adapted
protease with a vast representation amongst Antarctic genus, suggesting
therefore its evolutionary success in this cold environment. Likewise, important
sites for genetic potentiation have been identified, which are extrapolated to
other enzymes of the same kind.