Cloning, sequencing, expression, and characterization of thermostability of oligopeptidase B from Serratia proteamaculans, a novel psychrophilic protease

• The gene of Serratia proteamaculans oligopeptidase B was cloned and sequenced.
• The corresponding enzyme (PSP) was produced in Escherichia coli cells.
• PSP enzyme is psychrophilic; Ca2+ increases its destabilization at 37 °С.
• The stable complex (1:1) of E. сoli GroEL with active PSP was obtained.
• Glycerol and complex formation with GroEL stabilize PSP at 37 °С.

Protease from Serratia proteamaculans (PSP) is the first known psychrophilic oligopeptidase B. The gene of S. proteamaculans 94 oligopeptidase B was cloned, sequenced and expressed in Escherichia coli. The unfolding of PSP molecule following heat treatment at 37 °C by measuring fluorescence spectra was examined in parallel with the residual activity determination. The effect of PSP thermostabilization by glycerol at 37–50 °С was revealed. Calcium ions and buffer solution of low molarity cause the opposite effect – the acceleration of PSP inactivation at 37 °C. The thermal stability of PSP molecule in the presence of 0–100 mM CaCl2 was also investigated by means of high-sensitivity differential scanning calorimetry. The artificial reconstruction of the natural complex PSP-chaperonin from S. рroteamaculans was carried out: the stable complex (1:1) of chaperonin E. сoli GroEL with active recombinant enzyme PSP was obtained. It was shown that complex formation with chaperonin promotes PSP thermostability at 37 °C.